Circular dichroism of the “random” polypeptide chain

The circular dichroism (CD) spectrum of an unordered polypeptide chain does not correspond, as has been assumed heretofore, to that of a charged chain such as poly-L -glutamic acid or poly-L -lysine. The latter have been shown to have locally ordered structures with characteristic CD spectra. We have now obtained CD spectra of the unordered forms of the above synthetic, polypeptides, as well as of two fibrous proteins (collagen and feather keratin) and a globular protein (myoglobin). These spectra are all similar to that of unordered polyproline, having a negative band in the vicinity of 2000 mμ and no additional bands at longer wavelengths. The lack of structural uniqueness of the unordered polypeptide chain is emphasized by these studies.     

Vibrational circular dichroism spectra of three conformationally distinct states and an unordered state of poly(L-lysine) in deuterated aqueous solution

Fourier transform ir vibrational circular dichroism (VCD) spectra in the amide I′ region of poly(L-lysine) in D₂O solutions have confirmed the existence of three distinct conformational states and an unordered conformational state in this homopolypeptide. Characteristic VCD spectra are presented for the right-handed α-helix, the antiparallel β-sheet, an extended helix conformation previously referred to as the so-called “random coil,” and a completely unordered conformation characterized by the absence of any amide I′ VCD. VCD for the antiparallel β-sheet in solution and the unordered chain conformation are presented for the first time. Each of the four different VCD spectra is unique in appearance and lends weight to the view that VCD has the potential to become a sensitive new probe of the secondary structure of proteins in solution.     

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.     

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.     

Raman studies of the helix-to-coil transition in poly-L-glutamic acid and poly-L-ornithine

The conformational transitions in water and in the solid state of poly-L -glutamic acid (PGA) and poly-L -ornithine (PO) have been studied by Raman spectroscopy. The Raman spectra of PGA, PO, and the monomer, dimer, and trimer of PGA in aqueous solutions and solid state are presented. The Raman spectral changes of PGA and PO were followed through the helix-to-coil transition induced by p^H, temperature, and solvent composition. A hyperchromic shift in the intensity of the amide III line accompanying the helix-to-coil transition was observed. This hyperchromic intensity shift occurs abruptly as a function of p^H but more slowly with heat denaturation of the alpha helix indicating that the Raman spectrum is sensitive to the transition mechanism. The high-temperature coil and the charged coil may have different conformations as evidenced by different amide III frequencies but similar intensities in these two conformations.     

Spectroscopic characterization of poly(Glu-Ala)

Infrared linear dichroism and ultraviolet circular dichroism speetroscopy have been used to distinguish four conformational forms of the ionizable sequential polypeptide poly(Glu-Ala). Two of these conformations, the α helix and the β form, were observed for the unionized polypeptide in solution. The α helix appeared immediately upon neutralization of the side-chain carboxyl functions, whereas the β form was observed after the neutralized solution had been standing for several days. The β form was also observed for films cast from either high or low pH solutions. Ionization of the glutamyl residues resulted in a circular dichroism spectrum which has previously been observed for charged homopolymers and appears to result from an extended helical conformation. Further, heating either the α helical or the charged extended helix resulted in a transition to a disordered chain. These results are consistent with the results of conformational calculations presented elsewhere.     

Molecular orbital calculations on the conformation of polypeptides and proteins. 8. The conformational energy maps and stereochemical rotational states of the asparaginyl, glutaminyl aspartyl and glutamyl residues

Quantum-mechanical calculations on the conformational energy map and stereo-chemical rotational states of aminoacid residues by the PCILO method are extended to the asparaginyl, glutaminyl, aspartyl and glutamyl residues in their neutral form. One of the most outstanding features of the results is the occurrence of the global minimum (or of one of a few equivalent global minima) in the region of the left handed α-helix for the first three of the above mentioned residues. The results of the calculations are compared with experimental data from eight, globular proteins which confirm that these residues may exist, in fact, in this conformation. They also enable to understand the experimentally observed possibility of helix reversal in esters of poly-L -aspartic acid as a function of substitutions in the side chain.     

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.     

