Magnetic resonance imaging (MRI) is regularly used for stereotactic imaging of Gamma Knife (GK) radiosurgery patients for GK treatment planning. MRI‐induced thermal injuries have occurred and been reported for GK patients with attached metallic headframes. Depending on the specific MR imaging and headframe conditions, a skin injury from MRI‐induced heating can potentially occur where the four headframe screws contact the skin surface of the patient's head. Higher MR field strength has a greater heating potential. Two primary heating mechanisms, electromagnetic induction and the antenna effect, are possible. In this study, MRI‐induced heating from a 3T clinical MRI scanner was investigated for stereotactic headframes used in gamma radiosurgery and neurosurgery. Using melons as head phantoms, optical thermometers were used to characterize the temperature profile at various points of the melon headframe composite as a function of two 3T MR pulse sequence protocols. Different combinations of GK radiosurgery headframe post and screw designs were tested to determine best and worst combinations for MRI‐induced heating. Temperature increases were measured for all pulse sequences tested, indicating that the potential exists for MRI‐induced skin heating and burns at the headframe attachment site. This heating originates with electromagnetic induction caused by the RF fields inducing current in a loop formed by the headframe, mounting screws, and the region of the patient's head located between any of the two screws. This induced current is then resistively dissipated, with the regions of highest resistance, located at the headframe screw–patient head interface, experiencing the most heating. Significant heating can be prevented by replacing the metallic threads holding the screw with electrically insulated nuts, which is the heating prevention and patient safety recommendation of the GK manufacturer. Our results confirm that the manufacturer's recommendation to use insulating nuts reduces the induced currents in the headframe nearly to zero, effectively preventing heating and minimizing the likelihood of thermal injury.PACS numbers: 87.57.‐s, 87.61.‐c, 87.61.Tg, 87.57.c‐ Important MRI safety issues that may be new to the radiation oncology clinic include potential damage and injury to property, patients and staff from several sources: the rapid acceleration of nearby ferromagnetic objects by the high‐static magnetic field ;(–) the gradient fields, which have been shown to induce nerve stimulation in humans; the cryogenics, which can cause severe frostbite, suffocation, and substantial explosions if the pressure relief system of the cryogen containers become defective; and the radiofrequency (RF) fields, which are likely the primary source of MRI‐induced thermal injury.Magnetic resonance imaging (MRI) is increasingly used in radiation oncology departments for radiation treatment planning because of the excellent contrast for soft tissues and tumors.–3 MR and by our clinic's use of a dedicated 3T MR simulator that serves as an integral part of an active GK radiosurgery program. The causes of reported MRI‐induced thermal injuries and burns are often not well understood, are sometimes described as unknown or mysterious, and seem to originate with different heating mechanisms. In some cases, burns were associated with wires used with electronic monitoring equipment or implanted biomedical devices such as pacemakers, while in other cases, thermal injuries have occurred with no wires present near the patient, in the extremities or around tattoos, for instance. While the specific situations leading to these injuries may be difficult to pinpoint, the heating mechanisms causing them are not mysterious. They are the well‐understood physical phenomena of electromagnetic induction and the antenna effect, both originating with the radiofrequency (RF) outputs of the MRI machine.This study is motivated by the increasing number of reports of MRI‐induced patient thermal injuries, including burns,,–23 Beginning at approximately the same time, reports to the US Food and Drug Administration (FDA) documented thermal injuries due to MR‐induced heating for patients wearing stereotactic headframes.(–)In 2003, headframe and GK manufacturer, Elekta,  notified users of the availability of “insulated posts”, with the stated use for “high tesla MR units and high frequency MR sequences”.25 Beg The use of uninsulated posts is permitted for X‐ray‐only procedures, such as CT scans or biplanar projection angiography.The physical explanation of these reported thermal injuries has not been given. Thus, this study seeks to prevent MRI‐induced burns in GK patients by first understanding the physical mechanisms that could lead to these injuries and, subsequently, validating the technique recommended to prevent them. The manufacturer's recommended burn prevention technique is to replace the tapped holes at the GK headframe screw–post interface, a metal‐to‐metal junction, with snap‐in insulated nuts. The use of the insulated headframe posts is required for both 1.5 T and 3 T MRI scans.34 The 1) resistive heating from currents induced by direct electromagnetic induction, 2) the unlikely coincidental presence of a conducting loop arranged perpendicular to the RF field and containing the right combination of inductance and capacitance to result in a resonant frequency equal to that of the MRI RF field , and 3) the antenna effect, which is antinodal heating at the tips of wires or other conductors of appropriate length that act as antennas. A brief review of these potential heating mechanisms is presented.Previous studies of MRI‐induced burns considered three potential heating mechanisms:,16 1E¯ is the electric field, l¯ is the distance around the loop, B¯ is the magnetic flux density, and a¯ is the cross‐sectional area enclosed by a conducting loop. This can be stated more simply as:V is the voltage induced in the loop, B is the magnetic field, and t is time. In this case, the rapidly changing magnetic fields induce a current in loops of wire or other conducting material, with the area enclosed by the loop oriented perpendicular to the changing magnetic field. The voltage in turn induces a current:V is the voltage and R is the resistance of the loop. The induced current is then dissipated as heat at a rate:The first mechanism, electromagnetic induction, is described by Faraday's law: different diameter loops of copper wire were placed perpendicular to the RF field and the temperature rise was recorded. Larger diameter loops resulted in more heating, consistent with Faraday's law. No resistor was placed in the loop and, therefore, the resistive heating was distributed evenly around the loop, resulting in a temperature rise along the whole loop. Had a resistor been placed in the loop, most of the heat would have been dissipated locally at and near the resistor, resulting in a higher temperature rise over a much smaller region of the loop. Whether the induced currents originated with the RF field or with the gradient field was not resolved in the Dempsey study. However, direct measurement of the induced voltage waveform in a loop of wire showed that the source of the heating is primarily the RF field. Subsequent measurements confirmed this result, showing that reducing the magnitude of the RF signal reduces the induced voltage in the loop of wire.In the study by Dempsey et al.16 dif found very high temperature rises of up to 61°C in loops with appropriately valued inductance and capacitance to cause resonance. The resonant frequency of the loop is given by:Resonance heating is a second possible heating mechanism. Dempsey et al.16 fouIn practice, it is unlikely that the loops described above will by coincidence happen to have the appropriate values of inductance (L) and capacitance (C) so that the resonant frequency matches the frequency of the MRI machine; however, if they do, there will be substantial heating.~λ/2, an electromagnetic oscillation (resonance) will be produced with a node in the center of the wire and an antinode at either end. The maximum amplitude will occur at the antinodes, resulting in the ends of the wire heating to high enough temperatures for thermal injury. Dempsey and Condon showed that for a 1.5 T MRI machine with operating frequency of 63.87 MHz, the antenna effect occurred in a wire length of ~220 cm, in rough agreement with the theoretical expectation of λ/2 approximately equal to 235 cm. Local temperature increases of up to 63°C were observed at the wire tip, high enough to cause burns to the experimental apparatus, and certainly high enough to cause severe or ablative tissue injury. This mechanism is suspected in a number of cases of MRI induced patient burns, including pulse oximeter wires. It is extremely important to realize that the length of an ideal λ/2 antenna inside the human body is reduced by an order of magnitude, because the EM wavelength is significantly reduced due to the dielectric and electrical properties of human tissues. For instance, pacemaker leads in human‐equivalent soft tissue will resonate at lengths of ~20 cm for 1.5 T (64 MHz), or ~10 cm for 3 T, not the 220 to 235 cm in‐air values reported earlier.The third mechanism that can lead to heating is the antenna effect, which occurs when a wire of appropriate length is exposed to the RF frequency and acts as an antenna. This effect is exploited in half‐wavelength dipole antennas for receiving radio signals. If a length of wire or other conductor is nd Condon15 shoB¯ is the magnetic field, E¯ is the electric field, μ is the permeability, ɛ is the permittivity, and σ is the conductivity. The solutions to the resulting wave equations are plane waves, but with a complex wave numberThe antenna effect in conductive tissues is explained as follows. The human body is conducting, and thus the behavior of incident EM waves is described by Maxwell's equations applied to conducting media. For this case, Ampere's law is: It is also important to note that antenna effect heating depends on the angle of the conductor with respect to the applied EM wave and is maximized when the length of the conductor is parallel to the direction of the EM wave.The wavelength, λ, in the human body is thus given by: and head phantoms . For the brain, for 3 T, the wavelength is estimated to be 25.5 cm, suggesting that an implant length of ~12.75 cm would be susceptible to antenna effect heating. At 7 T, wavelength becomes 10.6 cm leading to potential antenna effect heating for 5.3 cm implants. These implant dimensions give an indication of possible conductor lengths of relevance for antenna effect heating for GK patients wearing headframes.The antenna effect for metallic implants has been studied in tissue equivalent body20 and~62 cm, is too short for antenna effect heating at 3 T  or 1.5 T . Also, the typical ~5−10 mm length of the screws embedded in the head surface is less than the ~13 cm length expected for the antenna effect in the human head to occur. Thus, the cause of heating is most likely due to the induction of currents in loops formed by the headframe and the tissue of the patient's head, with the area of the loop oriented perpendicularly to the rapidly changing magnetic fields. Because the heat source is resistive heating, P=i2R .16 Als=i2R Eq. , one neeThe following experimental results using melon phantoms show that heating occurs during standard MR brain scans near attached GK headframe screws, the heating mechanism is electromagnetic induction, the amount of heating depends on the material type of the headframe screws and posts, and the use of insulated