the parainfluenza virus 5 ( piv5 ) belongs 
 to the paramyxoviridae family , which 
 contains significant 
 pathogens to mammals such as measles , mumps , and hendra viruses .
two glycoproteins in the lipid envelope , a receptor - binding 
 protein ( hn , h , or g ) and a fusion protein ( f ) , are required for membrane 
 fusion .
the f protein , similar to the influenza 
 hemagglutinin ( ha ) and the hiv env protein , is synthesized as a homotrimer 
 and is activated by proteolytic cleavage , which creates a highly hydrophobic 
 n terminus called the fusion peptide ( fp ) that is essential for membrane 
 fusion .
the cleaved protein is anchored to the virus 
 envelope by a hydrophobic c - terminal transmembrane ( tm ) domain .
two heptad repeats , hra and hrb , lie next to 
 the fp and tm domains , respectively .
crystal structures of the 
 water - soluble portions of a number of 
 viral fusion proteins have been determined and have provided much of the 
 current understanding of the mechanism of protein - mediated virus  cell 
 membrane fusion .
it is known that fusion proteins undergo multiple 
 conformational changes to provide the necessary energy for membrane 
 fusion .
the conformations that have been observed 
 correspond to the prefusion states before and after cleavage , an extended 
 prehairpin state and the postfusion hairpin state .
the hairpins are 
 formed between two heptad - repeat domains common in class i fusion 
 proteins and give rise to a six - helix bundle ( 6hb ) that is characteristic 
 of the postfusion state of these trimeric proteins .
a consequence of this 6hb is that it enforces close proximity of 
 the neighboring fp and tm domains in the merged membrane , but no direct 
 structural evidence of this close packing in the membrane has yet 
 been reported . for the parainfluenza f protein ,
the crystal structures 
 of the uncleaved prefusion state , the 
 cleaved prefusion state , and the postfusion 
 state have been determined , and an extended 
 prehairpin structure was observed by electron microscopy . in comparison ,
structural information about 
 the membrane - bound fp and tm domains is still scarce .
solution 
 and solid - state nmr studies of the influenza and hiv fusion 
 peptides in detergent micelles and lipid bilayers have provided insights 
 into the mechanisms of virus  cell fusion .
the ha fusion peptide is predominantly -helical , but the 
 exact tertiary structure depends on the peptide length and the membrane - mimetic 
 environment .
a 20-residue construct adopts an obliquely inserted boomerang 
 conformation in detergent micelles , but in lipid bilayers at fusogenic ph , it also samples a small population 
 of a helical hairpin conformation .
a 
 23-residue construct that includes the conserved gxxxg and gxxg motifs 
 adopts a helical hairpin conformation already in detergent micelles , 
 with the hairpin stabilized by gly 
gly to ala mutation 
 at residue 8 results in a mixture of hairpin and boomerang structures .
it is -helical in detergent micelles but a -strand in lipid bilayers containing 
 more than 20% cholesterol .
solid - state nmr data indicate that both helical and strand conformations 
 of the hiv fp insert into the lipid membrane but cross - linked trimers 
 insert more deeply than monomers and are also more fusogenic .
the conformational polymorphism of these 
 viral fusion peptides 
 indicates the importance of the lipid environment in regulating membrane 
 fusion .
however , the lipid environment 
 is important not only for modulating the fp structure but also for 
 directly influencing the membrane curvature and hydration during fusion . 

a large number of computational analyses and experimental 
 studies have probed the structures of membrane intermediates during 
 fusion ; however , few studies have combined or correlated 
 the fp structure with the membrane - intermediate structure .
we 
 recently reported the first solid - state nmr structural study 
 of the piv5 fusion peptide in lipid bilayers .
we found that the peptide adopted an -helical conformation 
 in the negatively charged popc / popg membrane but a -strand 
 conformation on the surface of neutral popc and dmpc bilayers . in 
 the current study , we have determined the complete backbone conformation 
 of the popc / popg - bound piv5 fp using chemical shift constraints .
the dopc / dopg membrane retains the same negative surface charge as 
 the popc / popg membrane but increases the unsaturation and disorder 
 of the lipid chains .
surprisingly , this change did not increase the 
 fp mobility but converted the peptide from an -helical structure 
 to a partial -strand structure . in the dope membrane
, the piv5 
 fusion peptide mainly adopts a -strand conformation , similar 
 to its structure in neutral pc membranes , but the -strand is 
 inserted into the dope membrane rather than surface bound . moreover , 
 the peptide changes the phase behavior and hydration of the dope membrane . 

