peptide amphiphiles are a fascinating 
 class of bioactive molecules 
 in which one or more lipid chains are attached to peptides , either 
 bioderived , bioinspired , or designed .
the lipid chains drive self - assembly , 
 leading to nanostructures in which the functional peptide unit can 
 be presented at high density , for example , on the surface of nanofibrils .
the use of enzymes to modulate the self - assembly of peptide 
 amphiphiles 
 ( pas ) and other amphiphilic biomolecules such as amphiphilic peptides 
 and peptide  polymer conjugates has attracted considerable attention 
 due to potential applications in biomedicine as well as catalysis 
 and sensing .
enzymes have been evolved by nature to perform many essential 
 functions in biological systems .
they have excellent specificity and 
 activity under mild environmental conditions associated with living 
 organisms .
the topic of enzyme - controlled peptide self - assembly has 
 been the subject of a recent comprehensive review .
other reviews cover enzyme - driven ( biocatalyzed ) self - assembly 
 of peptide derivatives , leading specifically to hydrogelation and the influence of enzymes on the aggregation 
 of polymer - peptide conjugates . as well as enzyme - stimulated 
 transitions in nanostructure
, peptide systems can also undergo enzyme - mediated 
 disassembly and enzyme - triggered self - assembly . by design of the peptide substrate , responsiveness to a 
 wide variety of enzymes
we have demonstrated a concept whereby pegylated 
 peptide micelles 
 prepared from an amyloid -peptide derived heptapeptide containing a diphenylalanine ( ff ) sequence attached to peg were 
 enzymatically degraded by -chymotrypsin which selectively cleaves 
 peptide bonds between aromatic residues .
this released a hexapeptide ( containing a c - terminal f ) along with 
 peg with an n - terminal f residue . this proof - of - principle work established 
 a possible route to create an enzyme - responsive delivery system based 
 on peg - coated particles which have relevance to biological applications 
 since peg provides a sterically stabilized corona , enabling enhanced 
 in vivo stability . here
, we investigate the influence of the 
 model serine protease 
 -chymotrypsin on the self - assembly of the pa c16-kkffvlk .
the peptide amphiphile ( pa ) has been designed based on 
 the klvff core motif from the amyloid beta ( a ) peptide .
this contains two f residues at the c terminus which drive self - assembly 
 through hydrophobic and -stacking interactions .
two additional lysine residues are incorporated between the hexadecyl 
 lipid chain and the ffklvff motif to promote solubility and impart 
 amphiphilicity .
the serine protease -chymotrypsin is expected 
 to cleave preferentially between the two f residues , although as discussed 
 below an additional cleavage site is also identified .
the pas corresponding 
 to cleaved products c16-kkf and c16-kkff are 
 shown to self - assemble into spherical micelles , quite distinct from 
 the nanotubes / helical ribbons formed by c16-kkffvlk .
we 
 thus show that it is possible to develop enzyme - responsive pa systems 
 with pronounced nanostructure transitions , leading in turn to macroscopic 
 changes in the sample transparency .
peptide amphiphile c16-kkffvlk 
 was purchased from cs bio ( menlo park , ca ) . for the first batch ,
the 
 molar mass by electrospray - ionization mass spectrometry was 1146.97 
 da ( expected 1147.57 da ) .
the molar mass by es - msi was 1147.65 da and the 
 purity was 99.73% by hplc .
peptide amphiphile c16-kkff was synthesized by cs bio ( menlo park , ca ) and supplied as 
 a tfa salt .
the molecular weight was found to be 807.31 da ( expected 
 807.11 da ) as revealed by electrospray - ionization mass spectrometry . 

