Reversibility of Structural Changes of Polypeptides in Multilayer

Dec 14, 2007 - The various peptides studied exhibit a strong tendency to adopt a β sheet conformation in the .... University of Connecticut School of...
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Biomacromolecules 2008, 9, 185–191

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Reversibility of Structural Changes of Polypeptides in Multilayer Nanofilms Ling Zhang† and Donald T. Haynie*,†,‡,§ Artificial Cell Technologies, Inc., 5 Science Park at Yale, Third Floor, New Haven, Connecticut 06511, Department of Chemistry and Bionanosystems Engineering Laboratory, National Dendrimer and Nanotechnology Center, Central Michigan University, Mt Pleasant, Michigan 48859, and Center for Molecular Tissue Engineering and Department of Surgery, University of Connecticut School of Medicine, Farmington, Connecticut 06030 Received August 30, 2007; Revised Manuscript Received October 2, 2007

Structural properties of different polypeptide multilayer nanofilms fabricated at neutral pH have been analyzed by UV spectroscopy, circular dichroism spectroscopy (CD), and Fourier-transform infrared spectroscopy (FTIR). The various peptides studied exhibit a strong tendency to adopt a β sheet conformation in the films. Changes in film structure on dehydration are completely reversed on rewetting. The time scale of reversibility is, however, substantially shorter for the polymer backbone than the side chains, as in protein folding.

Introduction Polypeptides in nature exist as unstructured polymers, proteins, or supramolecular assemblies.1 Some peptides aggregate spontaneously into structures that display a remarkable degree of hierarchical organization, for example, filamentous structures of the cytoskeleton. Determining how peptide-based structures self-organize was made a major goal of biochemistry decades ago.2 Despite extensive effort, the molecular basis of the equilibrium conformation of a single polypeptide chain of arbitrary sequence, the kinetics of structure attainment, and the reversibility of structural changes remain largely unsolved puzzles. It may be that putting all pieces of the jigsaw in place will require combining key results from several rather different methods of inquiry, some of which are perhaps presently unknown. Layer-by-layer assembly is a straightforward, inexpensive, and versatile method of fabricating the nanostructured materials known as multilayer nanofilms.3 Many different chemical species are suitable for incorporation into films, so many different nanofilm architectures are possible. The species are generally deposited from aqueous solution, making the nanofilm fabrication process environmentally benign. The films can be formed on a surface of virtually any size, shape, or roughness. One intriguing feature of the method used to fabricate these films is its paradoxical character: the adsorbing chemical species undergo spontaneous but self-limited assembly, driven by Coulomb’s law and the second law of thermodynamics under externally imposed solution conditions during successive steps of an externally controlled cyclical process. Polyelectrolyte multilayer nanofilms are of widespread interest for fundamental studies and technology development.3 The simplest polyelectrolyte multilayer nanofilm consists of one species of polycation and one species of polyanion. The range of potential applications of polyelectrolyte nanofilms encompasses optical coatings, biosensors, drug delivery systems, and * To whom correspondence should be addressed. E-mail: [email protected]. Telephone: +1 (203) 772 3430. Fax: +1 (203) 567 8073. † Artificial Cell Technologies, Inc. ‡ Central Michigan University. § University of Connecticut School of Medicine.

other technologies.3,5,6 Multilayer nanofilms made of polypeptides are attractive for applications where chirality, biocompatibility, biodegradability, and specific functionality are relevant,4 for example, biotechnology and medicine.3 The present study concerns polypeptide multilayer nanofilms. Highly charged peptides that encode aromatic residues as spectral probes have been used to investigate nanofilm buildup, film structure, and changes in structure on dehydration. The data show that the behavior of aggregated peptides in multilayer nanofilms is surprisingly similar with respect to the behavior of individual peptides in protein folding.

Materials and Methods Sample Preparation. The polymers were 70.0 kDa poly(allylamine) hydrochloride (PAH), 70.0 kDa sodium poly(styrene sulfonate) (PSS), 38.0 kDa poly(L-lysine, L-tryptophan) 4:1 hydrobromide [poly(Lys,Trp)], 24.6 kDa poly(L-lysine, L-tyrosine) 1:1 hydrobromide [poly(Lys,Tyr)], poly(L-lysine) hydrobromide of 13.8 kDa (PLL-L), 45.8 kDa (PLLM), and 130 kDa (PLL-H), and sodium poly(L-glutamate) of 14.5 kDa (PLGA-L), 64.0 kDa (PLGA-M), and 95.3 kDa (PLGA-H). Key properties of these polymers are summarized in Table 1. PSS and PAH were from Aldrich (USA); all peptides were from Sigma (USA). Average polymer mass was determined by viscometry in each case. All polymers were used without further purification. The buffer was 10 mM potassium phosphate, pH 7.4. For FTIR experiments, deuterated monobasic potassium phosphate (Aldrich), deuterated dibasic potassium phosphate (C/D/N Isotopes, Inc., Canada), and 99.990% D2O (Aldrich) were substituted for the reagents in UVS and CD analysis, and pD was adjusted to 7.4 with NaOD (Aldrich) or DCl (Aldrich). All multilayer nanofilms were prepared at ambient temperature (21–25 °C) by electrostatic layer-by-layer assembly (Figure 1). The concentration of each polymer in adsorption solutions was 2 mg mL-1. In each case the polymer in the first adsorption step was a polycation. Each adsorption step had a duration of 15 min. Films were rinsed in three separate phosphate buffer baths following each polymer adsorption step. No film was dried at any point during fabrication. For dehydration experiments, films were dried with a gentle stream of air. Data collection on rewet films was begun 2 min to 24 h after film rewetting. For UV spectroscopy and circular dichroism spectrometry (CD) experiments, 15-layer films were prepared on thin plates of quartz

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Zhang and Haynie

Table 1. Polymer Properties

a The structure of lysine (Lys) is the same as in poly(L-lysine) (PLL). Both copolymers are random copolymers. Trp ) tryptophan, Tyr ) tyrosine, Glu ) glutamic acid. PLGA ) poly(L-glutamic acid). H ) “high” molecular weight. M ) “medium.” L ) “low.” PAH ) poly(allylamine). PSS ) poly(styrene sulfonate). b Dawson et al.30 c DP ) degree of polymerization. Counterions are assumed to contribute to the experimental determination of molecular weight (Na+ for Glu, Br- for Lys, Cl- for PAH, Na+ for PSS).

(Electron Microscopy Sciences, USA). Plates were cut into pieces prior to film fabrication and cleaned with hot 1% sodium dodecyl sulfate for 30 min, 1% NaOH in 50%/50% by volume 99.5% ethanol/water for 2–3 h, and with a hot mixture of 98% sulfuric acid and 27% hydrogen peroxide overnight. For FTIR experiments, 25-layer films were prepared on thin plates of CaF2 (International Crystal Laboratories, USA). A stream of air was used to remove small particulate matter from the plates immediately before film assembly. Polymers were adsorbed from solution onto one side of the plates. Evaporation of solution was limited by enclosing the wetted substrate in a small chamber. No film was allowed to dry during the fabrication process. Sample Analysis. All spectra were collected at ambient temperature. Wet and dry baseline spectra were obtained and all film spectra were processed accordingly. UVS spectra were collected with a Shimadzu UV mini-1240 spectrophotometer (Japan). The scanning rate was 10–15 nm s-1, the step size was 0.5 nm, and the spectral bandwidth was 5 nm. For CD and FTIR measurements, the sample chamber was purged continuously with nitrogen gas. CD data were collected with a Jasco

J-715 spectropolarimeter (Japan). Twenty scans were averaged for measurements in the far UV, 60 scans for the near UV. The scanning rate was 100 nm min-1, the step size was 0.5 nm, and the bandwidth was 1.0 nm. FTIR spectra were measured with a Nicolet 60SX spectrometer (USA). The detector was deuterated triglycine sulfate. 32 scans were collected, averaged, and further processed by Happ-Genzel apodization. The data range was 400–4000 cm-1, the mirror speed was 0.6329 cm s-1, and the resolution was 4 cm-1. The time required to acquire 32 scans was 40 s. Each film spectrum was processed by subtracting the water vapor spectrum collected immediately beforehand.

Results and Discussion Spectroscopic methods have been used to obtain information on the nanofilm buildup process for the peptide structures in Table 1. For example, spectra of films of different numbers of layers of poly(Lys,Trp) and PLGA-M are shown in Figure 2a.

Reversibility of Polypeptides in Multilayer Nanofilms

Figure 1. Polyelectrolyte layer-by-layer assembly. (a) Polymer deposition: A substrate bearing a surface charge (not shown) is dipped into a solution of oppositely charged polymers, creating the first layer. Loosely bound material is removed by rinsing with buffer. The substrate is then dipped into a solution of polymers of opposite sign to the first polymers, creating the second layer. The process can be repeated indefinitely and automated. (b) The resulting film: In essence, one layer of polymers is deposited per adsorption step for strong polyelectrolytes with a high absolute linear charge density, λ.10 Layers interpenetrate. The reversibility of polymer adsorption depends on λ and net charge. Polymers with a large degree of polymerization and large λ will bind irreversibly to a surface of high charge density.

A large absorption band, due to peptide bonds, is seen around 195 nm in the UV spectrum (inset). Peaks around 225 and 280 nm are from the Trp side chain.7 The large band in the CD spectrum near 220 nm, which is not present in spectrum of the free peptide in solution (not shown), indicates the formation of chiral structure on polymer adsorption.8 The magnitude of this band grows with an increasing number of peptide layers deposited. Overlaid spectra of films incorporating an increasing number of layers are approximately coincident near 208 nm. The increase in optical density with increasing layer number provides compelling evidence of polymer adsorption in successive steps of the film fabrication process. The general character of nanofilm buildup evident by UV absorption is consistent with that by CD (Figure 2b). Optical density grows approximately exponentially with increasing layer number. Migration of the position of the absorption band during film fabrication (inset) indicates that average film structure depends on film thickness, corroborating the incomplete coincidence in the CD signal near 208 nm (Figure 2a). Film properties will become increasingly independent of the electronic character of the substrate with an increasing number of layers.9,10 Spectra have been obtained to compare polypeptide multilayer nanofilms of different architecture. All films made of the

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Figure 2. Buildup of poly(Lys,Trp)/PLGA-M. (a) Spectral analysis: Far UV CD. Spectra of wet films were taken after adsorption of 3 (black), 6 (red), 9 (green), 12 (blue), and 15 (cyan) layers. Overlaid spectra after baseline subtraction are shown. Dichroic bands are seen at 195 and 220 nm; an apparent isosbestic point is evident at around 208 nm. The relatively high noise below around 205 nm, which is attributable to the presence of ions in the film and the relatively high scanning rate of the experiments, does not influence the interpretation of results in the present context. Inset, UV: Spectra were taken of wet films after adsorption of 3 (black), 6 (red), 9 (green), 12 (blue), and 15 (cyan) layers. Overlaid spectra after baseline subtraction are shown. Absorption bands are evident at 195 nm (peptide bond) and 225 and 280 nm (Trp side chain). (b) Comparison of CD and UV data: The negative CD signal at 220 nm (circles, peptide bonds) is plotted together with UV absorbance at 280 nm (squares, aromatic groups). The results of the CD and UV experiments are in good agreement. The red line is an exponential fit to one of the data sets. Inset: position of main absorption band extrema.

polymers in Table 1 show a UV absorbance maximum near 200 nm (Figure 3a). An aromatic absorption peak is apparent at 280 nm for poly(Lys,Trp)/PLGA-M and poly(Lys,Tyr)/ PLGA-M. The peak amplitude at this wavelength is substantially larger for the former than the latter, reflecting the comparatively large absorption dipole moment of the indole group.7 Poly(Lys,Trp)/PLGA-M, poly(Lys,Tyr)/PLGA-M, and PAH/PSS show characteristic aromatic absorption near 225 nm. Nanofilm optical density has a substantial dependence on molecular structure, in particular degree of polymerization (Figure 3a). PLL-L/PLGA-L < PLL-M/PLGA-M < PLL-H/ PLGA-H, as in previous work.8 Surprisingly, PLL-H/PLGA-L

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Figure 3. Wet film spectroscopy. (a) UVS spectra: All films were 15 layers thick. PLL-L/PLGA-L (black), PLL-M/PLGA-M (red), PLL-H/ PLGA-H(green),PLL-L/PLGA-H(blue),PLL-H/PLGA-L(cyan),poly(Lys, Tyr)/PLGA-L (magenta), poly(Lys,Trp)/PLGA-M (yellow), and PAH/ PSS (gold). Overlaid spectra after baseline subtraction are shown. A red-shift in the peak at 200 nm occurs with increasing optical density. PSS absorbs at 225 nm. Aromatic absorption peaks are evident around 280 and 225 nm in the poly(Lys,Tyr) and poly(Lys,Trp) films. The same films also show peaks around 225 nm. These peaks are not seen in any of the PLL/PLGA films. (b) Amide I region of wet poly(Lys,Trp)/PLGA-M: The film was 25 layers thick. The baseline and ambient water vapor are subtracted. The bands at 1612, 1645 cm-1 and 1681 cm-1 provide information on average backbone conformation.

> PLL-H/PLGA-H, but PLL-L/PLGA-H ≈ PLL-L/PLGA-L. The two comparisons suggest that the optical density of a PLL/ PLGA nanofilm will be determined more by the degree of polymerization of PLL than PLGA. The PLL side chain, which has two more methylene groups than the PLGA side chain, contributes the larger hydrophobic surface area per residue. For PLL-H films, PLGA-L increases thickness relative to PLGAH, whereas for PLL-L films, PLGA-L decreases thickness relative to PLGA-H. Such behavior could be due respectively to polymer migration in the film, as noted in previous studies11 or to soluble polyelectrolyte complex formation. Poly(Lys,Tyr)/ PLGA-L . PLL-L/PLGA-L, and poly(Lys,Tyr)/PLGA-M . PLL-M/PLGA-M. Evidently, the character of film buildup displayed by a given polymer structure of given average degree of polymerization is “context dependent.”12 Polypeptides differ from “common” polyelectrolytes of multilayer film studies, for example PSS and PAH, in that peptides can form secondary structures, for example, the R helices and β sheets in proteins and peptides in nature. Both R helices and β sheets are characterized by hydrogen bonds

Zhang and Haynie

between amide hydrogen atoms and carbonyl oxygen atoms in the polymer backbone.1 It seems plausible that secondary structures in polypeptide multilayer films, if present at all, will have at least a qualitative resemblance to secondary structures in proteins and peptides in nature. The results of the present work do indeed indicate a strong tendency for polypeptides to adopt a β sheet structure in multilayer films formed at neutral pH. PLL and PLGA in solution are effectively entirely disordered near neutral pH.13–18 The CD spectrum of each film in Figure 3a, by contrast, closely resembles the classical spectrum of a β sheet.13 (see Figure 2a.) The transition from random coil in solution to β sheet in a multilayer film mirrors the behavior of PLL and PLGA molecules on formation of a soluble complex in aqueous solution.19 The slight red-shift in the UV absorption peak maximum with increasing layer number (inset, Figure 2b) is consistent with a minor decrease in percentage random coil and a corresponding increase in percentage β sheet.14 The tendency for entropy to increase will favor the formation of β sheets over R helices in a polypeptide multilayer film because there are more ways to configure a peptide of given degree of polymerization into a β sheet than an R helix.20 The shape of the far UV CD spectrum of each film in Figure 3a is practically indistinguishable from that of poly(Lys,Trp)/PLGA-M (Figure 2b and Figure S3 in Supporting Information). FTIR spectra of PLL and of PLGA in the coil conformation show a pronounced absorption band at 1643–1644 cm-1 and a shoulder near 1670 cm-1.18 The amide I region of the spectrum of 25-layer poly(Lys,Trp)/PLGA-M, by contrast, shown in Figure 3b, closely resembles that of 14-layer PLL/PLGA.18 The peak at 1612 cm-1 is, however, less prominent for poly(Lys,Trp)/ PLGA-M than PLL/PLGA. The poly(Lys,Trp)/PLGA-M spectrum also resembles that of transthyretin amyloid fibrils,21 although the 1645 cm-1 peak is more prominent for poly(Lys,Trp)/ PLGA-M. X-ray fiber diffraction studies have shown that a large amount of β sheet structure but little or no R helix is adopted by many different proteins under conditions favoring amyloid fibril formation.22 The bands at 1612 and 1681 cm-1 in Figure 3b correspond to vibrational modes of hydrogen-bonded carbonyl groups and are indicative of antiparallel β sheets.15 The broad band near 1645 cm-1 is attributable to irregular structure, not R helix. These comparisons suggest that R structure is practically absent from poly(Lys,Trp)/PLGA-M and that β structure is present but at a lower proportion than in PLL/PLGA or amyloid fibrils of transthyretin. The present data support a model of polypeptide multilayer nanofilm structure in which little or no R helix is present at any stage of film buildup at neutral pH, only β sheet, random coil, and β turn. This view is corroborated by the R helix and β sheet stabilizing effects of amino acids relatiVe to alanine (Ala),23 the side chain of which is a methyl group. Lys and ionized Glu in solvent-exposed internal positions destabilize R helices by 1.3 and 2.3 kJ mol-1, respectively; whereas in β sheets, the amino acids are stabilizing by 1.5 and 1.0 kJ mol-1. Trp and Tyr destabilize R helices by 1.3 and 2.4 kJ mol-1 and stabilize β sheets by 4.2 and 6.8 kJ mol-1. For comparison, thermal energy is ∼2.5 kJ mol-1 under the conditions of the experiments. Further evidence in support of the β sheet model of polypeptide multilayer nanofilm structure is provided by the obvious appearance of a contribution of R helical structure to the CD spectrum when PLL/PLGA prepared at neutral pH is immersed in acidic buffer.24 Lyophilization of various proteins results in a reversible increase in β sheet, decrease in R helix, and overall increase in secondary structure content.25 Drying of PLL/PLGA results in

Reversibility of Polypeptides in Multilayer Nanofilms

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Figure 4. Reversibility of structural changes in poly(Lys,Trp)/PLGA-M. (a) Far UV CD: The difference spectrum between the wet and rewet film is plotted. The dashed lines represent a deviation of (5% of band magnitude at 220 nm (see inset). Agreement is very good above about 205 nm, where the signal-to-noise ratio of the spectra is excellent. Inset: wet film (black), dry film (red), and rewet film (green); overlaid spectra after baseline subtraction are shown. (b) Near UV CD: Wet film (black), dry film (red), and rewet film (green). Overlaid spectra after baseline subtraction are shown. The peak at 275 nm shifts to the red by about 12 nm on drying and increases in amplitude. Hydrogen bonding to the indole NH of Trp can red-shift one of the two allowed aromatic π f π* transitions by as much as 12 nm.7 The rewet film spectrum is a baseline-subtracted, 60-scan average collected 10–52 min after rewetting. Agreement between the wet and rewet film is very good in general. Inset: spectrum of poly(Lys,Trp) in solution prior to incorporation into a multilayer film. (c) FTIR: Overlaid spectra after subtraction of baseline and ambient water vapor are shown. Wet poly(Lys-Trp)/PLGA-M (magenta) has relatively large absorption bands around 1450 and 1565 cm-1, which provide information on side chains, and smaller bands at 1612, 1642 and 1681 cm-1, which provide information on secondary structure. On dehydration (blue), the peak at 1612 cm-1 shifts to 1620 cm-1 and becomes more distinct, whereas the peak at 1450 cm-1 diminishes in amplitude and splits into distinct asymmetric and symmetric stretching bands of COO-. The small peak at around 1718 cm-1, which is likely to be due to COOH, is evident in dry films only. Rewetting the film (2 min, cyan) resulted in the reversal of the structural changes on drying. Effectively complete reversibility of structure was attained within 2 h (purple). (d) Model of reversal of structural changes on film rewetting: Reversibility of backbone conformation (secondary structure) is “fast,” whereas reversibility of side chain conformation (tertiary structure) is “slow.” The middle species resembles the molten globule state in protein folding. Molten globules are non-native conformations in which secondary structures are intact but side chain interactions are disrupted. Molten globules are stable at equilibrium under mild denaturing conditions and transiently populated on the folding pathway under conditions favoring the native conformation of the protein.30,31

deswelling on the order of 75% but does not impair the adsorption of additional layers after film rewetting.26 Dry PLL/ PLGA is 1–2 nm per layer thick23 but a substantial amount of water is present.27 Spectroscopic methods have been used in the present work to investigate details of the reversibility of changes in molecular structure on dehydration of polypeptide multilayer films. Figure 4a presents the far-UV CD spectrum of poly(Lys,Trp)/ PLGA-M before and after drying. The far UV CD spectrum of a protein provides information on its secondary structure. The

amplitude of the large band near 220 nm more than doubles on film dehydration, and the extremum shifts to the blue by a few nanometers (inset). The spectrum collected over a period of 14 min following 10 min of film rewetting is practically indistinguishable from the original. The data are consistent with a model in which the conformation of the polypeptide backbone is essentially completely reversible on the time scale of the experiment. Near-UV CD has been used to study changes in average side chain orientation on polypeptide multilayer nanofilm drying. The near-UV CD spectrum of a protein provides information on its

