Delineation of a new structural motif involving NHN γ-turn.

Macromolecules are characterized by distinctive arrangement of hydrogen bonds. Different patterns of hydrogen bonds give rise to distinct and stable structural motifs. An analysis of 4114 non-redundant protein chains reveals the existence of a three-residue, (i - 1) to (i + 1), structural motif, having two hydrogen-bonded five-membered pseudo rings (the first, an NH···OC involving the first residue, and the second being NH∙∙∙N involving the last two residues), separated by a peptide bond. There could be an additional hydrogen bond between the side-chain at (i-1) and the main-chain NH of (i + 1). The average backbone torsion angles of -76(±21)° and - 12(±17)° at i creates a tight turn in the polypeptide chain, akin to a γ-turn. Indeed, a search of three-residue fragments with restriction on the terminal Cα ···Cα distance and the existence of the two pseudo rings on either side revealed the presence 14 846 cases of a variant, termed NHN γ-turn, distinct from the NHO γ-turn (2032 cases) that has traditionally been characterized by the presence of NHO hydrogen bond linking the terminal main-chain atoms. As in the latter, the newly identified γ-turns are also of two types-classical and inverse, occurring in the ratio of 1:6. The propensities of residues to occur in these turns and their secondary structural features have been enumerated. An understanding of these turns would be useful for structure prediction and loop modeling, and may serve as models to represent some of the unfolded state or disordered region in proteins.

A novel secondary structure based on fused five-membered rings motif.

An analysis of protein structures indicates the existence of a novel, fused five-membered rings motif, comprising of two residues (i and i + 1), stabilized by interresidue Ni+1-H∙∙∙Ni and intraresidue Ni+1-H∙∙∙O=Ci+1 hydrogen bonds. Fused-rings geometry is the common thread running through many commonly occurring motifs, such as ß-turn, ß-bulge, Asx-turn, Ser/Thr-turn, Schellman motif, and points to its structural robustness. A location close to the beginning of a ß-strand is rather common for the motif. Devoid of side chain, Gly seems to be a key player in this motif, occurring at i, for which the backbone torsion angles cluster at ~(-90°, -10°) and (70°, 20°). The fused-rings structures, distant from each other in sequence, can hydrogen bond with each other, and the two segments aligned to each other in a parallel fashion, give rise to a novel secondary structure, topi, which is quite common in proteins, distinct from two major secondary structures, α-helix and ß-sheet. Majority of the peptide segments making topi are identified as aggregation-prone and the residues tend to be conserved among homologous proteins.

Antiparallel Self-Association of a γ,α-Hybrid Peptide: More Relevance of Weak Interactions.

To learn how a preorganized peptide-based molecular template, together with diverse weak non-covalent interactions, leads to an effective self-association, we investigated the conformational characteristics of a simple γ,α-hybrid model peptide, Boc-γ-Abz-Gly-OMe. The single-crystal X-ray diffraction analysis revealed the existence of a fully extended ß-strand-like structure stabilized by two non-conventional C-H⋅⋅⋅O=C intramolecular H-bonds. The 2D (1) H NMR ROESY experiment led us to propose that the flat topology of the urethane-γ-Abz-amide moiety is predominantly preserved in a non-polar environment. The self-association of the energetically more favorable antiparallel ß-strand-mimic in solid-state engenders an unusual 'flight of stairs' fabricated through face-to-face and edge-to-edge Ar⋅⋅⋅Ar interactions. In conjunction with FT-IR spectroscopic analysis in chloroform, we highlight that conformationally semi-rigid γ-Abz foldamer in appositely designed peptides may encourage unusual ß-strand or ß-sheet-like self-association and supramolecular organization stabilized via weak attractive forces.

ω-Turn: a novel ß-turn mimic in globular proteins stabilized by main-chain to side-chain C−H···O interaction.

