Sequence-specific liquid crystallinity of collagen model peptides. I. Transmission electron...

Sequence-Specific LiquidCrystallinity of CollagenModel Peptides. I.Transmission ElectronMicroscopy Studies ofInterfacial Collagen Gels

Regina ValluzziDavid L. Kaplan

Tufts University,Department of Chemical

Engineering andBiotechnology Center, 4

Colby Street,Medford, MA 02155

Received 16 June 1999;accepted 25 October 1999

Abstract: The conformation, crystal structure and self-assembly behavior of three peptides withcollagen-like repetitive sequences [(1) peptide GAPGPP: (Glu)5(Gly–Ala–Pro–Gly–Pro–Pro)6(Glu)5; (2) peptide GVPGPP: (Glu)5(Gly–Val–Pro–Gly–Pro–Pro)6(Glu)5; and (3) peptideGAPGPA: (Glu)5(Gly-Ala-Pro-Gly-Pro-Ala)6(Glu)5] were compared. The peptides were charac-terized using transmission electron microscopy, electron diffraction, environmental scanning elec-tron microscopy, and Fourier transform ir spectroscopy in order to determine how the moleculargeometry dictated by each sequence affects the spontaneous generation of long-range orderedstructures. Samples of each peptide, at ambient temperature and at 5°C, were examined as filmsdried from aqueous solution, air–water interfacial films, and chloroform–water interfacial films.Peptide GAPGPP prepared at 5°C and dried from bulk solution was found to have a collagen-liketriple-helical structure. A sinusoidally textured gel, suggestive of cholesteric behavior was observedfor peptides GAPGPP and GVPGPP at the aqueous chloroform interface at 5°C. Peptide GAPGPAalso formed a gel, but less reproducibly and the sinusoidal texture was not as well defined. Theperiodicities of the sinusoidal textures were reproducibly 10mm for peptide GAPGPP, 7mm forpeptide GVPGPP, and 6mm for peptide GAPGPA. The differences in the periodicity of the bandedstructure and in the crystallization behavior of the three peptides is attributed to differences in thesymmetry of the preferred packing arrangement for each peptide, as evidenced by electrondiffraction from crystallites that coexist with the sinusoidal gel. These differences are believed to bea measure of the effective symmetry and shape of the molecular cross section.© 2000 John Wiley& Sons, Inc. Biopoly 53: 350–362, 2000

Keywords: collagen; liquid crystal; cholesteric; helicoid; interface; self-assembly; hierarchicallyordered material; liquid–liquid interface; conformation selection; electron diffraction; electronmicroscopy; Fourier transform ir

INTRODUCTION

The collagen proteins are present in almost everyanimal tissue. These proteins are composed of blocksof triple-helical and “nonhelical” conformation. The

triple-helical conformation imparts stiffness andstructural integrity to the molecule, and may also helpdirect liquid crystalline mediated self-assembly intofibrils, helicoidal structures, and the other collagenoustextures and morphologies found in tissues. The high

Correspondence to:David L. KaplanContract grant sponsor: National Science FoundationContract grant number: DMR-9708062, BES-972-7401

Biopolymers, Vol. 53, 350–362 (2000)© 2000 John Wiley & Sons, Inc.

350

proportion of conformationally restricted proline andhydroxyproline residues present in the collagen con-sensus sequence stabilizes the triple-helical conforma-tion, a characteristic structural motif of the collagenproteins. This consensus sequence is (Gly–X–Y),where X is often proline and Y is often hydroxypro-line. Each strand in the collagen triple helix is in aconformation that is slightly more compressed thanthe polyproline II threefold helical conformation,making three full turns in 10 residues as opposed tothree turns in 9 residues (three residues per turn).

Since the collagen triple-helical structure was elu-cidated, a number of researchers have studied theeffects of different tripeptide, hexapeptide, and non-apeptide repetitive sequences on the conformation andthe crystal structure of collagen. More recently, withthe advent of modern molecular biological and pep-tide synthesis techniques, researchers have begun toexamine the detailed effects of residue substitutionsand other sequence variations on the physical proper-ties of collagen and collagen-like molecules includingconformational effects, thermal stability, and solventinteraction effects.

The presence of amino rather than imino acids inthe 2nd and 3rd positions of the sequence (Gly–X–Y)results in a conformational change as the helix twiststo accommodate hydrogen bonding between theamino acid residue and the environment.1–3The effectis most pronounced with the amino acid in the Yposition, to the extent that the triple-helical confor-mation can even be destabilized, which can also occurwith substitution of any amino acid for the glycine inthe lst position.1,3–8 A correlation between the se-quence of a collagen-like polypeptide, triple-helicalsupercoiling,1,3,9–14and ir amide A band (3290–3400cm21) position has been noted.11,15As the proportionof hydroxyproline increases, the degree of supercoil-ing and the thermal stability of the collagen triplehelix increase.6,16–20However a collagen triple-heli-cal conformation can be attained without hydroxypro-line in the sequence,1,3,11–14,19,21and many inverte-brate collagens contain substantial proportions of pro-line, but only small amounts of hydroxyproline.22

Correlations between primary sequence, local con-formation, supercoiling of the triple helix, and phys-ical properties have been demonstrated.4,7,8,23–28

Based on this information, questions regarding theformation of supermolecular ordered structures suchas crystals, liquid crystal structures, and orientedpolycrystalline domains can begin to be addressed.For example, what influence do the sequence-specificconformational variations in collagen have on thepacking of the collagen molecules? Does the patternof residue side chains “decorating” the outside of thehelix affect its liquid crystalline behavior? The colla-

gen triple helix is known to participate in liquid crys-talline phases, especially in cholesteric phases.29–38

Some workers have even proposed liquid crystallineintermediates in collagen fiber formation.34,39,40Smallchanges in conformation and in the overall “shape” ofthe helix, resulting from modifications in the primarysequence, should be transmitted into the structuresformed by interhelical packing and thus be observableas morphological changes in these macromolecularassemblies.