Optical properties of the polyglycine II helix

The circular dichroism spectrum obtained from a dilute aqueous solution of poly (ala-gly-gly) resembles that described for charged polypeptides such as the salt form of poly glutamic acid. A similar spectrum is found for films cast from aqueous solution where x-ray studies reported elsewhere have indicated a poly-glycinc II conformation. Evidence is presented for a heat induced poly-glycine II to unordered state transition similar to that described for collagen. The interpretation of this, the first observation of the optical properties of a poly-amino acid in the poly glycine II conformation, is further rationalized on the basis of spectra obtained from a number of polypeptides whose conformation approaches that of a 3₁ helix.     

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.     

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.     

The circular dichroism of aromatic polypeptides: Theoretical studies of poly-L-phenylalanine and some para-substituted derivatives

We have calculated rotational strengths and circular dichroism (CD) curves for sidechain and backbone transitions in poly-L -Phenylalanine (PLP), POLY-p-amino-L -phenylalanine (PPALP), poly-p-chloro-L -phenylalanine (PPCLP), poly-o-acetyl-L -tyrosine (POALT), and poly-p-nitro-L -phenylanine (PPNLP), using methods applied previously to poly-L -tyrosine (PLT). Comparison of the theoretical CD curves with available experimental data for PLP and PPALP indicate that these polypeptides form right-handed helices with side-chain conformations similar to that of PLT. For PPNLP, where experimental data are also available, no conformational assignment could be made, as none of the calculated curves gave good agreement with experiment. Possible reasons for this lack of agreement are discussed. For the other two polypeptides, PPCLP and POALT, although no experimental data are yet available, the calculated curves indicate that an unambiguous assignment should be possible. For the conformations (RA and LA) in which the side chains are packed more loosely, there are strong similarities in the calculated CD curves of a particular conformation, regardless of the para substituent. In the tighter R1 and L1 conformations, few generalizations can be drawn, each derivative having a distinctive pattern. In PLP, PPCLP, and POALT, where the side-chain L^a band is in the 200–210 nm region, the L1 conformation exhibits a negative nπ* rotational strength, opposite to that expected for a left-handed helix. One must therefore be cautious about assigning the helix sense of aromatic polypeptides on the basis of the sign of the nπ* CD band. Side-chain nπ* transitions present in POALT and PPNLP were found to have small rotational strength.     

Conformational preferences of the sequential fragments of the hinge region of the human IgA1 immunoglobulin molecule

The mean solution conformation of tetrapeptide fragments spanning the hinge region of human IgA1 was investigated by CD and 13C-NMR methods. Distinct conformational differences for the partial sequences of IgA1 were found. In a series of tetrapeptides having the Thr-Pro-Pro-Thr sequence, the Pro-Pro fragment was ordered to the structure of a type II polyproline helix, but with unordered forms prevailing in the equilibria. In the case of the Pro-Pro-Thr-Pro sequence, a distinct preference for the beta-turn conformation was found. Acetylation of this tetrapeptide shifts the equilibrium towards unordered forms containing some elements of the type II polyproline helix. The peptide Thr-Pro-Ser-Pro exists predominantly in the beta-turn conformation whereas Pro-Ser-Pro-Ser-NH2 has, for the most part an unordered conformation.     

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.     

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.     

Conformational preferences of Leu-enkephalin in reverse micelles as membrane-mimicking environment

