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α-Helix peptide folding and unfolding activation barriers: A nanosecond UV resonance raman study

Lednev, IK and Karnoup, AS and Sparrow, MC and Asher, SA (1999) α-Helix peptide folding and unfolding activation barriers: A nanosecond UV resonance raman study. Journal of the American Chemical Society, 121 (35). 8074 - 8086. ISSN 0002-7863

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Abstract

We used UV resonance Raman spectroscopy to characterize the equilibrium conformation and the kinetics of thermal denaturation of a 21 amino acid, mainly alanine, α-helical peptide (AP). The 204-nm UV resonance Raman spectra show selective enhancements of the amide vibrations, whose intensities and frequencies strongly depend on the peptide secondary structure. These AP Raman spectra were accurately modeled by a linear combination of the temperature-dependent Raman spectra of the pure random coil and the pure α-helix conformations; this demonstrates that the AP helix-coil equilibrium is well-described by a two-state model. We constructed a new transient UV resonance Raman spectrometer and developed the necessary methodologies to measure the nanosecond relaxation of AP following a 3-ns T- jump. We obtained the T-jump by using a 1.9-μm IR pulse that heats the solvent water. We probed the AP relaxation using delayed 204-nm excitation pulses which excite the Raman spectra of the amide backbone vibrations. We observe little AP structural changes within the first 40 ns, after which the α-helix starts unfolding. We determined the temperature dependence of the folding and unfolding rates and found that the unfolding rate constants show Arrheniustype behavior with an apparent κ8 kcal/mol activation barrier and a reciprocal rate constant of 240 ± 60 ns at 37 °C. However, the folding rate constants show a negative activation barrier, indicating a failure of transitionstate theory in the simple two-state modeling of AP thermal unfolding, which assumes a temperature-independent potential energy profile along the reaction coordinate. Our measurements of the initial steps in the α-helical structure evolution support recent protein folding landscape and funnel theories; our temperature-dependent rate constants sense the energy landscape complexity at the earliest stages of folding and unfolding.


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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Lednev, IK
Karnoup, AS
Sparrow, MC
Asher, SAasher@pitt.eduASHER
Date: 8 September 1999
Date Type: Publication
Journal or Publication Title: Journal of the American Chemical Society
Volume: 121
Number: 35
Page Range: 8074 - 8086
DOI or Unique Handle: 10.1021/ja991382f
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Refereed: Yes
ISSN: 0002-7863
Date Deposited: 20 Mar 2013 16:43
Last Modified: 02 Feb 2019 16:55
URI: http://d-scholarship.pitt.edu/id/eprint/17791

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