headframe posts renders the induced currents and associated resistive heating negligible.R~1 MΩ within the outer shell of the melon, but decreasing to ~50−300kΩ if the inner pulp is penetrated by the meter lead , and 2) in order to induce increased heating, an enhanced 13 minute fast spin echo sequence . Temperature was measured using two MR‐compatible fiber optic thermometers, . The temperature is determined via a temperature sensitive phosphor located at the probe tip and energized by an LED pulse. Thermal connections to the headframe screws were made by careful adhesion of the more thermally conductive side (as opposed to the end) of the optical thermometer to the tip of the screw  nuts Fig. . These c~235 cm, which corresponds to λ/2~117 cm. The longest posts on the GK headframe are ~15 cm and the overall unwrapped length is ~62 cm, much shorter than the required ~117 cm necessary for antenna heating to occur in air. Antenna‐effect heating was experimentally ruled out based on temperature measurements that showed: 1) no heating for the assembled headframe suspended in air, and 2 no heating for screws of various lengths embedded at depths of 0.5 cm in the melon. This latter null result is expected based on the nominal EM wavelength produced by 3 T scans for soft tissue (~40 cm) and for brain tissue (~25 cm).The initial experiment was designed to determine which of the two most likely heating mechanisms, the antenna effect or electromagnetic induction, is responsible for the heating that occurs at the GK headframe screw–melon interface. Antenna‐effect heating can be theoretically ruled out because the dimensions of the GK headframe are not large enough for this type of heating to occur. For the 128 MHz RF field of the 3 T magnet, the RF wavelength is (~25 cm).,36Resonance‐induced heating is a remaining phenomenon that could possibly result in heating. We have measured negligible capacitances and inductances in the GK headframe–melon composite and there is no evidence of resonance heating occurring in this experiment.Temperature measurements versus time as a function of position between the two anterior screws show that heating is greatest at the tapered part or tip of the screw along the melon–screw interface and that heating decreases with increasing distance from the screw tip Fig. . Specifi~100 kΩ. Experiments repeated with the thermometers placed in this deeper region show significantly decreased heating, owing to the reduced resistance . The manufacturer's recommended method works because the heating mechanism is electromagnetic induction. It is important to note that all four posts should be insulated, because as shown in Fig. Replacing the uninsulated posts with the insulated posts prevented all but negligible heating of the melon headframe composite Fig. , which i~30−50 cm in air, and as small as just a few cm in the human body, depending on the type of tissue or organ where the implant is located, with the amount of heating dependent on the implant's depth below the surface and its angle with respect to the applied EM wave. This effect may be a major concern for patients with small implants in the human body, such as stents or aneurysm clips, that could pose a severe burn hazard at very high field strengths. Thus, with current MRI scanners operating at fields up to 7 T, the antenna effect may become the dominant heating hazard in the near future.The manufacturer's recommended heating prevention method, however, works only to prevent heating by electromagnetic induction and would not prevent antenna‐effect heating. An entirely different method would be required to prevent heating by the antenna effect. Fortunately, the GK headframe geometry is not a problem at 1.5 T or 3 T field strengths, because the dimensions are too small to heat by this method. However, if the static field operated at 7–10 T, the implant dimensions required for the antenna effect would be decreased to One of the key implications of this study is that therapists and other health professionals who image GK patients with headframes in MRI scanners and have not yet obtained insulated posts need to be aware of new safety regulations (such as the requirement to use only insulated posts for MRI scans of GK patients with headframes). It may be useful for GK health professionals to obtain additional training to understand how RF interacts with and potentially heats the human body through resistive heating in loops. It should be understood how current loops could potentially form through complex combinations of parts of a patient, a patient monitoring device, or instrument wiring via capacitive coupling, especially for higher field MRI units. The more complex heating mechanism of the antenna effect should also be understood by GK health professionals so that they are aware that thermal injuries can occur by mechanisms other than EM induction. Tables of conductivities and permittivities of different organs and tissues within the human body should be made readily available, along with dimensions of conducting implants that lead to antenna‐effect heating in various tissues in different field strength MRI scanner.This study shows that heating caused by the RF field of a 3T MRI scanner due to electromagnetic induction, as described by Faraday's law, occurs at the GK headframe screws when melon phantoms are mounted in the headframe during stereotactic MR imaging. Titanium screws combined with the uninsulated alumina posts result in maximum heating at the screw tips. This heating can be greatly reduced to negligible levels by the use of insulating nuts (the manufacturer's recommended procedure) that electronically separate the metallic screws from the posts. This method would, in principle, work for any conducting headframe post material .There is increased risk of thermal injury at field strengths higher than 3T. The antenna effect was ruled out as the cause of headframe screw heating. However, above 3T it poses an increased risk of internal heating for GK and other patients with metallic implants having lengths suitable for standing wave formation (such as pacemaker wires), because resonant length decreases with increasing field strength and RF frequency. Thus, injury may be possible with even small‐dimension implants, such as aneurysm clips and stents. Electromagnetic induction, the principal heating mechanism identified for GK patients with attached headframes, also poses an increased risk because higher RF frequencies at field strengths above 3T may create unintended current paths through unintended capacitances. Thus, at sufficiently high magnetic fields beyond 3T, the electrically insulating nuts may no longer protect against induced heating. Their effective protection will need to be validated before use for GK patients with headframes undergoing stereotactic very high‐field  MR imaging.The authors would like to thank S. Shave at General Electric Healthcare, O. Eriksson at Elekta, and Lisa Wilkins and Darrell Sloan at the Department of Radiation Oncology at Wake Forest University for their time and help with this project. This work is supported by the TRADONC post‐doctoral training program at Wake Forest University School of Medicine, funded by Grant No. NCI‐32 CA113267.


Heterogeneous nucleation of ice from supercooled liquid water by some atmospherically relevant nucleators is enhanced by ammonium salts and suppressed by alkali halides. 5. This concentration was chosen for a survey across multiple solutes–nucleant combinations since it had a minimal colligative impact on freezing and is relevant for activating cloud droplets. Other nucleators, for instance a silica gel, are unaffected by these ‘solute effects’, to within experimental uncertainty. This split in response to the presence of solutes indicates that different mechanisms of ice nucleation occur on the different nucleators or that surface modification of relevance to ice nucleation proceeds in different ways for different nucleators. These solute effects on immersion mode ice nucleation may be of importance in the atmosphere as sea salt and ammonium sulphate are common cloud condensation nuclei (CCN) for cloud droplets and are internally mixed with ice-nucleating particles in mixed-phase clouds. In addition, we propose a pathway dependence where activation of CCN at low temperatures might lead to enhanced ice formation relative to pathways where CCN activation occurs at higher temperatures prior to cooling to nucleation temperature.Heterogeneous nucleation of ice from aqueous solutions is an important yet poorly understood process in multiple fields, not least the atmospheric sciences where it impacts the formation and properties of clouds. In the atmosphere ice-nucleating particles are usually, if not always, mixed with soluble material. However, the impact of this soluble material on ice nucleation is poorly understood. In the atmospheric community the current paradigm for freezing under mixed phase cloud conditions is that dilute solutions will not influence heterogeneous freezing. By testing combinations of nucleators and solute molecules we have demonstrated that 0.015 M solutions (predicted melting point depression <0.1 °C) of several ammonium salts can cause suspended particles of feldspars and quartz to nucleate ice up to around 3 °C warmer than they do in pure water. In contrast, dilute solutions of certain alkali metal halides can dramatically depress freezing points for the same nucleators. At 0.015 M, solutes can enhance or deactivate the ice-nucleating ability of a microcline feldspar across a range of more than 10 °C, which corresponds to a change in active site density of more than a factor of 10 Ice nucleation is an important process in several fields. It has relevance to the atmosphere,aw.et al.As such, the role of solute molecules in the freezing of liquid water is a process of fundamental and applied interest. In the case of homogenous ice nucleation (ice nucleation in the absence of any INPs), it is generally, accepted that the ‘water activity criterion’et al.4)2SO4, organic compounds such as ethylene glycol and glycerol, organic acids and the polymer polyethylene glycol 300, among others. They found that an adapted form of the water activity criterionaw = 0.305 for homogenous nucleation whereas Δaw,het for heterogeneous nucleation ranged from 0.100 for the nonadecanol monolayers used to 0.195 for ATD. Several other studies with non-reactive solutes are consistent with the conclusion that heterogeneous freezing can be described by Δaw.Much of the ice nucleation which occurs in the troposphereIn contrast, there is evidence of acids causing reductions in the ice nucleation activity of certain mineral dusts that is greater than would be expected from the water activity of the acid solutions. These reductions are attributed to the destruction of, or coating of, ice-nucleating sites by the acid.Pseudomonas Syringae4I can enhance the ice nucleation activity of PbI2. Zobrist et al.4)2SO4, AgI exhibited enhanced nucleation properties. As their experimental technique involved synthesis of AgI in the presence of solute molecules they attributed this to changes in crystal habit caused by changes in concentration of Ag+ in the presence of (NH4)2SO4. Overall, there are enough exceptions to the water activity criterion approach for heterogeneous freezing in the literature to warrant further investigation.Reischel and ValiWe have tested the ice nucleation activities of various combinations of nucleators and solutes using the microlitre Nucleation by Immersed Particles Instrument (μl-NIPI). This instrument has been described in detail previouslyns(T), for most samples. Specific surface areas cannot be reasonably determined for humic acid, hence ns(T) cannot be determined. For the other materials, ns(T) can be calculated using:11n(T) is the number of droplets frozen at temperature (T), N is the total number of droplets in the experiment and A is the surface area of nucleant per droplet. ns(T) is a site specific measure of ice nucleation efficiency which does not account for the effects of cooling rate (i.e. time) dependence.