these results suggest the structural roles of the piv5 fusion peptide 
 during membrane fusion .
the fusion peptide used in this 
 study corresponds to residues 103129 of the piv5 f protein , 
 with the amino acid sequence of fagvviglaalgvataaqvtaavalvk . 
 to increase the peptide solubility ,
a lys tag kkkk was appended to 
 the c terminus through a flexible dioxa linker ( nh(ch2ch2o)2ch2co ) .
five c- , n - labeled peptides were synthesized by primm biotech ( cambridge , 
 ma ) : gval - fpk4 , igalv - fpk4 , gvtaa - fpk4 , vlaat - fpk4 , and aaqv - fpk4 
 ( table 1 ) .
the labeled residues cover all except 
 for four residues at the n and c termini ( f103 , a104 , v128 , and k129 ) 
 of the peptide .
fpk4 was reconstituted into 
 popc / popg ( 4:1 ) , dopc / dopg ( 4:1 ) , and 
 dope membranes at a peptide / lipid molar ratio of 1:20 .
briefly , the peptide was dissolved in trifluoroethanol ( tfe ) and 
 mixed with lipids in chloroform .
hcl , 1 mm edta , 1 mm nan3 , ph 7.5 ) or phosphate buffer ( 10 mm na2hpo4nah2po4 , 1 mm edta , 1 mm nan3 , ph 7.5 ) and dialyzed for a day .
the proteoliposomes were 
 centrifuged at 55  000 rpm at 4 c to obtain membrane pellets , 
 which were equilibrated to 3040 wt % water before being transferred 
 to 4 mm magic - angle - spinning ( mas ) rotors .
mas nmr experiments were 
 carried out on bruker avance-600 ( 14.1 t ) and dsx-400 mhz ( 9.4 t ) 
 spectrometers .
c chemical shifts were referenced to the 
 adamantane ch2 signal at 38.48 ppm on the tms scale , and 
 the n chemical shifts were referenced to the n - acetylvaline signal at 122.0 ppm on the liquid ammonia 
 scale .
p chemical shifts were referenced to the hydroxyapatite 
 signal at 2.73 ppm on the phosphoric acid scale .
two - dimensional 
 ( 2d ) c  c correlation spectra were 
 measured using a h - driven c spin diffusion 
 experiment with h irradiation ( darr ) during a mixing time of 2060 ms .
experimental temperatures 
 ranged from 233 to 303 k to investigate fpk4 conformation in both 
 the gel and liquid - crystalline ( lc ) phases of the membrane .
n  c correlation spectra were measured 
 using a redor - based pulse sequence with 
 a coherence transfer time of 857 s .
one - dimensional ( 1d ) static and 
 mas p spectra were measured between 273 and 313 k to 
 probe the membrane morphology and structure .
the size of the p chemical shift anisotropy ( csa ) is characterized by its 
 span , defined as the difference between the 0 edge and the 90 
 edge of the uniaxial powder pattern ,  = 0  90. a 2d p 
the depth of insertion of the fusion peptide was measured 
 using 
 2d h spin diffusion experiments in either the lc phase or the gel phase .
the lc - phase experiment 
 was applied to dopc / dopg - bound peptide with igalv and aaqv labels 
 using a h t2 filter of 0.81.0 ms and 
 a spin diffusion mixing time of 9625 ms .
spin diffusion buildup 
 curves were quantified after correcting for h t1 relaxation and were simulated using diffusion coefficients of 0.012 
 and 0.30 nm / ms for the lipid and peptide , respectively . 
 the water 
peptide interfacial diffusion coefficient ( dwp ) was 0.0020.003 nm / ms 
 while the lipid  peptide coefficient ( dlp ) was 0.00250.005 nm / ms .
the gel - phase 
 spin diffusion experiment was carried out on popc / popg- and dopc / dopg - bound 
 fpk4 .
the intensity ratios between the water and lipid ch2 cross peaks of each residue were measured to compare residue - specific 
 depths .
we recently reported that 
 popc / popg - bound fpk4 exhibited only -helical 
 chemical shifts for nine labeled residues , suggesting that this membrane promotes a single conformation of 
 the peptide . to obtain the complete backbone conformation in this 
 anionic membrane , we labeled additional residues ( table 1 ) .
figures 1 and 2 show the 2d c  c and n  c correlation spectra of fpk4 in gel - phase 
 popc / popg bilayers .
consistent with the previous study , most residues 
 exhibited -helical chemical shifts and a single set of signals . 

