Gel formation from solutions of single-chain gelatin

The changes in conformation undergone by α-gelatin molecules on quenching aqueous solutions to below the temperature at which they can gel have been monitored by nuclear magnetic resonance and dielectric relaxation techniques. The relative rates of these conformational transitions are compared with changes in rheological properties. The nmr spectral intensity changes for 0.2 and 0.5% w/v α-gelatin solutions correspond to a unimolecular process with k ～ 10^?2 min^?1 at 15°C; this process occurs independently of whether or not the solution is concentrated enough to form a gel. The process involves a slow intramolecular nucleation step, followed by a rapid conformational change of the whole molecule from random coil to a rigid stage. Comparison with other data suggests that the transition gives rise to a triple collagen-like helix. In dilute solution (but above the critical concentration for gel formation, e.g., 0.5% w/v), the gelatin process follows the formation of the rigid molecular species. It probably involves the formation of junction zones consisting of three polypeptide chains in a collagen-like triple-helical conformation. These junctions may form, at low concentrations, from a reorganization of previously formed, intramolecular, triple helices. Solutions below a concentration of about 0.4% w/v α-gelatin cannot gel by this mechanism, and only form viscous liquids.     

Raman spectra of D and L amino acid copolymers. Poly-DL-alanine, poly-DL-leucine, and poly-DL-lysine.

The Raman spectrum of poly-DL -alanine (PDLA) in the solid state is interpreted in terms of the disordered chain conformation, in analogy with the spectrum of mechanically deformed poly-L -alanine. The polymer is largely disordered with only a small α-helical content in the solid state. When PDLA is dissolved in water, the spectra suggest that short α-helical segments are formed upon dissolution. These helical regions might be stabilized by hydrophobic bonds between side-chain methyl groups. Addition of methanol to the aqueous PDLA solutions results in a Raman spectrum resembling that of solid PDLA. This result suggests that the methanol disrupts the helical regions by breaking the hydrophobic bonds. The Raman spectra of poly-DL -leucine (PDLL) and poly-L -leucine (PLL) are compared and only slight differences are observed in the amide I and III regions, indicating that PDLL does not have an appreciable disordered chain content. Significant differences are observed in the skeletal regions. The 931-cm^?1 lines in the PLL and PDLL spectra are assigned to residues in α-helical segments of the preferred screw sense, i.e., L -residues in right-handed segments and D -residues in left-handed segments (in PDLL). On the other hand, the 890-cm^?1 line in the spectrum of PDLL is assigned to residues not in the preferred helical sence, i.e., L -residues in left-handed segments and D -residues in right-handed ones. The Raman spectra of poly-DL -lysine and poly-L -lysine in salt-free water at pH 7.0 are compared. The Raman spectra of the two polymers are very similar. However, this does not negate the hypothesis of local order in poly-L -lysine because the distribution of the residues in poly-DL -lysine probably tends towards blocks, and the individual blocks may take up the 3₁ helix.     

Determining the depth of insertion of dynamically invisible membrane peptides by gel-phase 1H spin diffusion heteronuclear correlation NMR

Solid-state NMR determination of the depth of insertion of membrane peptides and proteins has so far utilized ¹H spin diffusion and paramagnetic relaxation enhancement experiments, which are typically conducted in the liquid-crystalline phase of the lipid bilayer. For membrane proteins or peptide assemblies that undergo intermediate-timescale motion in the liquid-crystalline membrane, these approaches are no longer applicable because the protein signals are broadened beyond detection. Here we show that the rigid-solid HETCOR experiment, with an additional spin diffusion period, can be used to determine the depth of proteins in gel-phase lipid membranes, where the proteins are immobilized to give high-intensity solid-state NMR spectra. Demonstration on two membrane peptides with known insertion depths shows that well-inserted peptides give rise to high lipid cross peak intensities and low water cross peaks within a modest spin diffusion mixing time, while surface-bound peptides have higher water than lipid cross peaks. Furthermore, well-inserted membrane peptides have nearly identical ¹H cross sections as the lipid chains, indicating equilibration of the peptide and lipid magnetization. Using this approach, we measured the membrane topology of the α-helical fusion peptide of the paramyxovirus, PIV5, in the anionic POPC/POPG membrane, in which the peptide undergoes intermediate-timescale motion at physiological temperature. The gel-phase HETCOR spectra indicate that the α-helical fusion peptide is well inserted into the POPC/POPG bilayer, spanning both leaflets. This insertion motif gives insight into the functional role of the α-helical PIV5 fusion peptide in virus-cell membrane fusion.     