–1). Uncertainty in ns(T) was calculated using simulations of possible site distributions propagated with the uncertainty in surface area of nucleator per droplet as described in Harrison et al.26In order to facilitate comparison of data presented here with other studies we have calculated the ice nucleation active surface-site density, 4)2SO4.We also conducted ice nucleation experiments by placing individual chips of a feldspar rich rock, which were approximately 1 mm along the longest dimension, onto a hydrophobic slide. We then pipetted 1 μl MilliQ water droplets onto them and conducted freezing experiments using μl-NIPI as usual. We removed the rock chips from the water droplets, dried them and repeated the experiment in a 0.015 M solution of (NH4)Cl, (NH4)2SO4 and (NH4)OH. These compounds were purchased from Sigma-Aldrich. Previous studies of the impact of solutes on heterogeneous freezing have focused on relatively concentrated solutions droplets relevant for cirrus conditions where there is a significant colligative melting point depression.4Cl and KCl4)2SO4 4)2SO4.For this study the solutes we used were KCl, NaCl, NaI, .In ns)/dT from the BCS376 ice nucleation activity parameterization in Atkinson et al.,ns of a factor of about 105.Clearly, ice nucleation by BCS 376 microcline is sensitive to the identity of solute ions and does not follow the water activity criterion at the solute concentration investigated here. The variation in freezing point depressions caused by the alkali metal halides has the order KCl > NaCl > NaI. The effect is striking, with KCl causing a decrease in freezing temperatures of about 8 °C and ammonium salts enhancing freezing by 3 °C, a spread of more than 10 °C. Taking dlog(4)2SO4 and NaCl on nucleation by BCS 376 microcline, the results are presented in 4)2SO4 there was little difference in the level of enhancement between the 1.5 × 10–2 M and 1.5 × 10–3 M solution while the enhancement in freezing activity was smaller for the 1.5 × 10–4 M solution, around 1.5 °C rather than 3 °C. This strongly suggests there is a limit to the extent to which the presence of ammonium salts can enhance ice nucleation efficiency, particularly when viewed in combination with the data in We have also examined the effect of varying concentration of (NH4)2SO4, KCl and NaCl. i.e. (NH4)2SO4 increased ice nucleation temperatures while NaCl and KCl reduced them (4)2SO4 did not significantly change freezing temperatures of ATD droplets, addition on KCl did reduce freezing temperatures at colder temperatures. We observed no temperature shift for experiments conducted using silica gel and humic acid as nucleators. The observation that solutes can influence nucleation by some materials, but not others is particularly interesting and will be addressed later. Also, the fact that the freezing temperatures do not shift for some materials confirms that the water activity of the solutions used in those cases is indeed equal to unity to within the sensitivity of μL-NIPI.We tested five other nucleators with 0.015 M solutions of NH2SO4, KClced them  whereas ced them . ATD shoet al.via a similar mechanism and our results are indeed compatible with the results of Zobrist et al.The impact of solutes on ice nucleation by silica and humic acid have been tested previously so some comparison to literature data is possible. The silica gel we have tested is amorphous, as were the silica balls tested by Zobrist et al.The humic acid we have tested is probably similar to humic substances tested by Knopf and Alpertet al.4)2SO4 but not with the results for KCl. It is interesting that only the lower temperature points for the ATD/KCl experiments are impacted strongly. It is not known what component of ATD is responsible for its ice-nucleating ability. It is certainly a complex mixture of many minerals.et al.et al.Droplets containing ATD were frozen with various solutes by Zobrist 4)2SO4, NH4Cl and NaCl on kaolin from Reischel and Vali4)2SO4 and NH4Cl of approximately 0.01 M. The other direct point of comparison, 0.01 M NaCl, does not agree well. We observed a shift of 5.5 °C to colder temperatures while Reischel and ValiReischel and ValiNevertheless, it is clear that at the concentrations we have investigated ammonium salts can enhance ice nucleation and alkali halides can inhibit it. Our work suggests that some nucleators are not affected by solutes however it is clear from Reischel and ValiIt has been suggested that aggregation of particles in droplet freezing experiments, leading to a loss of particles from suspension and a reduction of surface area within aggregates of particles, may be responsible for discrepancies between different instruments used for measuring ice nucleation.4)2SO4. Median freezing temperature shifted to –11.8 ± 0.4 °C. This demonstrates that the ice nucleation enhancing effect of (NH4)2SO4 does not depend on the nucleator being in powder form and that the solute effect on ice nucleation is not related to particle aggregation.To test this we took individual chips of a feldspar rich rock of approximately 1 mm diameter and placed them onto a hydrophobic slide. We then pipetted 1 μl Milli-Q water droplets onto them and conducted a freezing experiment using μl-NIPI as usual. The left hand panel of As there is no way in which aggregation can occur in this system we conclude that aggregation of particles is not the reason for solute effects on ice nucleation we have observed. We are therefore left to consider other mechanisms by which solutes may impact ice nucleation in ways beyond the water activity criterion.4)2SO4 and NaCl are common components of atmospheric aerosol,These results have significant and obvious atmospheric implications.  is that there is very little understanding of why any particular substance should nucleate ice efficiently. Traditionally, it has been assumed that a mixture of strong hydrophilicity and good lattice match to ice will cause a substance to nucleate ice efficiently.The results we present here suggest an avenue to help unpick the tricky problem of the mechanisms by which heterogeneous nucleation of ice from liquid water occurs. Clearly, various solutes influence different nucleators differently with respect to their ice nucleation properties. As such it may be possible to classify nucleators by their responses to the presence of solutes.2SO4 which will react with and cause the dissolution of some surface components). Hence, it seems that the presence of solutes either directly influences the formation of critical nuclei or alters the active sites on the surface in ways which promote ice nucleation. This alteration could change the strength of interaction of the nucleator with liquid water or could conceivably subtly alter the spatial location of surface groups on the nucleator, giving a better surface for ice nucleation.It is known that ice nucleation by feldspar minerals is strongly influenced by their ‘microtexture’ and most probably by associated topographical features.via different mechanisms.et al.It has been shown that in computational models different surfaces can nucleate ice In the light of these ideas about the how chemical effects can influence ice nucleation, we can see three broad categories of mechanism by which ammonium salts and alkali metal halides might influence ice nucleation by certain nucleators:4+ in the nucleant surface enhances ice nucleation by altering surface properties in a way conducive to efficient ice nucleation. Conversely, insertion of alkali metal cations into surfaces does the reverse, inhibiting ice nucleation. Nucleators that cannot readily incorporate cations into their structures are not affected in the same way.(1) Nucleant modification: replacement of cations with NH4+ onto the nucleator surface changes the strength of the interaction of water with the surface in a way favorable to ice nucleation. Conversely, adsorption of alkali metal cations to the surface inhibits ice nucleation or else metal cations fail to adsorb to the surface.(2) Adsorption on the nucleant: adsorption of NH4+ with water alters the nature of the critical ice nucleus in a way that encourages ice nucleation. Conversely, aqueous alkali metal cations disrupt critical nucleus formation. Different nucleators nucleate ice via different mechanisms, which are impacted differently by solutes. There are various potential mechanisms by which disruption or promotion of critical ice nuclei can occur, which also potentially apply to homogeneous ice nucleation. These are discussed below.(3) Aqueous phase interactions: the interaction of aqueous NHet al.4)2SO4, NaCl, KCl and NH4Cl. As far as we are aware no data is available for homogeneous nucleation in the presence of NH4OH or NaI. Overall, it appears that the water activity criterion is a good approximation for homogeneous nucleation and exceptions to this have been attributed to contamination of the solute compounds used.Any aqueous phase interaction which impacts heterogeneous ice nucleation might reasonably be expected to also play some role in homogeneous nucleation. Hence, it is perhaps surprising the water activity criterion appears to account for solute induced shifts in homogeneous ice nucleation temperatures. It might be expected that different solute species would influence interfacial tension between solution and the critical nuclei, and the diffusion activation energy for a water molecule to cross the solution–ice interface. For the solute compounds we have used in this study the seminal paper by Koop We now make some observations about the physical and chemical characteristics of several of the nucleators in order to suggest some possible explanations for our observations and link to the three proposed options for the mechanism by which solutes influence ice nucleation.4+ can substitute for the cations in feldspars to form ammonium feldspar• It is also known that NH4)2SO4 at warmer temperatures, relatively unaffected by (NH4)2SO4 at colder temperatures and suppressed by NaCl at colder temperatures) suggests that the two effects are not necessarily caused by the same mechanism with reversed influences from ammonium salts and alkali halides.• The split and more complex response to solutes observed for ATD  concentrations of ammonium salts and alkali halides can have a profound influence on the ice-nucleating efficiency of some nucleators, but not others. The current paradigm is that temperature shifts in heterogeneous nucleation temperatures due to solutes in a freezing solution depends entirely on the water activity of the solution, and does not depend on the specific solute species.This work is potentially of great importance for understanding ice nucleation in mixed phase clouds, which have a profound impact on climate,46Regardless of applications, the solute effects on ice nucleation provides a potential route to differentiating mechanisms of immersion mode ice nucleation by different nucleators and therefore a route to understanding these mechanisms. Further work looking at different solutes, different nucleators and concentration dependences may help to unpick the puzzle.There are no conflicts to declare.