modest conformational disorder was manifested at residues a118v121 
 as reduced intensities and peak multiplicity .
for example , 
 the q120 cc cross peak is 4-fold weaker than 
 the a118 and a119 peak ( figure 1c ) , suggesting 
 dynamic disorder at q120 .
a recent hexamer model of piv5 fusion peptide placed q120 
 in the interior of the hexamer and postulated that this residue may 
 be involved in intermolecular h - bonding . since oligomeric assembly and h - bonding should order and immobilize 
 the peptide , our data does not support this model for fpk4 in the 
 popc / popg membrane .
2d c  c correlation spectra 
 of 
 piv5 fpk4 in gel - phase popc / popg ( 4:1 ) bilayers .
shown at the top 
 is the amino acid sequence with labeled residues color - coded according 
 to samples .
( a ) gvtaa - fpk4 spectrum , measured at 253 k with 20 ms 
 mixing .
( b ) vlaat - fpk4 spectrum , coadded from two spectra measured 
 at 243 k with 20 ms mixing and 253 k with 60 ms mixing .
( c ) aaqv - fpk4 
 spectrum , measured at 253 k with 20 ms mixing .
 c correlation spectra of 
 piv5 fpk4 in gel - phase popc / popg ( magenta ) , dopc / dopg ( black ) , and 
 popc ( blue ) membranes .
the peptide shows predominantly -strand chemical shifts in 
 the popc membrane , -helical chemical shifts in the popc / popg 
 membrane , and mixed strand and helix chemical shifts in the dopc / dopg 
 membrane .
most residues in the gvtaa and vlaat samples show two sets 
 of chemical shifts in the dopc / dopg bilayer .
the aaqv sample shows 
 nearly identical -helical chemical shifts in the popc / popg 
 and dopc / dopg membranes .
the assigned c and n chemical shifts 
 of popc / popg - bound fpk4 ( table 2 ) allow us 
 to obtain a backbone conformational model of the peptide .
all 23 residues 
 ( g105l127 ) exhibit -helical chemical shifts as the 
 dominant signals , with positive c and co secondary shifts and 
 negative c secondary shifts ( figure 3a ) . using talos+
, we obtained backbone 
 (  ,  ) torsion angles ( table 3 ) , 
 which indicate a nearly ideal -helical conformation in the 
 popc / popg membrane .
chemical shifts were measured 
 from 2d spectra at 233253 k. italics indicate the second conformation .
c chemical shifts are referenced to tms , and n chemical shifts are referenced to liquid ammonia . from the gvtaa sample ( g105 , v106 , 
 t122 , a123 , and a124 ) . from 
 the vlaat sample ( v107 , l110 , 
 a111 , a116 , and t117 ) . from 
 the igalv sample ( i108 , g109 , 
 a112 , l113 , and v125 ) . from 
 the gval sample ( g114 , v115 , 
 a126 , and l127 ) . from the 
 aaqv sample ( a118 , a119 , 
 q120 , and v121 ) .
c and n secondary chemical shifts of 
 fpk4 in ( a ) popc / popg and ( b ) dopc / dopg membranes .
fpk4 shows clear 
 -helical chemical shifts ( red ) in popc / popg bilayers and mixed 
 helical and strand chemical shifts ( blue ) in dopc / dopg bilayers .
the 
 random coil values of zhang et al . were 
 used to calculate the secondary shifts .
the dopc / dopg values were predicted 
 from the main set of chemical shifts .
fpk4 undergoes intermediate - time scale motion in the lc phase of 
 the popc / popg membrane .
the resulting line broadening precludes the 
 lc - phase h spin diffusion experiment for measuring the insertion depth of the peptide .
therefore , 
 we carried out the gel - phase spin diffusion experiment , which resolves the water , lipid , and peptide h signals in the indirect dimension by h homonuclear 
 decoupling .
strong cross peaks between lipid protons and peptide c signals indicate deep insertion of the peptide into the 
 membrane .
in addition , well - inserted peptides exhibit similar h intensity patterns as the lipid chain carbons , while surface - bound 
 peptides exhibit different h cross sections , with much 
 higher water cross peaks than lipid cross peaks .
figure 4a shows representative gel - phase h spin 
 diffusion spectra of fpk4 in the popc / popg membrane . by 25 ms ,
the 
 peptide shows strong cross peaks with both lipids and water , and the 
 peptide c and lipid ch2 cross sections have similar h chemical shifts , linewidths , and intensity distributions 
 ( figure 4b ) , indicating that fpk4 is well inserted 
 into the hydrophobic region of the membrane . with a shorter
mixing 
 time of 4 ms , more residue - specific depth information is obtained , 
 since different residues give different relative intensities between 
 the water and lipid cross peaks ( figure 4c ) : 
 terminal residues such as g105 and a126 have higher water / lipid intensity 
 ratios than middle residues such as a112 and l113 , indicating that 
 the two termini are in closer contact with water .
the water / lipid 
 intensity ratios ( figure 4d ) are the lowest 
 between a111 and t117 ( 0.100.13 ) and higher for both the n 
 and c termini ( 0.170.35 ) , consistent with a membrane - spanning 
 topology of the peptide .
the intensity profile is asymmetric , with 
 the n terminus having higher values than the c terminus , indicating 
 that the n - terminal half of the peptide is more exposed to the membrane 
 surface .
depth of insertion of fpk4 in the popc / popg membrane from gel - phase 
 spin diffusion .
( a ) representative 2d spectra with 0 and 25 ms spin 
 diffusion mixing at 258 k. ( b ) h cross sections for the 
 peptide c peaks ( red ) and the lipid ch2 peak ( black ) . 

already at 4 ms , the peptide and lipid h cross sections 
 have similar intensity patterns , indicating that the peptide is well 
 inserted into the membrane .
( c ) c cross sections extracted 
 from the water ( blue ) and lipid ch2 ( black ) h chemical shifts from the 4 ms 2d spectra .
the n- and c - terminal 
 residues have higher water / lipid intensity ratios than the middle 
 residues .
( d ) water / lipid intensity ratios for all labeled sites . since fpk4 undergoes intermediate - time scale motion 
 in the popc / popg membrane at ambient temperature , we searched for 
 a different lipid membrane that may speed up the helix motion .
fast 
 motion not only gives higher - resolution nmr spectra but may also allow 
 helix orientation to be determined from motional order parameters 
 without requiring macroscopically aligned samples .
the most obvious choice is the dopc / dopg ( 4:1 ) membrane , since it 
 has the same membrane surface charge as the popc / popg bilayer while 
 having a 18 c lower gel - to - lc phase - transition temperature due 
 to the presence of a double bond in both acyl chains of each lipid .
surprisingly , the increased disorder and dynamics of the dopc / dopg 
 membrane did not speed up motion of the fusion peptide but changed 
 the peptide conformation .
1d c cp - mas spectra ( figure 5 ) show high - intensity -strand signals for 
 various residues at ambient temperature and few -helical signals . 

when the membrane is cooled to the gel phase , the -helix signals 
 become detectable and comparable in intensity as the -strand 
 signals .
thus , the remaining -helical conformation has similar 
 intermediate - time scale motion between the dopc / dopg and the popc / popg 
 membranes , but the new -strand structure is immobilized in 
 the lc phase .
the increased disorder of the lipid chains shifted the 
 conformational equilibrium of the fusion peptide toward -strand , 
 without changing the mobility of the -helical segment .
representative 
 1d c cp mas spectra of dopc / dopg - bound 
 fpk4 as a function of temperature .
the vlaat - fpk4 spectra are shown . 
 at high temperature , mainly -strand chemical shifts ( blue dotted 
 lines )
are observed , while at low temperature , both -helical 
 ( red dashed lines ) and -strand chemical shifts are detected .
figure 6 shows 2d c  c darr spectra of four 
 labeled peptides in the gel and lc 
 phases of the dopc / dopg membrane . in the gel phase ,
the exceptions are g105 , v106 , i108 , 
 and g109 , which exhibit only -strand signals , and q120 and 
 v125 , which display only -helical chemical shifts . increasing 

