At present, it is still uncertain whether single screw has the same stability as double screws in the treatment of ulnar coronal process basal fracture (Regan–Morry type III). So, we aimed to compare the pull‐out force and anti‐rotation torque of anterior single/double screw‐cancellous bone fixation  in this fracture, and further study the influencing factors on anatomical and biomechanical stability of smart screw internal fixations.A total of 63 adult volunteers with no history of elbow injury underwent elbow CT scanning with associated three‐dimensional reconstruction that enabled the measurements of bone density and fixed length of the proximal ulna and coronoid. The models of coronal process basal fracture, aSSBF and aDSBF, were developed and validated. Using the finite element model test, the sensitivity analysis of pull‐out force and rotational torque was carried out.The pull‐out force of aSSBF model was positively correlated with the density of the cancellous bone and linearly related to the fixed depth of the screw. The load pattern of pull‐out force of aDSBF model was similar to that of aSSBF model. The ultimate torque of aDSBF model was higher than that of aSSBF model, but the load pattern of ultimate torque of both models was similar to each other when the fracture reset was satisfactory, and the screw nut attaches closely to coronoid process. Moreover, with enhancement of initial pre‐tightening force, the increase of ultimate torque of both models was small.In addition to three pull‐out stability factors of smart screw fixations, fracture surface fitting degree and nut fitting degree are the other two important anatomical and biomechanical stability factors of smart screw fixations both for rotational stability. When all pull‐out stability and rotational stability factors meet reasonable conditions simultaneously, single or double screw fixation methods are stable for the treatments of ulnar coronoid basal fractures. Biomechanics of 12 finite element (FE) models of fractured and surgical elbows were simulated. The pull‐out force and rotation torque of screw fixations were ranked by finite element model (FEM). Two new stability factors of screw fixations were determined by FEM sensitivity test. Three‐dimensional anatomy and biomechanical FEM contribute to orthopaedic optimization. The aims of this study were: (i) to measure the cancellous bone mineral density (BMD) and fixed length of the proximal end of the ulna and coronal process by three‐dimensional reconstruction of CT images of volunteers with normal elbow joints; (ii) to compare the pull‐out force and anti‐rotation torque of anterior single screw‐cancellous bone fixation (aSSBF) and anterior double screw‐cancellous bone fixation (aDSBF) in ulnar coronal process basal fracture by the sensitivity analysis of the finite element (FE) models; (iii) to further study the influencing factors of pull‐out stability and rotational stability of smart screw internal fixations in ulnar coronal process basal fracture.This research was approved by the Ethics Committee of Our Medical University (2020‐028).We collected CT images of 63 normal adult elbow joints from January 2016 to August 2019 at the Medical Examination Center of the First Affiliated Hospital of Our Medical University. The CT scan slices ranged from 0.5 to 1.0 mm and were saved in DICOM format. There were 41 cases in the young group aged 15–44 years, including 22 men and 19 women; 22 cases in the middle‐aged and younger elderly group aged 46–68 years, including seven men and 15 women. Inclusion criteria: well‐developed healthy adult volunteers, whose height, weight, and body mass index (BMI) meet the standards and have no upper limb deformity. Exclusion criteria: history of trauma ; congenital malformations such as ulna and radius fusion, achondroplasia, rickets, and gigantism; bone hyperplasia, porosity, degeneration, osteoarthritis and other bone diseases; joint diseases caused by systemic diseases such as diabetes, rheumatism or rheumatoid arthritis, hypertension, pregnancy, other endocrine diseases, etc.ρu, ρc) of the proximal ulna and the coronal process located at the sagittal plane, proximal ulna sagittal diameter length (Lu) and coronal process sagittal diameter length (Lc), longitudinal height (Hc) and lateral width (Wc) at the base of the coronal process were measured as shown in Figure The 63 normal elbow‐joint CT images were imported into Mimics software  for 3D reconstruction. Then the 3D models were input into Geomagic software  for sectioning and measurements. According to the Regan–Morry classification of ulnar coronoid process fracture, 3D digital anatomy models were built, as shown in Figure The FE modeling research adopts the CT and magnetic resonance (MR) image datasets of the second‐generation Chinese Digitized Human (F2‐CDH) “female No. 24” .In the normal ulna model, according to a fracture case of Regan–Morry type III , the anterior screw, ulnar cancellous bone and its coronoid fracture block were assembled, and the FE modeling was completed. The calculation model simplifies cortical bone as constraints, the minimum and maximum mesh sizes of cancellous bone mesh were set to 0.15 and 1.1 mm, respectively, its element type was defined as quadratic tetrahedron elements (C3D10M). The minimum screw grid was set to 0.3 mm, its element type was defined as primary tetrahedral element (C3D4). It was assumed that the cancellous bone material is homogeneous and isotropic. Three kinds of bone mineral density which represent different degrees of osteoporosis were usedIn the numerical pull‐out test, the screw nut was rigidly coupled, and an axial load was applied to the center of the screw nut with a loading speed of 0.03 mm/s, four indexes are obtained when pulling out the anterior screw: (1) failure displacement (node displacement when the load does not increase with the displacement), (2) pull‐out force, (3) maximum von Mises stress of screw, and (4) maximum von Mises stress of cancellous bone.3),The anterior screw‐cancellous bone rotation model adopted the element types of the anterior screw‐cancellous bone pull‐out force model, but the meshes of the geometric contact area of the coronoid process were encrypted, with their minimum sizes reaching 0.15 mm. The material properties of cancellous bone were treated with a high‐density category (0.32 g/cmFor the rotation models with both sides in contact (BIC), three different pre‐tightening loads (PTL) states were set: PTL = 0 N, PTL = 20 N, and PTL = 40 N, which could tighten cancellous bone screw appropriately,In this numerical rotation test, four indexes were obtained when the anterior screw was rotated at failure state: (1) ultimate torque (torque load at the limit of the rotation angle); (2) screw cross section load ; (3) the maximum von Mises stress of screw; (4) the maximum von Mises stress of cancellous bone.The biomechanical experiments by Renani et al.According to the biomechanical experiment by Hackl et al.In addition, under the same axial compression experiment conditions , the fractured and dislocated elbow joint FE model Figure  predicteIn the study of low, medium, and high bony density,p < 0.05 was considered as the differences that achieved statistically significant level.All data management and analysis were conducted using IBM SPSS Statistics for Windows version 20.0 . The statistical outcomes of bone mineral densities, geometric structural parameters of proximal ulna and coronoid process were expressed as mean ± standard deviation (SD). The multivariate analysis of variance in the general linear model program was used to compare the differences of bone mineral densities, geometric structural parameters of proximal ulna and coronoid process in the sex and age categories. ρc between sex is not significant (p = 0.610), but for age, the difference is significant (p = 0.013); the difference of ρu between sex (p = 0.230) and age (p = 0.221) are not significant; the differences of Lc, Lu, Wc and Hc between sex are significant , but the differences between age are not significant (p > 0.05). According to the measured value of BMD, it can be classified as three types of bone density in the calculation: low density 0.16 g/cm3, medium density 0.24 g/cm3, and high density 0.32 g/cm3. According to Regan–Morry type III, the measured values of the proximal ulnar and coronal process sagittal diameter length, and the height and width of the coronal process base, two types of screw fixation length calculation types can be defined: aSSBF (12 mm), aDSBF .The BMD and fixed length of the proximal ulna and coronal process of 63 volunteers are shown in Table The pull‐out force and the failure displacement of aSSBF model and aDSBF model on the BMD, and the fixed length of the screw are shown in Table As seen from Table The ultimate torque and load of aSSBF model and aDSBF model under different reset and pre‐tightening conditions are shown in Table Table The present study found and confirmed that fracture surface fitting degree and nut fitting degree are the other two important anatomical and biomechanical rotational stability factors of screw fixations for Regan–Morry type III ulnar coronoid fractures in adults. Then, three existing biomechanical pull‐out stability factors  were added to form the “five‐factor of anatomical and biomechanical stability of screw fixation”. When total five main stability factors