In vitro studies showed DI to be a competitive inhibitor of ROCKII . This finding was supported by in silico molecular surface docking of DI with the ROCKII ATP-binding pocket. Time course analysis of myosin regulatory light chain (LC20) phosphorylation catalyzed by ROCKII in vitro revealed a significant decrease upon treatment with DI. ROCKII signaling was investigated in situ in human coronary artery vascular smooth muscle cells (CASMCs). ROCKII down-regulation using siRNA revealed several potential substrates involved in smooth muscle contraction  and actin cytoskeletal dynamics (cofilin). The application of DI to CASMCs attenuated LC20, Par-4, LIMK, and cofilin phosphorylations. Notably, cofilin phosphorylation was not significantly decreased with a novel ZIPK selective inhibitor (HS-38). In addition, CASMCs treated with DI underwent cytoskeletal changes that were associated with diminution of cofilin phosphorylation. We conclude that DI is not selective for ZIPK and is a potent inhibitor of ROCKII.The selectivity of (4Z)-2-(4-chloro-3-nitrophenyl)-4-(pyridin-3-ylmethylidene)-1,3-oxazol-5-one (DI) for zipper-interacting protein kinase (ZIPK) was previously described by in silico computational modeling, screening a large panel of kinases, and determining the inhibition efficacy. Our assessment of DI revealed another target, the Rho-associated coiled-coil-containing protein kinase 2 (ROCKII).  MLCK phosphorylates the regulatory light chains (LC20) of myosin II on Ser19, resulting in contraction of smooth muscle through increases in actin-activated myosin MgATPase activity and cross-bridge cycling62+]i is the primary determinant of VSM contraction, it is the balance between MLCK and MLCP activities that dictates the contractile activity of VSM. Indeed, MLCP functions independently of Ca2+-calmodulin and can be regulated by G protein-coupled receptors and downstream signaling modules. A variety of studies have demonstrated that MLCP activity (and hence Ca2+ sensitization) is regulated by a number of protein kinases that act to phosphorylate the myosin phosphatase-targeting subunit (MYPT1)62+ sensitization is not dependent on MLCP inhibition but rather on the direct phosphorylation of LC2052+-independent diphosphorylation of LC20 at both Thr18 and Ser19 induces force comparable to that evoked by MLCK-catalyzed phosphorylation at Ser19, but force is sustained due to a reduction in the rate of dephosphorylation of diphosphorylated compared to monophosphorylated LC202+ sensitization.Smooth muscle plays an important role in the regulation of vascular tone and many other biological functions. Of central importance to the development of vascular smooth muscle (VSM) tone is the variable nature of the relationship between cytosolic free Ca2+ sensitization phenomenon in VSM, with a prominent contribution of Rho-associated coiled coil-containing kinase (ROCK) revealed in the literature311121314161214LIN-11, ISL1 and MEC-3 (LIM) kinase, leading to the inhibition of the actin-severing protein cofilin1521Several protein kinases are linked to the Ca2+ sensitization and VSMC contractility2324252627282024252932232629353632The coordinated regulation of tone via protein kinases is a key functional property of VSM, and it is not surprising that VSMCs possess a variety of signal transduction mechanisms to regulate force development. Zipper-interacting protein kinase  is a Ser/Thr protein kinase that has emerged as a key regulator of Cain vitro characterization of the 2-phenyl-4-(3-pyridinylmethylene)-5(4H)-oxazolone inhibitor  suggested specificity and potency of this compound toward ZIPK41The potential cross-talk between ZIPK and ROCK activities4132P]-ATP was purchased from ICN Biomedical . Antibodies to ROCKII, pSer19-LC20, pThr155-Prostate-apoptosis response (Par)-4, pSer82-Heat shock protein (HSP)-27, pSer3-cofilin, pThr508-LIMK1/pThr505-LIMK2 and LIMK1 were from Cell Signaling ; LC20 and β-actin antibodies were from Santa Cruz Biotechnology ; anti-pThr265-ZIPK was from Abcam ; anti-pThr696-MYPT1 and anti-pThr853-MYPT1 were from EMD Millipore/Upstate Biotechnology ; anti-rabbit IgG coupled to horseradish peroxidase (HRP) was from Chemicon . The Enhanced Chemiluminescence (ECL) Kit was purchased from GE Healthcare , and the Phos-tag acrylamide reagent was from NARD Chemicals . All other chemicals were reagent grade and were obtained from Sigma Chemicals . Myosin regulatory light chains (LC20) were purified from chicken gizzard as previously describedThe DAPK3/ZIPK inhibitors (4Z)-2-(4-chloro-3-nitrophenyl)-4-(pyridin-3-ylmethylidene)-1,3-oxazol-5-one , (4Z)-2-ethylidene(phenyl)-4-(pyridin-3-ylmethylidene)-1,3-oxazol-5-one , and (4Z)-2-(3-methylphenyl)-4-(pyridin-3-ylmethylidene)-1,3-oxazol-5-one  were purchased from ChemDiv . The ROCK inhibitors, GSK269962A (PubChem CID: 16095342) and H1152 (PubChem CID: 448043), along with staurosporine, were acquired from Alexis Biochemicals . HS-38 (PubChem CID: 42938883/T6069135) was synthesized as previously described2, 200 μM ATP (containing 1 μCi [γ-32P]-ATP), and 100 μM LC20 peptide. MLCK reactions also contained purified bovine calmodulin (10 μg/mL) and 0.1 mM free CaCl2. Reactions were initiated by the addition of ATP solution and terminated after 15 min by spotting the mixture onto phosphocellulose P81 paper. After washing three times with 20 mM H3PO4, 32P incorporation was determined by scintillation counting. Kinetic parameters  were determined using the Henri-Michaelis-Menten equation and Lineweaver-Burk plots. In some cases, the phosphorylation of LC20 protein by ZIPK or ROCKII was analysed by Phos-tag SDS-PAGE as previously describedThe phosphorylation of LC20 substrate peptide (KKKRPQRATSNVF) by ZIPK (10 μg/ml), ROCKII (4 μg/ml) or MLCK (4 μg/ml) was assayed at 25 °C in a final volume of 50 μl. The standard kinase reaction mixtures contained 25 mM HEPES buffer , 2 mM MgCl5 CASMCs were seeded in 2 ml antibiotic-free normal growth medium supplemented with FBS. Scrambled- and ROCKII-siRNAs were diluted with siRNA transfection medium (sc-36868) to give a final concentration of 20 nM. The mixture was gently overlaid onto the cells and incubated for 16 h at 37 °C. Normal growth medium  was added, and the cells were incubated for an additional 24 h. Fresh SmGM-2 growth medium was applied, and cells were harvested for analysis after 48 h.Human coronary artery smooth muscle cells  were cultured as previously described5 in DMEM culture medium with 10% (v/v) FBS at 37 °C with 5% CO2. Cells were fixed in 4% (v/v) formaldehyde and then probed at 4 °C overnight with anti-pS3-cofilin (Thermo Scientific Pierce) diluted 1:100 in blocking serum. After washing with PBS, immunoreactivity was detected with goat anti-rabbit AlexaFluor488-conjugated secondary antibody. For F-actin visualization, AlexaFluor488-phalloidin (Molecular Probes) was diluted 1:40 in 1% (w/v) BSA in PBS and then incubated with the cells for 1 h at room temperature. Cells were rinsed with PBS, counterstained with DAPI for 5 min to detect nuclei, and then examined with an InCell 6000 Imaging System . For quantitation, the InCell 6000 Imaging System was programmed to complete whole-well scanning of four independent plates of cells. Ten random visual fields were analyzed from each well, and eight images were taken from each field. The F-actin fluorescence signal was quantified with ImageJ (https://imagej.nih.gov) by splitting the colour channels of the original image. The AlexaFluor488-phalloidin signal was identified by selecting cells of interest using the drawing tool and then applying the analysis parameters of the area and integrated density functions. For the pS3-cofilin signal, the intracellular immunofluorescence was quantified and then normalized to the DAPI nuclear counterstain.Cells were plated at 2 × 102HPO4, and 1.76 mM KH2PO4) for 45 min and then washed with TBST. Nonspecific binding sites were blocked with 5% (w/v) nonfat dry milk in TBST. Membranes were washed with TBST and incubated overnight with anti-LC20 at a 1:1000 dilution in 1% (w/v) nonfat dry milk in TBST. Membranes were incubated for 1 h with HRP-conjugated secondary antibody  and developed with ECL reagent.Whole cell extracts were prepared from CASMC cultures by first washing cells with PBS and then completing cellular lysis with addition of 2X sample buffer (2% (w/v) SDS, 100 mM DTT, 10% (v/v) glycerol, 0.01% (w/v) bromphenol blue, and 60 mM Tris-HCl, pH 6.8) with gentle rocking followed by heating at 95 °C for 5 min. Cellular extracts were resolved by SDS-PAGE and transferred to 0.2-μm nitrocellulose membranes in a Tris/glycine transfer buffer containing 10% (v/v) methanol. Non-specific binding sites were blocked with 5% (w/v) nonfat dry milk in Tris-buffered saline with Tween  Tween-20). Membranes were washed and incubated overnight with primary antibody at 1:1,000 dilution with 1% (w/v) nonfat dry milk in TBST. Membranes were then incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody  in TBST and developed with ECL reagent. LC20 phosphorylation was analyzed by Phos-tag SDS–PAGE as described previously1429In silico docking experiments of DI with the ROCKII (PDB: 2F2U) and ZIPK (PDB: 3BQR) catalytic domain structures were completed with Arguslab software . The Ascore algorithm was used to calculate the binding poses within a grid resolution of 0.4 Å. The docking search was completed without defining the target area or protein pocket, and the docking pose with the minimal energy was taken as the optimal binding mode.n indicating the number of independent experiments. Data were analyzed by Student’s t-test, and P < 0.05 was considered to indicate statistical significance. All statistical analyses were performed using the GraphPad Prism 6.0 program.Data are presented as the mean ± S.E.M., with 32P-phosphoryl transfer assays with LC20 peptide to examine the potential inhibitory effect of DI on contractile kinases, namely ZIPK, ROCKII and MLCK. A pronounced decrease in LC20 phosphorylation was detected upon treatment of constitutively-active ZIPK(1-320) and ROCKII(5-554) with increasing concentrations of DI -2-(4-chloro-3-nitrophenyl)-4-(pyridin-3-ylmethylidene)-1,3-oxazol-5-one (DI) molecule was originally developed by a structure-based screening strategy as a potential therapeutic inhibitor for the treatment of ischemic reperfusion injuryns of DI . Similar of DI 41. A more l/min/μg . The addt to ATP . In addif 130 nM .2+ coordinationGXGXXGXV) of the kinase domain that interface with the phosphate groups of ATPAs shown in LC20 mono- and di-phosphorylation by ROCKII were detected by Phos-tag SDS-PAGE with silver staining . Maximalin situ. The Ser19 phosphorylation site of LC20  toward ROCKII to be similar to that of Y27632 (Ki = 140 nM) and fasudil (Ki = 158 nM) and less than that of H1152 -2-(4-chloro-3-nitrophenyl)-4-(pyridin-3-ylmethylidene)-1,3-oxazol-5-one (DI) molecule is an ATP-competitive inhibitor developed by a structure-based screening strategy as a potential therapeutic agent to inhibit DAPK1 and ZIPK function(s). We set out to study the selectivity and potency of this compound to investigate the role of ZIPK in VSMC signaling pathways linked to cellular contractility and actin cytoskeletal dynamics. Surprisingly, the initial d]pyrimidines  display similar or greater potencies versus DAPK1/ZIPK and do not display any of the off-target liabilities of other published DAPK inhibitors against contractile kinases such as ROCKIId]pyrimidinone core are important for DAPK/ZIPK affinities and are thought to be sufficient to distinguish the selectivity profile of HS38 from other DAPK/ZIPK inhibitor scaffolds. Although HS38 is not associated with any compromising effects on ROCKII activity, the molecule does possess non-selective effects toward the PIM kinases and IRAK4 that must be considered58The first reported small molecule inhibitor of the DAPK family was based on an aminopyridazine scaffold41in vivo administration of DI to spontaneously-hypertensive rats was associated with decreases in blood pressure, reactive oxygen species (ROS) production, inflammatory responses, as well as vascular smooth muscle contractility and hypertrophyIn vitro treatment of human umbilical vein endothelial cells with DI could also suppress tumor necrosis factor (TNF)α-induced inflammatory responses via ROS-dependent mechanisms. In a second study, the DI compound was used in vitro to link ZIPK with PDGF-BB-induced vascular SMC proliferation and migration through p38MAPK and HSP27 pathwaysin vivo treatments with DI were shown to attenuate vascular structural remodeling during carotid neointimal hyperplasia. Both studies provide descriptions of unique roles for ZIPK in smooth muscle and signaling cascades leading to modulation of downstream targets, and in some cases, the influence of ZIPK was also validated in vitro with siRNA knock-down experiments. For example, TNFα-dependent effects on vascular cell adhesion molecule (VCAM)-1 expression, ROS production, and p38MAPK, Akt and JNK signaling were also linked to ZIPK with small interfering RNA treatments.The data presented herein reveal DI to possess similar efficacy toward both ZIPK and ROCKII activities; nevertheless, the DI compound has been used in two recent studies by Usui and colleagues to assess the role of ZIPK in cardiovascular disease31in vivo outcomes. These two studies of DI and ZIPK inhibition in vascular smooth muscle systems profiled the phosphorylation status of key regulatory proteins. TNFα activation was linked to ZIPK-dependent effects on VCAM-1, E-selectin, cyclooxygenase (COX)-2, as well as ROS production, LC20 phosphorylation, and cellular migration of endothelial cells. Some of these proteins have been previously identified as downstream targets of RhoA/ROCKII signaling. For example, previous reports suggest that the activation of ROCK during TNFα-induced inflammation in lung endothelial cells leads to increased LC20 phosphorylation and cellular permeability15in vivo DI treatment are restricted to ZIPK, a secondary effect of ROCKII signal attenuation, or some combination of the two signaling pathways.Regrettably, the concurrent inhibition of ROCKII during DI treatment could provide an equally rational mechanism for some A complex integration of cytoskeletal events coincident with the activation of the actomyosin system regulates contractile force development in VSMCs. We have employed small interference RNA to knockdown ROCKII in CASMCs and identified diminution in the phosphorylation status of LC20 at Ser19, MYPT1 at the inhibitory Thr853 site (but not Thr696), and cofilin at Ser3. These data were consistent with previously defined roles for ROCKII in VSMCsin vivo, others have broad target specificity and can impact upon multiple targets and regulate diverse cellular processes58The new availability of small molecule inhibitors provides an opportunity to examine aspects of ZIPK signaling in VSM that were previously not attainable. While some kinase inhibitors are highly specific and will only recognize a restricted number of target molecules How to cite this article: Al-Ghabkari, A. et al. A novel inhibitory effect of oxazol-5-one compounds on ROCKII signaling in human coronary artery vascular smooth muscle cells. Sci. Rep.6, 32118; doi: 10.1038/srep32118 (2016).


