Apparent Helix-Coil Transition for Poly(L-Alanine) as Measured by Nuclear Magnetic Resonance

Abstract

The previous contention by several groups that double peaks that arise in NMR spectra from NH and a-CH protons of poly L-alanines are caused by the coexistence of helical and random-coil structures in solution is disputed.Numerous reports have appeared showing that under certain circumstances many polypeptides exhibit NMR spectra with double peaks from NH and a-CH protons (2-8). The doubling of peaks was attributed initially by Ferretti to the coexistence of helical and random-coil structures in solution, in spite of the fact that dynamic measurements indicate that the average rate of the helix to coil transition (0.1-10 Asec) is extremely rapid on the NMR time scale (9-12). Such rates should lead to a single averaged peak. Explanations for the doubled peaks have been offered based on polydispersity (13), slow nucleation of the transition (14), and protonation of the peptide bonds (15). Scheraga and his coworkers (16) speculated that the a-helix peak should not be observable because of dipolar broadening. They suggested that double peaks arise from specific, slowly exchanging solvations of random-coil species in mixed solvents containing acids such as trifluoroacetic acid (CF3COOH). Recently, Bradbury and his associates (17) assigned the double peaks to the a-helix and random coils on the basis of comparisons of the areas for a-CH with the side-chain peaks. The Bradbury group concludes that rapid exchange between a-helix and random coil takes place, with the double peaks arising from polydispersity. NMR spectra of poly(L-alanine) were analyzed by Jardetzky (3), Bradbury (18), and Ferretti (3) and their associates. The second two groups found double peaks for the a-CH at 220 MHz. One peak at 4.5 ppm was attributed to the a-helix, while the other peak at 4.7 ppm was assigned to the random-coil conformation of the polypeptide chain.In this paper, we present results indicating that the double peaks seen for poly(L-alanine) may not arise from the helix to coil transition, and that the above explanations may not apply to the observed phenomena. We believe the lowfield peak arises from the a-CH of oligomeric peptides. The high-field peak represents a single averaged peak of helix and random coil. We performed measurements using a 220-MHz Varian spectrometer and very low concentrations of L-alaninederived peptides, necessitating time-averaging to improve the signal-to-noise ratios in solvents composed of deuterochloro- form (CDC13) and CF3COOH. We varied the molecular weight of the polymers, sample concentrations, solvent compositions, and temperature at which the experiments were performed. Although the spectra do not exhibit good signal-to-noise ratios (even after time-averaging), we were able to obtain clear and interpretable results.Double peakst at about 4.6 and 4.3 ppm are observed for poly(L-alanine)i-[average degree of polymerization (DP)37], as is shown in Fig. 1

Determination of the relaxation time of the helix–coil transition of poly(γ‐benzyl‐L‐glutamate) by the nmr technique

“…Ullman7 first carried out a simulation and found that the double peak appears even in the interconversion rate of the helix-coil transition, which is fast enough on the nmr time scale. Experimental confirmations of the polydispersity theory have been given by the present authors12,13 and Bradbury et al14 for poly(y-benzy1-Lglutamate), and Goodman et al 15 for poly(L-Ala). As far as the double peak is concerned, it is thought that the polydispersity in the sample is its conclusive origin.…”

A nuclear magnetic resonance study of the helix–coil transition of poly(L‐glutamic acid)

“…Many theoretical interpretations have been given to these observations in efforts to resolve this problem. [16][17][18][19][20][21][22][23][24][25][26] Polypeptides with ionizable side chains have appeared to be either a special case or an anomaly with respect to the above considerations. Heretofore, when the helix-coil transition was induced by changes in pH, no doubling of the resonances was observed with either lH or l3C nmr.27- 30 However, experiments such as polarimetric temperature jump,2 ultrasonic a b s o r p t i~n ,~-~~~J~J~ polarimetric electric field jump,14 and electric field pulse methodI5 with poly(L-glutamic) acid yield relaxation times of the order of sec.…”

Folding of the Transmembrane 25-Residues Influenza A M2 and Ala-25 Peptides

“…85 Synthetic Ala 14 -based peptides have been shown to undergo a transition from αhelical structures to β-sheet complexes in vitro, 86 mimicking the structural transition that is believed to be a prerequisite for fibril nucleation and growth. 18,87 These poly-alanine peptides form fibrils with βsheet structures, which are aqueous soluble, and stabilized by hydrophobic intersheet interactions. (b) Peptide hydrogen bonds.…”

“…16 Because Ala has a small side chain poly-alanine peptides are free of van der Waals interactions between side chains by Ala and helix formation is stabilized predominantly by the backbone peptide hydrogen bonding interactions. [16][17][18][19][20][21] Thus, the third aim of the simulations is to estimate, through the comparison with the behavior of the Ala 25 peptide in the same solvent, the relative α-helix formation propensity in the two 25-residue peptides.…”