SPECTROSCOPIC MONITORING OF ALPHA HELICAL UNIFORMITY

It is known that membrane-embedded a-helices are more uniform structurally than their aqueous counterparts. Despite this uniformity, distortions and localized unfolding are thought to be common in these proteins in order for them to conduct their cellular tasks. However, how amino acid sequence facilitates these conformational shifts remains unknown, as methods for investigating structural heterogeneity in transmembrane proteins are limited. Circular dichroism (CD) is often used to characterize the secondary structure of proteins, but the broadly overlapped spectral features limit its usefulness. Deep-ultraviolet resonance Raman spectroscopy (dUVRR) is an emerging structurally sensitive spectroscopic technique for analyzing membrane protein structure. The backbone amide modes are resonance enhanced in dUVRR spectra while the lipid features are not resulting in strong membrane protein spectral features in near native environments. Using model leucine-alanine peptides in increasingly dehydrated (aqueous, surfactant and bilayer) environments, hydration dependent changes in the amide modes were characterized. The amide I mode in the dUVRR spectra of these peptides increased with increasing dehydration, while the amide III decreased. These results indicate that the dehydration of the peptide backbone is accompanied by and increase in helical structure. Incorporation of helix breaking residues (HBR), proline or glycine, in these model peptides promoted helical instability in lipophilic environments.