C have been used to determine the location of the excess Mn in MnxGa (1.15 ≤ x ≤ 1.8). This information has then been used to constrain the fits to neutron powder diffraction patterns measured at ambient temperature and so determine unambiguously the Mn moments in this system. We find that Mn randomly occupies the two Ga sites (2a and 2b) in the I4/mmm structure and propose that it is more appropriate to use a simpler structure based on the P4/mmm space group with a reduced unit cell. In this structure the two Ga sites are formally equivalent (they occupy the 1a site while Mn occupies the 1d site). Our experimental observations are supported by DFT calculations. Below TC we find that the Mn(1d) moment is constant at 2.45(3) μB, while Mn on the 1a site carries a slightly larger moment (~3 μB) that is coupled antiparallel to the Mn(1d) moments, leading to the observed drop in magnetisation with increasing Mn content in MnxGa.Neutron powder diffraction patterns measured above T Mn frequently carries a large moment, has reasonable chemical stability, and comes at a moderate cost. One possible system is Mn-Ga. The predicted saturation magnetisation of stoichiometric MnGa is 116 J/T/kg2 and if a reasonably square loop could be achieved, this would yield an energy product in excess of 200 kJm−3. Unfortunately, the Mn-Ga binary phase diagram is extremely complex, lacking a single congruently-melting compound. As a result, sample preparation is complex and quality is often poor. The problem has been compounded by focusing on producing technically interesting materials before the intrinsic properties have been understood.Manganese-based magnetic compounds are of interest for two main reasons: (1) they avoid the use of rare earths, (2) they offer modest-cost/modest performance alternatives to the two dominant hard magnet technologies: NdxGa system3. Here we concentrate on establishing the basic crystal and magnetic structures; determining the location and ordering behaviour of the excess manganese.In a previous paper we described the excellent intrinsic magnetic properties of the Mnmmm structure  and the occupation of the 4d site by Mn in the I4/mmm structure imposes a further constraint on l: l = 2n, ∀ h, k. The result of these constraints is that it is essentially impossible to distinguish the P4/mmm and I4/mmm Mn-Ga structures on the basis of x-ray diffraction data. Indeed, unless there is a severe imbalance in the Mn/Ga occupations of the two Ga sites shown in Fig. xGa P4/mmm cell can be mapped directly onto an I4/mmm cell yielding the same (doubled cell) crystal structure and precisely the same diffraction pattern. Even if we assume perfect selectivity, the impact on the diffraction pattern is vanishingly small: Taking a composition of Mn1.66 Ga and placing all of the excess Mn on the Ga(2a) site in the I4/mmm cell  changes the intensity of the (002) peak from ~1% of the (112) peak (the strongest in the x-ray diffraction pattern) to ~1.7%. We adopted the I4/mmm cell as the starting point for our structural analysis but, as we show below, found that a much simpler structure based on the P4/mmm space group with a reduced size unit cell  What is the origin of the reduction in magnetisation seen on increasing x from 1 in MnxGa?Our interest here is threefold: (1) What is the correct space group and structure for this system? (2) What is the internal packing of the cell? xGa are almost indistinguishable from that of a uniform face-centred tetragonal (fct) cell, with the strongest fct-forbidden reflection ((001) for the P4/mmm cell) being of order 1% of the intensity of the primary (111) reflection4. In addition, magnetic order cannot be studied using conventional x-ray diffraction methods. Therefore, we turn to neutron diffraction where there is almost optimal contrast between Mn and Ga  = −3.75 fm and bc(Ga) = +7.288 fm), and we are also sensitive to the magnetic ordering. This latter sensitivity is important as the magnetisation of MnxGa decreases with increasing x3, suggesting that the additional Mn couples antiferromagnetically (AF) to the ferromagnetic (FM) Mn found in the parent MnGa cell.The poor x-ray contrast between Mn and Ga means that x-ray diffraction cannot be used to study the distribution of Mn within the cell. Indeed, the x-ray diffraction patterns of MnxGa (1.15 ≤ x ≤ 2.0) were coupled AF to those on the primary Mn site and that this was the origin of the reduction in magnetisation5. However, strong correlations between the cell packing and magnetic contributions to the diffraction patterns made an unambiguous determination of the basic structure impossible. Here we eliminate the correlation problem entirely by analysing neutron diffraction patterns taken above the ordering temperature of each material where the magnetic contribution is absent, and then using the cell packing information derived from these fits to constrain the analysis of the neutron diffraction patterns taken at ambient temperature. This approach is complemented by using density functional theory (DFT) calculations to investigate possible site preferences for the excess Mn in MnxGa, and to determine the magnitudes and orientations of the Mn moments on the three possible sites in the  I4/mmm cell.Our initial neutron diffraction work on this system confirmed that the moments on Mn atoms that occupied the Ga sites in MnxGa  alloys were prepared by induction melting high purity gallium (99.9%) and manganese (99.5%) in an argon atmosphere. To compensate for evaporation losses during melting, an extra 3 wt.% Mn was added to the alloys. The as-cast ingots were annealed in a tubular vacuum furnace at temperatures, Ta, ranging from 700 K to 900 K for one to seven days and then quenched into ice water. The annealing step is crucial for obtaining single-phase alloys, and the optimal annealing temperature range was found to be quite narrow and depended critically on the composition of the alloy3.A series of tetragonal MnKα x-ray diffraction. Magnetic properties were measured using a Quantum Design Physical Properties Measurement System (PPMS) magnetometer with a maximum magnetic field of 14 T. Neutron powder diffraction experiments were carried out at both ambient and elevated temperatures on the C2 800-wire powder diffractometer (DUALSPEC) at the NRU reactor, Chalk River Laboratories, Ontario, Canada, using neutron wavelengths (λ) of 1.32905(5) Å and 2.37047(8) Å to cover the widest range of scattering vectors (0.3 Å−1 ≤ q ≤ 8.0 Å−1). The multi-wire detector covers an 80-degree arc of 2θ at one time and the angle ranges used for the two wavelengths were chosen so as to have two strong diffraction peaks in common between the two patterns to facilitate co-refinement  data, shown in Fig. With the cell symmetry and packing established from the analysis of the high temperature patterns, the ambient temperature patterns  were then co-refined to determine the Mn moments, subject to the structural constraints derived from the high temperature fits.a and 2b sites in the I4/mmm cell, the total energy for MnxGa (1.0 ≤ x ≤ 2.0) with different occupancy configurations of Mn were calculated by a DFT  method. We set the super-cell with a nominal composition of Mn4(Mn2Ga2) with the four Mn atoms at the 4d site and the two Ga atoms at either the 2a or the 2b site. The remaining two Mn atoms were then placed on the vacant 2b or 2a site. The QUANTUM ESPRESSO package8, in the projected augmented wave (PAW) framework9, was employed to perform DFT calculations using the generalized gradient approximation (GGA) of Perdew-Burke-Ernzerhof10, 11 for the exchange correlation functional. The atomic PAW potentials were adopted from the PS library.1.0 generated by A. Dal Corso12. The wave functions were expanded in plane-wave basis sets truncated at a cutoff energy of 60 Ry. Brillouin zone integrations were performed on a 8 × 8 × 8 k-point grid, and Marzari-Vanderbilt broadening13 was applied with a smearing width of 5 mRy. The structural degrees of freedom were fully relaxed to obtain optimized structural parameters. In addition, the magnetic moments in MnxGa have been calculated using a linear muffin-tin orbital method with coherent potential approximation (LMTO-CPA)14, 15, 16.To understand the site distribution of the excess Mn at the 2mmm cell with Mn atoms on the 4d site and Ga on both the 2a and 2b sites giving an initial stoichiometry of MnGa. The two patterns for each composition were fitted by permitting vacancies on the 2a and 2b sites, partial filling of each site with Mn, or no vacancies with a mix of Mn and Ga on the 2a and 2b sites. In each case, statistically equivalent quality fits could be obtained, but only the model with the 2a and 2b sites filled (no vacancies) returned the expected stoichiometry. The other models were therefore rejected.Our starting point for the fits to the high temperature neutron diffraction patterns was the body-centred tetragonal I4/a site over the 2b site. However, if we reversed the starting point for the fits by exchanging the occupations of the 2a and 2b sites, we could obtain a fit with the same χ2 and precisely the same occupations (to four digits) but with Mn favouring the 2b site over the 2a site. Examination of simulated diffraction patterns with a stronger 2a:2b bias revealed that an occupation imbalance led to appearance of the (101) peak  but with the reduced-size unit cell originally proposed by Bither and Cloud17. Figure mmm structures. Placing the Mn atoms in the 1d site (located at a site (located at 0 0 0) yields precisely the same packing as our I4/mmm starting point, and the same calculated diffraction pattern. The (101) peak (in the I4/mmm cell) that could have signalled a difference in Mn occupation between the “two” Ga sites is forbidden, consistent with the sites now being equivalent. Refinements from this starting point yield fits that were fully equivalent to those obtained using the I4/mmm cell and returned the expected stoichiometry, but required fewer fitting parameters. Fitted patterns for the Mn1.60Ga sample taken at 500 °C and using the smaller P4/mmm cell are shown in Fig. Constraining the occupations of the 2a and 2b sites in the I4/mmm cell were indeed equivalent, we carried out DFT calculations looking at the energy associated with placing Mn on either the 2a or the 2b site. Our calculations showed that the binding energy for the excess Mn at the 2a site is precisely the same as that for Mn at the 2b site (−4.08 eV/atom). We therefore conclude that the two sites are indeed fully equivalent and that the P4/mmm space group is the correct one to use for MnxGa. As a final check, we confirmed that the energies associated with substituting Mn at each of the two Ga sites in the large-cell P4/mmm structure were also the same. The revised structural description is simpler  and the stoichiometry for the end-member (MnGa) is obtained directly.To further investigate whether the 2a site and the resulting stoichiometry are plotted in Fig. The Mn occupation of the 11.60Ga shown in Fig. 5. However, by determining the Mn locations above TC we sidestep this problem entirely, and can proceed to determining the magnetic moments free of ambiguity.The most striking feature of the ambient temperature patterns for Mnmm′m′ magnetic space group that allows for c-axis FM ordering of the Mn moments on the 1d site. This magnetic group also permits c-axis ordering at the 1d site where we have shown that the excess Mn substitutes for Ga. Only collinear magnetic structures were considered