the temperature decreased the intensities of the helix signals while 
 retaining the strand signals .
the position of the peptide at which 
 the helix and strand have comparable intensities is t117 .
the signals 
 of several ala residues partially overlap in the short - mixing - time 
 spectra but become resolved by inter - residue cross peaks at long mixing 
 times . for example
, the 300 ms 2d spectrum ( figure s1a , supporting information ) shows -strand 
 l110a111 cross peaks and -helical a116t117 
 cross peaks , indicating that a111 is primarily in the strand conformation 
 while a116 is mostly helical .
a118 and a119 show chemical shifts for 
 all three conformations , but the -helix intensity dominates 
 the strand and coil intensities ( figure 6 g , 
 h ) . finally , the n - terminal half of the peptide underwent a slow conformational 
 change from -helical to -strand in the dopc / dopg membrane : 
 v107 , l110 , and a111 initially showed -helical chemical shifts , 
 which converted to -strand chemical shifts at equilibrium ( figure 
 s1b , supporting information ) . however , 
 the more c - terminal a116 and t117 in the same vlaat sample remained 
 stably -helical .
2d c  c correlation 
 spectra of 
 dopc / dopg - bound fpk4 in the gel phase ( 233 or 243 k , left column ) 
 and the lc phase ( 303 k , right column ) .
c correlation spectra 
 ( figure 2 ) confirmed the mixed strand / helix 
 conformation 
 of the n- and c - terminal halves of the dopc / dopg - bound peptide .
two 
 sets of chemical shifts were observed for many residues , but residues 
 g105l113 show dominant -strand peaks while residues 
 a118v125 have dominant -helical peaks .
comparison of 
 the peptide spectra for three lipid membranes , popc / popg , dopc / dopg , 
 and popc , highlights the membrane - induced conformational polymorphism 
 of fpk4 .
the -helical chemical shifts of the c - terminal half 
 of the peptide are the same between the popc / popg and dopc / dopg membranes , 
 whereas the -strand chemical shifts of the n - terminal residues 
 differ between the popc and dopc / dopg membranes . for example , 
 the chemical shifts of i108 , a112 , and l113 in the dopc / dopg membrane 
 are intermediate between the corresponding chemical shifts in the 
 popc and popc / popg membranes ( figure 2c ) . on the basis of the cross - peak intensities in the low - temperature 
 2d c  c spectra , we quantified the 
 -helical content of each residue ( table s1 , supporting information ) .
residues up to l113 are less than 
 35% -helical , whereas residues a116v125 are greater 
 than 50% helical .
the increasing helicity toward the c terminus was 
 consistently observed for all labeled peptides , independent of minor variations 
 in the hydration and salt content of the samples .
the talos+ 
 predicted backbone (  ,  ) torsion angles 
 of the major conformer of dopc / dopg - bound fpk4 ( table 3 ) confirm the n - terminal -strand and c - terminal -helical 
 structures of the peptide .
for this mixed conformation , oligomerization , 
 if present , is expected to be parallel rather than antiparallel .
this 
 is consistent with the cross - peak pattern detected at long mixing 
 times .
the labeled residues within gvtaa- and igalv - fpk4 lie at the 
 two ends of the peptide .
thus , if antiparallel packing or a hairpinlike 
 structure were present , we would observe inter - residue cross peaks 
 between the n- and c - terminal residues .
the 500 ms 2d spectra ( figure 
 s2 , supporting information ) of these samples 
 show only sequential inter - residue cross peaks such as g105v106 , 
 t122a123 , i108g109 , and a112l113 but no long - range 
 cross peaks , thus ruling out antiparallel packing and the helical 
 hairpin conformation .
since fpk4 adopts a surface - bound -strand 
 structure in neutral pc membranes but 
 an inserted -helical structure in the anionic popc / popg membrane , 
 the topology of the partial -strand peptide in the anionic 
 dopc / dopg membrane is not immediately obvious .
we thus measured the depth 
 of the peptide in the dopc / dopg membrane , using both the lc - phase h spin diffusion experiment and the gel - phase experiment . 
 by 100 ms , the 2d h 
c correlation 
 spectra at 293 k ( figure s3 , supporting information ) showed clear cross peaks between lipid - chain protons and peptide c for both the -strand and -helical residues , 
 indicating that the entire peptide is inserted into the dopc / dopg 
 membrane .
this is confirmed by the fast lipid - to - peptide spin diffusion 
 buildup rates for both conformations ( figure s3c , f , supporting information ) .
more residue - specific depth information 
 is obtained from the gel - phase spin diffusion spectra obtained at 
 243 k. by 4 ms , the peptide h cross section is already 
 similar to the lipid h cross section ( figure 7a ) , indicating equilibration of the h magnetization among the peptide , lipid , and water .
similar to the 
 popc / popg case , fpk4 has higher water / lipid cross - peak intensity ratios 
 for the terminal residues than the central residues ( figure 7b , c ) , indicating that the peptide spans the bilayer 
 thickness .
but in contrast to the popc / popg - bound fpk4 , the c - terminal 
 -helical residues are significantly more exposed to water than 
 the n - terminal -strand residues ( figure 7c ) . the lc - phase spin diffusion spectra ( figure s3c , f , supporting information ) also exhibit slightly 
 faster lipid 
peptide spin diffusion buildup rates for the n - terminal 
 residues than the c - terminal residues .
it is not fully clear whether 
 it is the backbone conformation ( helix versus strand ) or the residue 
 position ( n or c termini ) that causes the different insertion asymmetry 
 between the popc / popg and dopc / dopg membranes .
however , the minor 
 -strand conformation of the c - terminal a123 and a124 has lower 
 water / lipid intensity ratios than the -helical counterpart , 
 while the minor -helical conformation of the n - terminal l110 
 has higher water exposure than -strand l110 ( figure 7c ) , suggesting that conformation may be the more 
 important determinant of depth : the -strand conformation is 
 more deeply inserted than the -helical conformation into the 
 dopc / dopg membrane .
depth of insertion of fpk4 in the dopc / dopg membrane from 
 gel - phase 
 spin diffusion spectra measured at 243 k. ( a ) h cross 
 sections of the peptide c peaks ( red ) and lipid ch2 peak ( black ) . by 4 ms ,
the peptide and lipid signals have equilibrated , 
 indicating that the peptide is well inserted into the membrane .
( b ) c cross sections from the water ( blue ) and lipid ch2 ( black ) h chemical shifts of the 4 ms 2d spectra .
the 
 c - terminal -helical residues have higher water cross peaks 
 than the n - terminal -strand residues , and the -helical 
 a123/a124 have higher water cross peaks than the -strand a123/a124 . 