In order to investigate the effect of modificationsof the collagen consensus sequence on the liquidcrystalline self-assembly of triple-helical molecules,three variants of the collagen consensus sequencewere chosen: (1) peptide GAPGPP: (Gly–Ala–Pro–Gly–Pro–Pro); (2) peptide GVPGPP: (Gly–Val–Pro–Gly–Pro–Pro); and peptide GAPGPA: (Gly–Ala–Pro–Gly–Pro–Ala). The core repetitive sequences in pep-tides GAPGPP and GAPGPA have been studiedpreviously by other workers.1,15,41The crystal struc-ture and conformation adopted by these hexapeptidesequences, and the tripeptides comprising thesehexapeptides, are known for a variety of condi-tions.10,13,15,25The third hexapeptide, peptide GAPGPA,is expected to have a lower triple-helical formingtendency than peptides GAPGPP and GVPGPP sincethe first trio of residues in the hexapeptide sequence(Gly–Ala–Pro) has been shown to destabilize the tri-ple-helical conformation, and this peptide lacks thetriple helix stabilizing (Gly–Pro–Pro). However, inthese earlier studies the crystalline habit resultingfrom this sequence and its characteristic liquid crys-talline features were not reported.1,10,11,14,42PeptideGVPGPP was designed to explore the influence ofsteric bulk and a more hydrophobic residue in the Xposition (of Gly–X–Y) on the liquid crystalline be-havior, crystallinity, and crystal habit of triple-helicalsamples prepared under the same conditions. It wasalso of interest to see whether the triple-helical con-formation would be stabilized at an aqueous peptide–chloroform interface, which represents an unusualenvironment for peptide structural studies.43 This typeof environment may mimic cell membrane interfaces.

The two peptides that readily adopted collagen-liketriple-helical conformations, peptides GAPGPP andGVPGPP, exhibited different liquid crystal-like tex-tures, different crystal habits, different crystal struc-tures, and other morphological features specific toeach sequence. Additionally, there is evidence of astrong concentration dependence on the behavior ofthe peptide solutions—behavior suggestive of lyo-tropic liquid crystallinity. It is also interesting to notethat the supermolecular structures formed by the twohexapeptides are quite different from each other undermost conditions studied. The notable exception is the

Sequence-Specific Liquid Crystallinity of Collagen Model Peptides. I351

aqueous chloroform interface samples prepared at5°C, where sinusoidally textured gels are observed forall three peptides. Peptide GAPGPP adopts a lesssupercoiled structure than peptides GVPGPP andGAPGPA at the aqueous peptide–chloroform inter-face. Peptide GAPGPP also forms a gel with sinusoi-dal characteristics at the 5°C aqueous chloroforminterface with a longer period than peptide GVPGPP,indicating that packing of triple helices is more par-allel for peptide GAPGPP.

The collagen triple helix has bulges and groovesrunning in a spiral “barber pole” around the moleculedue to the pattern made by the side chains of theglycine and proline residues. The chiral shape thusimparted to the molecule should generate cholestericliquid crystalline textures. One would expect changesin this shape, i.e., changes in the conformation of thetriple helix and in the triple-helical packing footprintin a crystal, to be reflected in the supermolecularstructures formed by the molecules as is observed forthese model peptides.

EXPERIMENTAL

Peptide Design

Three peptides were synthesized and purified by the TuftsUniversity Protein Chemistry Facility. The peptides weresynthesized using standard solid-phase 9-flourenylmethy-oxycarbonyl chemistry on an Applied Biosystems 431Ausing HBUTAT activation. Reverse phase high perfor-mance liquid chromatography (HPLC) using a C18 columnwas used to purify the peptides and the composition andpurity of each peptide were verified using mass spectrom-etry. The peptide sequences (with major changes in primarysequence noted in bold face) used were as follows:

1. Peptide GAPGPP: (Glu)5(Gly–Ala–Pro–Gly–Pro–Pro)6(Glu)5

2. Peptide GVPGPP: (Glu)5(Gly–Val–Pro–Gly–Pro–Pro)6(Glu)5

3. Peptide GAPGPA: (Glu)5(Gly–Ala–Pro–Gly–Pro–Ala)6(Glu)5

The purity of the peptides was checked using massspectrometry after HPLC purification. The peptideseach represent 12 repeats of the collagen tripeptideconsensus sequence. The glutamic acid blocks on theends were added to promote solubility in pure water,eliminating the need for buffers that introduce con-taminating salts. A similar strategy has been shown tobe an effective means of studyingb-sheet peptides inaqueous solution, and the triblock structure was be-lieved to minimize micellization and other interac-tions that might distort the helical conformation.44

Sample Preparation

Millipore purified water, 17 MV, was used to prepare 40, 7,and 1 mg/mL solutions of the purified peptides in purewater. No buffers, acids, or salts were added. Samples weremaintained at room temperature or cooled to 5°C. For eachset of conditions, sample preparation of all three peptideswas carried out in parallel, to avoid artifacts due to temper-ature fluctuations, relative humidity differences, and otherenvironmental factors. Substrate-free, SiO, and carbon sub-strate coated transmission electron microscopy grids wereused to collect samples under each condition studied. Whencoated grids were used to collect precipitate or interfacialfilms, excess liquid was removed from the grid by carefullytouching the grid edges with a piece of filter paper.

1. Coated and uncoated transmission electron micros-copy (TEM) grids were drawn through the solutionsat 5°C, in order to pick up small precipitates. Thesolutions were first shaken to suspend the precipi-tate. Solutions were also dropped onto ZnSe crystalschilled to 5°C and slowly dried at 5°C to obtaincomparable Fourier transform ir (FTIR) samples.