here. The magnetic space group adopted for the analysis here is essentially equivalent to the I4/mm’m’ magnetic space group used previously when the I4/mm’m’ structure was being considered5.The magnetic structure used as a starting point was based on the P4/a site have moments that couple antiparallel to those on the primary 1d site, broadly consistent with our earlier analysis5. In addition, we find that the size of the Mn(1d) moment is essentially independent of composition at 2.45(3) μB/Mn. The situation at the 1a site is somewhat less clear as the Mn occupation of this site never exceeds 0.25 and for the two lowest excess Mn samples (x = 1.15 and 1.20) the Mn occupation of the 1a site is only ~0.1. This leads to rather large uncertainties on the fitted moment, however it appears to be broadly constant, and at 3.0(1) μB/Mn, it is clearly larger than that on the 1d site. These results are plotted on the upper panel of Fig. d and 1a sites in the P4/mmm space group is only compatible with purely c-axis ordering, but leaving this issue of principle aside, any planar (ab−) component to the magnetic order would almost certainly lead to a severe mis-fit to the intensity of the (002) peak, especially given the significant magnetic contribution to the (110) peak. Any long-period modulation of the order  would be expected to contribute both magnetic satellites and a low-angle peak from the new periodicity. Neither are seen . It is clear from our fits to the magnetic structure that the decline in bulk magnetisation with increasing Mn in MnxGa is the result of antiparallel Mn moments on the 1a (Ga) sites  decline over the studied range, with an average value of 2.40(2) μB/Mn, while for the Mn on the 1a site we observe a similar, ~3%, decline with increasing Mn content and an average value of −3:24(3) μB/Mn. The former moment is in perfect agreement with the experimental value, while the latter value is only slightly outside experimental error for the average value and well within the experimental scatter  alloys. Both our high temperature diffraction patterns and DFT calculations show that there is no difference between the the 2a and 2b sites in the commonly used I4/mmm–based (D022) structure, or equivalently, 1a and 1c sites in the large-cell P4/mmm (L10) structure. We therefore propose a much simpler crystal structure based on the I4/mmm space group with a reduced unit cell, originally suggested by Bither and Cloud17. We find that the excess Mn in MnxGa replaces some of the Ga on the 1a site, and with the crystal structure as a constraint we are able to make an unambiguous determination of the magnetic structure and Mn moments. Antiparallel ordering between the 2.45(3) μB Mn moments on the 1d site and the 3.0(1) μB moments associated with the excess Mn on the 1a site leads to the observed decline in bulk magnetisation. These results are in full, quantitative, agreement with both DFT calculations and bulk magnetisation measurements.Neutron powder diffraction patterns measured above and below TxGa system, it will be necessary to find something that will substitute for Ga on the 1a site but that either carries a much smaller moment than the Mn (without causing a loss of anisotropy) or couples parallel to the Mn moments on the 1d site. Given the remarkable agreement between our experimental data and the DFT calculations, it would appear that a preliminary search using DFT calculations could reliably identify promising candidates and so greatly reduce the synthetic effort associated with the development of new materials in this system.If hard magnets are to be developed based on the Mn


The kallikrein-related peptidase (KLK) family of proteases is involved in many aspects of human health and disease. One member of this family, KLK4, has been implicated in cancer development and metastasis. Understanding mechanisms of inactivation are critical to developing selective KLK4 inhibitors. We have determined the X-ray crystal structures of KLK4 in complex with both sunflower trypsin inhibitor-1 (SFTI-1) and a rationally designed SFTI-1 derivative to atomic (~1 Å) resolution, as well as with bound nickel. These structures offer a structural rationalization for the potency and selectivity of these inhibitors, and together with MD simulation and computational analysis, reveal a dynamic pathway between the metal binding exosite and the active site, providing key details of a previously proposed allosteric mode of inhibition. Collectively, this work provides insight into both direct and indirect mechanisms of inhibition for KLK4 that have broad implications for the enzymology of the serine protease superfamily, and may potentially be exploited for the design of therapeutic inhibitors. KLK) gene family is the largest protease gene cluster in the human genome and encodes 15 serine proteases that have sequence identity varying from 40–80%, with a high degree of structural similarity around the active site3467The kallikrein (CTKSIPPICFPD14). The backbone cyclization coupled with an intramolecular disulfide linkage causes SFTI-1 to adopt a constrained structure in solution1 specificity pocket15The most direct way to inhibit KLK4 is by designing specific active site inhibitors. The Bowman-Birk family of protease inhibitors (Bowman-Birk Inhibitors (BBI)) are a class of proteins and peptides found in plants and plant seeds, where they are thought to play a role in defense against pathogensSFTI-1 has been shown to inhibit KLK4 in addition to trypsin and matriptase1212Allostery offers another route to KLK4 inhibition. KLK4, like the majority of serine proteases, is tightly regulated by conformational switches. Serine proteases are synthesized in an inactive zymogen state that has a distorted active site unable to efficiently support catalysis21In order to investigate further both direct and indirect mechanisms of KLK4 inhibition here we describe the structures of KLK4 in complex with SFTI-1, an engineered SFTI-1 variant, and with nickel (KLK4-Ni). The very high resolution (~1.0 Å) of the KLK4-SFTI-1 structures provides exquisite detail of KLK4-inhibitor interactions and a structural rationalization for inhibitor potency and selectivity. Analysis of the KLK4-Ni structure reveals conformational heterogeneity, as well as metals bound at the active site and at an indirect inhibitory site. Combining this structure with molecular dynamics simulations depicts an indirect mechanism of information transfer resulting in active site inhibition. Collectively, this work provides insight into direct and indirect inhibition of KLK4 that has broad implications for the enzymology of serine proteases.We previously showed that SFTI-1 potently inhibits KLK4 proteolytic activity13Interface interactions are dominated by only 4 SFTI-1 residues: P1 Lys5, P4 Arg2, P2′ Ile7, and Phe12 . Calculap-aminobenzamidine  substrate, with P4 and P2 residues affecting activity the mostFCQR , and resulted in a 18-fold increased inhibition of KLK4 (Ki = 3.59 nM)FCQR complex to 1.3 Å resolution. Compared with KLK4-SFTI-1 there is an overall increase in the buried surface area and number of protease-inhibitor interactions  simulation, loop III exhibits the most flexibility, compared to the rest of the molecule . Thus, Moop VIII . Our sim2+ is bound to the His25/Glu77 site, offering mechanistic insight into metal-mediated indirect inhibition of proteolytic activity.By assuming SFTI-1 residues P4-P2′ bind to KLK4 in a similar mode to a natural substrate, we can use the KLK4-SFTI structure to model the effect that MD-simulated loop motions would have on substrate binding. Superimposing the SFTI-1 structure onto the MD-generated ensemble of KLK4-apo simulations shows that the His25 and loop II, III and VIII motions do not interfere with SFTI-1 P4-P2′ binding and would not be inhibitory . Thus thIn order to explore the effect of metal-binding on large amplitude motions of KLK4 we calculated normal mode (NM) coordinates for each conformational state explored during the MD trajectories of the KLK4 in the presence and absence of nickel. Conformations explored during atomistic MD were projected onto the first five low-frequency NMs. With one key exception, the distribution of conformations was centered on the origin, showing limited sampling of rigid-body motions during MD  & S4. HoFCQR, the more potent inhibitor  with 9-fluorenylmethyl carbamate as semi-permanent protecting group using a Discover SPS Microwave System (CEM Corporation) to enhance conventional solid phase peptide synthesis. Peptide cyclisation was carried out in solution also using microwave enhancement as previously describedi) was determined by non-linear regression in GraphPad Prism (Morrison equation), as recently describedInhibition of KLK4 by SFTI-1 was assessed in competitive inhibition assays, and the inhibition constant  ratio of protein to mother liquor.p-aminobenzamidine (PABA)2 buffer.Crystallization conditions for KLK4 in complex with FCQR complexes, 20 mg/ml protein was incubated overnight with 3-fold molar excess of inhibitor (solubilized in 25% acetonitrile to the working concentration of 100 mM) at 4 °C. In order to identify initial hits, commercially available screens including Index (Hampton Research), JCSG+ and PACT (Qiagen) were usedFCQR crystals were obtained within a week by mixing 2 μL of protein-peptide solution with equal volume of crystallization buffer  and equilibrated at 18 °C. KLK4-SFTI-1 crystals were produced by mixing 2 μL of protein-peptide solution and 2 μL of reservoir buffer  and appeared after 9 months of incubation at 18 °C. Prior to crystallization experiments of the KLK4-SFTI-1 and KLK4-SFTI-1All datasets were collected at the Australian Synchrotron, Victoria, Australia on MX1For all analysis and MD simulations, missing atoms, side chains and residues were rebuilt using Modeller v9.10Calculations, modeling and simulations were performed on a range of computing resources: ORCHARD 800 core x86 cluster ; AVOCA/MERRI .In MD simulations, atomic coordinates were obtained from the following PDB entries: 4KGA (chain A), 4K8Y & 4K1E. Missing residues and atoms were rebuilt using MODELLER version 9.102+ ion bound at the His25 and Glu77 site.Each protein, with protonation states appropriate for pH 7.0656869After an energy minimization stage, and an equilibration stage, production simulations were performed in the NPT ensemble. Three independent replicates of each system were simulated for 200 ns each using NAMD 2.9ε = 2ri,j), to treat electrostatic interactions. Prior to NM calculations, the KLK4-apo structure was energy minimized using the steepest descent (SD) and conjugate-gradient (CG) methods followed by the Adopted Basis Newton-Raphson (ABNR) algorithm. The energy minimized structure presented 0.7 Å RMSD (backbone atoms) against the crystallographic conformation. Harmonic restraints were applied during the SD steps and were progressively decreased from 250 to 0 kcal mol−1 Å−2. Then, the system was further energy minimized with 1000 CG steps and the ABNR algorithm applied without positional restraints using a convergence criterion of 10−5 kcal mol−1 Å−1 RMS energy gradient. The first 100 low frequency normal modes and the atomic fluctuations were computed with the VIBRAN module of CHARMM. The first five low frequency NMs , 4K8Y (KLK4-SFTI-1) and 4K1E (KLK4-SFTI-1FCQR).How to cite this article: Riley, B. T. et al. Direct and indirect mechanisms of KLK4 inhibition revealed by structure and dynamics. Sci. Rep.6, 35385; doi: 10.1038/srep35385 (2016).