purity of the sample was 99.88% by analytical hplc in a 0.1% tfa water / acetonitirile 
 gradient .
peptide amphiphile c16-kkf was purchased 
 from cs bio 
 ( menlo park , ca ) and supplied as a tfa salt .
electrospray - ionization 
 mass spectrometry revealed a molecular weight of 659.73 da ( expected 
 659.94 da ) .
purity was 99.35% by analytical hplc in a 0.1% tfa water / acetonitirile 
 gradient and supplied as a tfa salt .
peptide fvlk was custom 
 synthesized by peptide synthetics ( peptide 
 protein research ltd .
the molecular weight 
 was found to be 505.641 da by electrospray - ionization mass spectrometry . 

the sample was purified in acetonitrile and water containing 0.1% 
 tfa prior to lyophilization and was supplied as a tfa salt .
the 
 sample was purified in acetonitrile and water containing 0.1% tfa 
 prior to lyophilization and was supplied as a tfa salt .
the enzyme -chymotrypsin , 
 which has a molecular weight of 25 kda , was purchased from sigma - aldrich 
 ( bovine pancreas extract ) . to characterize self - assembly in 
 water ,
solutions were made by 
 direct dissolution in ultrafiltered water , from a barnstead nanopure 
 system .
the ph of a 0.5 wt % solution of c16-kkffvlk was 
 4.18 , and the ph was 3.84 for a 1 wt % solution .
direct - infusion electrospray mass 
 spectra ( es - ms ) were obtained on a thermofisher scientific orbitrap 
 xl instrument .
the ms / ms experiments were performed with an isolated 
 target mass of 648 da ( isolation width of 3 da ) , and fragmentation 
 was achieved in the hcd trap with a fragmentation energy of 35 . a 
 mixture of 1 wt % c16-kkffvlk and 2 wt % -chymotrypsin 
 was prepared for mass spectrometry analysis .
a vial containing the solution was then placed in a 
 sonicator at 50 c for 1 h. this temperature was found to improve 
 the activity of -chymotrypsin ( previous work showed increasing 
 activity with temperature , but the highest temperature studied was 
 35 c ) .
the pa / enzyme mixture was 
 left to age for 4 months before mass spectrometry measurements were 
 performed .
spectra were recorded using 
 a nexus - ftir spectrometer 
 equipped with a dtgs detector and a multiple reflection attenuated 
 total reflectance ( atr ) system .
solutions of the pas and peptides 
 in d2o ( 0.5 , 1 , and 2 wt % ) were sandwiched in ring spacers 
 between two caf2 plate windows ( spacer 0.006 mm ) .
spectra 
 were recorded with a varian cary eclipse fluorescence spectrometer 
 with samples in 4 mm inner width quartz cuvettes .
the spectra were 
 recorded from 460 to 600 nm using an excitation wavelength ex = 440 nm .
measurements were performed 
 on stalks prepared by drying filaments of the pas or peptides , prepared 
 from 0.5 , 1 , or 2 wt % solutions .
solutions of the peptide were suspended 
 between the ends of wax - coated capillaries and dried .
the stalks were 
 mounted ( vertically ) onto the four axis goniometer of a raxis iv++ 
 x - ray diffractometer ( rigaku ) equipped with a rotating anode generator 
 and a saturn 992 ccd camera . the sample 
the x - ray wavelength was  
 = 1.54  . the wavenumber scale ( q = 4 
 sin / , where 2 is the scattering angle ) was geometrically 
 calculated using the size of each pixel in the detector screen ( 0.0898 
 mm ) and the sample  detector distance .
saxs data were collected 
 on the biosaxs beamline bm29 at the esrf , 
 grenoble , france .
solutions containing 0.5 , 1 , or 2 wt % c16-kkffvlk were loaded in pcr tubes in an automated sample changer . 