2. Aqueous–chloroform interfaces were prepared bychilling vials containing chloroform to 5°C, andthen adding peptide solution. The vials were thencapped to prevent evaporation and left at 5°C over-night. The resulting interfacial films were collectedonto prechilled carbon substrate TEM grids anddried at 5°C overnight.

3. Aqueous–chloroform interfaces were prepared atroom temperature by adding aqueous peptide andchloroform to vials that were then sealed to preventevaporation. The vials were left overnight, and thenthe interface was sampled using uncoated andcoated TEM grids. No interfacial film was detectedunder these conditions.

4. Air–water interfacial films were collected on un-coated TEM grids from solutions at room tempera-ture and at 5°C.

5. Peptide solution was dropped onto carbon substrateTEM grids or onto ZnS crystal at room temperature.

Characterization

A Bruker Equinox 55 FTIR spectrometer was used in trans-mission mode to examine samples prepared by drying roomtemperature peptide solutions on cold ZnS crystals. An irmicroscope attachment was used to obtain ir reflectionspectra from carbon substrate TEM grids with peptide sam-ples on them, prepared under different conditions. Interfer-ence from the periodic grid bars was alleviated by sputteringgold on the back of the substrate film (opposite side fromthe sample) after analysis in the electron microscope. Thisprovided an even, smooth reflective surface under the sam-ple for reflectance ir.

A JEOL 2000 Mark 2 transmission electron microscope,operated at 200 kV in TEM mode was used to image thesamples prepared on TEM grids. Electron diffraction wasused to characterize the conformation and crystal structure

352 Valluzzi and Kaplan

of the peptides in the areas imaged. Gold, sputtered onto theTEM support film on the opposite side from the sample, wasused as an internal diffraction standard. In a well-alignedTEM the effective camera length should be within;10% ofthe camera length calculated by the microscope software.The camera length recorded by the microscope was com-pared to the value calculated using the gold standards toensure that there were no errors in choosing gold reflectionson the recorded diffraction patterns. A cryogenic samplestage was used to prevent crystal melting and to minimizebeam damage. In addition, very low electron dosages wereused when locating interesting sample regions and record-ing data. Kodak DEF-5 x-ray film was used to recordimages and diffraction patterns. This film offers a roughlyfivefold increase in speed over TEM film, has a linearresponse to electrons over much of its range, and has a highdensity of halide grains making it more efficient than lowerdensity films at high electron voltages. A Philips ESEMExplorer, operated at 20 kV and 7.0 Torr, with a workingdistance of 8.0 mm was used to obtain images of thicksample film surfaces on the TEM grids. The relatively largeworking distance was necessary to avoid excessive samplecharging.

MODELING

A commercial software package, Sybyl, was used tomodel changes in conformation due to residue substi-tution. A minimized published model structure forpoly(Gly–Pro–Pro)45 was used as the starting pointfor conformational modeling. Three repeats of thehexapeptides—1: (Gly–Ala–Pro–Gly–Pro–Pro); 2:(Gly–Val–Pro–Gly–Pro–Pro); 3: (Gly–Ala–Pro–Gly–Pro–Ala); (4) (Gly–Leu–Pro–Gly–Pro–Pro)—wereconstructed using the “residue mutate” function inSybyl. This function replaces a residue in the se-quence with a different residue, while preserving thebackbone conformation. The sequence (Gly–Leu–Pro–Gly–Pro–Pro)3 was added to the modeling studybecause polypeptides substituting leucine for prolinein the (Gly–Pro–Pro)x sequence have been studiedcrystallographically. As a control, the starting se-quence (Gly–Pro–Pro–Gly–Pro–Pro)3 was also usedin parallel conformational minimization studies. Mod-eling results that include (Gly–Leu–Pro–Gly–Pro–Pro)3 and (Gly–Pro–Pro–Gly–Pro–Pro)3 can be com-pared to literature trends, providing sufficient datapoints to verify whether the simulation reproducesthose trends. Minimization was performed on eachstructure with Del Re charges, and the rise per residuewas measured. Each chain in the collagen triple helixmakes three turns in ten residues. Ten residue dis-tances from the second amide nitrogen to the eleventhamide nitrogen in the twelve-residue peptide chainswere measured and used to determine the average rise

per residue. Distances between interior residues (e.g.,not the first or twelfth) were measured to avoid non-representative chain end effects.

In a second, more sophisticated simulation, Sybylwas used to build a solvent shell around each mole-cule, using water as the solvent. The solvent mole-cules were minimized with the collagen model con-strained in order to remove bad contacts (extremelyunfavorable steric interactions, multiple atoms occu-pying the same point in space) with the solvent. Thenthe collagen model was reassigned new charges in thepresence of water and reminimized. The rise per res-idue for each sequence was measured.

RESULTS AND DISCUSSION

The peptides all dissolved readily in pure water. Atroom temperature, all preparations of the peptidesyielded samples that were in the polyproline II con-formation. Although there appeared to be some dif-ferences in the morphologies and oriented texturesobserved in air–water and chloroform interfacial filmsof the three peptides (Figure 1), there were no dis-cernible differences in the diffraction patterns ob-tained from the room temperature samples. The tex-tures observed were, however, all oriented polycrys-talline textures. Small chain conformation differencesbetween the peptides might be observed if sharp sin-gle crystal diffraction patterns can be obtained in thefuture.

Low Temperature Precipitates

When the solutions of peptides GAPGPP andGVPGPP were chilled to 5°C, a ribbon-like whiteprecipitate formed. This precipitate remained evenafter the solutions warm back to room temperature.Attempts at sampling this precipitate for characteriza-tion in the TEM revealed textured films for bothpeptide GAPGPP and peptide GVPGPP (Figure 1).Regularly twisted morphologies were common forpeptide GVPGPP (Figure 1b). Some sample regionscontained single crystallites. Samples of peptideGAPGPA did not form precipitates.

Single crystal diffraction patterns for peptideGAPGPP can be indexed on an orthorhombic unit cellcontaining two triple-helical collagen moleculespacked in a pseudohexagonal array leading to anorthorhombic unit cell with the dimensionsa 5 21.3Å, b 5 10.4 Å, c 5 30 Å. The interhelical distance,10.4 Å is similar to the 10.3–10.6 Å reported forhexapeptides with similar sequences in the collagentriple-helical conformation. The chain axis dimensionis also typical of a collagen triple helical type of


structure, with a rise per residue of 3.0 Å. The ob-servedd spacings and calculatedd spacings for pep-tide GAPGPP are summarized in Table I.

Polycrystalline diffraction data were obtained forpeptide GVPGPP. These data indicate a strong reflec-tion at 4.5 Å and a weak reflection at 5.1 Å. Nosmaller angle (longerd spacing) reflections were ob-served for this peptide under these conditions. Similarpeptides in a triple helical conformation tend to havestrong reflections at approximately 5.3–5.4 Å andweaker reflections at approximately 4.6 Å. Whenthese peptides are in the polyproline II conformation,a strong reflection at approximately 4.5–4.6 Å and aweak reflection at 5.1–5.2 Å are observed.3,12,46Thediffraction data thus support a polyproline II confor-mation for precipitates from this peptide.

Chloroform Interface

When, rather than simply chilling the peptide solu-tion, a sublayer of chloroform was added and thesesolutions were allowed to chill for 2 h, the white

ribbon-like precipitate did not form. Instead, a cloudyfilm formed at the peptide solution–chloroform inter-face. The peptide solutions then gradually becamecloudy as they remained at 5°C. The solutions clearedagain as they were warmed to room temperature.When chilled chloroform interface samples are pre-pared and the cloudy solutions generated are left (withchloroform present) at 5°C overnight, rewarming thesamples again results in clear peptide solutions, butthe cloudy film at the interface persists.

At a concentration of 40 mg/mL, the white filmsobtained for the peptides can be picked up onto car-bon substrate TEM grids, and have sinusoidal bandsof contrast in the TEM. Film formation for peptideGAPGPA was significantly poorer than for peptidesGAPGPP and GVPGPP, and the films obtained forthis peptide covered only small portions of the TEM

FIGURE 1 Precipitate from bulk solution of peptideGVPGPP at 5°C. Uniform diameter twisted fibrils can beobserved at the edges of the precipitate. Patterned precipi-tate morphologies are also observed for this peptide.

Table I Unit Cell and Observed d Spacings ofPeptide GAPGPP at Aqueous ChloroformInterface and Precipitated at 5°C

ConditionSpacing

(Å) Index Intensityb

1. Chloroform interface(cluster) 10.35 100 v w

8.54 102 w7.20 103 m w5.71 110 m w4.73 202 v w4.53 203 v s4.36 106 m s2.81 130 m

a 5 b 5 11.9 Å andc 5 29.5 Å2a. Precipitate (sample 1) 8.48 202 m s

6.98 212 m s4.76 220 m s4.00 306 m3.58 420 v s2.92 330 m w2.60 040 w2.34 440 s

13.3 102 Streak2b. Precipitate (sample 2) 6.03 310 m

5.12 120 m4.56 220 s3.77 420 m s3.07 330 m w2.75 040 m w2.56 140 m w2.14 640 m w

a 5 21.3 Å, b 5 10.4 Å, c 5 30 Å

a The two sets of values for the precipitate are from differentcrystallites at different orientations.

b v w 5 very weak, w5 weak, m w5 moderately weak, v s5 very strong, m s5 moderately strong, m5 moderate, s5 strong


grid. Higher magnification images of these bands re-veal a gel-like structure with sinusoidal characteristicsas shown in Figure 2. The sinusoidal pattern observedfor peptide GAPGPP, has a period of 10mm. Thebands observed for peptide GVPGPP, Figures 2a (lowmagnification image) and 2b (higher magnification),have a period of 7mm, which is consistent for thissequence. Films obtained from peptide GAPGPAhave an apparent sinusoidal pattern with a period of 6mm, but the sinusoidal pattern is not as well defined.The consistency of these banded patterns is even moremarked in portions of the film obtained for peptideGAPGPP that were too thick to image in the TEM,shown here in an environmental scanning electronmicroscopy (ESEM) image (Figure 2c). The film inFigure 2c exhibited a remarkably consistent pattern ofbands that cover the entire TEM grid (thus the TEMgrid cannot be used to calibrate the magnification ofthe sample). The image in Figure 2c is from a samplethat had been aged on the grid several months prior toobservation and appears to contain several layers ofthe texture, whereas the TEM images are for singlelayers of banded gels.

At a concentration of 7 mg/mL peptide GVPGPPagain forms a sinusoidal gel with a period of 7mm,but films of peptide GAPGPP break up into “islands”of precipitated peptide when dried on a carbon sub-strate TEM grid. At 1 mg/mL, interfacial films areobserved for peptides GAPGPP and GVPGPP, but thefilms appear featureless when dried on either carbonor SiO substrate coated TEM grids. Peptide GAPGPAdid not exhibit significant film formation under any ofthese conditions. Films of all three peptides picked uponto uncoated grids dry to form clusters of crystal-lites. In this manner, single crystals of the peptideshave been obtained from samples prepared at 5°C atthe aqueous chloroform interface. Crystallites frompeptide GAPGPP were observed primarily in smallclusters making the habits of individual crystalliteshard to discern. An apparent single faceted lamellawith an irregular habit is shown in Figure 3a, a crys-tallite of peptide GAPGPP. These irregular lamellaeyield diffraction patterns, as shown in Figure 3b,containing arcs, rings or multiple zones, indicatingthat the apparent lamellae are polycrystalline clustersor possibly mosaic crystal structures.

This diffraction pattern can be indexed on thepseudohexagonal unit cell reported by Segal et al.46

for crystalline poly(Gly–Ala–Pro–Gly–Pro–Pro) inthe collagen triple-helical conformation using x-raydiffraction techniques, wherea 5 b 5 11.9 Å andc5 29.5 Å. They reportd spacings and relative inten-sities for the 100, 103, 110, 200, and 203 reflectionsthat are the same as those observed in the presentstudy. This is not surprising considering that the two

polypeptides have the same repetitive sequence. Be-cause the reflections in single crystal electron diffrac-tion data are generally much sharper and more easilydistinguished from the background than in a polycrys-talline x-ray diffraction experiment, a few additionalreflections are observed in the current study. TheMiller Index assignments and observedd spacings arelisted in Table I. It is interesting to note that the riseper residue observed in the aqueous chloroform inter-face structure, at 2.95 Å is slightly shorter than thedistance of 3.0 Å obtained from the bulk precipitate at5°C, and the packing of the triple helices is differentas well.

The crystallites observed from peptide GVPGPP,(Gly–Val–Pro–Gly–Pro–Pro), were found in clusteredmasses making individual crystal habits hard to dif-ferentiate. However, facets appeared to be present.The diffraction pattern from these crystallites can beindexed on an orthorhombic unit cell with dimensionsa 5 22 Å, b 5 11.5 Å, c 5 28.8 Å, suggesting twotriple-helical molecules packed in a pseudohexagonalarray with an 11.5 Å intermolecular distance. Assign-ments of Miller Indices and measuredd spacings aresummarized in Table II. Previous studies on poly-hexapeptides such as poly(Gly–Ala–Pro–Gly–Pro–Pro), poly(Gly–Pro–Ala–Gly–Pro–Pro), and other tri-ple-helical polyhexapeptides comprised of glycine,alanine, and proline with the collagen consensussequence have intermolecular packing distancesof 10.4 –10.6 Å.9,10,12,13,42 For triple helices ofpoly(Pro–Leu–Gly) this distance is 11.4, and for(Leu–Pro–Gly) it is 12.5 Å.3 Since peptide GVPGPPcontains valine, which is intermediate in size betweenalanine and leucine, an intermolecular distance of 11Å is consistent with a triple-helical conformation forpeptide GVPGPP. Crystallites of peptide GAPGPAare infrequent, and some crystallites with a polypro-line II structure are obtained in addition to collagen-like triple-helical structures. In the 5°C, 40 mg/mLaqueous peptide–chloroform interface sample (theonly condition where this peptide formed an interfa-cial film), crystallites were obtained which had anorthorhombic unit cell with the parametersa 5 11.5Å, b 5 12.4 Å, c 5 28.7 Å. Thea and c unit celldimensions obtained strongly suggest a triple-helical,collagen-like conformation. The diffraction data forpeptide GAPGPA are summarized in Table III.

Molecular Modeling

In order to separate the steric effects of residue sub-stitutions on the collagen triple-helical conformation,in the absence of preferential solvent interactions,minimum energy structures obtained without solventwere compared for the sequences used in the experi-


mental study with the addition of (Gly–Leu–Pro–Gly–Pro–Pro)3 and (Gly–Leu–Pro–Gly–Pro–Pro)3 forcomparison. The rise per residue values predicted bymolecular mechanics energy minimization in vacuumwith calculated Del Re charges using theSybylforce

FIGURE 2 Banded gel morphology observed at the aque-ous (40 mg/mL) peptide–chloroform interface. (a) PeptideGVPGPP; the circled area (arrow) appears to be a typical21/2 defect and is sketched in the lower left-hand corner.(b) Close-up of banded gel of peptide GVPGPP showinggel-like structure in bands (c) ESEM image of the surface ofa thick film region of peptide GAPGPP showing a typicaltexture observed covering the entire TEM grid.

FIGURE 3 Crystallite and diffraction pattern for peptideGAPGPP obtained at the aqueous chloroform interface at5°C. (a) Crystallite. (b) Diffraction pattern.

Table II Unit Cell and Observed d Spacings ofPeptide GVPGPP at Aqueous Chloroform Interface

Spacing Index Intensity

9.18 111 v w7.99 210 m w4.91 220 s3.85 130 m2.92 620 m2.70 630 m w

a 5 11.5 b 5 21.6 c 5 28.8


field are listed in Table IV. A trend is observed in themolecules with sequences having the pattern (Gly–X–Pro–Gly–Pro–Pro) whereX is proline or a hydropho-bic residue. As thesize of the hydrophobic residueincreases, the predicted rise per residuedecreases, asthe triple helix becomes wider to accommodate theincreased steric bulk. The notable exception is thecase of (Gly–Leu–Pro–Gly–Pro–Pro)3. Leucine islarger than valine; however, it has two methyl groupsattached to theb-carbon, placing much of the sidechain away from the triple-helical main chains. Valinehas two methyl groups attached to thea-carbon, closeto the main chain, where steric crowding will beexacerbated.

A similar trend is observed in the correlation be-tween the hydrophobicity of substituted residues andthe published rise per residue in Gly–X–Pro se-quences (These sequences can also be written X–Pro–Gly, depending on how the sequence was synthe-sized). When a hydrophobic residues is incorporatedat the X position, there is a correlation between thesize and hydrophobicity of the X residue and the riseper residue observed for the triple-helical conforma-tion. Larger more hydrophobic residues cause thetriple helix to become more supercoiled, with a con-comitant decrease in the rise per residue. For theseries (Pro–Gly–Pro)n, (Ala–Pro–Gly)n, (Leu–Pro–Gly)n the rise per residue, determined by x-ray dif-fraction, decreases from 2.87 to 2.85 to 2.82.3,11–13,46

When triple-helical structures formed by poly(G-APGPP) are compared with poly(GPPGPP), thepoly(GAPGPP) triple helix is extended relative to thepoly(GPPGPP) triple helix, having a rise per residueof 2.95 rather than 2.87 Å.3,11–13,46The extension ofthe triple helix observed for poly(GAPGPP) has beenattributed to the presence of water and the concomi-tant conformational changes that result when the triplehelix deforms to enable hydrogen bonding betweenamino acid residues (in the X position, Gly–X–Y) andthe solvent.3,11–14,17,18,24,27,46,47This extension is notobserved in the solvent-free simulation; rather, a de-

crease in the rise per residue for (GXPGPP) peptidesis observed that can be rationalized in terms of sterics.

Unlike poly(GAPGPP), the triple-helical con-formation reported for poly(GAPGPA) does not ex-hibit an extension of the triple-helical conforma-tion.3,11–13,46In this case, the simulation predicts amore supercoiled conformation for peptide GAPGPAthan the other peptides even in the absence of water.The short rise per residue reported for this peptide isattributed to an available packing arrangement thatclusters the hydrophobic alanine residues together,resulting in a more favorable interaction betweencomponent chains in the triple helix. The model didnot incorporate this clustering, and it may be thatadditional steric effects contribute to the supercoilingin poly(GPAGAP) as well as hydrophobic clustering.

Simulations carried out in the presence of waterreveal a slightly different trend, as can be seen fromTable V. When water is added to the simulation, thedegree of triple-helical supercoiling is correlated withthe hydrophobicity of the residue in the X position. Inboth simulations the distances obtained are only qual-itatively similar to the reported crystal structure pa-rameters for polypeptides with these repetitive se-quences. However, the simulations were not carriedout on periodic crystalline lattices, and the Triposforce field is a general force field for biomolecules. Aforce field incorporating parameters more specific tothe collagen class of proteins might give more accu-rate results.

IR Spectroscopy

Samples of the peptides dried on ZnSe crystals atroom temperature and at 5°C were also characterizedusing FTIR. The results for the three peptides at roomtemperature, 5°C, and at an aqueous chloroform in-terface at 5°C are summarized in Table VI, listing theresulting amide A, amide I, and amide II positions.The amide A and amide I bands shift to higher fre-quencies when peptides GAPGPP and GVPGPP are

Table IV Modeling Results: 10 Residue Distances forSimulated Triple-Helical Peptides Minimizedin Vacuum

SequenceAverage Rise for

10 Residues Standard Deviation

GPPGPP 30.09 0.30GAPGPP 29.34 0.23GVPGPP 29.27 0.24GLPGPP 29.57 0.29GAPGPA 27.89 0.55

Table III Unit Cell and Observed d Spacings ofPeptide GAPGPA at Aqueous Chloroform Interface

Spacing Index Intensity

5.81 200 w5.52 120 s6.20 020 v w7.36 103 v w3.11 040 s3.56 303 vs

a 5 11.5 b 5 12.4 c 5 28.7


prepared at 5°C, compared to the room temperaturesamples. No shift is observed for peptide GAPGPA.These data suggest a change in conformation is in-duced when peptides GAPGPP and GVPGPP arechilled to 5°C, but that peptide GAPGPA remains inits room temperature conformation. The amide Aband shift is most pronounced for peptide GAPGPP,from 3292 cm21 at room temperature to 3328 cm21 at5°C.

In previous studies, some differences in the irspectra were noted for collagen-like polypeptides inthe threefold helical and triple-helical states. The mostconsistent and pronounced of these tendencies was ashift in the amide A band to a larger frequency in thetriple-helical conformation. This is attributed to sta-bilization of the triple-helical structure by hydrogenbonding. Similarly, in spectroscopic and structuralstudies of other model polypeptides that adopt ex-

tended chain conformations in the twofold to three-fold helical region, a change in the amide I bandposition has been noted for the more extended two-fold helical conformation, at 1621–1626 cm21, andthe more compressed threefold helical conformation,at 1655–1665 cm21. For peptides GAPGPP andGVPGPP prepared at 5°C this band also shifts tohigher frequencies, again suggesting that a slightlymore compressed conformation is being adoptedwhen compared to the room temperature conforma-tion. The large shift in amide A frequency that occurswhen peptide GAPGPP is chilled to 5°C suggests thatthis peptide supercoils to adopt a triple-helical con-formation under these conditions.

These findings agree with the diffraction data ob-tained for the precipitates from solutions of peptidesGAPGPP and GVPGPP obtained at 5°C, which cor-roborate a collagen triple-helical conformation for

Table V Modeling Results: 10 Residue Distance for Simulated Triple-Helical Peptides Minimized in Water

Sequence GPPGPP GAPGPP GLPGPP GVPGPP GAPGPA

Chain 1 30.01 29.80 30.00 29.84 26.23Chain 2 30.17 29.54 29.63 29.59 28.95Chain 3 30.16 29.77 29.41 29.32 26.02

Average 30.11 29.70 29.68 29.58 27.07

Kyte and Doolittle hydropathya

Proline Alanine Leucine Valine

21.6 1.8 3.8 4.2

a Kyte and Doolittle (1982).55

Table VI Summary IR Results for the Three Synthetic Peptides at Room Temperature, 5°C, and at the AqueousChloroform Interface at 5°C