Mimicry of structural motifs is a common feature in proteins. The 10-membered hydrogen-bonded ring involving the main-chain C − O in a ß-turn can be formed using a side-chain carbonyl group leading to Asx-turn. We show that the N − H component of hydrogen bond can be replaced by a C(γ) -H group in the side chain, culminating in a nonconventional C − H···O interaction. Because of its shape this ß-turn mimic is designated as ω-turn, which is found to occur ∼ three times per 100 residues. Three residues (i to i + 2) constitute the turn with the C − H···O interaction occurring between the terminal residues, constraining the torsion angles ϕi + 1, ψi + 1, ϕi + 2 and χ'1(i + 2) (using the interacting C(γ) atom). Based on these angles there are two types of ω-turns, each of which can be further divided into two groups. C(ß) -branched side-chains, and Met and Gln have high propensities to occur at i + 2; for the last two residues the carbonyl oxygen may participate in an additional interaction involving the S and amino group, respectively. With Cys occupying the i + 1 position, such turns are found in the metal-binding sites. N-linked glycosylation occurs at the consensus pattern Asn-Xaa-Ser/Thr; with Thr at i + 2, the sequence can adopt the secondary structure of a ω-turn, which may be the recognition site for protein modification. Location between two ß-strands is the most common occurrence in protein tertiary structure, and being generally exposed ω-turn may constitute the antigenic determinant site. It is a stable scaffold and may be used in protein engineering and peptide design.

Self-association behavior of a novel nonproteinogenic ß-strand-mimic in an organic solvent.

The self-association behavior of a newly characterized ß-strand-mimic, presented by an achiral nonproteinogenic model system Boc-γ-Abz-NHMe (1: Boc = tert-butyloxycarbonyl; γ-Abz = γ-aminobenzoic acid; NHMe = N-methylamide), have been investigated using (1)H NMR and FT-IR absorption spectroscopy, in combination with computational ab initio calculations. The concentration dependence of (1)H NMR chemical shifts of the amide-NHs in CDCl3 exhibited noncooperative behavior of self-association, whereas the variable temperature (1)H NMR chemical shifts data of the amide-NHs, i.e., temperature-coefficient (Δδ/ΔT) values, could be accounted for by significant enhancement of self-association, i.e., aggregates higher than dimers. In the absence of N-H···O intramolecular H-bond in 1, the intense FT-IR absorption bands in informative amide-A region, i.e., N-H stretches at ∼3465 and 3438 cm(-1) in chloroform solution, could be interpreted in terms of intermolecular H-bonding. The ab initio quantum mechanical calculations performed on two discrete isolated antiparallel H-bonded duplexes with a face-to-face and an edge-to-edge aromatic-aromatic interaction provided strong support for their relative importance to stabilize favorable dimeric structures. The thermodynamic parameters deduced from van't Hoff plots, constructed from variable temperature (1)H NMR data of the amide-NHs in CDCl3, also substantiated the effectiveness of aromatic-aromatic interactions for dimer formation and higher-order self-association. In view of the enormous structural importance of ß-strand-like building blocks in peptide design, we highlight intrinsic self-associating potentials of the readily available γ-Abz moiety, besides the fact that such planar secondary structural mimics are presumed to offer greater prospective for constructing peptidomimetics and therapeutically relevant small molecules.

Unusual folding propensity of an unsubstituted ß,γ-hybrid model peptide: importance of the C-H⋠⋠⋠O intramolecular hydrogen bond.

The single-crystal X-ray diffraction analysis of a ß,γ-hybrid model peptide Boc-ß-Ala-γ-Abu-NH2 revealed the existence of four crystallographically independent molecules (A, B, C and D conformers) in the asymmetric unit. The analysis revealed that unusual ß-turn-like folded structures predominate, wherein the conformational space of non-proteinogenic ß-Ala and γ-Abu residues are restricted to gauche-gauche-skew and skew-gauche-trans-skew orientations, respectively. Interestingly, the U-shaped conformers are seemingly stabilised by an effective unconventional C-H⋅⋅⋅O intramolecular hydrogen bond, encompassing a non-covalent 14-membered ring-motif. Taking into account the signs of torsion angles, these conformers could be grouped into two distinct categories, A/B and C/D, establishing the incidence of non-superimposable stereogeometrical features across a non-chiral one-component peptide model system, that is, "mirror-image-like" relationships. The natural occurrence of ß-Ala and γ-Abu entities in various pharmacologically important molecules, coupled with their biocompatibilities, highlight how the non-functionalised ß,γ-hybrid segment may offer unique advantages for introducing and/or manipulating a wide spectrum of biologically relevant hydrogen bonded secondary structural mimics in short synthetic peptides.