( c ) water / lipid intensity ratios of all labeled residues in the dopc / dopg 
 membrane ( blue and red symbols ) .
open symbols 
 indicate the minor conformation . for comparison , the popc / popg - bound 
 fpk4 data are also shown ( black open symbols ) .
to investigate whether fpk4 causes curvature and dehydration 
 to 
 the dopc / dopg membrane , we measured the static and mas p spectra ( figure s4a , b , supporting information ) .
fpk4 displayed little perturbation of the structure of the dopc / dopg 
 membrane : the lamellar - bilayer powder pattern is retained , and the 
 isotropic chemical shift is unchanged .
however , the mas isotropic 
 line width is significantly broadened by the peptide ( from 30 to 130 
 hz ) , and the p transverse relaxation times of dopc and 
 dopg decreased from 18.3 and 19.4 ms , respectively , for the peptide - free 
 membrane to 2.4 and 2.0 ms for the peptide - bound membrane ( figure 
 s4c , supporting information ) .
thus , the 
 apparent p linewidths are largely homogeneous , and the 
 fusion peptide slows down the lipid headgroup motion without changing 
 its average conformation .
finally , the 2d p  h correlation spectrum shows clear water  lipid cross 
 peaks ( figure s4d , supporting information ) for both dopc and dopg , indicating that fpk4 retains the hydration 
 of the membrane surface .
the p mas spectrum ( figure 
 s4b , supporting information ) exhibits a 
 small isotropic peak at 
 2.2 ppm .
this peak can be assigned to the phosphate buffer , since 
 samples prepared in tris or hepes buffer did not show this peak ( data 
 not shown ) .
we previously observed the same isotropic peak in static 
 and mas p spectra of fpk4-containing popc and dmpc membranes , 
 and the peak intensity increased with the peptide concentration .
the 
 latter led to the erroneous conclusion that this peak resulted from 
 a peptide - induced high - curvature isotropic phase .
we now attribute the concentration dependence of this p peak to electrostatic attraction between the cationic lys 
 tag and the phosphate ions .
similar cases of phosphate buffer interactions 
 with membrane peptides have been reported in the literature .
thus , the -strand fpk4 that binds to 
 the surface of the popc membrane does not cause curvature on the sub-10 
 nm scale .
however , this does not exclude the possibility that the 
 peptide may cause curvature on larger length scales of 50100 
 nm , which would not manifest as a narrow peak in the static p spectra . to further investigate whether 
 fpk4 induces membrane curvature , 
 we studied the structure and lipid interactions of dope - bound fpk4 . 

the small headgroup of dope and its disordered acyl chains create 
 spontaneous negative curvature to the membrane , causing an inverse 
 hexagonal phase ( hii ) in a wide temperature range .
the 
 dope phase diagram has been measured using nmr and x - ray diffraction , and the lamellar ( l)hii transition 
 temperature ( th ) is known to depend on 
 the hydration : above 16 water molecules per lipid , the membrane 
 converts to the hii phase by 283 k. if fpk4 causes 
 membrane curvature , then th will be affected : 
 positive curvature generation by the peptide increases th while negative curvature generation lowers the transition 
 temperature .
figure 8 shows the static p spectra of dope membranes without and with fpk4 from 273 
 to 313 k. at 273 k , pure dope membrane shows an l-phase powder pattern with a chemical shift anisotropy span of + 44.5 
 ppm . above 273 k ,
the p spectrum shows increasing intensities 
 of a narrower line shape with a span of 21.5 ppm , which is 
 inverted from the l line shape around the isotropic p chemical shift .
the pure dope 
 membrane fully converted to the hii phase by 283 k ( figure 8a ) , consistent with the reported th value . upon fpk4 binding , the lhii transition shifted to higher 
 temperatures and was complete only by 293 k , indicating that 
 fpk4 exerted positive membrane curvature .
in addition , an isotropic 
 peak appeared in the spectra ( figure 8b ) . in 
 principle
moreover , small - angle x - ray diffraction data of dope containing 
 the ha fusion peptide indicated the presence of inverted bicontinuous 
 cubic phases as well as an increase of the lhii transition temperature , and 
 independent md simulations also predicted the same effect .
thus , the isotropic p peak seen 
 here is most likely due to cubic - phase formation in the dope membrane , 
 which suggests that the piv5 fusion peptide causes both positive and 
 negative curvatures ; that is , the peptide generates negative gaussian 
 curvature .
also known as saddle - splay curvature , negative gaussian 
 curvature results from the product of positive and negative principal 
 curvatures and is present at membrane pores and protrusions formed 
 during membrane budding and scission .
( a , b ) static p spectra of the 
 membrane without ( a ) and with ( b ) fpk4 from 273 
 to 313 k. fpk4 increased the l-to - hii phase transition temperature and caused a small isotropic peak . 

