Nonlinear Infrared Spectroscopy of Protein Conformational Change during Thermal Unfolding

Hoi Sung Chung, Munira Khalil,† and Andrei Tokmakoff*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
J. Phys. Chem. B, 2004, 108 (39), pp 15332–15342
DOI: 10.1021/jp0479926
Publication Date (Web): August 25, 2004
Copyright © 2004 American Chemical Society
†

 Present address:  Department of Chemistry, University of California, Berkeley, California.

,
*

 Corresponding author. E-mail:  tokmakof@mit.edu.

Abstract

Femtosecond two-dimensional infrared (2D IR) spectroscopy, dispersed vibrational echo (DVE) spectroscopy, and dispersed pump−probe (DPP) spectroscopy of the amide I vibrations are used to follow the thermal denaturing of ribonuclease A. Each experiment measures a spectrum with features that reflect vibrational couplings between amide I transitions of the protein backbone and is therefore more sensitive to protein conformation than traditional FTIR spectroscopy. As the temperature is raised, 2D IR spectra show the disappearance of cross peaks between two vibrational transitions arising from antiparallel β-sheet structure as well as the blue shift to a diagonally elongated inhomogeneous spectrum. DVE and DPP spectra can be related to projections of the complex 2D IR correlation spectrum and also show clear signatures of β-sheet structure in the native state. Singular value decomposition of the nonlinear spectra reveal a pretransition at 47 °C that precedes the melting of β sheets in the primary denaturing transition at 65 °C. Residual secondary structure is observed in the thermally denatured state. These experiments suggest that nonlinear infrared spectroscopy is an effective probe of protein conformation that can be used to probe the equilibrium thermodynamics and nonequilibrium kinetics and dynamics of protein folding.

View: Full Text HTML | Hi-Res PDF

Tools

Accession Codes

History

  • Published In Issue September 30, 2004
  • Received May 10, 2004
    Revised July 6, 2004

Recommend & Share

Related Content

Other ACS content by these authors: