Transient 2D IR spectroscopy of ubiquitin unfolding dynamics
-
Edited by Robin M. Hochstrasser, University of Pennsylvania, Philadelphia, PA, and approved April 25, 2007 (received for review February 2, 2007)
Abstract
Transient two-dimensional infrared (2D IR) spectroscopy is used as a probe of protein unfolding dynamics in a direct comparison of fast unfolding experiments with molecular dynamics simulations. In the experiments, the unfolding of ubiquitin is initiated by a laser temperature jump, and protein structural evolution from nanoseconds to milliseconds is probed using amide I 2D IR spectroscopy. The temperature jump prepares a subensemble near the unfolding transition state, leading to quasi-barrierless unfolding (the “burst phase”) before the millisecond activated unfolding kinetics. The burst phase unfolding of ubiquitin is characterized by a loss of the coupling between vibrations of the β-sheet, a process that manifests itself in the 2D IR spectrum as a frequency blue-shift and intensity decrease of the diagonal and cross-peaks of the sheet's two IR active modes. As the sheet unfolds, increased fluctuations and solvent exposure of the β-sheet amide groups are also characterized by increases in homogeneous linewidth. Experimental spectra are compared with 2D IR spectra calculated from the time-evolving structures in a molecular dynamics simulation of ubiquitin unfolding. Unfolding is described as a sequential unfolding of strands in ubiquitin's β-sheet, using two collective coordinates of the sheet: (i) the native interstrand contacts between adjacent β-strands I and II and (ii) the remaining β-strand contacts within the sheet. The methods used illustrate the general principles by which 2D IR spectroscopy can be used for detailed dynamical comparisons of experiment and simulation.
Footnotes
- †To whom correspondence should be addressed. E-mail: tokmakof{at}mit.edu
-
Author contributions: H.S.C. and Z.G. contributed equally to this work; H.S.C., Z.G., K.C.J., and A.T. designed research; H.S.C., Z.G., and K.C.J. performed research; H.S.C., Z.G., K.C.J., and A.T. analyzed data; and H.S.C., Z.G., K.C.J., and A.T. wrote the paper.
-
The authors declare no conflict of interest.
-
This article is a PNAS Direct Submission.
-
This article contains supporting information online at www.pnas.org/cgi/content/full/0700959104/DC1.
- Abbreviations:
- DVE,
- dispersed vibrational echo;
- MD,
- molecular dynamics;
- T-jump,
- temperature jump.
- © 2007 by The National Academy of Sciences of the USA