Peptide Sequence

Amide Bands

ConformationA I

Peptide 1 GAPGPA 3303 1651 PPIIPeptide 2 GAPGPP 3292 1643 PPIIPeptide 3 GVPGPP 3300 3228ha 1647 PPIIPeptide 1b GAPGPA 3303 1646Peptide 2b GAPGPP 3328 1654 CollagenPeptide 3b GVPGPP 3306 1675 1647Peptide 1c GAPGPA Poor film formation CollagenPeptide 2c GAPGPP 3313 1661 1629 CollagenPeptide 3c GVPGPP 3332 1675 1655 Collagen

a h: Shoulder; s: strong; w: weak; b: broad.b 5°C, bulk dried solution.c 5°C, aqueous peptide–chloroform interface.


peptide GAPGPP and a polyproline II conformationfor peptide GVPGPP. It is also interesting to note thatwhile the amide A band for peptide GVPGPP did notshift markedly, the amide I band did shift almost 30cm21, from 1647 to 1675 cm21. At room temperature,this peptide is soluble at all three concentrations from1 to 40 mg/mL, yet at 5°C it precipitates from a 7mg/mL solution. Similarly, significant crystallinitywas observed in samples of peptide GVPGPP pre-pared at room temperature, while almost no crystal-linity was observed for the 5°C precipitate. These dataindicate that while this peptide does not appear toadopt a supercoiled triple-helical structure under theseconditions, it does undergo a conformational transi-tion that affects its supermolecular self-assembly be-havior. Diffraction data for peptide GAPGPA do notindicate a triple-helical conformation under these con-ditions, FTIR data do not indicate any detectableconformational change, and the solubility and crystal-linity of peptide GAPGPA also remain essentiallyunchanged at the lower temperature.

At 5°C at the aqueous chloroform interface, pep-tides GAPGPP and GVPGPP form interfacial filmsthat can be sampled. Films from peptide GAPGPA donot coat TEM grids or other substrates well enough toallow FTIR characterization. FTIR microscopy wasused to characterize regions of aqueous chloroforminterfacial films prepared from 40 mg/mL peptidesolutions, on the same TEM grids used for electrondiffraction analysis. Regions were selected where thepeptide film was thickest to avoid sampling solutionthat had dried onto the grid in the process of collectingand drying the interfacial film. These regions had abanded appearance and reveal a pronounced shift inthe amide A band position for peptide GVPGPP, anda more pronounced shift in the amide I band forpeptide GAPGPP. This suggests that the triple helicalconformation is also present at the aqueous chloro-form interface. The lower amide I and amide A fre-quencies observed for peptide GAPGPP suggest aslightly more extended conformation than for peptideGVPGPP.11 This observation is corroborated by thecrystallographic studies that indicate a 2.95 Å rise perresidue for peptide GAPGPP at the 5°C aqueous chlo-roform interface and a 2.88 Å rise per residue forpeptide GVPGPP. The molecular modeling data alsoindicate a shorter rise per residue for PeptideGVPGPP.

There is a shift in the amide I band of roughly 10cm21 from 1654 to 1661 cm21 when the peptide isobtained at a 5°C aqueous chloroform interface asopposed to being precipitated from a 5°C bulk solu-tion. This suggests that the triple-helical conformationadopted by peptide GAPGPP is more compressed orsupercoiled at the aqueous chloroform interface.

Again this observation is corroborated by the diffrac-tion data that indicate a rise per residue of 3.0 Å forthe crystallites of triple-helical bulk precipitate, ascompared to 2.95 Å at the aqueous chloroform inter-face.

Conformation Selection

At this point, it is not completely clear why there issuch a noticeable difference in the behavior exhibitedby peptides GAPGPP and GVPGPP. The data fromthe bulk state suggest that the peptide GAPGPP triplehelix is more stable than the peptide GVPGPP triplehelix. Rather than forming a triple helix bysupercoil-ing when cooled to 5°C, peptide GVPGPP adopts amore tightly coiled structure. Since the only differ-ence between the samples of peptides GAPGPP andGVPGPP is the substitution of valine for alanine, theincreased hydrophobicity of the valine residue mayprevent the hydrogen-bonding interactions with wa-ter, which are widely believed to cause the peptideGAPGPP helix to extend. At the 5°C aqueous chlo-roform interface all three peptides supercoil to formtriple-helical structures, but the valine-containing he-lix is more compressed. The peptides require differentsolution concentrations to form films of collagen-liketriple helices at this interface, with peptide GAPGPArequiring the most concentrated solution, and peptideGVPGPP requiring the lowest concentration. The ala-nine-containing triple helix is more compressed at thisinterface than it is in the bulk at the same temperature.In summary, the peptide GAPGPP triple helix appearsto be more inherently more stable, but the peptideGVPGPP triple helix experiences a greater stabilityenhancement at the aqueous chloroform interface.Peptide GAPGPA appears to experience the smalleststability enhancement at the interface.

The “3.3-fold” helices comprising the collagen tri-ple-helical structure, when combined with thehexapeptide repetitive sequences used in both pep-tides, would result in very hydrophobic regions foreach chain which slowly wind around the circumfer-ence of the helix like stripes on a barber pole as inFigure 4. It may be that the 3.3-fold conformation isnucleated at the interface through interactions with thehydrophobic side chains and the hydrophobic chloro-form solvent. A hydrophobicity-driven conforma-tional rearrangement would be expected to be morepronounced for residues with a greater hydrophobic-ity.