h correlation spectrum of 
 fpk4-bound dope membrane with a spin diffusion mixing time of 225 
 ms .
( d ) h cross sections from the 2d p  h spectra of peptide - free and peptide - bound dope membranes , 
 compared with the 1d h single - pulse spectrum ( top ) .
the equilibrium fpk4 conformation in the dope membrane is 
 predominantly 
 -strand , after the transient existence of a mixed strand / helix 
 conformation ( figure 9a , b ) .
the -strand 
 shows clear cross peaks with lipid ch2 protons in the 2d h 
c correlation spectra ( figure 9c ) , indicating that the peptide is embedded in the 
 hydrophobic region of the hexagonal - phase cylinders ( figure 10e ) .
the 2d p  h 
 correlation spectrum of the fpk4-bound dope membrane shows a much 
 weaker water 
p cross peak than the peptide - free 
 membrane ( figure 8c , d ) , indicating that the 
 -strand fpk4 dehydrates the dope membrane in addition to causing 
 curvature to this membrane . conformation and depth of fpk4 in the dope membrane . 

( a ) 2d c  c correlation spectrum of 
 a fresh gvtaa - fpk4 
 sample at 243 k. the peptide exhibits both helix and strand signals . 

( b ) c cp - mas spectra of the initial and equilibrated gvtaa - fpk4 
 at 246 k. at equilibrium , most residues exhibit -strand chemical 
 shifts .
( c ) 100 ms 2d c  h correlation 
 spectrum at 293 k , in the hii phase membrane .
lipid  peptide 
 cross peaks are observed , indicating that the -strand peptide 
 is inserted into the hydrophobic region of the dope membrane .
piv5 fusion peptide conformations in 
 lipid membranes from solid - state 
 nmr and outside the membrane from crystal structures .
( a ) fusion peptide 
 is fully -helical in popc / popg bilayers but adopts a mixed 
 strand / helix conformation in dopc / dopg bilayers .
the peptide is inserted 
 into both membranes , but the depicted tilt angle is hypothetical . 

the structures were built using (  ,  ) torsion angles 
 predicted by talos+ .
( b ) prefusion crystal structures of the piv5 
 f protein in the uncleaved ( green ) and 
 cleaved ( red ) states .
the fusion peptide 
 domain has similar conformations before and after cleavage and has 
 a bend near t117 .
( c ) prefusion crystal structures of the influenza 
 ha in the uncleaved ( green ) and cleaved 
 ( red ) states .
the n - terminal half of 
 the fusion peptide is rotated around n12 before and after cleavage . 

seven residues ( t122v128 ) of the fusion 
 peptide are detected and show -helical structure extended from 
 hra .
( e ) schematic of the piv5 fusion peptide conformation in the 
 dope membrane .
the lipid cylinders and water radius are drawn to scale 
 using 1518 water molecules per lipid based on the dope phase 
 diagram .
( f ) the hemifusion stalk intermediate showing 
 both negative and positive membrane curvatures and dehydration between 
 two opposing bilayers .
the present solid - state 
 nmr data indicate at least four distinct 
 conformations and membrane topologies of the piv5 fusion peptide . 
 in the popc / popg membrane ,
the peptide adopts a membrane - spanning 
 -helical conformation ( figure 10a ) . 

the popc / popg membrane has a hydrophobic thickness of 27  
 at 30 c based on x - ray scattering data .
the full -helix has a length of 34  from g105 
 to l127 c based on the talos+ structural model .
thus , the helix 
 may be tilted by 3540 to achieve optimal hydrophobic 
 match between the popc / popg bilayer thickness and the peptide length . in the dopc / dopg membrane
, fpk4 adopts a mixed conformation with 
 an n - terminal -strand ( residues 105113 ) and a c - terminal 
 -helix ( residues 116125 ) .
no chemical shift 
 constraints were measured for residues g114 , a115 , a126 , and l127 . 

the current structural model assumed a126l127 to be similarly 
 helical as in the popc / popg - bound fpk4 and g114 and a115 to be random 
 coil due to its position near a likely bend ( see below ) .
the overall 
 dimension of the mixed helix / strand conformation is not known without 
 long - range distance constraints .
however , since the -strand 
 is much more extended than the -helix , the peptide is likely 
 to be significantly tilted to match the hydrophobic thickness of the 
 dopc / dopg bilayer , which is similar to that of the popc / popg bilayer . in the dope membrane ( figure 10e ) , the chemical 
 shift constraints suggest a predominantly -strand peptide , 
 which is inserted into the hydrophobic region between the lipid cylinders . 

finally , fpk4 adopts a surface - bound -strand structure in neutral 
 popc and dmpc membranes , as we showed previously .
the four conformations and topologies of fpk4 suggest 
 several principles 
 for the influence of the lipid membrane on the fusion peptide structure . 