Isostructural unbranched alkyl-chains as tools for stabilizing ß-turn structure.

To investigate the structural role played by isostructural unbranched alkyl-chains on the conformational ensemble and stability of ß-turn structures, the conformational properties of a designed model peptide: Plm-Pro-Gly-Pda (1, Plm: H3 C-(CH2)14-CONH-; Pda: -CONH- (CH2 )14 -CH3) have been examined and compared with the parent peptide: Boc-Pro-Gly-NHMe (2, Boc: tert-butoxycarbonyl; NHMe: N-methylamide). The characteristic (13)C NMR chemical-shifts of the Pro C(ß) and C(γ) resonances ascertained the incidence of an all-trans peptide-bond in low polarity deuterochloroform solution. Using FTIR and (1) H NMR spectroscopy, we establish that apolar alkyl-chains flanking a ß-turn promoting Pro-Gly sequence impart definite incremental stability to the well-defined hydrogen-bonded structure. The assessment of (1)H NMR derived thermodynamic parameters of the hydrogen-bonded amide-NHs via variable temperature indicate that much weaker hydrophobic interactions do contribute to the stability of folded reverse turn structures. The far-UV CD spectral patterns of 1 and 2 in 2,2,2-trifluoroethanol are consistent with Pro-Gly specific type II ß-turn structure, concomitantly substantiate that the flanking alkyl-chains induce substantial bias in enhanced ß-turn populations. In view of structural as well as functional importance of the Pro-Gly mediated secondary structures, besides biochemical and biological significance of proteins lipidation via myristoylation or palmytoilation, we highlight potential convenience of the unbranched Plm and Pda moieities not only as main-chain N- and C-terminal protecting groups but also to mimic and stabilize specific isolated secondary and supersecondary structural components frequently observed in proteins and polypeptides.

Intramolecular C---H···O hydrogen-bond mediated stabilization of a cis-DPro imide-bond in a stereocontrolled heterochiral model peptide.

The X-ray diffraction analysis of a stereocontrolled heterochiral designed model peptide Boc-(D) Pro-Thr-OMe (1) revealed the existence of an unusual folded molecular structure, stabilized via an effective unconventional C---H…O type intramolecular hydrogen-bond, encompassing a noncovalent 12-membered ring-motif. Together with an uncommon type a disposition of the urethane moiety, the tightly folded topology is compounded with a cis-(D) Pro imide-bond. The overall conformation is suggested to be the reminiscent of specific type VI ß-turn structures, hitherto, characterized across the Aaa-cis-Pro peptide-bonds in globular proteins and polypeptides. The (13) C NMR spectrum of 1 in an apolar CDCl(3) environment revealed the presence of approximately an equal population of cis and trans isomers unexpectedly, analogous to Pro side-chain, the (13) C NMR chemical-shifts of Thr C(ß) -resonance is observed to be sensitive toward cis-trans isomerization. In conjunction with solid-state FT-IR spectral data, we established that a network of complex intermolecular hydrogen-bonds stabilize a self-complementary noncovalent helical hexagonal self-assembly and crystallographic supramolecular aggregate. The results incline us to highlight that the stabilization of cis-(D) Pro peptide-bond in crystalline state may be driven by the favorable energy of formation of an unconventional weak C---H…O intramolecular hydrogen-bond.

