UV Raman Studies of Peptide Conformation Demonstrate That Betanova Does Not Cooperatively Unfold

Abstract

We used UV resonance Raman spectroscopy (UVRR) excited within the peptide bond π f π* electronic transitions and within the aromatic amino acid π f π* electronic transitions to examine the temperature dependence of the solution conformation of betanova, a 20-residue -sheet polypeptide [Kortemme, T., Ramirez-Alvarado, M., and Serrano, L. (1998) Science 281, 253-256]. The 206.5 nm excited UVRR enhances the amide vibrations and demonstrates that betanova has a predominantly -sheet structure between 5 and 82°C. The 229 nm excited UVRR, which probes the tyrosine and tryptophan side chain vibrations, shows an increase in the solvent exposure of the tryptophan side chains as the temperature is increased. Our results are consistent with the existence of an intermediate state similar to that calculated by Bursulaya and Brooks [Bursulaya, B. D., and Brooks, C. L. (1999) J. Am. Chem. Soc. 121, 9947-9951] and exclude the previously proposed two-state cooperative folding mechanism. Betanova's structure appears to be molten globule over the 3-82°C temperature range of our study.The completion of the human genome sequence makes available the primary sequence of all proteins synthesized by the human body. This represents only the first stage in the development of an understanding of the protein structural basis of disease. The next stage in this development requires an ability to predict the structure and function of proteins from their primary sequences. At present this is impossible, since not enough is known about the dynamics and thermodynamics of protein folding (1-5).The understanding of protein folding mechanisms is advancing as experimentalists develop better strategies for the de novo design of model peptides and better methodologies for characterizing equilibrium secondary structures, as well as the dynamics of folding. To date, synthetic schemes have been successfully developed for the synthesis of R-helix-forming peptides, and much has been uncovered about their folding dynamics (6-13). In contrast, only limited progress has been made toward understanding the synthesis of and the dynamics of -sheet peptides. Primarily, studies have focused on -hairpins rather than complete sheet structures (14-18). Successful synthesis of three-stranded -sheet structures has been accomplished only recently. However, these model peptides only form complete -sheet structures under limited conditions, in the presence of organic solvents and with the use of unnatural amino acids (19)(20)(21)(22). Thus, there remains much to learn about -sheet folding.Significant advances have also occurred in the theoretical understanding of protein folding (23-28). Such work includes molecular dynamics simulations of energy landscapes that describe folding thermodynamics. Currently, some of these models accurately predict experimental results. Thus, theory can now impact how experimental data are interpreted. It seems likely that the most powerful insights into the rules of protein folding will result from combined experimental and theoreti...

The structural basis for biphasic kinetics in the folding of the WW domain from a formin-binding protein: Lessons for protein design?

“…To further an understanding of the elements responsible for stability in ␤-sheets, de novo design methods have, in two cases, been used to construct three-stranded antiparallel ␤-sheets (8,9), which have subsequently become the subject of both theoretical (10)(11)(12) and experimental (13,14) folding studies. To ensure the generality of results toward natural proteins, however, we opt to study a series of three-stranded antiparallel ␤-sheet domains found in a variety of proteins: the WW domains (Fig.…”

Conformations of Betanova in aqueous trifluoroethanol

“…14 It has been suggested that contributions of the aromatic side chains of Betanova in the 200-230 nm region will frustrate attempts to analyze such spectra in terms of the relative contributions of canonical b-sheet and a-helical peptide backbones. 13 Our translational diffusion measurements indicate that the hydrodynamic radius of Betanova both in water and in 42% TFE/water is around 11 Å . Danielsson et al 41 provide an empirical scaling law [Eq.…”

“…14 The observed insensitivity of the CD spectra to temperature suggests that little change takes place in the relative populations of secondary structures of the peptide over the temperature range examined, as concluded by Boyden and Asher. 13 There are modest effects of TFE on the ellipticity near 200 nm that are temperature-sensitive, but these effects are not large and are not consistent with a substantial enhancement of the populations of conformations containing b-structures or helical elements. The effects observed appear not to be specific to TFE since ethanol present at the same concentration produces essentially the same spectral changes.…”

“…The spectra are similar to those reported by Kuznetsov et al 14 and by Boyden and Asher. 13 Kuznetsov et al 14 concluded that the CD spectrum of Betanova in water indicates that the majority of peptide conformations present are best characterized as being random coils. 14 The observed insensitivity of the CD spectra to temperature suggests that little change takes place in the relative populations of secondary structures of the peptide over the temperature range examined, as concluded by Boyden and Asher.…”

“…Boyden and Asher 13 used UV resonance Raman spectroscopy to examine structural changes in Betanova as the temperature was varied over the range where the earlier work suggested a folding-unfolding transition. No evidence for such a transition was provided by these experiments and the authors suggested that Betanova may be a molten globule with virtually the same conformational properties over the entire temperature range studied.…”