meet reasonable conditions simultaneously, single or double screw fixation methods are stable for the treatments of ulnar coronoid basal fractures in adults.3), while the BMD of the ulnar bone posterior to the coronoid region of middle‐aged and elderly women is close to the low‐density value of artificial cancellous bone (0.16 g/cm3).The pull‐out force of a cancellous bone screw is an important index for predicting screw loosening and fracture block displacement.The anti‐rotation torque of a cancellous bone screw is another important index for predicting screw rotation and rotational displacement of fracture block.In this study, based on the existing three pull‐out stability factors ,In this study, in addition to studying the pull‐out stability of the single screw and double screw fixation of the FE models, we also studied the rotational stability of the single screw and double screw fixation of the FE models and confirmed two important factors of anatomical and biomechanical stability of the rotational stability.Although the simulation results in this paper can be well‐verified, the FE models of aSSBF and aDSBF in this paper still have some reasonable simplifications. The FE models of aSSBF and aDSBF have been simplified to some extent, and the minor influence of the ulnar ligaments on pull‐out force has not been taken into account. When defining bone materials as continuum materials,Based on the existing three biomechanical pull‐out stability factors of screw fixations , two new anatomical and biomechanical rotational stability factors  were confirmed and added to form the “five‐factor anatomical and biomechanical stability of screw fixation”. When the five main stability factors meet reasonable conditions simultaneously, aSSBF or aDSBF methods are stable for the treatment of ulnar coronoid basal fractures.All authors listed meet the authorship criteria according to the latest guidelines of the International Committee of Medical Journal Editors.All authors are in agreement with the manuscript.Hao Ye: Finite element analysis, data curation, model validation, funding acquisition. Yongchao Yang: case collected, model validation, clinical guidelines, resources. Tingyang Xing: Data curation, statistical analysis, model validation. Guirong Tan: Digital anatomy, 3D measurement, statistical analysis. Shuxun Jin: 3D measurement, statistical analysis. Zhichao Zhao: 3D measurement, statistical analysis. Weikang Zhang: 3D measurement, statistical analysis. Yanyan Li: 3D measurement, statistical analysis, funding acquisition. Lei Zhang: case collected, model validation, clinical guidelines, resources. Jianshun Wang: case collected, model validation, clinical guidelines, resources. Rongmei Zheng: 3D measurement, project administration. Yun Lu: Case collected, model validation, clinical guidelines. Lijun Wu: Conceptualization, methodology, investigation, finite element analysis, Writing—original draft, visualization, resources, funding acquisition, supervision.This work is supported by the National Natural Science Foundation of China , Wenzhou Medical University Scientific Development Foundation of Zhejiang, China (Grant no.: QTJ06012), Basic Scientific Research Project of Wenzhou City of China (Grant No.: Y20210398), Zhejiang Province Science and Technology Plan Research and Xinmiao Talent Program, China (Grant No.: 2021R413036).The authors declare that they have no conflict of interest.This research was approved by the Ethics Committee of Wenzhou Medical University (2020‐028).


Antibody–antigen interactions are central to theimmuneresponse. Variation of protein antigens such as isoforms and post-translationalmodifications can alter their antibody binding sites. To directlyconnect the recognition of protein antigens with their molecular composition,we probed antibody–antigen complexes by using native tandemmass spectrometry. Specifically, we characterized the prominent peanutallergen Ara h 2 and a convergent IgE variable region discovered inpatients who are allergic to peanuts. In addition to measuring theantigen-induced dimerization of IgE antibodies, we demonstrated howimmunocomplexes can be isolated in the gas phase and activated toeject, identify, and characterize proteoforms of their bound antigens.Using tandem experiments, we isolated the ejected antigens and thenfragmented them to identify their chemical composition. These resultsestablish native top-down mass spectrometry as a viable platform forprecise and thorough characterization of immunocomplexes to relatestructure to function and enable the discovery of antigen proteoformsand their binding sites. The covalent bonds of isolates arethen fragmented, usually through collisions with neutral gas, electroncapture, or a combination of these activation methods.5 Comparisons of observed and theoretical fragment ion masses thenidentify the species and localize the placement of various modificationswithin the full-length protein sequence.Proteoforms are the many protein end productsoriginating froma single gene, considering their modifications, sequence permutations,and isoform variants.6 However,IP elution typically denatures noncovalent complexes and dissociatesbound metals and cofactors, which means that neither the immunocomplexnor any multiproteoform complexes enriched as antigens can be directlyidentified. Alternatively, “native” MS preserves endogenousnoncovalent interactions for characterization, simply by using buffersolutions at neutral pH containing little or no organic solvents.7 While there is substantial precedent in the literaturefor native MS characterization of noncovalent immunocomplexes,10 there is limited literature on antigen ejection as a consequenceof activation in the gas phase (“antigen ejection”).11 Furthermore, the cited example of antigen ejectionwas conducted at low resolution and stopped short of characterizingand confirming the antigen’s identity through fragmentation.Another work, by Zhang et al., showcases direct fragmentationof an immunocomplex (using only the fragment antigen binding regionas opposed to the entire antibody), introducing ambiguity in caseswhere multiple antigens are bound.12When TDMS is coupledto immunoprecipitation enrichment , this workflow identifies proteoformsafter elution off the antibody.13 of peanut allergen Ara h 2 and the convergentlyevolved variable region of an IgE, with picomolar binding affinityfor Ara h 2, found in a clonal family of six plasmablasts from twounrelated peanut-allergic patients.14 Asdepicted in Here we put forth the first demonstration of proteoform-specificantigen identification from immunocomplexes via native TDMS, a maturationof concepts introduced and explored in the works referenced above.We analyzed the interactions between the four major isoformsTable S1), we subjected synthetic peptideantigens bound toa convergent set of IgE complementarity-determining region sequences,presented using an IgG1 scaffold , to native MS on a Q Exactiveultra-high-mass range mass spectrometer (Table S2). The basic peptide contains a sequence with two bindingsites (“two-site peptides”), known to be conserved acrossall four Ara h 2 isoforms. A detailed experimental methods sectionis found in the Supporting Information.An 8.5 μM:10 μM mixture of two-site peptides with theantibody (Table S3)15 with sometrimerization (8.3%). Two-site peptides bound to the antibody monomerat stoichiometries of 0 (5.0%), 1 (40%), 2 (41%), and 3 (14%), andthe peptides also bound to the antibody dimer in stoichiometries of2 (60%), 3 (33%), and 4 (7.7%). The antibody trimer exhibited two-sitepeptide binding stoichiometries of 3 (63%), 4 (36%), and 5 (1.1%).We hypothesized that since the antibody dimers bound to a minimumof two peptides as opposed to zero peptides in the monomeric case,the peptides may act as a mediator for antibody dimerization. Otherwise,lower peptide stoichiometries would be prominent in the antibody dimer.To probe the interactions between the antibodyand antigen . A one-site peptide experimentexhibited 88% monomeric antibody form and 12% dimerization , demonstrating different conformationsand, therefore, modes of dimerization .18 These data also show that allfour Ara h 2 isoforms may be capable of mediating antibody dimerization,as the sequence containing both binding sites in the two-site peptideis conserved across all four isoforms. The two binding sites are separatedby a single glutamine residue; therefore, their sequence proximitydoes not appear to be a barrier for multiple binding events. In otherwords, the peptide-mediated dimerization predicts antigen-mediateddimerization for all four isoforms given that the protein’stertiary structure does not obstruct binding regions. As antibodydimers in the two-site peptide experiment only exhibited peptide stoichiometriesof 2, 3, and 4, antigen-mediated complexation coincides with bothbinding regions of at least one antibody being occupied. While somestoichiometries exceed the number of available binding sites in anantigen-mediated model, such as three peptides on the antibody monomeror four peptides on the antibody dimer, these less frequent casesare likely due to nonspecific binding as shown in a “zero-site”peptide experiment . Therefore,most antibody complexation is site-directed, reinforcing the uniquedimerization of the two-site experiment.The antibody dimerization exhibitedduring the two-site peptideexperiment was