Measuring unfolding of proteins in the presence of denaturant using fluorescence correlation spectroscopy

¹ Washington University
² Harvard Medical School
³ Washington Univ. Sch. of Med.
⁴ Washington Univ. School of Medicine

^* To whom correspondence should be addressed. E-mail: frieden{at}biochem.wustl.edu.

Submitted on September 17, 2004
Revised on November 4, 2004
Accepted on 5 November 2004

	Abstract

The intestinal fatty acid binding protein (IFABP) is a small (15 kDa) protein consisting mostly of antiparallel beta-strands that surround a large cavity into which ligands bind. We have used fluorescence correlation spectroscopy (FCS) to show that the native protein, labeled with fluorescein, exihibits dynamic fluctuation with a relaxation time of 35 microsec [Chattopadhyay et al. (2002) Proc. Natl Acad Sci 99, 14171-14176]. 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 UV circular dichroism. We also show that the magnitude of the 35 microsec phase, reflecting the conformational fluctuation in the native state, decreases sharply as the concentration of denaturant increases and the protein unfolds. While 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.

Key Words: fluorescence, protein dynamics, protein folding, refractive index, unfolded state

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