Enthalpic and entropic stages in alpha-helical peptide unfolding, from laser T-jump/UV Raman spectroscopy.
The alpha-helix is a ubiquitous structural element in proteins, and a number of studies have addressed the mechanism of helix formation and melting in simple peptides. However, fundamental issues remain to be resolved, particularly the temperature (T) dependence of the rate. In this work, we report application of a novel kHz repetition rate solid-state tunable NIR (pump) and deep UV Raman (probe) laser system to study the dynamics of helix unfolding in Ac-GSPEA3KA4KA4-CO-D-Arg-CONH2, a peptide designed for helix stabilization in aqueous solution. Its T-dependent UV resonance Raman (UVRR) spectra, excited at 197 nm for optimal enhancement of amide vibrations, were decomposed into variable contributions from helix and coil spectra. The helix fractions derived from the UVRR spectra and from far UV CD spectra were coincident at low T but deviated increasingly at high T, the UVRR curve giving higher helix content. This difference is consistent with the greater sensitivity of UVRR spectra to local conformation than CD. After a laser-induced T-jump, the UVRR-determined helix fractions defined monoexponential decays, with time-constants of approximately 120 ns, independent of the final T (Tf = 18-61 degrees C), provided the initial T (Ti) was held constant (6 degrees C). However, there was also a prompt loss of helicity, whose amplitude increased with increasing Tf, thereby defining an initial enthalpic phase, distinct from the subsequent entropic phase. These phases are attributed to disruption of H-bonds followed by reorientation of peptide links, as the chain is extended. When Ti was raised in parallel with Tf (10 degrees C T-jumps), the prompt phase merged into an accelerating slow phase, an effect attributable to the shifting distribution of initial helix lengths. Even greater acceleration with rising Ti has been reported in T-jump experiments monitored by IR and fluorescence spectroscopies. This difference is attributable to the longer range character of these probes, whose responses are therefore more strongly weighted toward the H-bond-breaking enthalpic process.
Balakrishnan
Gurusamy
G
Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
Hu
Ying
Y
Bender
Gretchen M
GM
Getahun
Zelleka
Z
DeGrado
William F
WF
Spiro
Thomas G
TG
D002942:Circular Dichroism; D019277:Entropy; D006358:Hot Temperature; D008956:Models, Chemical; D008968:Molecular Conformation; D010455:Peptides / Q000737:chemistry*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence / Q000379:methods; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013059:Spectrum Analysis, Raman; D013696:Temperature; D013816:Thermodynamics
D010455:Peptides