antigen-mediated. In addition to the discrepancy indimer abundance, the highest-abundance charge state of the antibodydimer in the two-site peptide experiment (37+) was higher than inthe one-site peptide experiment (34+) and the control (34+) . We mixed Ara h 2 purified from peanutflour and with the IgE construct described above (Table S1) in a 1:1 ratio; this yielded both antibody monomers than the primary distribution (77%). This secondary distributionis comprised of two antibodies and a single antigen, primarily a lightisoform (74%). Most of the two-antigen complexes in the activatedspectrum included one heavy isoform and one light isoform (53%), followedby two light isoforms (40%) and two heavy isoforms (7.8%). Despitethe improved spectral clarity, the mass differences among isoformswithin the same weight group were typically not resolved. The 34+charge state, for example, did have partially resolved peaks for allfour isoforms , but this resolutionwas not enough to provide satisfactory deconvolution. This experimentdemonstrates that intact native MS is insufficient for deep characterizationwith even modest levels of antigen heterogeneity. However, the secondarydistribution in the activated spectrum indicates that HCD had ejecteda single antigen that carried away a disproportionate amount of charge(“asymmetric charge partitioning”).21 This trendvalidates that antigen ejection and subsequent characterization arepossible, which would provide a direct and unambiguous link betweenantibody and antigen.While the syntheticpeptide data predict that all four Ara h 2isoforms can mediate antibody dimerization, we sought to prove thisby probing immunocomplexes containing the Ara h 2 antigens S4. We mi23 (“VRF”)that markedly enhances the signals of high-mass complexes while simultaneouslyfiltering out unwanted low-mass components such as unbound antigens.With VRF, ions are accelerated into and decelerated out of a high-pressureregion near the inlet of the instrument. Unlike their complexed counterparts,unbound antigens lose too much of their kinetic energy through collisionsin the high-pressure region to proceed, giving rise to a filteringeffect far before the excess antigens can substantially interferewith the complexes via space charge effects. Without VRF, all fourisoforms were represented , and the immunocomplex was detected at <10%spectral intensity  showing proof-of-concept for confident antigen identification.24 The masses of all C-terminal fragment ions supporta slightly elongated terminus, consistent with the two additionalresidues present in isoforms 1 and 2. Furthermore, the C-terminalfragment ions localized a 14 Da loss to E130 , which is consistent with the E130 → D130substitution detected exclusively in isoforms 2 and 4 . The ability to distinguish isoform2 from three other isoforms of similar mass directly from the antibody–antigencomplex solidifies native TDMS as a viable method for the precisecharacterization of the immune response.We next conducted native TDMS on an OrbitrapEclipse to controllablydisassemble the immunocomplex 3, which ntensity 3a, top. ntensity 3c. Disul25 Whether the antibody consistsof a convergent variable region on a monoclonal IgG scaffold (as wasdone here) or a polyclonal population, the profile of ejected antigensreveals which of their proteoforms are recognized by immunocapturereagent(s). The ability to directly link antigens to the immune responsewith proteoform-level resolution can reveal how specific functionsare triggered in the adaptive immune response including aberrant recognitionof endogenous proteins underlying a diverse set of autoimmune disorders.This native MS proof-of-concept establishesa basis with whichnative TDMS can be used to inform our understanding of the immuneresponse. The power in this approach lies in the ability to characterizeboth the stoichiometry of immunocomplexes and the ejected antigens,whose subtle mass differences typically cannot be resolved by gel-or fluorescence-based readouts. Immunoglobulin therapeutics can beevaluated against the antigens to which they were designed to targetas well as disrupters of those immunocomplexes. Differences betweenthe antigens actually bound and the bulk antigen population can revealhow and to what extent a therapeutic can bind—even in complexmixtures.


Here we investigatehigh molecular weight polyproline as an ice recrystallization inhibitor(IRI). Low molecular weight polyproline is known to be a weak IRI.Its activity is hypothesized to be due to the unique PPI helix itadopts, but it has not been thoroughly investigated. Here an open-to-airaqueous N-carboxyanhydride polymerization is employedto obtain polyproline with molecular weights of up to 50000 g mol–1. These polymers were found to have IRI activity downto 5 mg mL–1, unlike a control peptide of polysarcosine,which did not inhibit all ice growth at up to 40 mg mL–1. The polyprolines exhibited lower critical solution temperaturebehavior and assembly/aggregation observed at room temperature, whichmay contribute to its activity. Single ice crystal assays with polyprolineled to faceting, consistent with specific ice-face binding. This workshows that non-vinyl-based polymers can be designed to inhibit icerecrystallization and may offer a more sustainable or environmentallyacceptable, while synthetically scalable, route to large-scale applications.Ice-binding proteins(IBPs) from extremophile organisms can modulateice formation and growth. There are many (bio)technological applicationsof IBPs, from cryopreservation to mitigating freeze–thaw damagein concrete to frozen food texture modifiers. Extraction or expressionof IBPs can be challenging to scale up, and hence polymeric biomimeticshave emerged. It is, however, desirable to use biosourced monomersand heteroatom-containing backbones in polymers for  By choice of initiator, the polymerizationcan be controlled, allowing for an extensive range of molecular weights,dispersities and architectures to be synthesized and implemented intobiomedical applications.40 Poly(amino acid) materials offeran appealing solution to degradable and biosourced materials to replaceoil-derived monomers.41 For instance, Wooleyand co-workers showed polypeptide-based, recyclable, redox batteries.40In the design of polymer mimics for future 43 suggests (to a first approximation) that simple homo-/copolypeptideswith IRI activity can be discovered. Knight et al.,44 and later Cameron and co-workers,45 reported that poly(hydroxyproline) had IRI activity. This is anexciting target, as the antifreeze glycoproteins  show a secondary structure similar to apolyproline II (PPII) helix.47 This also suggeststhat the PPII is a valuable design target, although several PPII designedpeptides failed to show IRI activity.48 In 2017, Gibson and co-workers explored how short polyprolines,49 rather than poly(hydroxyprolines) ,could lead to activity, supporting the emerging hypothesis that hydrophobicfaces (rather than hydroxyls) engage ice in many IBPs.50 Although the oligoprolines were relatively weakin terms of IRI , they were found useful for cryopreservation.51 It is important to note that this is distinct from the effect ofthe amino acid l-proline in cryopreservation as a protectiveosmolyte.53 Alanine/lysine copolymers have also beenreported to show IRI activity;55 for biomedical applications,the net cationic charge may lead to cytotoxicity. Poly(serine) hasbeen tested for IRI,24 but the magnitudereported was essentially identical with a negative control of PEG.23 High molecular weight polyproline has not beenexplored for IRI as it is challenging to obtain by NCA polymerizationsdue to proline NCA’s unique bicyclic structure which typicallyleads to slow polymerization kinetics and relatively low molecularweight materials until copolyemerized.57 Recent advancesin proline NCA polymerizations now make high molecular weight polyprolineaccessible.58 Lu and co-workers have reportedthe water-assisted controlled ROP of ProNCA in ACN/water mixture affordingwell-defined polyproline II helices.59The relatively simple primary amino acidsequence of antifreezeglycoproteinsN-carboxyanhydride polymerization, giving rise to polypeptides ofup to 50 kg mol–1. The peptides were found to bepotent ice recrystallization inhibitors, with the highest molecularweight peptides inhibiting all growth below 5 mg mL–1. The peptides were found to specifically bind ice crystals, as shownby cryomicroscopy.Considering the above, this work explores the synthesis and ice-bindingprotein-mimetic activity of polyproline as a step toward identifyingbiosourced and scalable ice recrystallization inhibitors. Polyprolinewas synthesized by aqueous open-air ring-opening All chemicals were used as supplied unlessotherwise stated. Boc-proline, Boc-hydroxyproline, and triphosgenewere purchased from Fluorochem. Hexylamine, epichlorohydrin, and acetonitrilewere purchased from Sigma-Aldrich. Formvar-carbon-coated (300 mesh)copper grids were purchased from EM Resolutions. Ultrapure water usedfor buffers was Milli-Q grade (18.2 mΩ resistance).1H NMR and 13C NMR spectra were recorded at 400 MHz on a Bruker Advance spectrometer,with chloroform-d (CDCl3), DMSO-d6 ((CD3)2SO), and D2O as the solvent. Chemical shifts of protons are reportedas δ in parts per million (ppm) and are relative to tetramethylsilane(TMS) at δ = 0 ppm when using CDCl3 or solvent residualpeak (H2O δ = 4.79 ppm/DMSO δ = 2.50 ppm). 