Effect of temperature on the circular dichroism spectra of polypeptides in the extended state

The circular dichroism (CD) spectra of poly-L -proline and of poly-L -glutamic acid and poly-L -lysine in their charged states have been studied as a function of temperature. The variation of CD spectra with temperature is inconsistent with the assignment of the spectrum of such charged polypetides to an unordered chain conformation, but does support our earlier assignment to a locally ordered structure—what we have called the extended helix conformation. These results also strengthen our previous assignment of the CD spectrum of an unordered chain, and indicate that three conformational states (α-helix, extended helix, and unordered) should be incorporated in our thinking about conformational transitions in polypeptides.     

Theoretical calculation of the circular dichroism of unordered polypeptide chains

A calculation has been done of the circular dichroism (CD) spectrum of an unordered polypeptide chain. This has been based on a Boltzmann averaging over a dipeptide conformational CD map. This is shown to be valid by comparing the CD spectra of 28-mer oligopeptides with those generated by summing dipeptide CD spectra. The calculated CD spectrum of an unordered polypeptide chain is found to agree with the assignment proposed by Tiffany and Krimm from experimental studies.     

The calculated circular dichroism of polyproline II in the polarizability approximation

The circular dichroism (CD) spectrum of polyproline II (PPII) has heretofore been moderately well calculated from exciton theory only at the expense of assuming unreasonable chain conformations and accepting a conservative spectrum in the 180–250-nm region (which is not observed). We have incorporated far uv transitions in the polarizability approximation and, together with the π₂π* transition, have calculated the resulting correction to the exciton model. This has been accompanied by a modified assignment of the ππ* transition in PPII, and a simultaneous calculation of the absorption and CD spectra of the α-helix, β structure, PPI, and PPII. We obtain good agreement with the observed CD spectrum of PPII in the 180–250-nm region for acceptable chain conformations. In addition, we predict a negative CD into the far uv, in agreement with recent experimental observations. Our calculations also reproduce features of the far uv CD spectrum of the α-helix, and are in agreement with the CD spectra of the β chain and PPI. The calculated CD of the unordered polypeptide chain is not significantly influenced by far uv contributions, indicating that our previous calculation is valid for such a system. These results demonstrate the importance of incorporating far uv transitions in order to achieve an adequate theoretical explanation of the CD spectra of polypeptides.     

Intrinsically unstructured proteins by design—electrostatic interactions can control binding,folding, and function of a helix‐loop‐helix heterodimer

Intrinsically disordered proteins that exist as unordered monomeric structures in aqueous solution at pH 7 but fold into four‐helix bundles upon binding to recognized polypeptide targets have been designed. NMR and CD spectra of the monomeric polypeptides show the hallmarks of unordered structures, whereas in the bound state they are highly helical. Analytical ultracentrifugation data shows that the polypeptides bind to their targets to form exclusively heterodimers at neutral pH. To demonstrate the relationship between binding, folding, and function, a catalytic site for ester hydrolysis was introduced into an unordered and largely inactive monomer, but that was structured and catalytically active in the presence of a specific polypeptide target. Electrostatic interactions between surface‐exposed residues inhibited the binding and folding of the monomers at pH 7. Charge–charge repulsion between ionizable amino acids was thus found to be sufficient to disrupt binding between polypeptide chains despite their inherent propensities for structure formation and may be involved in the folding and function of inherently disordered proteins in biology. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Raman scattering of some synthetic polypeptides: poly(gamma-benzyl L-glutamate), poly-L-leucine, poly-L-valine, and poly-L-serine

The Raman spectra of poly-γ-benzyl-L -glutamate, poly-L -leucine, poly-L -valine, and poly-L -serine are reported. For the α-helical polymers, the conformationally sensitive amide I, II, and III modes are observed in the Raman as, well as the infrared. For the β form, the Raman effect, supplies the infrared inactive inphase motion which is useful for the determination of a parallel or antiparallel chain alignment. Modes characteristic of the specific polypeptide are also observed which are insensitive to conformation.     

Optical properties of polypeptides in the β-conformation

The rotational strengths and oscillator strengths of the nπ* band and ππ* exciton bands have been calculated for antiparallel and parallel β-structures of varying length and width. The results are compared with experiment and with previous theoretical treatments of β-structures. The generally good agreement of calculations on the antiparallel β-structure with experimental results on poly-L -lysine and poly-L -serine indicates that these systems are indeed in the antiparallel conformation. It is found that the exciton component strongest in absorption shifts to longer wavelengths as the width of an antiparallel structure increases, and it is suggested that the position of the ππ* absorption band may be a useful criterion of sheet width. The results also reconcile the linear dichroism measurements of Rosenheck and Sommer on poly-L -lysine films with an anti-parallel structure. Calculations on parallel β-structures indicate that the CD spectra of this form will be rather similar to that of the antiparallel form. However, the major absorption band in the antiparallel form is associated with a small positive CD band, while in the parallel form it coincides with a large negative CD band. Finally, it is pointed out that the large positive CD bands predicted for single-stranded parallel and antiparallel β-structures at about 200 mμ render unlikely the suggestion that random-coil polypeptides contain a substantial fraction of extended chain.     

Raman spectroscopic study of mechanically deformed poly-L-alanine

Raman spectroscopy has been used in investigating the conformational transitions of poly-L -alanine (PLA) induced by mechanical deformation. We see evidence of the alpha-helical, antiparallel beta-sheet, and a disordered conformation in PLA. The disordered conformation has not been discussed in previous infrared and X-ray diffraction investigations and may have local order similar to the left-handed 3₁ poly glycine helix. The amide III mode in the Raman spectrum of PLA is more sensitive than the amide I and II modes to changes in secondary structure of the polypeptide chain. Several lines below 1200 cm^?1 are conformationally sensitive and may generally be useful in the analysis of Raman spectra of proteins. A line at 909 cm^?1 decreases in intensity after deformation of PLA. In general only weak scattering is observed around 900 cm^?1 in the Raman spectra of antiparallel beta-sheet polypeptides. The Raman spectra of the amide N–H deuterated PLA and poly-L -leucine (PLL) in the alpha-helical conformation and poly-L -valine (PLV) in the beta-sheet conformation are presented. Splitting is observed in the amide III mode of PLV and the components of this mode are assigned. The Raman spectrum of an alpha-helical random copolymer of L -leucine and L -glutamic acid is shown to be consistent with the spectra of other alphahelical polypeptides.     

Structural composition of betaI- and betaII-proteins

Circular dichroism spectra of proteins are sensitive to protein secondary structure. The CD spectra of alpha-rich proteins are similar to those of model alpha-helices, but beta-rich proteins exhibit CD spectra that are reminiscent of CD spectra of either model beta-sheets or unordered polypeptides. The existence of these two types of CD spectra for beta-rich proteins form the basis for their classification as betaI- and betaII-proteins. Although the conformation of beta-sheets is largely responsible for the CD spectra of betaI-proteins, the source of betaII-protein CD, which resembles that of unordered polypeptides, is not completely understood. The CD spectra of unordered polypeptides are similar to that of the poly(Pro)II helix, and the poly(Pro)II-type (P2) structure forms a significant fraction of the unordered conformation in globular proteins. We have compared the beta-sheet and P2 structure contents in beta-rich proteins to understand the origin of betaII-protein CD. We find that betaII-proteins have a ratio of P2 to beta-sheet content greater than 0.4, whereas for betaI-proteins this ratio is less than 0.4. The beta-sheet content in betaI-proteins is generally higher than that in betaII-proteins. The origin of two classes of CD spectra for beta-rich proteins appears to lie in their relative beta-sheet and P2 structure contents.     

Spectroscopic studies of random chain and -helical polypeptides in hexafluoroisopropanol

The infrared, ultraviolet, circular dichroism, and optical rotatory dispersion spectra of five synthetic polypeptides dissolved in hexafluoroisopropanol are reported. This solvent is useful because it dissolves most proteins and non-ionic polypeptides and also is transparent in spectral regions critical for polypeptide conformational diagnoses. Poly-γ-morpholinylethyl-L -glutamamide has random chain type spectra in this solvent, whereas the spectra of poly-γ-methyl-L -glutamate, poly-L -methionine, poly-ε, N -Carbo-benzoxy-L -lysine, and poly-L -homoserine indicate that these four polypeptides are α-helical. Small but significant variability between the different α-helical polypeptides is seen in their circular dichroism spectra and optical rotatory dispersions. An argument is presented that these differences may be due to slight geometry differences between different α-helices.     

The conformation of poly-L-alanine in hexafluoroisopropanol

High-molecular-weight poly-L -alanine dissolved in hexafluoroisopropanol exhibits infrared, ultraviolet, circular dichroism, and optical rotatory dispersion spectra which are unique and unlike any other previously reported polypeptide spectra. Strong evidence that a helical conformation is present is shown by the high degree of hypochromism in the 187mμ absorption peak and by the positions of the amide infrared bands. The CD and ORD spectra are also similar to those of α-helical polypeptides, though important qualitative and qualitative differences are observed. To explain the novel spectra, which are not mixtures of the spectra of previously reported polypeptide conformations, a new α-helix-like conformation is proposed. The postulated conformation (a doubly hydrogen-bonded helix) is a distorted α-helix in which the peptide carbonyl groups point slightly out from the helix axis and are hydrogen bonded simul taneously both to the NH of the fourth peptide residue to the carboxyl terminal side (as in the classical α-helix), as well as to a solvent molecule's hydroxyl hydrogen.     

Interactions between chondroitin-6-sulfate and poly-L-lysine in aqueous solution: Circular dichroism studies

The interactions between chondroitin-6-sulfate (chondroitin sulfate C) and poly-L -lysine have been studied as models for investigation of possible complex formation between fibrous proteins and mucopolysaccharides. Results obtained using circular dichroism spectroscopy show that poly-L -lysine adopts the α-helical conformation in dilute aqueous salt solution at pH 7 when mixed with chondroitin-6-sulfate, rather than the “charged-coil” observed in the absence of this mucopolysaccharide. This conformation-directing interaction is at a maximum when the ratio of lysine to disaccharide residues is 1 : 1. Changes in the CD spectrum of a 1 : 1 mixture following increase in the salt concentration, or addition of non-polar solvents, indicate that the interaction is ionic in nature. No such effects are observed for non-sulfated mucopolysaccharides mixed with poly-L -lysine, suggesting that, for chondroitin-6-sulfate, it is the sulfate groups rather than the carboxyls which interact with the amine groups of the polypeptide. Elevation of the temperature leads to disruption of the interactions between the polypeptide and polysaccharide species. A sharp melting transition occurs at 47.0 ± 1.0°C, when the poly-L -lysine reverts to the “charged-coil” conformation. The sharp transition suggests that regular ionic bonds are formed between the polypeptide and polysaccharide. These results suggest that a conformation-directing interaction may occur between sulfated mucopolysaccharides and the polar regions of collagen and other fibrous proteins.     

Raman spectra of imino acids and poly-L-hydroxyproline

The Raman spectra of poly-L -hydroxyproline in the solid state and aqueous solution have been obtained. From the spectra, the conformations of the polypeptide in the solid and aqueous solution are found to be the same. Major differences in the spectra between the solid and solution are traced to environmental influences of the pyrrolidine group as indicated by studies of the monomers. The Raman spectra of the imino acids, proline and hydroxyproline, as the dipolar ions, the hydrochlorides, and the sodium salts are also reported.     

Synthesis and conformational analysis of salivary proline‐rich peptide P‐B

The 57‐amino acid human salivary polypeptide P‐B has been synthesized by the solid‐phase method using 9‐fluorenylmethoxycarbonyl (Fmoc) strategy. The circular dichroism (CD) spectroscopy, Fourier‐transform infrared spectroscopy (FTIR) and molecular modeling methods have been used for conformational studies of P‐B. Examination of the CD spectra of P‐B showed the content of the secondary structure to be independent of temperature over the range 0–60 °C at pH = 7 as well as over the pH range of 2–12 at 37 °C. P‐B adopts predominantly unordered structure with locally appearing β‐turns. The cumulative results obtained using the CD and FTIR spectroscopic techniques indicate the percentage of the polyproline type‐II (PPII) helix being as low as about 10%. Similarly, the molecular dynamics (MD) simulations reveal only a short PPII helix in the C‐terminal fragment of the peptide (Pro⁵¹–Pro⁵⁴), which constitutes 7%. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational studies on poly-L-tryptophan: circular dichroism and x-ray diffraction studies

Circular dichroism (CD) measurements were carried out on various copolymers of L -tryptophan and γ-ethyl L -glutamate in ethylene glycol monomethyl ether as the solvent. On increasing the L -tryptophan content of the copolymers a gradual change in the CD spectra was observed. The typical spectrum of the right-handedα-helix becomes more and more evident as the L -tryptophan content decreases. On the basis of these results we assumed that no conformational transition occurs on proceeding from pure poly (γ-ethyl L -glutamate) to pure poly-L -tryptophan in ethylene glycol monomethyl ether: therefore the conformation of poly-L -tryptophan should be that of a right-handed α-helix. Moreover we observed that the change in the CD spectra of the copolymers is gradual but not linear on increasing the tryptophan content. The deviations from linearity were attributed to interactions among side-chain chromophores whose contributions to the optical activity are not simply additive. An x-ray analysis carried out on oriented films of poly-L -tryptophan casted from solutions of the polymer in dimethylformamide shows conclusively that the solid-state conformation of the polymer is that, of an α-helix.     

Differentiation between the α-helical and coillike conformations of poly(L-glutamic acid) in aqueous solution through binding of pinacyanol

Pinacyanol in the presence of an excess of poly(L -glutamic acid) [polymer/dye ratio (P/D) > 10] exhibits different absorption spectra in the visible region when bound to the slightly charged polypeptide in the α-helical conformation or to the nearly completely dissociated polypeptide in the coillike conformation. These spectra reveal aggregation of the dye bound to the macromolecular chain in both conformations, although in the coillike one different kinds of aggregates may be present. Dye binding is accompanied by the appearance of CD bands in the visible region which are also different for the α-helical and the coillike conformations. The aggregates formed in the presence of the latter change slowly in time and seem to induce some conformational changes in the polypeptide chain. Furthermore, they have been found to be, at least partially, stable with respect to a subsequent reversal to the α-helical conformation. All results could be qualitatively interpreted assuming that in the coillike conformation, ordered regions exist along the chain as proposed by Krimm and Tiffany.     

Solvent effects on the conformation and far UV CD spectra of gramicidin

Solvent effects on the far-uv CD spectra of the polypeptide gramicidin have been studied systematically in a series of alcohols of increasing chain length, ranging from methanol to dodecanol. The effects observed are of two types: primary, involving a change in the equilibrium mixture of conformers present, and secondary, involving a shift in the spectral peak positions as a function of solvent polarizability. To quantitate the primary effect, the ratio of the individual conformers present was estimated by deconvolution of the spectra into their component species. For short chain length alcohols, both parallel and antiparallel double helices are found in considerable abundance. As the solvent chain length is increased and its polarity is decreased, the left-handed antiparallel double helical species is favored. For all alcohols with chain lengths of four or more carbon atoms, the ratio of the conformers present remains relatively constant. To quantitatively examine the secondary effect, the magnitudes of the spectral shifts on the dominant conformer (species 3) have been correlated with the dielectric constants and refractive indices of the solvents, thereby indicating what underlying physical properties are responsible for these shifts. This work thus demonstrates that for gramicidin, a flexible polypeptide, the solvent effects on the CD spectra can be resolved into two types: changes due to the mixture of conformers present and shifts in the spectral characteristics. Both effects need to be considered when interpreting CD spectra in terms of secondary structure for this and other polypeptides in nonaqueous solutions.     

Nuclear magnetic resonance investigation of the helix in random coil transformation in poly-alpha-amino acids. I

High-resolution nuclear magnetic resonance spectra at 100 MHz and 220 MHz have been obtained on two samples of poly-L -alanine of differing molecular weights (2500 and 42 500) in the chloroform–trifluoroacetic acid system under various conditions of solvent composition, temperature, and polypeptide concentration. Separate helix and random coil peaks are observed for the α-CH and peptide NH backbone proton resonances, thereby permitting the determination of helix content. This observation of separate peaks demonstrates that the lifetimes of the helix and random coil portions of poly-L -alanine have lower limits of about 10^?1 sec. It is suggested that solvent–peptide versus peptide–peptide hydrogen bond competition, coupled with a destabilizing effect of the trifluoroacetic acid on the helix, is responsible for the helix–random coil transformation.     

Molecular dynamics simulations of a winter flounder “antifreeze” polypeptide in aqueous solution

A winter flounder antifreeze polypeptide (HPLC-6) has been studied in vacuo and in aqueous solution using molecular dynamics computer simulation techniques. The helical conformation of this polypeptide was found to be stable both in vacuum and in solution. The major stabilizing interactions were found to be the main-chain hydrogen bonds, a salt-bridge interaction, and solute–solvent hydrogen bonds. A significant bending in the middle of the polypeptide chain was observed both in vacuo and in solvent at 300 K. Possible causes of the bending are discussed. From simulations of mutant polypeptide molecules in vacuo, it is concluded that the bend in the native polypeptide was caused by side chain to backbone hydrogen bond competition involving the Thr 24 side chain and facilitated by strains on the helix resulting from the Lys 18-Glu 22 salt bridge. © 1993 John Wiley & Sons, Inc.     

The circular dichroism spectrum of poly-L-acetoxyproline

We report the circular dichroism (CD) spectrum of poly-L acetoxyproline in trifluoroethanol, a solvent in which the form I to form II conformational isomerization occurs slowly enough to permit observation of the spectral changes. A comparison is made to poly-L proline. As judged by the similarity of the CD spectra, the conformations of the corresponding forms of the two polymers must be nearly the same. Transition assignments are proposed; these are shown to agree with the theoretical calculations of Pysh. There is a serious unexplained discrepancy between our solution data and those of Fasman for poly-L acetoxyproline.     

Far-infrared spectra of poly(-α-amino acids) with basic alkyl group side chains

Far-infrared spectra in the region from 700 to 60 cm^?1 have been measured for the α-helix structures of poly(L -α-amino-n-butyric acid), poly-L -norvaline, poly-L -norleucine, and poly-L -leucine and for the β-form structures of poly(L -α-amino-n-butyric acid), poly-L -valine, poly(DL -amino-n-butyric acid), poly-DL -norvaline, and poly-DL -norleucine. The changes of the spectra on N-deuteration have been measured in the region between 700 and 400 cm^?1. It is concluded that, the α-helix has characteristic bauds near 690, 650, 610, 380, 150, and 100 cm^?1, and that the β-form has characteristic bands near 700, 240, and 120 cm^?1. The main-chain vibrations in the region from 600 to 200 cm^?1 are strongly coupled with the side-chain deformation vibrations.