CFTR, the chloride channel mutated in cystic fibrosis (CF) patients, is opened by ATP binding to two cytosolic nucleotide binding domains (NBDs), but pore-domain mutations may also impair gating. ATP-bound NBDs dimerize occluding two nucleotides at interfacial binding sites; one site hydrolyzes ATP, the other is inactive. The pore opens upon tightening, and closes upon disengagement, of the catalytic site following ATP hydrolysis. Extent, timing, and role of non-catalytic-site movements are unknown. Here we exploit equilibrium gating of a hydrolysis-deficient mutant and apply Φ value analysis to compare timing of opening-associated movements at multiple locations, from the cytoplasmic ATP sites to the extracellular surface. Marked asynchrony of motion in the two ATP sites reveals their distinct roles in channel gating. The results clarify the molecular mechanisms of functional cross-talk between canonical and degenerate ATP sites in asymmetric ABC proteins, and of the gating defects caused by two common CF mutations. Cystic Fibrosis (CF) is an incurable, devastating inherited disease caused by mutations in the CF Transmembrane Conductance regulator (CFTR) chloride ion channel . A largeCFTR belongs to the large family of ATP-Binding Cassette (ABC) proteins , most ofviolet loops), drives the major conformational rearrangements of the substrate translocation pathway  and a cytosolic nucleotide binding domain (NBD). In CFTR these two canonical halves are linked by the unique regulatory (R) domain which must be either phosphorylated by cyclic AMP-dependent protein kinase (PKA) , or dele pathway . Upon fo pathway , whereas pathway . The pre pathway , but mut pathway , affect  pathway , and maykCO) as a function of the closed-open equilibrium constant (Keq = kCO/kOC) for a series of point substitutions (Brønsted plot). For the opening step of a simple idealized two-state channel with a single intervening T state, Φ is a measure of how far the conformation of the position has progressed along the reaction coordinate in the T state (0≤Φ ≤1). For a position that moves very early, and has therefore reached its open-like conformation in the T state (Φ = 1), perturbations affect T-state and open-state stabilities to similar extents, and thus impact only opening but not closing rate. At the other extreme, for a position that moves very late, and is still in its closed-like conformation in the T-state (Φ = 0), perturbations affect closing but not opening rate  state for channel opening may shed light on the sources of molecular strain that underly the large Gibbs energy of this highest-energy intermediate conformation, which rate limits the overall transition . Such T-ing rate . In realing rate . The terred star) that disrupts ATP hydrolysis at catalytic site 2 (red cross), thus reducing gating in saturating ATP to a simple closed-open equilibrium . These conditions are satisfied in a background construct  near the NBD1 signature sequence: the R555 and T1246 side chains span the site 2 interface (lower dotted ellipse) to form a hydrogen bond in open CFTR channels (o) in saturating (10 mM) ATP was reduced by up to 10-fold (Keq) was caused predominantly by mutational effects on mean interburst durations (τib) which were prolonged by up to 10-fold (kCO = 1/τib). Importantly, since 10 mM ATP remained saturating for all of the mutants (red bars), this reduced rate kCO indeed reflects slowing of the C1→O1 step (b) were shortened by less than twofold (kOC = 1/τb). Correspondingly, the slope of the Brønsted plot  in the first extracellular loop, where mutations affect gating and are associated with CF -ATP (P-dATP) which markedly stimulated channel open probability . Replacement of the native histidine at position 1348 with an alanine increased Po, whereas substitutions by methionine, tyrosine, or glutamate decreased it . Positions 460 and 1348 flank non-catalytic site 1 (upper dotted ellipse) from two sides. Relative to the native threonine at positions 460, the alanine, serine, and proline substitutions markedly reduced Po (o again reflected coupled opposing changes in mean burst and interburst durations (orange bars).To target the opposing, NBD1 surface of non-catalytic site 1, we chose position 460 in the NBD1 Walker A motif, corresponding to position 1246 in ATP site 2 , orange.duced Po . As withurations . Correspurations  revealedurations , 10 mM Aviolet helices), since CH1 and CH2 are located closer to non-catalytic site 1, whereas CH3 and CH4 closer to catalytic site 2 (violet helices).The large Φ of positions in catalytic site 2 but low-to-intermediate Φ of positions in non-catalytic site 1 implies that timing of movements in the two ATP sites during pore opening is highly asymmetric: whereas catalytic site 2 has already adopted its open-like conformation in the T state, non-catalytic site 1 is still on the move. If the movements completed in non-catalytic site 1 between the T state and open state are large-scale rearrangements, then a similarly pronounced temporal asymmetry of motions might be detectable one structural level further along the axis, at the level of the four coupling helices (purple). At positions 172, 961, and 1068  substitutions caused both increases and decreases in open probability , 961 inbability , and thebability : a clearbability –3. Thus,bability , indicatbability .deep purple), the locus of the most common (>70%) CF mutation, is found in a surface cleft of NBD1 which accommodates CH4 to form a joint-like interface in which the F508 side chain interacts with aromatic CH4 residues, including F1068 (deep purple bars). Channels could be efficiently counted at the end of each experiment by exposure to dATP or P-dATP . Whereas X-ray or cryo-EM structures may capture stable closed  and numbers (right)). Thus, unlike catalytic site 2 which has already adopted its open-like conformation  in the T state, non-catalytic site 1 is still on the move between its closed- and open-like conformations . Interestingly, such pronounced asymmetry in the timing of motions at one end of the dimer interface compared to the other cannot be detected at the level of the four coupling helices, which are all characterized by Φ values of 0.5–0.6 (violet residues (left) and numbers (right)). Thus, the movements that take place in non-catalytic site 1 between the T state and the open state likely remain confined to the site-1 interface.In striking contrast to the large Φ values of positions on both sides of the dimer interface in catalytic site 2, positions flanking non-catalytic site 1 display low-to-intermediate Φ values (top current trace), and such ‘spontaneous’ openings reflect occasional dimerization of empty, unliganded NBDs (top cartoon). Comparison of such spontaneous gating of WT channels with the equilibrium gating of a hydrolysis-deficient mutant, such as the NBD2 Walker A lysine mutant K1250R, in saturating ATP (bottom current trace) reveals two robust effects of bound ATP: a shortening of closed interburst durations (green bars above current traces) and a lengthening of open burst durations (red bars below current traces), both by >100 fold (black vs. orange standard Gibbs energy profiles).ATP bound at the two composite sites acts as molecular glue that stabilizes the open-pore conformation by bonding the NBD interfaces together . Our datded NBDs  : that bonding readily explains the reduction in the energetic barrier for opening, that is, stimulation of opening rate, by ATP. Whether stably bound ATP in non-catalytic site 1 might also contribute to speeding opening, by maintaining some contact across the site-1 interface even in the closed state and hence preventing complete NBD disengagement, is still unsettled . By selectively stabilizing the open state relative to the T state, the site-1 glue increases the height of the barrier for non-hydrolytic closure, explaining the observed prolongation of non-hydrolytic bursts by ATP.Which of the two bound ATP molecules affects non-hydrolytic closing rate? Because it is bonded both in the T and O1→O1) whereas ATP bound at non-catalytic site 1, by slowing rate O1→C1, ensures progress through ATP hydrolysis: such unidirectional cycling is essential for ABC proteins to mediate uphill transport. In asymmetric ABC proteins flipping to the outward-facing, NBD-dimerized conformation is followed by a step in which the consensus site becomes committed to ATP hydrolysis . Moreover, it is in contact with the conserved Q-loop glutamine Q1291 (yellow)   which ac binding . Thus, r binding . Whethernding 1, , or is cnding 1, , might btiming of movements in the two ATP sites during pore opening, clarifying the extent of these movements during each step in the channel opening/closing cycle will require comparisons among high-resolution structures of CFTR trapped in phosphorylated closed- and open-channel states. The recent structures of unphosphorylated closed  , and thoXenopus laevis [RRID:NXR_0.0080] following a IACUC-approved protocol. Oocytes were defolliculated by treatment with Type II collagenase  and stored at 18°C in a frog Ringer's solution supplemented with 1.8 mM CaCl2 plus 50 μg/ml gentamycin sulfate (Sigma). Recordings were done 1–3 days after co-injection with 0.1–10 ng cRNA for both CFTR segments.Ovarian lobes were removed from anaesthetized 2, 5 HEPES, pH = 7.4 with NMDG, bath solution contained 134 NMDG-Cl, 2 MgCl2, 5 HEPES, 0.5 EGTA, pH = 7.1 with NMDG. The bath solution was continuously flowing, and could be exchanged with a time constant of ~100 ms. MgATP (3 or 10 mM) and 2'-deoxy-ATP sodium salt  (Sigma) were added from 400 mM and 100 mM aqueous stock solutions, respectively (pH = 7.1 with NMDG); dATP was supplemented with equimolar MgCl2. 25–50 μM 2'-deoxy-N6-(2-phenylethyl)-ATP sodium salt  was added from a 10 mM aqueous stock solution. Unitary CFTR currents in 10 mM MgATP were recorded at −80 mV (−100 mV for position 117 mutants)  digitized at 1 kHz , and lowpass-filtered at 50 Hz using a Gaussian filter. Single-channel patches were identified as very long  recordings without superimposed channel openings. For the low-Po mutants targeting positions 117, 508, and 555 strong stimulation by dATP or P-dATP at the end of each experiment was used to facilitate correct estimation of the number of active channels in the patch (CFTR unitary currents were recorded in excised inside-out patches at 25°C as described earlier . Pipettehe patch . Macrosche patch . For diskCO) and closing (kOC) rates were defined as the inverses of the mean interburst (τib) and burst (τb) durations, respectively, and Keq as kCO/kOC. Plots of ln(kCO) as a function of ln(Keq) (Brønsted plots) were fitted by linear regression (SigmaPlot 8 [RRID:SCR_003210]).Bursts and interbursts were reconstructed as described earlier . In brieAll data are given as mean ± SEM of measurements from ≥3  long segments of single-channel recordings, from 3 to 15 patches for each mutant. In the face of alternating periods of lower and higher activity typical to CFTR , several In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Richard Aldrich (Senior Editor) and three reviewers, one of whom, Kenton J Swartz (Reviewer #1), is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Roderick MacKinnon (Reviewer #3).Thank you for submitting your article "Asymmetry of movements in CFTR's two ATP sites during pore opening serves their distinct functions" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This interesting manuscript from the Csanady lab presents