The conformation of an alamethicin in methanol by multinuclear NMR spetroscopy and distance geometry/simulated annealing

The solution conformation of the antibiotic peptide alamethicin was investigated using multi-nuclear spectroscopy and the distance geometry/simulated annealing algorithms from the program DSPACE. ¹H-, ¹³C-, and ¹⁵N-nmr chemical shifts and homonuclear ¹H coupling constants suggest that the molecule is flexible in the vicinity of Gly-11 and Leu-12. The temperature dependence of the amide proton chemical shifts indicates that there is flexibility in the middle of the 20 residue peptide and provides evidence that, at the very N-terminus, the molecule adopts a 3₁₀-helical conformation. The large differences in the ¹³C chemical shifts of the pro-R and pro-S methyls of the α-aminoisobutyric acid residues were used to constrain those residues to the right-handed helical conformation in the distance geometry/simulated annealing algorithms. A family of 24 structures was generated but did not converge to a common conformation when superimposed over the entire polypeptide sequence. The molecules did converge to a helical conformation over residues 1–10 and residues 13–18. The lack of convergence when the entire lengths of the molecules are superimposed is explained by the flexibility of the peptide near Gly-11/Leu-12. The results suggest that the protein consists of two helices connected by a flexible “hinge.” The flexibility of the molecule is discussed with respect to the macrodipole model of voltage gating. © 1995 John Wiley & Sons, Inc.     

Steady-state and pulsed NMR studies of gelation in aqueous agarose

Steady-state and pulsed NMR techniques have been used to investigate molecular motion in sols and gels of agarose. In passing through the sol–gel transition, the molecular mobility of water molecules is reduced only by a small amount, whereas motion of the polymer chains is greatly attenuated. The results are discused in terms of the network theory of gelation, with references to the role of water in the process and the nature of the “junction zones” between polymer chains. T₂ and line-width measurements are dominated by exchange broadening. The effects of exchange rate and differences in relaxation time between the exchanging sites are discussed. The temperature hysteresis behavior of agarose gels has been investigated and the effects of “ageing” correlated with changes in nuclear relaxation times. The synergistic increase in gel strength obtained on adding locust bean gum (LBG) to agarose has been investigated. The results indicate that LBG does not form double-helix junctions and may decrease rates of gelation by steric effects. At high agarose concentration, the LBG remains mainly in solution in interstitial water, but at low agarose concentration, it is suggested that the LBG can link gel aggregates together into a self-supporting structure, producing a synergistic increase in gel strength. Comparisons have been made between the nature of the agarose–LBG interaction and agarose–cellulose interactions in biological systems.     

Influence of isopropanol-water solvent mixtures on the conformation of poly-L-lysine

The helix–coil transition of poly-L -lysine (PLL) in water–isopropanol solvent mixtures has been investigated at room temperature by circular dichroism measurements. Within the range of 70%–80% isopropanol concentration (by volume), the polymer undergoes a sharp transition, characterized by the formation of a fully charged α-helical structure. On the basis of some experimental evidence the role of the organic component in solution appears more complicated than that of strengthening the intramolecular hydrogen bonds in the polymer. By analogy with the distribution of the components of alcohol–water mixtures in simple ionic systems, it is thought that only an high co-solvent concentration brings about an extensive and possible cooperative depletion of the clusters of firmly-bound water molecules in the domain of the polylelectrolyte, favoring the transition to the α-helical structure. On the other hand, CD spectral patterns show that the addition of NaCl in the alcohol-rich–water mixtures of charged poly-L -lysine gives rise to a transition from the α-helical to a β-structures conversion obeys a first-order rate law at all times, with a rate constant dependent on solvent composition and ionic strength. In these conditions, the rate of the process is close to that found for the thermally induced α–β transition. Higher polymer concentration and/or ionic strength cause a phase separation of β-PLL, suggesting that in this case interchain reactions (where hydrogen bonding should play the major role) predominate. Titration experiments on charged α-helical poly-L -lysine in 85% or 90% isopropanol mixtures confirm the occurrence of a conformational transition, which takes place within a degree of dissociation α of 0.2–0.75. The transition is accompanied by a visible turbidity, which increases as the titration proceeds. Implications of the solvent distribution around the macroion on the observed conformational phenomena are also discussed.     