Determination of an unusual secondary structural element in the immunostimulating tetrapeptide rigin in aqueous environments: insights via MD simulations, (1)H NMR and CD spectroscopic studies.

An immunomodulating tetrapeptide, rigin (H-Gly-Gln-Pro-Arg-OH), has been examined for its secondary structure preferences through combined use of high-temperature unrestrained MD simulations in implicit water and 1D and 2D 1H NMR spectroscopy.The distribution of backbone torsion angles revealed the predominance of trans conformers across the Xaa-Pro peptide bond. The results of MD simulations revealed that of the five predicted families A-E, the predominant families, family A (92 structures), family C (63 structures) and family D (31 structures), could be complemented by extensive 1D and 2D 1H NMR parameters acquired in aqueous PBS solution. A survey of specific inter- and intraresidue NOEs substantiated the predominance of an unusual type VII beta-turn structure, defined by two torsion angles, i.e. psiGln approximately 155 degrees and psiPro approximately -65 degrees across the Gln-Pro segment. The proposed semi-folded kinked topology precluded formation of any intramolecular interaction, i.e. hydrogen bond or electrostatic interaction. Far-UV CD spectral characteristics of rigin in aqueous PBS solution and non-aqueous structure promoting organic solvents, TFE and TMP, revealed its strong solvent dependence. However, in aqueous PBS solution, the presence of a weak negative shoulder at approximately 234 nm could be ascribed to a small population with ordered, semi-folded topology.We propose that the plausible structural attributes may be exploited for design and rigidification of the bioactive conformation of this immunomodulator toward improved immunopharmacological properties.

Crystallographically observed folded topology of an unsubstituted γ-aminobutyric acid incorporated in a model peptide: importance of a C--H···O interaction.

To validate the existing hypothesis put forward by Navarro et al., we performed single crystal X-ray diffraction structural analysis of a designed model peptide incorporating an unsubstituted achiral γ-aminobutyric acid: Boc-Pro-γ-Abu-OH (1) lacking C-terminal amide group. The analysis established existence of an overall unusual tightly folded topology stabilized by a conventional N(i)···H--N(i + 1) and an unconventional C(i)--H···O(i) type intramolecular hydrogen bonding interactions, encompassing a five-membered and a six-membered ring motifs, respectively. Moreover, in conjunction with Fourier transform infrared (FT-IR) absorption study in solid KBr, the results provided evidence that two conventional and one unconventional noncovalent intermolecular interaction stabilize a right-handed helical architecture generated via molecular self-assembly by translating the symmetry related molecules along the crystallographic b axis. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 927-931, 2010.

Distinctive influence of two hexafluoro solvents on the structural stabilization of Bombyx mori silk fibroin protein and its derived peptides: 13C NMR and CD studies.

Employing high-resolution (13)C solution NMR and circular dichroism (CD) spectroscopic techniques, the distinctive influence of two intimately related hexafluoro solvents, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and hexafluoroacetone trihydrate (HFA), on the structural characteristics of Bombyx mori (B. mori) silk fibroin, the chymotrypsin precipitate (C(p)) fraction, and two synthetic peptides, (AGSGAG)(5) and (AG)(15), is described. The observed (13)C solution NMR and CD spectra of these polypeptides in HFIP and HFA revealed a distinctive influence on their conformational characteristics. The (13)C NMR spectra, as analyzed from the unique chemical shifts of C(alpha) and C(beta) resonances of constituent residues revealed that fibroin largely assumes helical conformation(s) in both solvents. However, the peak shifts were greater for the samples in HFIP, indicating that the types of helical structure(s) may be different from the one populated in HFA. Similar structural tendencies of these polypeptides were reflected in CD spectra. The observed CD patterns, i.e., a strong positive band at approximately 190 nm and negative bands at approximately 206 and 222 nm, have been attributed to the preponderance of helical structures. Of the two prevalent helical structures, alpha-helix and 3(10)-helix, the evidence emerged for the fibroin protein in favor of 3(10)-helical structure stabilization in HFIP and its significant disruption in HFA, as deduced from the characteristic R1 (=[theta](190)/[theta](202)) and R2 (=[theta](222)/[theta](206)) ratios, determined from the CD data. Conversely, the native polypeptides and synthetic peptide fragments derived from highly crystalline regions of the silk fibroin protein sustained predominantly an unordered structure in HFA solvent.