further 
 experiments ( including kinetics measurements ) on a 1 wt % sample were 
 performed on beamline id02 at the esrf , grenoble , france .
saxs data 
 were collected with a frelon kodak ccd with a 1.2 m sample  detector 
 distance , and waxs data were measured simultaneously with an aviex 
 ccd .
solutions were injected 
 using a syringe into enki ki - beam thin ( 0.05 mm ) wall 1.85 mm diameter 
 polycarbonate capillaries which optimize background subtraction .
heat / cool experiments were performed 
 with the sample mounted in a quartz capillary instead of the enki 
 polycarbonate capillary .
cryo - tem 
 was carried out using a field emission cryo - electron microscope ( jeol 
 jem-3200fsc ) , operating at 200 kv voltage ( except for sample vlk for 
 which the voltage was 300 kv ) .
images were taken in bright field mode 
 and using zero loss energy filtering ( omega type ) with slit width 
 of 20 ev .
vitrified specimens were prepared using an automated 
 fei vitrobot device using quantifoil 3.5/1 holey carbon copper grids 
 with the hole size of 3.5 m . just prior to use ,
grids were 
 plasma cleaned using a gatan solarus 9500 plasma cleaner and then 
 transferred into an environmental chamber of a fei vitrobot at room 
 temperature and 100% humidity .
thereafter , 3 l of sample solution 
 ( 2 wt % concentration ) was applied to the grid and it was blotted 
 twice for 5 s and then vitrified in a 1/1 mixture of liquid ethane 
 and propane at temperature of 180 c . the most viscous 
 gel ( 5 wt % concentration )
was blotted four times for 5 s. the grid 
 with vitrified sample solution were maintained at liquid nitrogen 
 temperature and then cryo - transferred into the microscope .
our objective was to examine the effect of 
 -chymotrypsin 
 ( c ) in cleaving the pa c16-kkffvlk and to investigate 
 the self - assembly of the product peptides and peptide amphiphiles . 

figure 1 shows an electrospray 
 ionization mass spectrum for the pa / enzyme mixture , which reveals 
 sharp peaks with m / z values of 660.50 
 and 506.33 da .
these peaks are identified as c16-kkf and 
 fvlk , respectively , since these values correspond to the expected 
 molar masses , listed in the experimental section ( i.e. , ions with z = 1 are produced ) .
these two 
 fragments are expected to be released from c16-kkffvlk 
 in the presence of c as the enzyme selectively catalyzes the 
 hydrolysis of peptide bonds on the c - terminal side of phenylalanine , 
 which in our peptide corresponds to cleavage between the two f residues . 

unexpectedly we observed an additional two peaks with high abundances 
 with m / z values of 807.57 and 359.27 
 da .
the former is ascribed to c16-kkff and the latter as 
 the tripeptide vlk , since the m / z values correspond to the expected molar masses for these species 
 ( again z = 1 ) .
ms / ms fragmentation was employed to 
 confirm the nature of the two unexpected fragments by sequentially 
 knocking off amino acid residues from the c - terminus as shown in figure 2 . as the amino acids k then l and then v are removed 
 from the c - terminus , a peak at 789.56 da
ms / ms fragmentation indicates that c cleaves at an additional 
 site between f and v residues and confirms the existence of the two 
 fragments ( c16-kkff and vlk ) .
es - ms for a mixture of 
 1 wt % c16-kkffvlk and 2 wt % 
 -chymotrypsin .
ms / ms fragmentation spectrum for c16-kkffvlk , sequentially 
 removing lysine , leucine , and valine from the c - terminus . to further elucidate the enzymatic degradation 
 products of the 
 pa by -chymotrypsin ,
figure 3 shows spectra for the pa and the enzyme alone , 
 as well as the mixture .
it is clear that the cd spectrum from the 
 mixture is completely different from either of the two components 
 and can not be expressed as a superposition of the spectra from the 
 species in the mixture .
the spectrum for c alone is 
 consistent with that previously reported by ourselves and others .
the spectrum for c16-kkffvlk is dominated by electronic transitions associated with the 
  stacking of the f residues , as discussed elsewhere . the expected cd 
 spectrum for -sheet structures , with a negative minimum near 
 216 nm and a positive maximum in the 195202 nm range ,
we provide evidence in a recent paper that this pa does form -sheet fibrils 
 and this is supported by further data provided herein , to be discussed 
 shortly .
cd spectra for 1 wt % c16-kkffvlk and 1 wt % -chymotryspin 
 and their mixture ( 1 and 1 wt % ) .
cd was also used to examine the self - assembly of peptides 
 and pas 
 corresponding to the cleavage products , c16-kkf and fvlk 
 and c16-kkff and vlk , respectively .
the spectra for the two pas ( figure 4a , b ) do not show features of canonical secondary 
 structure such as -sheets .
the spectra contain contributions 
 arising from  stacking of phenylalanine residues 
 which as shown in our papers on phenylalanine - rich peptides and peptide conjugates can give rise 
 to a positive maximum in the cd spectrum at 210220 nm .
there 
 is also a contribution from polyproline ii ( ppii ) structure in this 
 region .
the presence of a deep minimum in a cd spectrum at around 
 190 nm , along with a broad positive maximum at around 210 nm , is a 
 signature of ppii ( collagen - like ) secondary structure .
the spectra for vlk and fvlk in figure 4c 
 and d show these features .
the spectra for flvk also show features 
 in the 240270 nm range associated with vibronic transitions 
 of the phenylalanine chromophore .
cd spectra for solutions of 0.5 , 1 , and 2 wt % for ( a ) 
 c16-kkff , ( b ) c16-kkf , ( c ) vlk , and ( d ) 
 fvlk .
spectra covering the amide i and amide ii 
 regions from 0.5 , 1 , and 2 wt % solutions of the pas and peptides 
 are shown in figure 5 .
the spectra for c16-kkffvlk show a peak at 1626 cm which 
 is due to -sheet structure , along with 
 a peak at 1672 cm which is due to bound tfa counterions .
the spectra for c16-kkff and c16-kkf do not 
 show peaks in the 16151635 cm range typical 
 of -sheet structures but have broad peaks near 1641 cm .
peak positions in this range are typically ascribed 
 to random coil structure ; however , as 
 discussed elsewhere , ppii structures can also give a peak in this 
 region .
all of the pas also exhibit broad 
 peaks in the amide ii region centered on 1580 cm .
the spectra for peptides fvlk and vlk in figure 5d , e show broad shoulder peaks in the 16401650 cm region due to random coil / ppii structure ( along with 
 the peak due to bound tfa counterions ) .
peaks in the amide i region are absent for the peptides indicating 
 a lower extent of ordering of n 
ftir spectra 
 for solutions of 0.5 , 1 , and 2 wt % for ( a ) c16-kkffvlk , 
 ( b ) c16-kkff , ( c ) c16-kkf , 
 ( d ) fvlk , and ( e ) vlk .
fiber x - ray diffraction was also used to examine peptide 
 secondary 
 structure .
diffraction patterns from dried stalks are shown in supporting information figure 1 along with meridional 
 and equatorial intensity profiles .
the peak positions were used to 
 calculate d spacings and these are listed in supporting information table 1 . the pattern for 
 c16-kkffvlk ( supporting information figure 1a ) shows features of a cross- structure with meridional reflections from a d = 4.83  spacing ( corresponding to the -strand spacing ) 
 along with a series of equatorial reflections from the -sheet 
 stacking distance and lateral packing of the pa molecules .
a series 
 of off - axis reflections close to the equator are due to the helical 
 twisting of nanoribbons .
the xrd pattern 
 shows a high degree of alignment and multiple bragg peaks confirming 
 the regular ordering of the pa within the nanotubes / ribbons .
in contrast , 
 the xrd patterns for c16-kkf and c16-kkff do 
 not show orientation , and only present a small number of reflections . 

the pattern for c16-kkff indicates the lack of secondary 
 structure for this pa since cross- features are not observed , 
 just sharp peaks corresponding to the packing of the lipid chains 
 along with a broad 5.7  ring due to the spacing of the molecules . 