1H-DOSY was recorded on the previously described spectrometer,and diffusion coefficients are reported in cm2 s–1, obtained using the MestReNova 6.02 software Bayesian method. Diffusioncoefficients were calculated from the intensity of the pyrrole (∼3.8ppm) signal of the l-Pro side chain using a monoexponentialdecay equation, which is a simplified version of the Stejskal–Tannerfunction.2O:ACN mixture (80:20) + 0.1 M NaNO3. Column oven and detector temperatures were regulated to40 °C, at flow rate 1 mL/min. Poly(ethylene oxide) standards were used for calibration between 100 and 500000g mol–1. Analyte samples were filtered through ahydrophilic GVWP membrane with 0.22 μm pore size before injection.Number-average molecular weights (Mn),weight-average molecular weights (Mw),and dispersities (ĐM = Mw/Mn) were determined by conventionalcalibration and universal calibration using Agilent GPC/SEC software.SEC analysis ofpolyproline (PPro) homopolymers was performed on an Agilent TechnologiesInfinity 1260 MDS instrument equipped with differential refractiveindex (DRI), light scattering (LS), and viscometry (VS) detectors.The column set used was a PL Aquagel-OH MIXED-M column. The mobilephase used was a H–1 in 1,1,1,3,3,3-hexafluoro-2-propanol(HFiP). The system was calibrated against Agilent Easi-Vial linearpoly(methyl methacrylate) (PMMA) standards and analyzed by PSS winGPCUniChrom.All SEC samples were prepared using a concentration of 2 mg mL–1 and were filtered through a 0.2 μm Milliporefilter prior to injection.SEC analysis of polysarcosine homopolymers was performed usinga PSS SECurity GPC system equipped with a PFG 7 μm 8 ×50 mm precolumn, a PSS 100 Å, 7 μm 8 × 300 mm column,and a PSS 1000 Å, 7 μm 8 × 300 mm column in seriesand a differential refractive index (RI) detector at a flow rate of1.0 mL min–1 and analyzed using OMNIC software.FTIRspectroscopy measurements were performed using an A PerkinElmer Spectrum100 spectrometer in the range 650–4000 cmDh) and size distributions of PPro polymersamples were determined by DLS using a Malvern Zetasizer Nano ZS witha 4 mW He–Ne 633 nm laser module operating at 25 °C. Measurementswere performed at an angle of 173° (backscattering), and resultswere analyzed using Malvern DTS 7.03 software. All determinationswere repeated 5 times with at least 10 measurements recorded for eachrun.Hydrodynamic diameters, equipped with a Canon DSLR 500Ddigital camera. Images were taken of the initial wafer  and again after 30min. Image processing was conducted using ImageJ. In brief, the numberof ice crystals in the field of view was measured for each photograph.The average (mean) of these three measurements was then calculatedto find the mean grain area (MGS). The average value and error werecompared to that of PBS solution, as appropriate, as a negative control.Splatcooling assays were performed as previously described.59 Boc-l-proline  and epichlrohydrin  weredissolved in acetonitrile (50 mL) at 0 °C, and triphosgene  was added in one portion. The reaction proceeded opento air at 0 °C for 2.5 h until no solids remained; the solutionwas filtered and reduced in vacuo at 40 °C for30 min. The flask of the remaining solution was filled with N2(g) using a cannula, then precipitated into a large excessof hexane, and stored overnight at −18 °C. The resultingsolution was reduced to 1/3rd volume in vacuo undervigorous stirring. The crude NCA oil was dissolved in ethyl acetateand reprecipitated into hexane twice to afford a solid. The NCA wasdried overnight over P2O5 to afford a whitepowder . 1H NMR  δ: 4.33 , 3.79 , 3.33 ,2.32 , 2.17 , 1.95 . 13C NMR  δ: 168.9, 155.0, 63.17, 56.7, 27.26,and 27.0Based on previous method.2O (1:1 v/v) at a concentration of50 mg mL–1 in a small vial open to air. Hexylaminewas added from a stock solution acetonitrile/H2O (1:1 v/v)directly in one portion, and the reaction proceeded as gas bubbleswere evident. Once the NCA peaks at 1823 and 1764 cm–1 had disappeared, the reaction was quenched by addition of excessof H2O. The solution was dialyzed (MWCO = 3.5 kDa) for3 days and lyophilized.Proline NCA was dissolvedin an acetonitrile/HMw, respectively. 1H NMR  δ: 4.70 (1H), 3.68 (2H), 2.30 (1H), 2.03–1.87(3H). 13C NMR  δ: 171.6,58.51, 47.55, 27.86, 24.50.The previous procedure was adapted so that NCA prolinewas addedportionwise in 2 or 5 doses for the 20K and 50K targeted 59 prolineNCA was synthesized from N-Boc l-prolineusing triphosgene and epichlorohydrin asan HCl scavenger  which is characteristicof NCA formation. The lack of FTIR peaks at 1640 and 1630 cm–1 indicates that the absence of polymeric side reactions and residualstarting material, respectively. Full characterization is includedin the Supporting Information . One drawback of using NCA monomers to obtain high-Mw polypeptides is their moisture sensitivity,thereby requiring stringent conditions to achieve a controlled polymerization.Recently, the ability to suppress or outpace the hydrolysis by tuningthe environment has been reported. Song et al. utilized a biphasicDCM/aqueous system which was shown to produce high molecular weightpolypeptides with narrow distributions.61 Alongside this the development of ROPISA has allowed for NCA monomersto polymerize within a basic aqueous environment to outpace the hydrolysis.62 With regard to NCA-proline the methodology reportedby Hu et al. was remarkable, using DFT calculations it was shown thatthe presence of water was promoting polymerization, and hence thismethod is employed here.58 In this methodacetonitrile/water mixtures are used which leads to fast kineticsand high yields and prevents precipitation of growing polyprolinechains  at 1 mg mL–1 in phosphate buffered saline (PBS) . While dry-state TEMimaging showed the presence of needle-shaped fibers, cryo-TEM suggeststhe presence of aggregates solely, and hence the dry-state TEM couldbe a drying effect rather than representative of the solution state.This aggregation does not prevent their later application, but isimportant to consider when measuring any properties that assembliesare present, not just freely dissolved individual chains. In fact,for the later IRI testing, hydrophobicity is known to be crucial,and hence this limit of solubility may be useful, as seen for faciallyamphiphilic glycopolymers.26 It was recentlydemonstrated that polyproline shows large hysteresis in its LCST transitionsmatching the observations found here.71Polymers featuring lactams (cyclic amides) areknown to displaythermoresponsive LCST  behaviorin aqueous solution,ne (PBS) 2B. The lMw < 10 kg mol–1) reported weak ice recrystallizationinhibition (IRI) activity, limiting growth to 50% MGS at 20 mg mL–1.49 It was hypothesized that theamphiphilic PPII backbone was a crucial feature, but this has notbeen widely explored. Other IRI active polymers typically show increasedactivity as molecular weight increases,54 and hence it was important to explore the IRI ofthe present polyproline library to determine if simply increasingthe molecular weight would lead to enhanced properties. To assessIRI, the splat assay was used. This assay allows separation of nucleationand growth effects by seeding a polycrystalline ice wafer which isthen monitored for growth while held at subzero temperatures.44 In the absence of IRI activity the crystals grow, but if an IRIactive agent is present, the ice growth  is suppressed.The data are reported as mean grain size (MGS) relative to a negativeof the buffer alone. It is crucial to note that saline (or other additives)is essential to form a eutectic phase to prevent false positives,and hence phosphate buffered saline was used ([NaCl] = 0.137 M).2349 The higher molecular weights were more activeinhibiting growth below 5 mg mL–1, placing themin the medium activity range of antifreeze protein mimetics (lessactive than PVA or antifreeze proteins).23 It is important to also note that due to the propensity for thepolyprolines to aggregate, the similar activity seen across the panelcould be due to larger aggregates dominating the observed activity. Supporting Information for thesynthetic procedure, Table S1 and Figures S5–S8). For all molecular weights,polysarcosine was less active, and it also highlighted a key featureof IRI testing that any macromolecule of sufficiently high concentrationwill slow ice growth and hence the importance of negative controls.2351 and demonstrates thata simple homopolypeptide can be deployed to prevent ice recrystallization.This may offer advantages compared to all-carbon backbone polymersin the context of making these degradable and from biosourced monomers.A previous report on oligoprolines .After 30 min annealing at −20 °C (note: this assay useslower temperatures than the splat) the control sample containing noadditives showed extensive recrystallization , but 10 mg mL–1 of PPro50k limited the ice growth. This secondary validation confirmsthat polyproline has the potential to be a potent IRI agent, so longas the Mw is sufficiently high.A second assay was also employed to further validatethe aboveIRI observations—the “sucrose sandwich”.73 as do some synthetic polymers21 and self-assembled structures.25 However,the magnitude of IRI activity varies, separately from the extent ofother properties associated with ice binding74 and some materials can display IRI without any evidence of ice binding.75 In short, the macroscopic properties of IRI,thermal hysteresis and dynamic ice shaping76 may be induced by different molecular level interactions.1450k. In this assay a single ice crystal is obtainedby melting polycrystalline ice until a single crystal remains andthen cooled to promote ice growth. In the absence of an ice bindercircular crystals are expected, but facets (different shapes) canbe seen if there is specific ice face binding. As can be seen, asthe ice crystal grows (and before burst growth occurs) faceting canbe seen with the formation of pits where there is no ice growth. Similarmorphologies are obtained for PVA which binds to the prism planesof ice.20 This experiment confirms thatpolyproline can bind to ice crystals faces. No thermal hysteresiswas observed, due to the rapid onset of growth upon cooling. Thissupports our hypothesis that the PPII helix adopted by polyprolinecan mimic the PPII-type helix found in antifreeze glycoproteins,47 where the hydrophobic face engages the ice face,50 which is distinct from e.g. polywhich forms hydrogen bonds to the ice.22 This is particularlyimportant as it suggests that a simple homopolypeptide sequence issufficient to bind ice, although the magnitude of effect seen herecould be due to the above-discussed aggregates. We note that a 14amino acid cyclic sequence  is very potent.77Ice binding proteins (IBPs) function by binding to specific icecrystal planes, which results in their macroscopic properties of icerecrystallization inhibition, thermal hysteresis, and dynamic iceshaping,N-carboxyanhydride(NCA) polymerization, enabling access to polymers with molecular weightsas high as 50000 g mol–1, which is challenging toachieve with standard NCA polymerization of proline. Because of therigid nature of the PPII helix, a DOSY NMR method was required toobtain molecular weights of the larger polyprolines, whereas the shortercould be analyzed by conventional size exclusion chromatography. Thepolyprolines were observed to show molecular weight dependent lowercritical solution temperature and potentially assembled at higherconcentrations. Ice recrystallization inhibition activity was assessedusing both the “splat” and “sucrose sandwich”assays demonstrating polyproline is a potent IRI. The highest Mw polyproline was able to inhibit all ice growthbelow 5 mg mL–1, which is significantly more activethan the previously reported proline oligomers which failed to fullyinhibit ice growth even at 20 mg mL–1. Single icecrystal shaping assays showed faceting, confirming that polyprolinecould bind ice crystal faces. These results demonstrate that structuralsimplification of ice binding proteins to (single) amino acid homopolymerscan retain the key IRI activity and even ice-crystal face recognitionproperties, without a complex primary sequence. These polypeptidesare easy to obtain and crucially can be prepared from biosourced startingmaterials, although it is important to note that a sustainable polymerizationmethod was not explored here, but alternatives such as step-growthor enzymatic polymerization could be employed. The polypeptides alsohave heteroatoms in their backbone and hence have potential to behydrolyzed, unlike other IRI active polymers such as poly.Here we report the synthesis and applicationof high molecularweight polyproline as a biomimetic ice recrystallization inhibitor(IRI), demonstrating that potent IRI activity could potentially beintroduced into materials derived from renewable sources, as opposedto previously reported polymeric IRIs. Our hypothesis is based onthe observations that antifreeze glycoproteins have a polyprolineII helix and that the amphipathic structure is a key motif for activity.We have previously reported that oligoprolines showed weak IRI activitylimited by the molecular weight. High molecular weight polyprolineswere obtained here by aqueous, open to air,


Megasphaera elsdenii. We find that a neutral FADH• semiquinone accumulates transiently during both reduction with sodium dithionite and with NADH in the presence of catalytic concentrations of EtfAB. In both cases full reduction of bcd to the hydroquinone is eventually observed, but the accumulation of FADH• indicates that a substantial portion of reduction occurs in sequential one-electron processes rather than a single two-electron event. In rapid-reaction experiments following the reaction of reduced bcd with crotonyl-CoA and oxidized bcd with butyryl-CoA, long-wavelength-absorbing intermediates are observed that are assigned to bcdred:crotonyl-CoA and bcdox:butyryl-CoA charge-transfer complexes, demonstrating their kinetic competence in the course of the reaction. In the presence of crotonyl-CoA there is an accumulation of semiquinone that is unequivocally the anionic FAD•− rather than the neutral FADH• seen in the absence of substrate, indicating that binding of substrate/product results in ionization of the bcd semiquinone. In addition to fully characterizing the rapid-reaction kinetics of both the oxidative and reductive half-reactions, our results demonstrate that one-electron processes play an important role in the reduction of bcd in EtfAB–bcd.We have investigated the equilibrium properties and rapid-reaction kinetics of the isolated butyryl-CoA dehydrogenase (bcd) component of the electron-bifurcating crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase (EtfAB–bcd) from  The flavin half-potentials in bifurcating systems are highly crossed, with the SQ/HQ half-potential being much higher than the midpoint potential for the two-electron process and the Q/SQ half-potential much lower. As a result, the very low-potential semiquinone oxidation state is highly unstable thermodynamically, a condition considered critical for bifurcation. In the course of bifurcation, the first electron leaving the fully reduced HQ, having been reduced by, e.g., NAD(P)H, has a high reduction potential, with the second, low-potential electron remaining as the unstable FAD•−  component. ETFs from a variety of prokaryotes and eukaryotes, including humans, have been extensively studied, although most are not involved in bifurcation. These simpler ETFs are αβ dimers, with FAD in the α subunit and AMP in the β subunit; the flavin half-potentials are highly uncrossed with the semiquinone oxidation state, typically the FAD•urcation , 2. The − of the bifurcating flavin that is generated in the course of bifurcation. The butyryl-CoA generated from the high-potential pathway is ultimately converted to caproyl-CoA, an intermediate in reverse β-oxidation that occurs in the course of the synthesis of straight-chain fatty acids and other metabolites.The bcd component of these systems has a third equivalent of FAD that is the site of crotonyl-CoA reduction, with ferredoxin being reduced directly by the low-potential FAD•C. difficile 4 core. The quaternary structure of the enzyme is a dimer of dimers, with the bcd subunits of one dimer interacting with one another to a greater extent than with the corresponding bcd subunits of the other pair. The domain containing the electron-transferring FAD (hereafter et FAD) is oriented with its flavin in close proximity to that of its partner bcd subunit, and >30 Å from the bifurcating FAD of the EtfAB. This orientation contrasts with that seen in the crystal structure of EtfAB alone from A. fermentans .The bcd of rsulfide . Mechanirsulfide , 14. A rA couple . BindingA couple , but thaA couple . Finallyzyme FAD . The si Surprisingly, no comprehensive study of the rapid-reaction kinetics of the isolated bcd has yet been reported. In order to characterize the kinetic behavior of the EtfAB–bcd system, we have characterized the reaction of NADH with EtfAB in previous work , 19 and As reported previously , 19, durWe next performed reductive titrations of oxidized bcd in the presence of a catalytic amount of EtfAB with both sodium dithionite and NADH at pH 7.5. As seen in A). This is the reverse of the physiological direction but can be driven, if incompletely, by mass action in the presence of high concentrations of butyryl-CoA. Although the long-wavelength absorbance in the 500 to 600 nm range . In addition, there is a significant absorbance increase at 377 nm in comparison with the absorption decrease at 450 nm at the end of the reaction , consistent with the accumulation of the anionic semiquinone, FAD•− rather than the neutral FADH• seen in the course of dithionite titrations. This suggests, as verified by EPR below, that substrate/product binding results in ionization of the semiquinone oxidation state.Lastly, we titrated reduced bcd with crotonyl-CoA, mpletion B. In addi.e., four-electron) reduced EtfAB, the reoxidation of reduced bcd by crotonyl-CoA, and the reduction of bcd by butyryl-CoA, all by stopped-flow spectrophotometry. Previous work had shown that reduction of EtfAB by NADH is fast with a hyperbolic dependence of kobs on [NADH] and limiting kred at high NADH of 590 s−1 . The magnitude of the absorbance increase indicates that some 40% of the total reduced bcd FAD is in the semiquinone form at the end of the reaction. This in turn means that a significant amount of the electron transfer from the reduced EtfAB to bcd occurs in a one-electron process. Forty percent undoubtedly underestimates the extent of one-electron transfer as some FADH• is very likely reduced subsequently on to the hydroquinone in a second one-electron process. Given that the reduction potentials of the Q/HQ of the et FAD in EtfAB are approximately 136 mV more negative than the SQ/HQ bcd FAD it stands to reason that the two-electron transfer would be thermodynamically quite favorable . The bcdred:crotonyl-CoA charge-transfer complex observed in the reductive titrations described above was fully formed in the mixing dead time of the stopped-flow apparatus, as shown in Figure 5B (blue), indicating extremely rapid and tight binding of crotonyl-CoA to bcd. The difference spectrum between the spectrum obtained immediately after mixing and that of the fully reduced enzyme is shown in the inset to Figure 5B to illustrate formation of the charge-transfer complex (black); the difference spectrum for the overall spectral change is also shown (blue). When reduced bcd is reacted with excess crotonyl-CoA, full reoxidation of the FAD is observed with loss of the charge-transfer complex occurring at a comparable rate, as shown in Figure 5C.The kinetics of the oxidative half of the reaction were next examined, reacting prereduced bcd with crotonyl-CoA, the experiment being performed at 10 °C given the fast kinetics that were observed. The observed kinetics were monophasic with a rate constant that was independent of [crotonyl-CoA] over the range 10 to 110 μM, with an apparent rate of 30 s−1 A. The bcA, hyperbolic fits of the plot of kobsversus [butyryl-CoA] yielded an apparent Kd of 60 μM and limiting kred of 1400 s−1. Given the unfavorable thermodynamics of the reaction noted above, the extent of reduction at lower concentrations of butyryl-CoA increased due to a mass action effect, but above 100 μM butyryl-CoA the extent of reduction became independent of butyryl-CoA. The reduction potential of the crotonyl-CoA/butyryl-CoA is −10 mV , the reaction does not go to completion even at the highest concentrations of butyryl-CoA used, and a charge-transfer complex of butyryl-CoA with the substantial amount of enzyme that remains oxidized is seen at the end of reaction, as reflected in the extended absorption increase above 500 nm (B).Finally, we examined the kinetics of bcd reduction by butyryl-CoA, working again at 10 °C given the fast rates that were observed. The transients were again monophasic, but unlike the reaction with crotonyl-CoA, the observed rate constant exhibited a hyperbolic dependence on [butyryl-CoA]. As shown in Figure 6s −10 mV , and conyryl-CoA A, the ree 500 nm B.Figure − can be differentiated on the basis of their different EPR linewidths (A), which in conjunction with the observed long-wavelength absorbance (− semiquinone (B). The resulting EPR signal, shown in Figure 7B, exhibits a linewidth of 1.6 mT, indicating that FAD•− does indeed form in the presence of crotonyl-CoA. The intensity of the signal represents approximately 20% of the total enzyme as the FAD•−. The extent of formation of FAD•− in the bound state also agrees well with the observed extent of reaction as shown in C. difficile ; there was no EPR evidence for FADH•, although a small amount of a signal with a linewidth of 16 G, representative of a very small percentage of FAD•− (∼2%) formed during the course of reduction by butyryl-CoA, was detected (C). This indicates that the transient absorbance increases seen in Figures 5B and 6B are due to charge-transfer complexes and not neutral semiquinone.Finally, in an attempt to establish whether the long-wavelength absorbance seen during the reduction of bcd by butyryl-CoA was due to the neutral semiquinone or a charge-transfer complex, we reduced 100 μM of bcd with 1 mM butyryl-CoA C; there detected C. This iC), the observed accumulation of FADH• is approximately 40% of the total enzyme flavin, and this undoubtedly underestimates the proportion of one-electron transfer occurring as some of the FADH• generated undoubtedly reduced on to the hydroquinone in a second one-electron event. In oxidative titrations with crotonyl-CoA and reductive titrations with butyryl-CoA, long-wavelength absorption again accumulates, although in both cases this is due to the formation of bcdred•crotonyl-CoA and bcdox•butyryl-CoA complexes. Furthermore, while significant (20%) flavin semiquinone accumulates, it is the anionic FAD•− rather than the neutral FADH• (see B), as confirmed by EPR. Similarly, long-wavelength absorption accumulates in the course of titrations of reduced bcd with crotonyl-CoA, again due to formation of charge-transfer complexes, and again the semiquinone that accumulates is FAD•− rather than FADH•.In the present work, and consistent with earlier work , we see Fig. 4ADH• see B, as con− in their presence indicates that their binding results in ionization of the semiquinone. Similar behavior has been observed with, e.g., trimethylamine dehydrogenase, where the anionic semiquinone is observed when the enzyme is radiolytically reduced while the neutral form is formed after the enzyme is inactivated by ferricenium and subsequently reduced  contrasts with its significant accumulation in the course of the titration of reduced bcd with crotonyl-CoA (B) deserves comment, particularly given that the final concentrations of crotonyl-CoA in the two experiments are the same (100 μM). Having confirmed that the observations in both the titration and stopped-flow experiments are fully reproducible, we have performed two additional experiments. In the first, equal volumes of solutions of reduced bcd and crotonyl-CoA were mixed into an anaerobic cuvette, mimicking after a fashion the conditions of the stopped-flow experiment. No semiquinone, anionic or neutral, was observed, and as in the stopped-flow experiment the reduced bcd was fully oxidized in a two-electron process . In the second experiment, reduced bcd was mixed with varying small volumes of concentrated crotonyl-CoA in the stopped-flow apparatus, in a manner that mimicked additions in the course of the oxidative titration. As in the case of the titration experiment, a significant amount of anionic semiquinone was indeed observed, as reflected in the transient increase in absorbance at ∼380 nm and confirmed by EPR , indicating again a substantial amount of bcd reduction is a one-electron event. Sequential one-electron transfer events to bcd would serve to gate electron transfer so as to prevent low-potential reducing equivalents from progressing down the high-potential pathway . As has been hypothesized , it is 0.29 s−1 C, indicathesized , the enz−1 and is independent of [crotonyl-CoA] above 10 μM. The reaction is monophasic at 450 nm, but at 600 nm there is clear evidence of the formation and decay of bcdred:crotonyl-CoA and bcdox:butyryl-CoA charge-transfer complexes. While previously observed  direction.The oxidation of reduced bcd by crotonyl-CoA occurs with an observed rate constant of 30 sobserved , 20, theC. difficile structure and the structure of the free bcd from the crystal structure of M. elsdenii are virtually identical, as illustrated in α between the two structures of 0.585 Å. Given the lack of conformational changes in bcd on complex formation with EtfAB, we conclude that the observed kinetics of isolated bcd with crotonyl-CoA and butyryl-CoA are relevant to the complex as well.We note that the structure of the bcd component in the M. elsdenii bcd (Mels_2128) and EtfAB (Mels_2126 + Mels_2127) and a C-terminal Strep II tag were constructed as described by Chowdhury et al., 2015. Plasmids were transformed into Escherichia coli BL21 cells with growth conditions as reported  containing the reported . Cells fvia 1 to 2 passages through a French Pressure Cell at 10,000 psi. To remove cell debris, the lysate was centrifuged for 60 min at 200,000g. The supernatant was then flash frozen in liquid nitrogen and stored at −80 °C. Thawed supernatant was bound batchwise with 6 ml of Strep-Tactin XT Superflow resin (IBA GmbH) pre-equilibrated with 50 mM Tris-HCl, 150 mM NaCl, pH 7.5. The column was washed with 30 column volumes of buffer A with 100 μM FAD. After washing, elution was accomplished using 50 mM biotin in 50 mM Hepes (pH 7.5) at 0.5 ml/min. Protein was then concentrated to a final volume of less than 1 ml via Amicon Ultra 4 (Millipore). After concentration, bcd was made anaerobic as described  column equilibrated in said buffer A. Purified protein was then aliquoted, frozen, and stored at −80 °C for future use.Purification of EtfAB and bcd was performed at 0 to 4 °C under aerobic conditions. A procedure described  was modiescribed  via fivefide CoA . The red450 = 21.1 mM−1 cm−1 and ε450 = 11.8 mM−1 cm−1, respectively, as reported  and bovine liver catalase  were made anaerobic via the Argon dry train on ice. After each sample was made anaerobic, glucose was added from the sidearm to a final concentration of 2 to 10 mM, which would scrub the solution of any residual O2 as described  or photomultiplier tube (PMT) detection with the acquisition software ProData SX 2.2.5.6. Samples containing bcd and EtfAB were made anaerobic in buffer A with catalytic amounts of glucose oxidase and catalase. Anaerobicity was achieved in an iced tonometer on an anaerobic train before mounting onto the stopped-flow instrument. Using the diode array detector, spectra in the range 230 to 740 nm were obtained. Kinetic transients at a given wavelength, typically at 450 nm, were extracted from the PDA data set and used for rate determination. With the photomultiplier detector, kinetic transients at specific wavelengths were obtained for the