Possible implications of serine and tyrosine residues and intermolecular interactions on the appearance of silk I structure of Bombyx mori silk fibroin-derived synthetic peptides: high-resolution 13C cross-polarization/magic-angle spinning NMR study.

Bombyx mori silk fibroin molecule is known to exist in two distinct structural forms: silk I (unprocessed silk fibroin) and silk II (processed silk fibroin). Using synthetic peptides, we attempt to explore the structural role played by Ser and Tyr residues on the appearance of silk I structural form of the fibroin. Twelve selected peptides (1-12) incorporating Ser and Tyr residues in the (Ala-Gly)(n) copolypeptide, that is, the sequences mimicking the primary structure of B. mori silk fibroin molecule, have been investigated under the silk I state, employing high-resolution (13)C cross-polarization/magic-angle spinning (CP/MAS) NMR spectroscopy. To acquire the silk I structural form, all the peptides were dissolved in 9 M LiBr and then dialyzed extensively against water, as established previously for the synthetic (Ala-Gly)(15) copolypeptide and B. mori silk fibroin. The diagnostic line shape of the Ala C(beta) peaks and the conformation-dependent (13)C chemical shifts of Ala and Gly resonances are presented to analyze and characterize the structural features. The results indicate that the incorporation of one Ser and/or one Tyr residue(s) at selected position in the basic (Ala-Gly)(15) sequence tend to retain predominantly the silk I structure. Conversely, the repeat pentameric and octameric Ala-Gly-Ser-Gly-Ala-Gly sequences, for example, (Ala-Gly-Ser-Gly-Ala-Gly)(5) or (Ala-Gly-Ser-Gly-Ala-Gly)(8), preferred predominantly the silk II form. The peptide sequences incorporating Ser and Tyr residue(s) into repeat Ala-Gly-Ser-Gly-Ala-Gly sequences, however, adopted the silk II structure with certain content structural heterogeneity or randomness, more pronounced for specific peptides studied. Interestingly, the crystalline Cp fraction of B. mori silk fibroin, when mixed with (Ala-Gly-Ser-Gly-Ala-Gly)(5) sequence in a 5:1 molar ratio, dissolved in 9 M LiBr, and dialyzed against distilled water, favor the silk I form. The finding tends to suggest that the less stable silk I form in (Ala-Gly-Ser-Gly-Ala-Gly)(n) sequences is likely to be induced and facilitated via intermolecular interactions with the Cp fraction, which predominantly prefers the silk I form under similar conditions; however, the hydrogen-bond formation involving O(gamma)H groups of the Ser residues may have some implications.

Beta-Ala containing peptides: potentials in design and construction of bioactive peptides and protein secondary structure mimics.