unexpectedly , c16-kkf shows more order in its xrd pattern 
 than c16-kkff with some cross- features ( i.e. , 4.79/4.65 
  spacings and a 13.4  peak ) although the 5.7  ring 
 and the 4.11  peak from the packing of the lipid chains predominate .
the self - assembled nanostructures of the pas ( c16-kkffvlk 
 and pas corresponding to the fragments produced after enzymatic degradation ) 
 and peptide fragments were examined by cryogenic transmission electron 
 microscopy ( cryo - tem ) .
c16-kkffvlk forms nanotubes coexisting 
 with helical ribbons ( figure 6a and ref ( 22 ) ) .
saxs data ( figure 7 ) confirm this assignment , as discussed in detail 
 elsewhere .
in complete contrast , c16-kkff and c16-kkf self - assemble into small ( approximately 
 5 nm diameter ) micelles .
this was confirmed by saxs ( figure 8) since the intensity profiles were fitted by form 
 factors corresponding to uniform spheres , yielding radii of 2.5 and 2.9 nm for c16-kkf and c16-kkff , respectively .
cryo - tem did not reveal a significant 
 extent of nanostructure formation for peptide fragments fvlk and vlk . 

there was some evidence for sparse fibril clusters for fvlk ( figure 6d ) , and for vlk occasional raftlike film structures 
 were observed ( figure 6e ) .
however , saxs on 
 solutions of these two peptides ( data not shown ) revealed no scattering 
 above background .
the lack of significant structures for these two 
 peptides is also consistent with ftir .
the observed raftlike film 
 structures for vlk may be due to the surface activity of this peptide .
cryo - tem 
 images for solutions containing 1 wt % ( a ) c16-kkffvlk , 
 ( b ) c16-kkff , ( c ) c16-kkf , ( d ) fvlk , 
 and ( e ) vlk .
saxs profiles fitted to a spherical shell form factor model for 
 ( a ) 0.5 wt % c16-kkf and ( b ) 0.5 wt % c16-kkff . the difference in morphology of 
 the aggregates of c16-kkffvlk and the fragments c16-kkf and c16-kkff 
 leads to changes in sample transparency as shown in figure 9 .
the large nanotube / ribbon structures formed by 
 c16-kkffvlk cause light scattering and a partly cloudy 
 appearance , whereas the micellar structures of c16-kkf 
 and c16-kkff show higher transparency .
confirmation that 
 addition of enzyme to a c16-kkffvlk solution leads to a 
 clear solution due to the cleavage process producing very small micelles 
 is provided by the image shown for the mixture in figure 9 .
image of samples ( 1 wt % ) in vials from left to right : 
 c16 - kkffvlk , c16-kkff , c16-kkf , 
 and c16-kkffvlk with added c ( 2 wt % ) . as discussed in more detail elsewhere
, c16-kkffvlk undergoes a thermoreversible 
 transition from 
 nanotubes coexisting with helical ribbons ( unwound nanotubes ) at 20 
 c to twisted tapes at higher temperature ( 55 c ) .
the oscillations in the data at 20 c are characteristic 
 of a nanotube / ribbon bilayer structure as analyzed by form factor 
 fitting .
these disappear on heating , showing the break - up of the nanotubes / ribbons . 

however , on recooling , the oscillations develop slowly over a period 
 of hours .
the enzymatic cleavage of a pa using the model protease -chymotrypsin 
 has been demonstrated .
es - ms reveals that c attacks two cleavage 
 sites within the pa and preferentially cleaves between the two f residues 
 as expected , leading to a higher abundance of both c16-kkf 
 and fvlk .
the second cleavage process occurs at the c terminus of 
 the second f residue in c16-kkffvlk to produce c16-kkff and vlk .
we have shown that the pas corresponding to 
 the cleavage products 
 c16-kkf and c16-kkff assemble into very different 
 structures than the parent pa which forms nanotubes and helical ribbons 
 at room temperature .
in contrast to c16-kkffvlk , the two pas with shorter peptide  headgroups  
 lack substantial defined secondary structure ( there were some signs 
 of -sheet content in the xrd pattern for c16-kkf , 
 possibly due to reduced hydrophobicity compared to c16-kkff ) . 

