High Pressure FTIR Studies on Model α-Helical Peptides

Abstract

High-pressure conditions force water into internal cavities of proteins; the presence of water in a hydrophobic interior can destabilize the tertiary structure resulting in protein unfolding. This effect has been predicted in molecular dynamics simulations for model alpha-helical peptides at high pressures, where water is forced into closer contact with backbone carbonyls (as measured by an increased in backbone hydration). According to simulations, this increase hydration depends on local sequence and the impact that specific side chains have on backbone conformation. We have investigated the effects of pressure on a series of 20-residue peptides. Model peptides based on the alanine and lysine repeats form water soluble, stable alpha helices (1). The sequence (AAAAK)3-AAAAY is a well-characterized, synthetic polypeptide, ideal for the study of helix properties(2). Using the model peptides, we experimentally confirm that the helical content is conserved under pressure. Perturbations were monitored using a probe of secondary structure, the amide I’ band, with infrared (IR) spectroscopy and a diamond anvil cell. Amide I’ mode shifts to a lower frequency with the increase of pressure. Local information is obtained by measuring amide I'bands in 13C labeled peptides, where 13C alanines are placed at different positions relative to the lysines within the peptide. We examined the shielding effects of lysine at the various positions and confirm that shielding of backbone carbonyls occurs.1. Starzyk, A., Barber-Armstrong, W., Sridharan, M., and Decatur, S. M. (2005) Spectroscopic evidence for backbone desolvation of helical peptides by 2,2,2-trifluoroethanol: an isotope-edited FTIR study, Biochemistry 44, 369-376.2. Paschek, D., Gnanakaran, S., and Garcia, A. E. (2005) Simulations of the pressure and temperature unfolding of an alpha-helical peptide, Proc Natl Acad Sci U S A 102, 6765-6770.