In recent years, there has been increasing interest in de novo design and construction of novel synthetic peptides that mimic protein secondary structures, i.e., turns, helices and sheets. The unique structural influences exerted by unsubstituted, non-coded, non-chiral beta-amino acid, i.e., beta-alanine (beta-Ala; 3- or beta- aminopropionic acid) on peptide backbone, when inserted into peptide chain comprised alpha-amino acids, offer an excellent opportunity to design and construct diverse well-defined three-dimensional structures. Our current understanding of folding-unfolding behavior of the beta-Ala residues relies primarily from an examination of conformational preferences of a large number of short cyclic- as well as acyclic beta-Ala containing peptides investigated using single crystal X-ray diffraction analysis. In addition, theoretical conformational energy calculations and different spectroscopic techniques: 1H NMR, FT-IR and CD, have also been employed although, to a lesser extent. The obtainable results tend to reveal overwhelming preferences of the beta-Ala moiety for the folded gauche (mu approximately +/-65+/-10 degrees conformation in cyclic- and for an extended trans (mu approximately +/-165+/-10 degrees) as well as gauche (mu approximately +/-65+/-10 degrees) orientations in acyclic beta-Ala containing peptides. The results also indicate that in short linear beta-Ala containing peptides, the specific influence of selective neighboring side-chain substituents e.g. linear- or cyclic symmetrically C(alpha,alpha)-disubstituted glycines and other conformational constraints, may be significant in controlling the overall folded-unfolded topographical features across the two methylene units (-CbetaH2-CalphaH2-) of the beta-Ala residue. Taking into consideration the wide occurrence of beta-Ala moiety in animal and plant kingdoms and the remarkable structural versatility of the peptides incorporating beta-Ala residue(s), together with appreciable resistance towards enzymatic degradation, hold strong promise for biophysicists and biochemists not only to design molecules that fold to mimic protein secondary structures but also to develop potent peptide analogs and peptidomimetics displaying unique pharmaceutical properties.

Structural analysis of Bombyx mori silk fibroin peptides with formic acid treatment using high-resolution solid-state 13C NMR spectroscopy.

Bombyx mori silk fibroin fiber is a fibrous protein produced by the silkworm at room temperature and from an aqueous solution whose primary structure is highly repetitive. In this study we analyzed the structural characteristics of native peptides, derived from B. mori silk fibroin, with formic acid treatment using high-resolution solid-state 13C NMR. We establish that the Ser residue bearing a short polar side chain has the ability to stabilize the conformation formed in the model peptides due to its ability to form intermolecular hydrogen bonds involving its hydroxyl group as a donor and the carbonyl groups of other residues as acceptors. On the other hand, insertion of Tyr residue in the basic (AG)n and (AGSGAG)n sequence motifs usually exhibited disruptive effects on the preferred conformations. Moreover, the environmental effect was investigated by mixing the native Cp fraction with the model peptides, showing that there is no significant structural difference on the Ser-containing peptides, while structural transformation was observed on the peptides containing the GAAS unit. This may be attributed to the fact that the Cp fraction promotes the formation of an antiparallel beta-sheet in the Ala-Ala unit. Such periodically disrupted ordered structures in the semicrystalline region of B. mori silk fibroin may be critical not only for facilitating the conformational transformation from silk I to silk II structural form but also for having some correlation with the unique properties of the silk materials.

An observation of non-superimposable stereogeometrical features in a non-chiral one-component beta-Ala model peptide.

This paper describes the chemical synthesis and crystal molecular conformation of a non-chiral beta-Ala containing model peptide Boc-beta-Ala-Acc5-OCH3. The analysis revealed the existence of two crystallographically independent molecules A and B, in the asymmetric unit. Unexpectedly, while the magnitudes of the backbone torsion angles in both molecules are remarkably similar, the signs of the corresponding torsion angles are reverse therefore, inclining us to suggest the existence of non-superimposable stereogeometrical features in a non-chiral one-component beta-Ala model system. The critical mu torsion angle around CbetaH2-CalphaH2 bond of the beta-Ala residue represents a typical gauche orientation i.e., mu = 67.7 degrees in A and mu = -61.2 degrees in B, providing the molecule an overall crescent shaped topology. The observed conformation contrasts markedly to those determined for the correlated non-chiral model peptides: Boc-beta-Ala-Acc6-OCH3 and Boc-beta-Ala-Aib-OCH3 signifying the role of stereocontrolling elements since the stereochemically constrained Calpha, alpha-disubstituted glycyl residues (e.g., Acc5, Acc6, and the prototype Aib) are known to strongly restrict the peptide backbone conformations in the 3(10)/alpha-helical-regions ( phi approximately +/-60+/-20 degrees, psi approximately +/-30+/-20 degrees) of the Ramachandran map. Unpredictably, the preferred, phi, psi torsion angles of the Acc5 residue fall outside the helical regions of the Ramachandran map and exhibit opposite-handed twists for A and B. The implications of the semi-extended conformation of the Acc5 residue in the construction of backbone-modified novel scaffolds and peptides of biological relevance are highlighted. Taken together, the results indicate that in short linear beta-Ala containing peptides specific structural changes can be induced by selective substitution of non-coded linear- or cyclic symmetrically Calpha,alpha-disubstituted glycines, reinstating the hypothesis that in addition to conformational restrictions, the chemical nature of the neighboring side-chain substituents and local environments collectively influences the stabilization of folding-unfolding behavior of the two methylene units of a beta-Ala residue.