new results continuing work started in a previous paper analyzing the open/closed transition state of the CFTR chloride channel. This channel, related to the ABC transporter family, undergoes a highly complex gating process which requires channel phosphorylation in the R domain as well as ATP binding and hydrolysis at the NBDs to drive irreversible gating. Though much progress has occurred in understanding these processes, key questions remain open. Here the authors dissect the final gating step by using transition state analysis of a mutant that is claimed to introduce a simple, two-state open closed reaction for the opening of the channel. Such a two-state system is required for the analysis to be valid. The authors analyze the effects of mutations at a series of positions on the open/closed equilibrium that complement the previous set, focusing now on areas around the ATP1 binding site and the outside of the pore. They find that the timing of the opening wave reveals propagation from ATP2 to ATP1, which is roughly simultaneous with movement of the interfacial coupling helices. A position at the outside end of the pore moves only very late in the reaction. Notably, the timing differences between the ATP binding sites seem not to persist through the interfacial helices, which all seem to move nearly simultaneously. The work reported here is careful and thorough, and the single channel recordings are really nice. The presentation is also excellent, very clearly discussing a difficult topic. This kind of work is highly complementary to the spate of emerging structures and provides information that will be difficult to obtain using other methods. We do have several concerns that the authors should address before we are fully convinced by their arguments. The authors should be aware that the reviewers have somewhat divergent opinions about the most interesting new conclusions in this manuscript.Essential revisions:all the mutants equally well fit by a single tau each? The reported tau values are said to be from single exponential fits, but I think it would be worthwhile to establish this formally. Perhaps the authors could show the dwell time histograms (or at least examples) and fits in a supplemental figure.1) The single-step assumption. Critical to the argument presented here is that the equilibrium is a single step reaction, and that the transitions we observe are between two discrete states. However, our impression is that the gating reflects the binding of ATP (at least to site 2), dimerization of the NBDs, and the channel conformational change. From the collective Phi's measured in this and the previous paper, and their apparently smooth gradation from slopes of near zero to near 1, we could be convinced that this is a single reaction, but we think the burden is on the authors to substantiate this a bit more. How single exponential are the results? Are 2) We are unclear of the data in the second part of 3) We think the authors should be more circumspect in their interpretation about how Phi analysis relates to timing of movements. The analysis speaks to where in the transition mutants at specific sites cause a perturbation. It may be reasonable to infer from this that this result is because those regions undergo dynamic rearrangements at different times in the transition, but it would seem more appropriate to separate the specific conclusion from the inference.after those of ATP1? I guess the model in our head is that the NBDs are partially 'glued' by ATP1 and ready to close upon ATP2 binding. Is this incorrect? If not, what is happening to ATP1 that makes the movements around it happen later?4) We were somewhat confused by the cartoon in  Essential revisions:1) The single-step assumption. Critical to the argument presented here is that the equilibrium is a single step reaction, and that the transitions we observe are between two discrete states. However, our impression is that the gating reflects the binding of ATP (at least to site 2), dimerization of the NBDs, and the channel conformational change.Because opening indeed requires binding of ATP to site 2 followed by the channel isomerization step, it is critical that all experiments be performed in saturating ATP to make the isomerization step rate limiting. We have verified that this condition is met for all of our mutants .From the collective Phi's measured in this and the previous paper, and their apparently smooth gradation from slopes of near zero to near 1, we could be convinced that this is a single reaction, but we think the burden is on the authors to substantiate this a bit more. How single exponential are the results? Are all the mutants equally well fit by a single tau each? The reported tau values are said to be from single exponential fits, but I think it would be worthwhile to establish this formally. Perhaps the authors could show the dwell time histograms (or at least examples) and fits in a supplemental figure.o  the chosen examples represent the mutants at the two extreme ends of the REFER plot.For our background construct we have demonstrated the validity of the single-step assumption in our earlier study . In a series of new supplementary figures  we now icrit) according to the algorithm of Magleby and Pallotta (1983). Panels D (I) and E (J) display the distributions of the durations of reconstructed bursts and interbursts, resulting from suppression of brief "flickery" closures, and reasonable fits of the distributions of reconstructed bursts by single-exponential probability density functions. We note, however, that the distributions of reconstructed interburst durations will never be truly single-exponential: first, the left tail of the true interburst distribution is truncated, and second, the right tail of the distribution of flickery closures will survive – this is particularly apparent for the high-Po mutants for which the two closed-time components overlap substantially . However, the choice of tcrit assures that the mean duration of reconstructed interburst events matches that of the true interburst events . We would like to point out that the values of τb and τib were not derived from single-exponential fits , but reflect simple arithmetic averages of burst and interburst dwell-time durations . All this is explained in the legend of the first histogram figure (Panels B (G) and C (H) show distributions of simple closed and open times, and the choice of the burst delimiter (tm figure .2) We are unclear of the data in the second part of Our original aim was to characterize Φ for position 464, which has been extensively studied by several groups in the past. However, unfortunately most of the mutants we generated failed to form functional channels, with the exception of K464C and K464R for which we did manage to obtain a few recordings. On the assumption that Φ values for nearby positions will be similar, we therefore sought to solidify the K464 data by combining them with data obtained from two mutants of nearby position 460 . We acknowledge that this approach depends on the above assumption.Specifically, we would rather not see pooled data sets (from 460 and 464). Since the most important result in this paper is the discovery of different Phi values for catalytic and non-catalytic sites, we would be more confident in seeing what the Phi values are for multiple positions within each site Done. Because we were unable to obtain a reliable Φ value for position 464, we have omitted the 464 data altogether. Instead, we have generated and functionally characterized three additional 460-position mutants, and now provide a clean REFER plot for position 460. The obtained Φ value of 0.36 ± 0.07 for position 460 is only slightly different from the Φ obtained earlier for the pooled 460-464 data (0.26 ± 0.06), and is quite similar to that of position 1348 located at the opposing surface of site 1 (0.42 ± 0.04). Thus, our conclusions are now based on the Φ values of two positions within each of the two ATP sites.Further, we are puzzled by the minimal discussion of the complexity of the data in The complexity of the notation (each construct denoted by a pair of letters) was due to the pooled nature of the original data set – in the present version this problem has been eliminated.3) We think the authors should be more circumspect in their interpretation about how Phi analysis relates to timing of movements. The analysis speaks to where in the transition mutants at specific sites cause a perturbation. It may be reasonable to infer from this that this result is because those regions undergo dynamic rearrangements at different times in the transition, but it would seem more appropriate to separate the specific conclusion from the inference.We now clarify this point in the Introduction:"For the opening step of a simple idealized two-state channel with a single intervening T state, Φ is a measure of how far the conformation of the position has progressed along the reaction coordinate in the T state (0≤Φ≤1). […] The terms "early" and "late" will be used here to reflect sequentiality of movements based on this interpretation."4) We were somewhat confused by the cartoon in initiated by site 2 necessarily means that site 2 must undergo some movement first, before site 1 also reacts: causality must imply sequentiality, otherwise site 1 would have to be able to "look into the future", to anticipate ATP binding-induced rearrangements in site 2.We do not think that there is anything surprising about this finding. It is clear that opening is initiated by ATP binding to site 2, because channels immediately cease to reopen once ATP is removed from the bath solution, even though site 1 likely keeps ATP bound for some tens of seconds . The mere fact that opening is I guess the model in our head is that the NBDs are partially 'glued' by ATP1 and ready to close upon ATP2 binding. Is this incorrect?Indeed, data from the Hwang laboratory  and from our group  have provided evidence for some interaction across site 1 which is maintained even in the closed state . If true, then this effect might also contribute to speeding opening (relative to unliganded gating) by preventing the NBDs from distancing themselves from each other as far as seen in unliganded cryo-EM structures . However, this issue is still unsettled, as work from the Gadsby laboratory  suggested complete separation of both ATP sites during each channel closing event.We have now added a sentence to the Discussion to briefly expose this controversial topic: "Whether stably bound ATP in non-catalytic site 1 might also contribute to speeding opening, by maintaining some contact across the site-1 interface even in the closed state and hence preventing complete NBD disengagement, is still unsettled ."binding" that initiates opening: in the closed channel ATP binding/unbinding to site 2 is a rapid equilibrium process, whereas opening rate is extremely slow (~1 s-1) even in saturating ATP. Thus, the movement at site 2 which initiates opening must occur after ATP2 binding, and has been shown to reflect closure of the site-2 NBD interface .Furthermore, we would like to point out that it is not "ATP2 If not, what is happening to ATP1 that makes the movements around it happen later?Regardless of whether site 1 separation is complete or incomplete in the closed state, the causality argument discussed above does not leave much room for any alternative explanation. The exact nature of these movements is obviously beyond the scope of our study.