This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.104.053199v1 88/2/1413    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chattopadhyay, K. Articles by Frieden, C. Search for Related Content PubMed PubMed Citation Articles by Chattopadhyay, K. Articles by Frieden, C.

Measuring Unfolding of Proteins in the Presence of Denaturant Using Fluorescence Correlation Spectroscopy

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

Correspondence: Address reprint requests to Carl Frieden, Dept. of Biochemistry and Molecular Biophysics, Box 8231, Washington University School of Medicine, 660 S. Euclid Ave. St. Louis, MO 63110. Tel.: 314-362-3344; Fax: 314-362-7183; E-mail: frieden{at}biochem.wustl.edu.

IFABP is a small (15 kDa) protein consisting mostly of antiparallel ß-strands that surround a large cavity into which ligands bind. We have previously used FCS to show that the native protein, labeled with fluorescein, exhibits dynamic fluctuation with a relaxation time of 35 µs. Here we report the use of FCS to study the unfolding of the protein induced by guanidine hydrochloride. Although the application of this technique to measure diffusion coefficients and molecular dynamics is straightforward, the FCS results need to be corrected for both viscosity and refractive index changes as the guanidine hydrochloride concentration increases. We present here a detailed study of the effects of viscosity and refractive index of guanidine hydrochloride solutions to calibrate FCS data. After correction, the increase in the diffusion time of IFABP corresponds well with the unfolding transition monitored by far ultraviolet circular dichroism. We also show that the magnitude of the 35 µs phase, reflecting the conformational fluctuation in the native state, decreases sharply as the concentration of denaturant increases and the protein unfolds. Although FCS experiments indicate that the unfolded state at pH 2 is rather compact and native-like, the radius in the presence of guanidine hydrochloride falls well within the range expected for a random coil.

This article has been cited by other articles:

				R. Engel, A. H. Westphal, D. H. E. W. Huberts, S. M. Nabuurs, S. Lindhoud, A. J. W. G. Visser, and C. P. M. van Mierlo Macromolecular Crowding Compacts Unfolded Apoflavodoxin and Causes Severe Aggregation of the Off-pathway Intermediate during Apoflavodoxin Folding J. Biol. Chem., October 10, 2008; 283(41): 27383 - 27394. [Abstract] [Full Text] [PDF]

				E. Sherman, A. Itkin, Y. Y. Kuttner, E. Rhoades, D. Amir, E. Haas, and G. Haran Using Fluorescence Correlation Spectroscopy to Study Conformational Changes in Denatured Proteins Biophys. J., June 15, 2008; 94(12): 4819 - 4827. [Abstract] [Full Text] [PDF]

				H. T. Tran and R. V. Pappu Toward an Accurate Theoretical Framework for Describing Ensembles for Proteins under Strongly Denaturing Conditions Biophys. J., September 1, 2006; 91(5): 1868 - 1886. [Abstract] [Full Text] [PDF]

				K. Chattopadhyay, E. L. Elson, and C. Frieden The kinetics of conformational fluctuations in an unfolded protein measured by fluorescence methods PNAS, February 15, 2005; 102(7): 2385 - 2389. [Abstract] [Full Text] [PDF]