Determining dihedral angles and local structure in silk peptide by 13C-2H REDOR.

13C-2H REDOR NMR experiments were performed on 30-residue (AlaGly)15 silk I mimics of Bombyx mori silk fibroin to gain structural details about the elusive structure of the silk I conformation. 13C,2H-labeling strategies are illustrated for measuring individual dihedral angles in peptides and for determining local structure by REDOR. A major turn of type II character is found in the region Gly(14)-Ala(17).

Structural characterization and artificial fiber formation of Bombyx mori silk fibroin in hexafluoro-iso-propanol solvent system.

High-resolution solution (13)C-NMR and CD studies of Bombyx mori silk fibroin revealed the presence of an ordered secondary structure 3(10)-helix, in hexafluoro-iso-propanol (HFIP). The solid-state structure of the silk fibroin film prepared by drying it gently from the HFIP solution still keep the structure, 3(10)-helix, which was studied with high-resolution solid state (13)C-NMR. The structural transition from the 3(10)-helix to silk II structure, heterogeneous structure including antiparallel beta-sheet, occurred during the artificial spinning from the HFIP solution. The wide-angle x-ray diffraction and differential scanning calorimetry thermograms of the artificial spinning fiber after postspinning treatments were observed together with the stress-strain curves. The results emphasize that the molecular structures, controlled morphology, and mechanical properties of the protein-based synthetic polymers can be modulated for enhancing biocompatibility.

Comparative structure analysis of tyrosine and valine residues in unprocessed silk fibroin (silk I) and in the processed silk fiber (silk II) from Bombyx mori using solid-state (13)C,(15)N, and (2)H NMR.

The solid-state (13)C CP-MAS NMR spectra of biosynthetically labeled [(13)C(alpha)]Tyr, [(13)C(beta)]Tyr, and [(13)C(alpha)]Val silk fibroin samples of Bombyx mori, in silk I (the solid-state structure before spinning) and silk II (the solid-state structure after spinning) forms, have been examined to gain insight into the conformational preferences of the semicrystalline regions. To establish the relationship between the primary structure of B. mori silk fibroin and the "local" structure, the conformation-dependent (13)C chemical shift contour plots for Tyr C(alpha), Tyr C(beta), and Val C(alpha) carbons were generated from the atomic coordinates of high-resolution crystal structures of 40 proteins and their characteristic (13)C isotropic NMR chemical shifts. From comparison of the observed Tyr C(alpha) and Tyr C(beta) chemical shifts with those predicted by the contour plots, there is strong evidence in favor of an antiparallel beta-sheet structure of the Tyr residues in the silk fibroin fibers. On the other hand, Tyr residues take a random coil conformation in the fibroin film with a silk I form. The Val residues are likely to assume a structure similar to those of Tyr residues in silk fiber and film. Solid-state (2)H NMR measurements of [3,3-(2)H(2)]Tyr-labeled B. mori silk fibroin indicate that the local mobility of the backbone and the C(alpha)-C(beta) bond is essentially "static" in both silk I and silk II forms. The orientation-dependent (i.e., parallel and perpendicular to the magnetic field) solid-state (15)N NMR spectra of biosynthetically labeled [(15)N]Tyr and [(15)N]Val silk fibers reveal the presence of highly oriented semicrystalline regions.