Analysis of local conformation within helical peptides via isotopeedited vibrational spectroscopy

Abstract

Short, alanine-rich peptides that form stable helices in aqueous solution are classic model systems for studying helix stability [1], the details of helix structure and conformation [2], and the physical properties of helices [3]. The most commonly applied spectroscopic tools for studying these peptides are ultraviolet circular dichroism (UV-CD) and NMR Quantitative analysis of UV-CD spectra produces a description of the overall helix content of the peptide, but cannot be used to assess conformation at the residue level. In a fully assigned NMR spectrum, the helix content at the residue level can be determined by interpretation of chemical shifts or measurement of proton exchange rates; however, pool spectral dispersion hinders the assignment process, and the dynamics of the helix-coil transition are too rapid for study by NMR. Vibrational techniques offer an alternative to UV-CD and NMR, and vibrational spectra of alanine peptides have been reported in the literature [4,5]. Vibrational spectroscopy has been widely used to probe the structure of proteins and peptides. Within a polypeptide, the amide I vibrational modes (primarily the stretching of the backbone C=O) of the of individual residues are coupled, and the observed frequencies and intensities of the amide I band are dependent on the backbone geometry (secondary structure) of the polypeptide. Conformationally dependent coupling between amide I modes can also be measured via vibrational circular dichroism spectroscopy (VCD), with different secondary structures giving rise to unique and distinguishable VCD spectral patterns [6]. Yet the coupling between vibrations of individual residues into delocalized modes is a double-edged sword; the vibrational spectra report on overall secondary structure content of the peptide but cannot probe local, residue-level conformational changes. One approach to obtaining residue-specific IR probes is to introduce isotope label! at specific sites within the peptide backbone [7,8]. Substituting into the backbone carbonyl shifts the amide I vibrational frequency by nearly giving rise to a band in the IR spectrum resolvable from the amide I band. The spectral features of the new amide I band report on the conformation of specific residues within the peptide sequence; description of conformation as a function of residue position can then be obtained through analysis of a series of isotope substituted peptides. We have applied this isotope-edited technique to the characterization of helical peptides. Using a combination of FTIR, VCD, and ab initio calculations, we have demonstrated that the specific isotope labels serve as effective residue-level probes of conformation within alanine-rich peptides.