first , while the entire sequence of the fusion peptide is capable 
 of conformational polymorphism , the c terminus has a higher propensity 
 for the -helical structure .
second , anionic membranes promote 
 the -helical conformation , as shown by the difference between 
 the popc / popg membrane and the popc membrane and by the difference 
 between dopc / dopg and dope membranes .
third , more disordered membranes 
 shift the peptide conformational equilibrium toward -strand , 
 as shown by the difference between the popc / popg and dopc / dopg membranes . 
 the third observation ,
while initially unexpected , can in fact be 
 understood by the fact that lipid unsaturation not only changes membrane 
 dynamics but also membrane curvature .
cone - shaped lipids ( with negative 
 intrinsic curvature ) such as oleic acids , cis - unsaturated 
 lipids , and phosphatidylethanolamine promote stalk formation , whereas 
 inverted - cone shaped lipids ( with positive curvature ) such as lysophosphocholine 
 inhibit fusion by preventing stalk formation .
thus , the more 
 unsaturated dopc / dopg lipids change the membrane curvature in addition 
 to membrane dynamics compared to the popc / popg lipids .
the higher 
 -strand content of the fusion peptide in the dopc / dopg membrane 
 thus suggests that the -strand structure may be the active 
 form in hemifusion intermediates .
our chemical shift analysis 
 indicates that the piv5 fusion peptide 
 has a higher conformational disorder in the middle of the sequence , 
 near g114t117 .
this region is not only the transition point 
 between the strand and helix segments in the dopc / dopg - bound peptide 
 but also has multiple conformations and residual dynamics in the popc / popg 
 membrane .
the prefusion crystal structures of several viral fusion 
 proteins and the nmr structures of other fusion peptides suggest that 
 conformational disorder in the middle of fusion peptide domains may 
 be general .
for example , in the uncleaved piv5 f protein , the c - terminal part ( a118v128 ) of the 
 buried fusion peptide shows an -helical structure extended 
 from hra , whereas the n - terminal part ( f103t117 ) has a mixed 
 conformation of random coil ( f103i108 ) , -helix ( g109l113 ) , 
 and -strand ( v115a116 ) ( figure 10b ) .
after 
 cleavage , the first four residues of 
 the fp undergo an orientational change while the other residues are 
 mostly unaffected . in the prefusion
ha crystal structures , the fp 
 is unstructured in both uncleaved and cleaved states , but the n - terminal segment undergoes a large - amplitude rotation 
 with respect to the rest of the protein after cleavage ( figure 10c ) . isolated ha fusion peptides bound to dpc micelles 
 exhibit -helical conformations , but the middle of the peptide 
 is disordered and forms the bend of the helical hairpin in the 23-residue construct and the bend of the 
 boomerang structure in the 20-residue construct .
the exact significance of this mid - domain disorder for 
 membrane fusion is not clear , but we speculate that the disorder may 
 be useful for controlling oligomerization , the degree of peptide insertion 
 into the membrane , and membrane hydration .
for example , if the -sheet 
 conformation is indeed more effective in dehydrating the lipid membrane 
 than the -helical conformation , as suggested by the current 
 dope data and the previous popc and dmpc data , then a mixed strand / helix 
 conformation may be useful for dehydrating one of the two surfaces 
 of the lipid bilayer .
mutagenesis data of the fusion peptide domain 
 of the piv5 f protein 
 indicated a competition between protein transport , surface expression , 
 and membrane fusion , but the n - terminal residues f103v115 
 appear to be more important for membrane fusion than other functions .
mutations of g105 , g109 , and g114 to ala reduced 
 protein expression but increased membrane fusion .
g109a and g114a 
 mutants showed 25% lower expression levels but 10-fold higher membrane 
 fusion than the wild - type protein .
while 
 fusion peptides are generally rich in gly and ala residues , in piv5 fp , all three gly residues 
 are located in the n - terminal region while over half of the ala residues 
 are located in the c - terminal region .
in contrast , ha and hiv fusion 
 peptides have a more uniform distribution of gly residues . the high 
 gly content of the n - terminal half of the piv5 fusion peptide may 
 be one of the reasons for the stronger -strand propensity of 
 the n - terminal half .
meanwhile , the helix propensity of the c - terminal 
 segment may be related to the neighboring -helical hra domain , 
 as seen in the postfusion crystal structure of piv5 f , which shows 
 -helical t122v128 in the fp domain .
since the n - terminal domain is more important for membrane 
 fusion and has a stronger propensity for the -strand conformation , 
 the -strand conformation may be more critical for membrane 
 fusion .
this is also consistent with the ability of the -strand 
 conformation in causing membrane dehydration , as discussed below .
static p nmr spectra indicate that the 
 piv5 fusion peptide neither causes curvature nor dehydration 
 to the popc / popg and dopc / dopg membranes but causes curvature to the 
 dope membrane .
the peptide increased the lhii transition temperature by about 10 k and generated a small 
 amount of an isotropic phase .
we attribute this signal to a cubic 
 phase , which would suggest that the fp promotes negative gaussian 
 curvature . increasing experimental evidence and simulations 
 indicate that generation of negative gaussian curvature may be a common 
 property of viral fusion peptides .
recent small - angle x - ray diffraction 
 data of the ha fusion peptide in methylated 
 dope showed that the peptide shifted the lhii phase transition to higher temperatures and additionally 
 promoted the formation of inverted bicontinuous cubic phases , lm3 m and pn3 m , which possess negative gaussian curvature .
this result revises 
 earlier literature that concluded that the ha fusion peptide promoted 
 only negative curvature .
these earlier studies 
 were based on differential scanning calorimetry experiments , which 
 may not be able to resolve the cubic phases from the l and hii phases , and on p nmr spectra that 
 showed clear isotropic peaks but poor - sensitivity powder patterns 
 that can not be definitively assigned to either the l phase or the hii phase .
therefore , these earlier data 
 can not be interpreted as stabilization of the hii phase 
 by the ha fusion peptide , but they do indicate 
 the generation of an isotropic phase , which is consistent with cubic - phase 
 formation .
molecular dynamics simulations of membranes containing 
 the ha fusion peptide indicate that the peptide systematically shifted 
 the lipid phase diagram toward more positive mean curvature and bicontinuous 
 cubic phases . for the hiv gp41 fusion 
 peptide ,
p nmr spectra and cryo - tem micrographs of dope - containing lipid membranes showed the presence of an isotropic 
 phase .
thus , all reliable evidence converges to indicate that influenza , 
 hiv , and piv5 fusion peptides cause negative gaussian curvature to 
 pe - rich membranes . on the basis of the intensity of the p isotropic peak ,
the piv5 fpk4 construct used here has weaker curvature - generating 
 ability than the influenza and hiv fusion peptides .
in addition 
 to generating membrane curvature , fpk4 also partially 
 dehydrated the dope membrane ( figure 8c , d ) . 