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tertiary structure. A significant dichroic band in poly(Lys,Trp)/ PLGA-M, due to Trp, is evident in the wet film near 275 nm (Figure 4b).7 Film drying results in a red-shift of this band by about 12 nm. The spectrum after 10 min of rewetting and 42 min of data collection differs little from the spectrum before drying. There was no change in the rewet spectrum after an additional 24 h of wetting (data not shown). The data support a model in which film structure is largely if not completely reversible on a time scale shorter than about 30 min. Independent evidence of reversibility of film structure has been acquired by FTIR. In Figure 4c, the spectrum of wet poly(Lys,Trp)/PLGA-M (magenta curve) shows large and pronounced absorption bands around 1450 and 1565 cm-1 (ionized and neutral Glu side chain, respectively) and less distinctive bands at 1612, 1642, and 1681 cm-1 (backbone conformation). A significant change in the film spectrum, indicative of a change in film structure, occurs on dehydration (blue curve). The peak at 1612 cm-1 shifts to 1620 cm-1 and becomes more pronounced, the resonance at 1565 cm-1 diminishes in amplitude and broadens, and the large peak at 1450 cm-1 shrinks dramatically and splits in two. A very small peak becomes noticeable around 1740 cm-1, suggesting the presence of small proportion of protonated Glu side chains in the dehydrated film. Rewetting for 2 min and data collection for 40 s yielded the spectrum shown in cyan. The peaks in the 1600–1700 cm-1 range and the peak at 1565 cm-1 are recovered on this time scale, but not the large peak at 1450 cm-1. After 2 h of rewetting (purple curve), however, the 1450 cm-1 peak is indistinguishable from the original, indicating essentially complete reversibility of film structure by this time, consistent with CD analysis. The CD and FTIR results obtained for the reversibility of structure in poly(Lys,Trp)/PLGA-M have also been found for the other peptide films shown in Figure 3 (Figure S4 in Supporting Information). Several of these films, however, namely PLL-L/PLGA-L, PLL-M/PLGA-M, and PLL-H/PLGAL, showed essentially complete reversibility within 10 min. PLLL/PLGA-H was reversible on a longer time scale. The far UV CD spectrum of PAH/PSS is flat: neither polymer backbone is chiral, and higher-order chirality is not induced in this wavelength range by film fabrication (Figure S3 in Supporting Information). The reversibility of polypeptide multilayer nanofilm structure on dehydration contrasts with the apparent irreversibility of PLL/PLGA on temperature shift and on pH shift9,24,26 but is consistent with refractive index measurements on dehydration/rehydration of PLL/PLGA.27 The spectral data for polypeptide multilayer nanofilms are consistent with a model in which secondary structure equilibrates substantially faster than tertiary structure after film rewetting (Figure 4d). Changes in local film organization, that is secondary structures, are apparently cooperative throughout a polypeptide multilayer nanofilm; changes in global film organization are apparently less cooperative (cf. Figure 4c). The rapid, substantial, and stochastic fluctuations in the refractive index displayed by PLL/PLGA during dehydration,26 however, will presumably depend more on tertiary than secondary structure, suggesting that some aspects of side chain reordering may be cooperative on the scale of the entire film. The relative rate of secondary and tertiary structure remodeling in a polypeptide multilayer film during rehydration resembles the behavior of small globular proteins in refolding experiments. Apomyoglobin at pH 5.3, which is just one of many possible examples, shows much more rapid formation of secondary structure than tertiary structure on the refolding pathway.28

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It has been suggested that polypeptide multilayer nanofilms could be useful as coatings for cell culture for control over cell behavior in vitro, coatings for functional biological cells or groups of cells to improve immunocompatibility, coatings for implant devices for control over cell behavior in vivo, artificial cells for local or systemic delivery of small molecules or biologics, and synthetic vaccines.4,18,20,26,29 Commercialization of the cell culture coating technology is likely to have the practical requirement of freeze-drying and therefore film rewetting prior to cell seeding.

Conclusion The polypeptide multilayer films of the present study are stabilized by electrostatic interactions and hydrophobic interactions between side chains. Nearly all residues adopt a β sheet or coil conformation in films prepared at neutral pH. Secondary structure equilibrates rapidly (