fastest of the reactions. All data were collected at 25 °C except for the reoxidation kred, as well as the apparent dissociation constant, Kd. The reaction of the prereduced EtfAB and oxidized bcd was performed and analyzed as described above. EtfAB was prereduced with sodium dithionite and reacted with oxidized bcd in two schemes at 25 °C: first at a 1:1 ratio of both proteins in the concentration range of 2 to 70 μM and second with 3 μM of prereduced EtfAB with increasing concentrations of oxidized bcd in the range of 3 to 24 μM. The reduction of bcd was fit to a single exponential.The resultant fit allowed for the determination of the limiting rate constant for reduction, ca. 1 ms) and the presence of the charge-transfer complex between oxidized bcd and butyryl-CoA was observed at longer wavelengths (maximum at 571 nm). The full wavelength spectral changes of the reaction of reduced bcd (prereduced with sodium dithionite) and crotonyl-CoA were monitored with the diode array detector. To determine the endpoint of the reaction, kinetic traces extracted from the PDA dataset at 450 and 571 nm were used to monitor the reoxidation of FAD and the decay of the charge-transfer complex, respectively. Both traces were well described by single exponential fits.The reaction of oxidized bcd with butyryl-CoA and that of reduced bcd with crotonyl-CoA were each monitored at 10 °C given the faster kinetics of the former. The photomultiplier detector was used to follow reduction of bcd with increasing concentrations of butyryl-CoA in order to more accurately observe the fastest portion of the reaction. With the photodiode array detector, the majority of the reaction with excess butyryl-CoA was complete in the mixing dead time of the stopped-flow instrument (russ.hille@ucr.edu).Data not contained in the article  are available on request to the corresponding author (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The Totten Glacier in East Antarctica, with an ice volume equivalent to >3.5 m of global sea-level rise, is grounded below sea level and, therefore, vulnerable to ocean forcing. Here, we use bathymetric and oceanographic observations from previously unsampled parts of the Totten continental shelf to reveal on-shelf warm water pathways defined by deep topographic features. Access of warm water to the Totten Ice Shelf (TIS) cavity is facilitated by a deep shelf break, a broad and deep depression on the shelf, a cyclonic circulation that carries warm water to the inner shelf, and deep troughs that provide direct access to the TIS cavity. The temperature of the warmest water reaching the TIS cavity varies by ~0.8 °C on an interannual timescale. Numerical simulations constrained by the updated bathymetry demonstrate that the deep troughs play a critical role in regulating ocean heat transport to the TIS cavity and the subsequent basal melt of the ice shelf. The Totten Glacier in East Antarctica is grounded below sea level and vulnerable to ocean forcing. Observations and simulations demonstrate warm water access from offshore to the glacier, facilitated by deep topography off the Sabrina Coast. The greatest discharge of ice from the East Antarctic Ice Sheet occurs via the Totten Glacier, which drains the Aurora Subglacial Basin in Wilkes Land. The Totten Glacier has an ice volume equivalent to >3.5 m of global sea-level rise3 and is grounded below sea level5, making the Aurora Subglacial Basin vulnerable to oceanic thermal forcing. The Totten Ice Shelf (TIS) experiences an area-averaged basal melt rate of 10.5 ± 0.7 m yr‒1, the highest of all East Antarctic ice shelves, and a basal melt amount of 63.2 ± 4 Gt yr−1 for 2007‒2008 (ref. 6).While the contribution to global sea-level rise from West Antarctica has received the most attention , East Antarctica has also been a major contributor (4.4 ± 0.9 mm over the same period)8. East Antarctic continental shelves are typically cold and fresh9 because access of warm CDW to the shelf is limited by the presence of the Antarctic Slope Current (ASC) associated with the Antarctic Slope Front (ASF) over the upper continental slope10. Knowledge of the factors that regulate the transport of ocean heat from offshore to the ice shelf cavity is essential to assess the vulnerability of the Totten Glacier and its large potential contribution to sea level rise. Substantial gaps remain because most of the continental shelf near the Totten Glacier has not been sampled due to heavy sea-ice cover. Previous observations on the Totten continental shelf are limited to the relatively shallow Dalton Polynya region and a single voyage to the front of the TIS12. Those observations showed that relatively warm modified CDW (mCDW) is present on the continental shelf and reaches the TIS12, through deep troughs that provide access to the ice shelf cavity3. However, from these limited observations, it was not possible to determine the pathways and processes regulating the transport of warm water from the offshore ocean to the TIS cavity, nor was it possible to assess variability in the temperature of mCDW.Ocean heat transport to the ice shelf cavity drives basal melt of ice shelves. High melt rates and ice loss from the West Antarctic Ice Sheet are driven by intrusions of warm Circumpolar Deep Water (CDW)Our observations span the Totten embayment, including broad helicopter-based measurements, detailed multibeam bathymetric surveys of the deep troughs that provide warm water access to the cavity, and hydrographic measurements from the unsampled western side of the ice front, where glacial meltwater is speculated to leave the cavity. We collected comprehensive observations of bathymetry and water properties off the Sabrina Coast during three cruises  extending from the upper continental slope/shelf break across the continental shelf to the TIS front (see Methods) and including many regions not previously sampled. Here we combine our bathymetric data with existing measurements to create a more realistic and detailed bathymetric dataset for the region  depression  connect the Sabrina Depression to the TIS cavity  on the eastern flank of the Sabrina Depression and weaker northward flow further west. Our comprehensive observations confirm the presence of a cyclonic gyre on the shelf, consistent with the circulation inferred from limited float data in an earlier study18, as well as satellite-derived sea surface velocity ) overlying mCDW  near the seafloor. These contrasting water masses are separated at ~500‒600 m depth by a sharp interface in temperature and salinity  Fig. . A zonal11. Each trough receives mCDW with different temperatures, with the warmest water (0.24 °C) observed in C-TT  is typically at ~500‒600 m depth in the Totten region  mCDW is only observed where the water depth exceeds 600‒700 m   Figs. b, 4. The2) Figs. . Salinit2) Figs. . Overall2) Figs. , suggest2) Figs. , and the22,) in the Totten region, at least at the sub-seasonal scale.The time series measurements at E-TT provide further evidence that the interaction between topography and thermocline depth regulates the temperature of water reaching the TIS. Fluctuations in temperature at the mean depth of the WW/mCDW interface are roughly synchronous with those in the near-bottom mCDW layer . Stable oxygen isotopes in seawater are sensitive tracers of freshwater origin in polar waters and are especially sensitive to input of glacial meltwater as it is the primary source of isotopically light freshwater25. The δ18O along the TIS front was lower throughout the water column in the west than at the central ice front  to the western ice front . The WW layer at the western ice front (stations T2 and T3) is also warmer and lower in DO ‒1 for the 1991‒2020 climatology, Supplementary Fig. −1)6. The simulated spatial distributions of near-bottom temperature . Further offshore, the simulated circulation is characterized by the presence of multiple semi-permanent cyclonic eddies  centered at the eastern side of C-TT. Lower salinity (less dense) waters exit the cavity on the western side of the TIS front. The results from the observations  in front of the Sabrina Coast were filled in . A comparison of the simulations with and without the deep troughs  clearly demonstrates the importance of the C-TT as a conduit for ocean heat transport to the TIS cavity. In the CTRL case Fig. , the mod11 based on a snapshot in 2015. During the circulation in the TIS cavity, the mCDW melts the TIS from below, mixes with the resulting glacial meltwater, and becomes lighter (0.1‒0.2σθ lower in density). The mixture flows along the base of the ice shelf to the north and finally exits the cavity, reducing the salinity and density of the water column at the western TIS front  in the west , a vertical displacement of only ~60 m relative to the shallowest sill in the trough could explain the ~0.8 °C range between the years with coldest and warmest mCDW in C-TT , thus altering the temperature and the thickness of the mCDW layer on the shelf. In the Totten region, wind-driven changes in the strength of upwelling over the upper continental slope37, of the westward flow of the ASC31, and of the eastward undercurrent on the upper continental slope18 have been linked to changes in the transport of mCDW onto the Totten continental shelf. Once on the continental shelf, the temperature and thickness of the mCDW layer may be altered by mixing or water mass