The behavior of the banded gel, possibly choles-teric in origin, also is interesting. The gel has a texturecontaining numerous defects that are commonly ob-served in chiral mesophases such as cholesterics (Fig-ure 2a, arrow), and the textured gel spreads to form a


film with no evident texture on hydrophilic surfacessuch as SiO. These observations strongly suggestsboth liquid behavior and some form of low dimen-sional chiral long-range order as the origin for the

(dried, solid) textures observed, such as a cholestericliquid crystalline phase. There have been numerousobservations of biopolymer liquid crystallinity andcholesteric behavior. For solid state biological liquidcrystalline analogues, a theory has been advanced byRudall48–50 suggesting that the helical shape of themolecules allows them to pack “bulge to groove,”causing the observed twist. While this explanationmay hold true in a few very specific cases, it wouldentail rotations of several degrees between helices.For molecular helices this would result in muchshorter cholesteric periods than the order of 100s to1000s of nm periods typically observed. Similarly,alterations in cholesteric pitch correlated to supercoil-ing–conformational changes have been observed forcondensed phases of DNA. In the DNA studies thecholesteric pitch was larger in less supercoiled, or“straighter” molecules. The collagen peptides in thecurrent study show a similar trend, although of thethree sequences two, peptide GVPGPP and peptideGAPGPA, have experimentally identical degrees ofsupercoiling. Three different periodicities are ob-served in the banded gels. However, in the DNAstudies, alterations in the degree of supercoiling wereachieved through substitution of nucleotides of the“wrong” handedness, or chirality. While some form ofshape recognition may be at work for the collagenpeptides, a tight bulge to groove packing does notadequately account for the large gel period, or for thechanges in the period observed.

Various extensions of the Meier–Saupe theory ex-ist to try to correlate the influence of various environ-mental factors on the cholesteric phases formed bychiral molecules. One relatively new treatment exam-ines the effects of molecular chirality, defined as amolecular biaxiality, on the cholesteric behavior ofchiral helical biopolymers.51–54 This theory predictsthat an increase in the biaxiality of a chiral helicalmolecule, resulting from a longer conformation forexample, would result in a larger cholesteric pitch andshorter cholesteric period. A possible experimentalobservation of molecular biaxiality is the apparentdeviation from hexagonal packing observed when themolecules crystallize. As can be seen from Table VII,

FIGURE 4 (a) Peptide GVPGPP in a collagen 10/3 helix(one chain). The collagen triple-helical conformation, a3.3-fold helix, results in clustering of the hydrophobic va-line residues. (b) Staggering of three GVPGPP sequencesinto a triple helix results in even stronger clustering ofvaline residues.

Table VII Cholesteric Period is Correlated with Packing Asymmetry Observed in Corresponding Crystals

PeptideRepetitiveSequence

Rise PerResidue

Value of g if PseudohexagonalPacking Were Present

Period ofBanded Gel

Texture

Percent SDBand

Distance

GAPGPP 2.95 120 10m 4.3GVPGPP 2.88 110 7m 3.4GAPGPA 2.87 106 6m 4.3


when the unit cell parameters are used to calculate theangle (g) between nearest neighbor molecules or thedeviation from a trigonal lattice (perfect cylinders),the period observed for the banded gel can be corre-lated with the experimental crystallographic data. Thelargest deviation from hexagonal packing is observedfor peptide GAPGPA, which has the smallest bandedgel texture periodicity. Similarly, peptide GAPGPPpacks into a pseudohexagonal lattice and has thelargest observed banded gel periodicity. Smallchanges in the packing asymmetry result in smallchanges in the periodicity of the gel morphology, ascan be seen by comparing peptide GAPGPA to pep-tide GVPGPP. The large change in crystalline pack-ing asymmetry observed when comparing peptideGAPGPP to the other two peptides is reflected as alarge difference in the periods of the banded gels,which are most probably a cholesteric texture.

The peptides used in this study, along with similardesigned peptides, offer a unique opportunity to ex-amine the effect of molecular conformation and asym-metry on cholesteric behavior. They are water-solubleand possess the same ratio of glycine, proline, and ahydrophobic group (alanine and valine), and the samenumber and kind of acid residues is used to solubilizeeach. They have the same molecular weight, and canbe prepared at identical concentrations and tempera-tures. Thus the variables often used to alter cholestericand other mesophasic behavior: solvent dielectric,concentration, temperature, and molecular weight canbe eliminated leaving only the degree of supercoilingand the details of the conformation as variables instudying changes in this behavior. Previous studies onthe effects of conformation on liquid crystalline be-havior in biopolymers have focused primarily onDNA, and the effects of the environment on super-coiling and on liquid crystallinity were difficult todecouple. Thus careful characterization of tailoredmodel peptides can conceivably provide very valuableexperimental model systems to test and develop ourunderstanding of liquid crystalline, and analogouschiral solid state, behavior in general.

CONCLUSIONS

The sequence of a collagen-like peptide can be used toinfluence the liquid crystalline behavior of the peptidein a threefold helical conformation. An oil–water(aqueous chloroform) interface can be used to nucle-ate or stabilize the triple-helical conformation if thisconformation results in thermodynamically favorableclustering of hydrophobic residues at the interface.The long-range periodic texture, possibly cholestericin origin, of collagen model peptides with similar

hexapeptide repetitive sequences is correlated withthe deviation from perfect hexagonal packing mea-sured in crystallites obtained under the same condi-tions as the long-range periodic texture. This devia-tion from hexagonal packing is a measure of theeffective asymmetry of the molecular cross section, orbiaxiality of the molecule. The results obtained indi-cate an increase in the period of the textured gel as theeffective molecular cross section becomes moreasymmetric, in agreement with recent theoreticalwork on the conformation and liquid crystalline be-havior of chiral biopolymers.51–54

Funding support from the National Science Foundation(DMR-9708062, BES-972-7401) and facilities support fromthe NSF MRSEC core facility at University of Massachu-setts Amherst, the W. M. Keck Foundation BiomimeticMaterials Characterization Laboratory (Tufts University)and the W. M. Keck Foundation Polymer MorphologyLaboratory (University of Massachusetts Amherst) aregratefully acknowledged. The Tufts University MedicalSchool Protein Core Facility provided peptide syntheses.We would also like to acknowledge helpful discussions withDavid Birk.

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