indeed , 
 a phase diagram assembled for pas on the basis of molecular dynamics 
 computer simulations anticipates the formation of spherical micelles 
 when hydrogen bonding interactions are weak .
the near absence of intermolecular hydrogen bonded -sheets 
 for c16-kkf or c16-kkff presumably reflects 
 the insufficient length of the peptide headgroup , i.e. there are insufficient 
 residues to participate in extensive hydrogen bonding networks .
the 
 change in nanostructure from c16-kkffvlk to c16-kkf and c16-kkff leads to macroscopic changes in sample 
 appearance which might be useful for a simple enzyme sensing system , 
 although more sensitive methods may be available .
we have recently 
 investigated the self - assembly of the pa c16-kttks which 
 forms extended -sheet nanotapes .
considering in addition 
 the findings reported here , our results on lysine - rich pas suggest 
 that a minimal sequence capable of forming -sheets may require 
 around five residues , provided that the peptide has both highly soluble 
 hydrophilic residues along with hydrophobic residues .
however , this 
 is not a general rule since , for example , we have recently shown that 
 a pa c16-ah with only a dipeptide -alanine - histidine 
 headgroup can self - assemble into -sheet fibrils in water .
neither of the peptide fragments vlk nor 
 fvlk form extensive self - assembled 
 nanostructures , although tripeptides are able to do so provided they 
 have sufficient amphiphilicity ( e.g. , if they contain two hydrophobic 
 phenylalanine residues ) .
our own work 
 also shows that a hexapeptide with one terminal phenylalanine does 
 not self - assemble whereas a homologous heptapeptide with two c - terminal 
 phenylalanines is capable to form -sheet fibrils .
one interesting application of our lysine - rich 
 pas may be antimicrobial 
 agents . the antimicrobial activity of pas c16-kxk ( where 
 x is a , g , l or k or k , i.e. d - lysine ) and c16-kk and c16-k has been investigated , and this was correlated 
 to their self - assembly properties .
the 
 most active pa ( against microbes ) c16-kkk formed very small 
 oligomers in contrast to c16-kgk and c16-klk 
 which formed fibrils and c16-kak which formed 10 nm diameter 
 micelles .
our results highlight the ability of enzymes to modulate 
 the self - assembly 
 of pa systems and the ability to tune nanostructure through use of 
 enzymatic triggers .
our proof - of - concept work may be extended to create 
 other enzyme - responsive assemblies or in applications where release 
 of peptide or pa fragments is beneficial .
specifically , release of 
 micelles in response to enzymatic degradation of extended nanostructures 
 ( which have been shown to offer enhanced circulation 
 times in vivo ) may be useful in delivery of actives .
 the enzymatic cleavage 
 of a peptide amphiphile ( pa ) is investigated . 
 
 the self - assembly of the cleaved products is distinct from that of 
 the pa substrate . 
 the pa c16-kkffvlk is cleaved by -chymotrypsin 
 at two sites leading to products c16-kkf with fvlk and 
 c16-kkff with vlk . 
 the pa c16-kkffvlk forms 
 nanotubes and helical ribbons at room temperature . both pas c16-kkf and c16-kkff corresponding to cleavage products 
 instead self - assemble into 56 nm diameter spherical micelles , 
 while peptides fvlk and vlk do not adopt well - defined aggregate structures . 
 
 the secondary structures of the pas and peptides are examined by ftir 
 and circular dichroism spectroscopy and x - ray diffraction . 
 only c16-kkffvlk shows substantial -sheet secondary structure , 
 consistent with its self - assembly into extended aggregates , based 
 on pa layers containing hydrogen - bonded peptide headgroups . 
 this pa 
 also exhibits a thermoreversible transition to twisted tapes on heating . 