transformation. For example, changes in buoyancy forcing associated with sea ice production in the Dalton Polynya may also drive changes in stratification and ocean heat supply to the TIS38. Input of glacial meltwater can increase the temperature and thickness of the warm mCDW layer by inhibiting deep convection that could vent heat from the mCDW layer before it reaches the ice shelf27. As observations are at present limited to four summer snapshots, it is not yet possible to determine which of these potential driving factors are most important to driving variability in ocean-driven basal melt of the Totten Glacier.Various processes may contribute to driving variability in temperature and thermocline depth on the Antarctic continental shelf. Remote or local changes in wind forcing can drive changes in circulation on the slope and shelf, in the exchange between the open ocean and the continental shelf, and in the depth of the thermocline  sources of meltwater.Glacial meltwater input keeps WW on the Totten continental shelf relatively fresh, inhibiting the formation of Dense Shelf Water (DSW)nt Figs. , 7a‒c. W41. These changes may reflect an increase in ocean heat transport to the TIS cavity, but this hypothesis cannot be tested with observations because oceanographic measurements at the TIS front (ref. 11. and this study) span a short time interval. However, long-term warming of CDW over the East Antarctic continental slope 42 and a significant southward shift in the Southern Boundary of the Antarctic Circumpolar Current, especially off the Sabrina Coast at ~120°E43, may have increased the reservoir of heat available offshore of the Sabrina Coast’s ice shelves. If the warmer offshore waters were transported to the deep troughs by the mechanisms described above and illustrated in Fig. 44.Satellite observations show that the Totten Glacier has thinned and the grounding line has retreated in recent decadesOur study provides valuable insights into the physical processes that control the melt rate of the Totten Glacier, now and in the future. In particular, our observations from the TIS front suggest that changes in depth of the thermocline drive variability in thermal forcing on a broad range of timescales . This further implies that changes in future forcing or regional circulation that alter the thickness and warmth of the mCDW layer will drive changes in basal melt. While our study provides comprehensive observations spanning nearly the full Totten embayment and robust evidence of temporal variability in the inflow of warm mCDW to the TIS cavity, further observations are needed to better understand the drivers of mCDW variability and the exposure of the Totten Glacier to ocean-driven melt, now and in the future. The large interannual variability observed in the temperature of mCDW reaching the TIS Fig.  highlighShirase” (AGB-5003). This observational campaign was conducted during the 61st Japanese Antarctic Research Expedition (JARE61), under the “Research of Ocean-ice BOundary InTeraction and Change around Antarctica” (ROBOTICA) project of the National Institute of Polar Research. In March 2018 (JARE59) and March 2022 (JARE63), we made the shipborne hydrographic and bathymetric observations at the TIS front and continental shelf regions under the same project.In December 2019 and February/March 2020, we made comprehensive ship- and airborne observations to obtain hydrographic and bathymetric data from the upper continental slope/shelf break, continental shelf, and ice front regions around the TIS on the Sabrina Coast from the icebreaker “Shirase’s helicopter  with a DO sensor   for scientific use  installed on the icebreaker N.B. Palmer (survey number 14-02)46 and the EM122/EM710 (Kongsberg) installed on the R/V Investigator cruise (survey number IN2017-V01)47. The N.B. Palmer data was obtained through Marine Geoscience Data System (MGDS) hosted by Lamont-Doherty Earth Observatory of Columbia University, then manually processed with the same methods as applied to the Shirase data. The Investigator data was provided in a processed format by the CSIRO National Collections and Marine Infrastructure.The multibeam sonar data from JARE observations were compiled with two sets of previously collected multibeam data  , and 2014/15-V2 cruise (AA2014) of RSV 16  relative to the geoid height and consists of the baroclinic (steric) and the barotropic (bottom pressure term) components. We employ a monthly mean DOT dataset (with 0.2° × 0.2° grid spacing) over the Southern Ocean , derived from multiple satellite radar altimeter datasetsThe DOT-derived velocity, which can be obtained at the sea surface only, reflects both vertically varying baroclinic and vertically homogeneous barotropic components. Meanwhile, hydrographic XCTD measurements provide the geostrophic velocity vertical profile, containing only the baroclinic component and not the barotropic component. We then obtain the vertical profiles of absolute velocity, including both baroclinic and barotropic components, by shifting the whole vertical profile of geostrophic velocity estimated with XCTD hydrographic profiles to such that the geostrophic velocity at the surface agrees with the DOT-derived velocity. We used the monthly mean DOT-derived velocity in February 2020, the closest to the observation period, for calculating the absolute velocity shown in Fig. δ18O can be used as conservative parameters to quantify the respective freshwater contribution from glacial and sea-ice meltwaters. Assuming that observed salinity and δ18O , meteoric water , and mCDW . Standard deviation of our δ18O measurement was estimated to be 0.016‰. The largest uncertainty comes from the δ18O endmember of meteoric meltwater. The melt line drawn on δ18O-salinity diagram for the data in Fig. δ18O of −32 ‰ at zero salinity from the values of mCDW, which is consistent with the inland surface accumulation value27. Assuming the uncertainty of 5% (ref. 25), the error in meteoric water fraction would be about 0.5% for a typical seawater of the upper layer. Although this error range is seemingly large, practical error in spatial difference in meltwater fraction should be much less among nearby stations because of the systematic nature of the error assumption.Salinity and oxygen isotope ratio https://ccsr.aori.u-tokyo.ac.jp/~hasumi/COCO/), with an ice-shelf component36. The horizontal grid of the model was discretized on an orthogonal, curvilinear, horizontal coordinate system, and we can regionally enhance the horizontal resolution around the Totten Glacier region by placing the two singular points on the longitude of the focal area . With this treatment, the horizontal resolution near the Totten Glacier region was 3‒4 km, keeping the model domain to the circumpolar Southern Ocean with an artificial solid boundary north of 43.2°S (depending on the longitude).We utilized an ocean–sea ice model, COCO  for the surface layers, 20 m (59 grid levels) for the 20‒1200-m range, 40 m (30 grid levels) for the 1200‒2400-m range, and 100 m (31 grid levels) for the 2400‒5500-m range. The maximum ocean depth in the model was set to 5500 m. The regionally high horizontal resolution enabled us to capture the realistic coastline and icescape. We used the General Bathymetric Chart of the Oceans51 as the background bottom topography for the Southern Ocean. We replaced the bottom topography around the Totten Glacier region in the following steps:The vertical coordinate system of the ocean model was a (1) The bottom topography in the region  was replaced with the detailed topography derived in this study and described in the Methods section for bathymetry above (Note that multibeam sonar bathymetric data from the 2021/22 season was not incorporated into the model bathymetry due to the unavailability at the time when we prepared the model configuration.);5;(2) The topography under the ice shelves and the ice-shelf draft were calculated from BedMachine Antarctica(3) In the coastal region within 20 km of the ice-shelf front, we blended our compiled data and BedMachine’s topography, selecting the deeper topography of the two.53. Because sea ice processes are strongly controlled by the positions of the icescape, we introduced the landfast sea ice areas into the model with a constant thickness (5 m) for ice-shelf grid cells, as in our previous studies36. We assumed a steady-shaped ice shelf in the horizontal and vertical directions in the model. A three-equation scheme with velocity-independent coefficients54 for the thermal and salinity exchange velocities (−1 and −1) was used to estimate the freshwater flux at the base of the ice shelves.The icescape in this region was characterized by well-developed landfast sea ice56 throughout the water column, with a damping time scale of 10 days. Outside of the focal region where the horizontal resolution is larger than 4.3 km  and then performed a hindcast simulation for the period from 1951‒2021 with interannually variable forcing (“CTRL” case).Near the artificial northern boundary or north of 40°S, water properties  were restored to the monthly climatology of the World Ocean Database 2018An additional experiment of the NOTR case was performed to quantify the roles of deep troughs in front of the ice-shelf cavities. In the NOTR case, the areas deeper than 500 m along the Sabrina Coast (purple areas in Fig. Supplementary InformationPeer Review File