for the hii - phase dope , this means a reduction of the water - core 
 diameter of the cylinders .
figure 10e depicts 
 the hexagonal cylinders , where the relative dimensions of the water 
 pore and the hydrophobic chains match the values reported from x - ray 
 diffraction data of dope at 1518 
 water molecules per lipid , which is the hydration level of the fpk4-containing 
 dope membrane . at this hydration ,
the water channel radius is 19 
  , while the distance between the centers of two cylinders in 
 adjacent layers ( dhex ) is 70  , 
 whose hydrophobic portion ( 32  ) is traversed by the 
 -strand fpk4 .
this peptide location is consistent with x - ray 
 scattering data that showed that the 
 hiv fusion peptide increased the dhex value 
 of dope at peptide concentrations above 2 mol % .
the increased hexagonal 
 spacing results from a compensatory effect of an increased hydrocarbon 
 volume , which implicates the hiv fusion peptide to be embedded in 
 the hydrocarbon region , and a decreased water volume , which agrees 
 with the dehydration seen in the current 2d p  h correlation spectra of the dope membrane .
thus , the piv5 
 and hiv fusion peptides exert similar changes to the dope membrane . 

the cross section of the inverse - hexagonal phase dope , in which the 
 opposed lipid chains of different cylinders experience negative curvature 
 ( figure 10e ) , is similar but not identical 
 to the cross section of the hemifusion stalk intermediate ( figure 10f ) , since the latter also contains 
 lipids experiencing positive curvature .
the hemifusion stalk is topologically 
 more similar to inverted bicontinuous cubic phases , which are the likely cause of the isotropic peak in the p spectra .
the lipids that experience positive curvature in the cubic 
 phase should correspond to lipids in the distal leaflet of the opposing 
 membranes .
on the basis of the conformation , 
 topology , and lipid and water 
 interactions of piv5 fpk4 in the four lipid membranes obtained from 
 solid - state nmr , we propose the following relations between the fp 
 structure and viral membrane fusion .
when the fp is released from 
 the globular head of the f protein , it inserts into the target cell 
 membrane in an -helical structure .
as the protein rearranges 
 its structure and hra forms a coiled - coil trimer in the prehairpin 
 intermediate , the fusion peptides of the three proteins interact in 
 the cell membrane to form a homotrimer .
when several trimers cluster 
 in regions of the membrane containing high concentrations of unsaturated 
 lipids with their ensuing negative intrinsic curvature , the gly - rich 
 n - terminal half of the fusion peptide converts to a -strand 
 conformation , which dehydrates the membrane surface and exerts negative 
 gaussian curvature to the membrane . at this point , depending on the 
 local lipid composition , the fusion peptide may be partially -strand 
 ( in pc - rich membranes ) or fully -strand ( in pe - rich membranes ) , 
 and the peptide is well inserted unless the local membrane composition 
 is predominantly neutral pc .
when the water - soluble ectodomain completes 
 its conformational change to a six - helix bundle , the fp and tm domains 
 are forced into close proximity , which may revert the fusion peptide 
 to the -helical conformation , which may in turn reduce membrane 
 curvature and increase membrane hydration .
multiple lines of evidence 
 obtained here suggest the -strand conformation of the fusion 
 peptide to be the most relevant structure in hemifusion intermediates , 
 responsible for remodeling the membrane to acquire the curvature and low hydration necessary for progression 
 to complete fusion .
 viral 
 fusion proteins catalyze the merger of the virus envelope 
 and the target cell membrane through multiple steps of protein conformational 
 changes . 
 the fusion peptide domain of these proteins is important 
 for membrane fusion , but how it causes membrane curvature and dehydration 
 is still poorly understood . 
 
 we now use solid - state nmr spectroscopy to investigate the conformation , 
 topology , and lipid and water interactions of the fusion peptide of 
 the piv5 virus f protein in three lipid membranes , popc / popg , dopc / dopg , 
 and dope . 
 these membranes allow us to investigate the effects of lipid 
 chain disorder , membrane surface charge , and intrinsic negative curvature 
 on the fusion peptide structure . 
 chemical shifts and spin diffusion 
 data indicate that the piv5 fusion peptide is inserted into all three 
 membranes but adopts distinct conformations : it is fully -helical 
 in the popc / popg membrane , adopts a mixed strand / helix conformation 
 in the dopc / dopg membrane , and is primarily a -strand in the 
 dope membrane . 31p 
 nmr spectra show that the peptide retains 
 the lamellar structure and hydration of the two anionic membranes . 
 
 however , it dehydrates the dope membrane , destabilizes its inverted 
 hexagonal phase , and creates an isotropic phase that is most likely 
 a cubic phase . 
 the ability of the -strand conformation of the 
 fusion peptide to generate negative gaussian curvature and to dehydrate 
 the membrane may be important for the formation of hemifusion intermediates 
 in the membrane fusion pathway . 