A Study of α-Helix Hydration in Polypeptides, Proteins, and Viruses Using Vibrational Raman Optical Activity

Abstract

A vibrational Raman optical activity (ROA) study, supplemented by protein X-ray crystal structure data, of α-helices in polypeptides, proteins, and viruses has suggested that ROA bands in the extended amide III spectral region may be used to distinguish between two types of right-handed α-helix. One type, associated with a positive ROA band at ∼1300 cm-1, dominates in hydrophobic environments and appears to be unhydrated; the other, associated with a positive ROA band at ∼1340 cm-1, dominates in hydrophilic environments and appears to be hydrated. Evidence is presented to support the hypothesis that unhydrated α-helix corresponds to the canonical conformation αc and hydrated α-helix to a more open conformation αo stabilized by hydrogen bonding of a water molecule or a hydrophilic side chain to the peptide carbonyl. α-Helical poly(l-lysine) and poly(l-ornithine) in aqueous solution and poly(l-alanine) in dichloracetic acid display both bands, but α-helical poly(l-glutamic acid) in aqueous solution and poly(γ-benzyl l-glutamate) in CHCl3 display only the ∼1340 cm-1 band and so may exist purely as αo due to enhanced stabilization of this conformation by particular side chain characteristics. The ROA spectrum of poly(β-benzyl l-aspartate) in CHCl3 reveals that it exists in a single left-handed α-helical state more analogous to αo than to αc.

The development of biomolecular Raman optical activity spectroscopy

“…Each has several distinct features characteristic of the type of structure. The average all-α ROA spectrum is very similar to that of model polypeptides in the α-helical conformation [10,11,21,76]. Although highly characteristic of β-sheet proteins, the average all-β ROA spectrum does not show quite such a close correspondence with that of a model β-sheet peptide [26,75].…”

“…Since then, hundreds of protein ROA spectra have been recorded and interpreted. It was soon apparent that, in addition to signatures of secondary structure like α-helix [76] and β-sheet [75], characteristic signals from loops and turns also appear which means that, rather than dwelling on ROA bands characteristic of secondary structure elements and how the corresponding percentages may be extracted, as is commonly done for other spectroscopic techniques, basic structural types or even motifs and folds in some cases might be recognized from the overall ROA band patterns. This is illustrated by the ROA spectra for a set of proteins with different folds displayed in Fig.…”

“…An example of how protein ROA spectra contain motif or fold information is provided by the negative band at ∼1374 cm −1 assigned to β-turns in the ROA spectrum of the α + β protein bovine ribonuclease A in which the β-sheet is antiparallel: this band is absent from the ROA spectrum of the α/β protein subtilisin Carlsberg because the ends of the parallel β-strands within its Rossman fold are connected by extended α-helix sequences rather than by β-turns. Detailed vibrational band assignments are discussed elsewhere [9,11,20,26,75,76,105,106]. The large number of resolved structure-sensitive bands in protein ROA spectra makes them suitable for the application of multivariate analysis (pattern recognition) techniques to extract structural information.…”

“…Additional details provided by ROA include discrimination between hydrated and unhydrated α-helix [76,102] and possibly β-sheet [75], and side chain conformation [20,26,105,106] including the absolute stereochemistry of side chains such as tryptophan [33,63].…”

“…Even more remarkable is that valuable information such as the folds of the major coat proteins can simply be 'read off'! For example, the ROA band pattern for the filamentous bacteriophage fd at the top is highly characteristic of an extended helix and looks very similar to that of α-helical polypeptides [10,11,21,76,105,106]; that of the second, tobacco mosaic virus, is characteristic of proteins with a helix bundle fold; those of the third and fourth, satellite tobacco virus and the MS2 virus capsid, are characteristic of up-down antiparallel β-sheet [75], with that of STMV containing additional bands characteristic of a significant amount of disordered structure from the many long strands. The first virus ROA spectra were reported for filamentous bacteriophages [31]; these data facilitated the identification of ROA bands associated with unhydrated and hydrated α-helix since large amounts of both types are present in the overlapping extended helical coat proteins in the intact viruses.…”