A pulsed NMR study of lipids,bound water and sodium ions in macroscopically-oriented lecithin/water lyotropic liquid crystal model membrane systems

The temperature and orientation dependence of pulsed NMR ‘free induction decay’ signals have been studied in detail for lipid bilayers macroscopically-oriented between glass slides. Results for the lipid molecules (¹H, ³¹P), bound water (²H₂O) and ions dissolved in the aqueous phase (²³Na) are presented. Bilayers of egg-lecithin, dimyristoyl lecithin and potassium oleate have been investigated. In the liquid crystal phase all the signals, including those from bound water and ions exhibit a |3 cos²? ? 1| dependence on orientation of the bilayer normal to the magnetic field. In the case of DML samples, some orientation dependence of both ¹H and ²H signals persists in the gel phase, indicating that the lipid molecules retain a degree of reorientational freedom about their long axes in this phase. At the gel-liquid crystal transition the ²H quadrupole spittings undergo a discontinuous change. Results are interpreted in terms of a model in which water molecules are bound to individual lipid head groups and reorient with them, while sodium ions are located in the aqueous channel between bilayers.     

Importance of the tryptophans of gramicidin for its lipid structure modulating activity in lysophosphatidylcholine and phosphatidylethanolamine model membranes. A comparative study employing gramicidin analogs and a synthetic α-helical hydrophobic polypeptide

The importance of the tryptophan residues of gramicidin for the lipid structure modulating activity of this pentadecapeptide was investigated by studying the interaction of gramicidin analogs A, B, C (which have a tryptophan, phenylalanine and tyrosine in position 11, respectively) and tryptophan-N-formylated gramicidin (in which the four tryptophan residues have been formylated) with several phospholipid systems. In addition in α-helical model pentadecapeptide (P15) was studied to further test the specificity of the gramicidin-lipid interaction. DSC experiments showed that all the gramicidin analogs produced a significant decrease in the gel to liquid-crystalline transition enthalpy of dipalmitoylphosphatidylcholine. The P15 peptide was much less effective in this respect. In dielaidoylphosphatidylethanolamine the gel → liquid-crystalline transition enthalpy was much less affected by the incorporation of these molecules. In this lipid system tryptophan-N-formylated gramicidin was found to be the most ineffective. ³¹P-NMR and small angle X-ray diffraction experiments showed that the ability of the peptides to induce bilayer structures in palmitoyllysophosphatidylcholine and H_II phase promotion in dielaidoylphosphatidylethanolamine systems follows the order: gramicidin A′ (natural mixture) ≈gramicidin A > gramicidin B ≈ gramicidin C > tryptophan-N-formylated gramicidin > P15. These results support the hypothesis that the shape of gramicidin and its aggregational behaviour, in which the tryptophan residues play an essential role, are major determinants in the unique lipid structure modulating activity of gramicidin.     

Segment TM7 from the cytoplasmic hemi-channel from VO-H-V-ATPase includes a flexible region that has a potential role in proton translocation

A 900-MHz NMR study is reported of peptide sMTM7 that mimics the cytoplasmic proton hemi-channel domain of the seventh transmembrane segment (TM7) from subunit a of H⁺-V-ATPase from Saccharomyces cerevisiae. The peptide encompasses the amino acid residues known to actively participate in proton translocation. In addition, peptide sMTM7 contains the amino acid residues that upon mutation cause V-ATPase to become resistant against the inhibitor bafilomycin. 2D TOCSY and NOESY ¹H-¹H NMR spectra are obtained of sMTM7 dissolved in d₆-DMSO and are used to calculate the three-dimensional structure of the peptide. The NMR-based structures and corresponding dynamical features of peptide sMTM7 show that sMTM7 is composed of two α-helical regions. These regions are separated by a flexible hinge of two residues. The hinge acts as a ball-and-joint socket and both helical segments move independently with respect to one another. This movement in TM7 is suggested to cause the opening and closing of the cytoplasmic proton hemi-channel and enables proton translocation.     

Proton transfer and polarizability of hydrogen bonds in proteins coupled with conformational changes. I. Infrared investigation of poly(glutamic acid) with various N Bases

The nature of hydrogen bonds formed between carboxylic acid residues and histidine residues in proteins is studied by ir spectroscopy. Poly(glutamic acid) [(Glu)_n] is investigated with various monomer N bases. The position of the proton transfer equilibrium OH…?N ? O^?…?H⁺N is determined considering the bands of the carboxylic group. It is shown that largely symmetrical double minimum energy surfaces are present in the OH…?N ? O^?…?H⁺N bonds when the pK_a of the protonated N base is two values larger than that of the carboxylic groups of (Glu)_n. Hence OH…?N ? O^?…?H⁺N bonds between glutamic and aspartic acid residues and histidine residues in proteins may be easily polarizable proton transfer hydrogen bonds. The polarizability of these bonds is one to two orders of magnitude larger than usual electron polarizabilities; therefore, these bonds strongly interact with their environment. It is demonstrated that water molecules shift these proton transfer equilibria in favor of the polar proton boundary structure. The access of water molecules to such bonds in proteins and therefore the position of this proton transfer equilibrium is dependent on conformation. The amide bands show that (Glu)_n is α-helical with all systems. The only exception is the (Glu)_n-n-propylamine system. When this system is hydrated (Glu)_n is α-helical, too. When it is dried, however, (Glu)_n forms antiparallel β-structure. This conformational transition, dependent on degree of hydration, is reversible. An excess of n-propylamine has the same effect on conformation as hydration.     

Multiple Molecular Dynamics Simulations of TEM β-Lactamase: Dynamics and Water Binding of the Ω-Loop

The Ω-loop of TEM β-lactamase is involved in substrate recognition and catalysis. Its dynamical properties and interaction with water molecules were investigated by performing multiple molecular dynamics simulations of up to 50 ns. Protein flexibility was assessed by calculating the root mean-square fluctuations and the generalized order parameter, S². The residues in secondary structure elements are highly ordered, whereas loop regions are more flexible, which is in agreement with previous experimental observations. Interestingly, the Ω-loop (residues 161-179) is rigid with order parameters similar to secondary structure elements, with the exception of the tip of the loop (residues 173-177) that has a considerably higher flexibility and performs an opening and closing motion on the 50-ns timescale. The rigidity of the main part of the Ω-loop is mediated by stabilizing and highly conserved water bridges inside a cavity lined by the Ω-loop and residues 65-69 of the protein core. In contrast, the flexible tip of the Ω-loop lacks these interactions. Hydration of the cavity and exchange of the water molecules with the bulk solvent occurs via two pathways: the flexible tip that serves as a door to the cavity, and a temporary water channel involving the side chain of Arg¹⁶⁴.     

Directional Information Transfer in Protein Helices

IN the course of studies on the relation between conformation and primary sequence in globular proteins, it has become clear that the choice a residue makes between a right handed α-helical conformation (H?) and any other conformation (H) is determined mainly by that residue and its near neighbours in the primary sequence^1–9. When looking for physical mechanisms to explain these findings it is important to know whether the influence of one residue on the conformational state of a neighbouring residue has any directional characteristics. The possibility of certain residues exerting helix-forming influence in either the COOH-terminal or NH₂-terminal direction preferentially is suggested by the non-random distribution of amino-acid residues between the two ends of helical regions in globular proteins^4,10. Ptitsyn's analysis⁴ suggested that a group of residues containing alanine and leucine tends to occur within helical regions, while positively and negatively charged side chain residues are distributed preferentially at the carboxyl-and amino-terminal ends, respectively, of helices.     

Electron Microscopic Evidence in Support of α-Solenoid Models of Proteasomal Subunits Rpn1 and Rpn2

Rpn1 (109 kDa) and Rpn2 (104 kDa) are components of the 19S regulatory complex of the proteasome. The central portions of both proteins are predicted to have toroidal α-solenoid folds composed of 9-11 proteasome/cyclosome repeats, each ∼ 40 residues long and containing two α-helices and turns [A. V. Kajava, J. Biol. Chem. 277, 49791-49798, 2002]. To evaluate this prediction, we examined the full-length yeast proteins and truncated versions thereof consisting only of the repeat-containing regions by gel filtration, CD spectroscopy, and negative-staining electron microscopy (EM). All four proteins are monomeric in solution and highly α-helical, particularly the truncated ones. The EM data were analyzed by image classification and averaging techniques. The preponderant projections, in each case, show near-annular molecules 6-7 nm in diameter. Comparison of the full-length with the truncated proteins showed molecules similar in size and shape, indicating that their terminal regions are flexible and thus smeared to invisibility in the averaged images. We tested the toroidal model further by calculating resolution-limited projections and comparing them with the EM images. The results support the α-solenoid model, except that they indicate that the repeats are organized not as symmetrical circular toroids but in less regular horseshoe-like structures.     

Chemical shift assignments of the connexin45 carboxyl terminal domain: monomer and dimer conformations

Connexin45 (Cx45) is a gap junction protein involved in cell-to-cell communication in the heart and other tissues. Here we report the ¹H, ¹⁵N, and ¹³C resonance assignments for the monomer and dimer conformations of the Cx45 carboxyl terminal (Cx45CT) domain and provide evidence of dimerization using diffusion ordered spectroscopy. The predicted secondary structure of the Cx45CT domain based on the chemical shifts identified one region of α-helical structure, which corresponds to the residues that broadened beyond detection in the dimer confirmation. Previous biophysical studies from our laboratory characterizing the CT domain from the other major cardiac connexins, Cx40 and Cx43, suggest that the amount of α-helical content may translate into the ability of a protein to dimerize. Even though the CT domain is thought to be the main regulatory domain of most connexins, the physiological role of CT dimerization is currently unknown. Therefore, these assignments will be useful for determining the intermolecular interactions that mediate Cx45CT dimerization, information that will be used to characterize dimerization in functional channels, as well as characterizing the binding sites for molecular partners involved in Cx45 regulation.     

Microcalorimetric studies of solvent-induced conformational change of sodium and cesium salts of poly(L-glutamic acid) in aqueous media

Organic solvent-induced coil → helix conformational change of poly(sodium) L -glutamate (NaPLG) and poly(cesium L -glutamate) (CsPLG) in solution in aqueous mixed solvents have been studied at 25°C. Heats of dilution of NaPLG in the water–dioxane pair have been measured as a function of polymer concentration and solvent composition. The results indicate that the overall chain conformation in the disordered form is not too different from that in the α-helical form. Heat capacity measurements by flow microcalorimetry have also been done. The apparent monomolar heat capacity at constant pressure of the polymer, C_{p, ?}, decreases with dilution similarly to other strong polyelectrolytes in aqueous media. In the water–dioxane pair, C_{p, ?} increases with the dioxane content due to partial desolvation of ionic species resulting from increasing ionic association. In the case of the water-2-chloroethanol (CE) pair, the transition takes place at low CE content and results show a fast decrease in C_{p, ?} when the α-helical conformation predominates. It is believed carboxylate groups and CE molecules associate themselves into a complex formation responsible for the transition. The size of the cation plays a significant role in the thermodynamic properties of these polyelectrolytes in solution since sodium ions are more strongly bound to the chain than cesium ions.     

Structure of human salivary histatin 5 in aqueous and nonaqueous solutions

The solution structure of human salivary histatin 5 (D-S-H-A-K-R-H-H-G-Y-K-R-K-F-H-E-K-H-H-S-H-R-G-Y) was examined in water (pH 3.8) and dimethyl sulfoxide solutions using 500 MHz homo- and heteronuclear two-dimensional (2D) nmr. The resonance assignment of peptide backbone and side-chain protons was accomplished by 2D total correlated spectroscopy and nuclear Overhauser effect (NOE) spectroscopy. The high J values (≥7.4 Hz), absence of any characteristic NH-NH(i, i + 1) or C^αH-C^βH(i, i + 3) NOE connectivities, high dδ/dT values (≥0.004 ppm K⁻¹) and the fast ¹H/²H amide exchange suggest that histatin 5 molecules remain unstructured in aqueous solution at pH 3.8. In contrast, histatin 5 prefers largely α-helical conformation in dimethyl sulfoxide solution as evident from the J values (≤6.4 Hz), slow ¹H/²H exchange, low dδ/dT values (≤0.003 ppm K⁻¹) observed for amide resonances of residues 6–24, and the characteristic NH-NH(i, i + 1) and C^αH-C^βH(i, i +3) NOE connectivities. All backbone amide ¹⁵N-¹H connectivities fall within 6 ppm on the ¹⁵N scale in the 2D heteronuclear single quantum correlated spectrum, and the restrained structure calculations using DIANA suggest the prevalence of α-helical conformations stabilized by 19 (5 → 1) intramolecular backbone amide hydrogen bonds in polar aprotic medium such as dimethyl sulfoxide. The interside-chain hydrogen bonding and salt-bridge type interactions that normally stabilize the helical structure of linear peptides in aqueous solutions are not observed. Histatin 5, unlike other naturally occurring antimicrobial polypeptides such as magainins, defensins, and tachyplesins, does not adopt amphiphilic structure, precluding its insertion into microbial membranes and formation of ion channels across membranes. Electrostatic (ionic type) and hydrogen bonding interactions of the positively charged and polar residues with the head groups of microbial membranes or with a membrane-bound receptor could be the initial step involved in the mechanism of antimicrobial activity of histatins. © 1998 John Wiley & Sons, Inc. Biopoly 45: 51–67, 1998     

Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H-V-ATPase

Vacuolar (H⁺)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an α-helical conformation for peptide MTM7 and in DMSO three α-helical regions are identified by 2D ¹H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an α-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Assignment of the helical structure in neuropeptide Y by HPLC studies of methionine replacement analogues and 1H-NMR spectroscopy

The HPLC retention behavior of three complete single methionine and methionine sulfoxide replacement sets of two 18-mer model peptides and neuropeptide Y (NPY) were investigated. All peptides were prepared by multiple solid-phase peptide synthesis. Plotting the retention time differences between methionine and methionine sulfoxide analogues vs the position of replacement shows that potentially α-helical peptides become helical on binding during reversed-phase high performance liquid chromatography. In the case of an amphipathic α-helix, the retention time differences change periodically with a 3–4 repeat pattern, which allow the location of amphipathic helical structures. Replacements in nonamphipathic α-helical domains cause local preferential binding areas and lead to sequence-dependent retention time profiles. Methionine replacement studies of NPY suggest an unstructured or extended conformation from Tyr¹ to Ala¹² connected to a well-defined amphipathic α-helix from Pro¹³ to Arg³⁵. The assignment is confirmed by comparison of nuclear Overhauser effects based two-dimensional ¹H-nmr spectroscopy and utilization of the C^αH shift index method in 50% trifluoroethanol/50% water. © 1996 John Wiley & Sons, Inc.     

Flow-induced conformational changes and phase behavior of aqueous poly-L-lysine solutions

Poly-L -lysine exists as an α-helix at high pH and a random coil at neutral pH. When the α-helix is heated above 27°C, the macromolecule undergoes a conformational transition to a β-sheet. In this study, the stability of the secondary structure of poly-L -lysine in solutions subjected to shear flow, at temperatures below the α-helix to β-sheet transition temperature, were examined using Raman spectroscopy and CD. Solutions initially in the α-helical state showed time-dependent increases in viscosity with shearing, rising as much as an order of magnitude. Visual observation and turbidity measurements showed the formation of a gel-like phase under flow. Laser Raman measurements demonstrated the presence of small amounts of β-sheet structure evidenced by the amide I band at 1666 cm⁻¹. CD measurements indicated that solutions of predominantly α-helical conformation at 20°C transformed into 85% α-helix and 15% β-sheet after being sheared for 20 min. However, on continued shearing the content of β-sheet conformation decreased. The observed phenomena were explained in terms of a “zipping-up” molecular model based on flow enhanced hydrophobic interactions similar to that observed in gel-forming flexible polymers. © 1998 John Wiley & Sons, Inc. Biopoly 45: 239–246, 1998     

Conformational characteristics of polyglutamic acid esters: Persistence of orientational correlation along the side chain flanking the α-helical backbone

The side chain conformations of α-helical poly(L -glutamic acid) esters $ \rlap{--}[NHCH(CH_2 CH_2 COOR)CO\rlap{--}]_x $, carrying a homologous series of ester residues such as R = ? (CH₂)_n? with n = 1–3, have been studied in the lyotropic liquid crystalline state (chloroform 20 v/v%) by the deuterium nmr method. In order to study the surface chirality of the molecule, the phenyl groups situated at the terminal of the side chain have been deuterated. From the observed deuterium quadrupolar splittings, the average inclination θ_p of the para-axis of the phenyl group with respect to the α-helical backbone was elucidated. A distinct odd–even oscillation in the quantity such as 〈 cos² θ_p〉 was observed with the number of methylene units n. A rotational isomeric state analysis has indicated that the observed orientational correlation arises from the interdependence of the neighboring bond rotation along the side chain. Preference of the “extended” conformations is also enhanced by the mutual conformational exclusion of neighboring side chains.