Enkephalin represents one of the bioactive peptide molecules most extensively investigated both in solution and in the crystal state. Depending upon the environment chosen for such studies, three main conformational states were identified for this flexible, linear pentapeptide—i.e., an extended conformation, a single-bend, and a double-bend structure. Since CD and Fourier transform ir (FTIR) spectra of Leu-enkephalin solubilized in negatively charged reverse micelles of bis (2-ethylhexyl)sulfosuccinate sodium salt/isooctane/water were supportive of a restricted conformational space of the aromatic side chains and of a bended type fold, we have analyzed by nmr the conformational preferences of Leu-enkephalin in reverse micelles using a synthetic [¹³C, ¹⁵N]-backbone-labeled sample. The overall conformation derived from nuclear Overhauser effect spectroscopy (NOESY) and ¹⁵N-filtered rotating frame NOESY (ROESY) spectra and by distance geometry calculations is a double-bend fold of the backbone that is comparable to one of the known x-ray structures. Thereby the tyrosine side chain is inserted into the hydrophobic core of the reverse micelles in a restrained conformational space as well evidenced by NOEs between the aromatic ring protons and the surfactant. The proximity of the aromatic rings of tyrosine and phenylalanine indicate a preferred structure consistent with the postulated conformation of the opioid peptide in the δ-receptor-bound state. These results confirm the interesting and promising properties of reverse micelles as membrane mimetica. © 1997 John Wiley & Sons, Inc. Biopoly 41: 591–606, 1997     

Conformation of poly-L-tyrosine in trimethyl phosphate solution

Absorption, circular dichroism (CD), and optical rotatory dispersion (ORD) measurements were carried out on poly-L -tyrosine in trimethyl phosphate solution over the spectral range 185–600 mμ. There is evidence in the CD spectrum for side chain-side chain interactions in poly-L -tyrosine. ORD and CD data in dimethylformamide and pyridine closely parallel those in trimethyl phosphate, indicating a similarity in conformation of the polymer in all three solvents. In the polarized infrared spectrum both position and polarization of amide A, I, and II bands are characteristic of α-helical polypeptides. Bands corresponding to side chain also exhibit dichroism, suggesting that the side chains are not randomly oriented. Viscosity and light-scattering studies are consistent with α-helical structure for the polymer that, remains rigid over a temperature range of 15–50°C and becomes somewhat flexible at higher temperatures. Optical rotatory properties were found to vary gradually and continuously with temperature over the range of ?30 to +100°C. This suggested that all three electronic transitions of tyrosyl side chain are optically active, and that the side chains have some freedom of motion that decreases with decreasing temperature, disappearing only at about ?30°C.     

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.     

Circular dichroism of collagen, gelatin, and poly(proline) II in the vacuum ultraviolet.

Circular dichroism spectra for acid-soluble calfskin collagen, gelatin, and poly(proline) II in solution have been extended into the vacuum ultraviolet region. The extended spectrum of gelatin reveals that the circular dichroism of this unordered polymer is more closely related to the spectrum of charged polypeptides than might be evident from near ultraviolet work. A short-wavelength band is found at about 172 nm, which corresponds in position, magnitude, and sign to a band recorded earlier for poly(L -glutamic acid) at pH 8.0. This band is observed in a helical structure for the first time in the vacuum ultraviolet circular dichroism and absorption spectra of poly(proline) II. Both circular dichroism and absorption spectra point to the assignement of this band as the nσ*. Neither the nσ* nor the expected positive lobe of the ππ* helix band is observed in the extended circular dichroism spectrum of collagen. We postulate that these two bands cancel here in analogy to the case of α-helical poly(L -glutamic acid).     

A CD strategy for the study of polypeptide folding/unfolding. A synthetic foot-and-mouth disease virus immunogenic peptide.

The circular dichroism spectrum of the 20-residue immunogenic peptide from the foot-and-mouth disease virus (VP1; 141-160 of serotype A, subtype 12) was solvent- and temperature-dependent. Careful solvent titration revealed two isodichroic points and plateaux consistent with stepwise unfolding of specific stable conformations. Variable temperature studies in cryogenic solvents and urea perturbation were consistent with the existence of three conformational moieties, the left-handed extended helix, the alpha-helix, and the 3(10) helix. The number of residues in each helix was confirmed by CD spectral simulations. The strategy described here can be used to determine the components of a conformational equilibrium and their statistical weights, to study peptide folding and unfolding and to determine the bioactive conformation(s) of linear peptides. The conclusions were supported by 2D-NMR studies. A new mechanism for the stabilization of left-handed extended helices and destabilization of alpha-helices by urea is proposed. The structure of the peptide as resolved by CD spectroscopy is of particular significance since the conformation of this antigenic sequence in situ has so far not been solved by X-ray crystallography.