help button home button Biophys. J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

Biophysical Journal 69: 202-213 (1995)
© 1995 the Biophysical Society

This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lindgren, M
Right arrow Articles by Jonsson, B H
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lindgren, M
Right arrow Articles by Jonsson, B H

Characterization of a folding intermediate of human carbonic anhydrase II: probing local mobility by electron paramagnetic resonance.

M Lindgren, M Svensson, P O Freskgård, U Carlsson, P Jonasson, L G Mårtensson and B H Jonsson

Department of Chemical Physics, Linköping University, Sweden.

ABSTRACT

The spin-labeling method was used to investigate human carbonic anhydrase, HCA II, undergoing unfolding induced by guanidine-HCI (Gu-HCI). The spin-probe, N-(2,2,5,5-tetramethyl-1-yloxypyrrolidinyl-3-yl)iodoacetamide, was attached covalently to the single cysteine (position 206) in the enzyme. The electron paramagnetic resonance spectrum of the folded structure showed the characteristic slow motional spectra. When the concentration of the denaturing agent, Gu-HCI, was gradually increased, new spectral components with narrower lines evolved to give complex electron paramagnetic resonance spectra, apparently containing superimposed contributions from several components of different mobility. By a differentiation technique, it was possible to follow the relative increase of the narrow components as a function of Gu-HCI concentration. The amplitude of difference spectra versus Gu-HCI concentration showed two distinct maxima, indicating the existence of a folding intermediate state/structure. The results were found to agree with optical absorption data, which showed similar transitions at the same Gu-HCI concentrations. From line-shape simulations assuming a Brownian diffusion model, the rotational diffusion constants for the spin-label in the folded, folding intermediate, and unfolded structures were determined. The relative abundances of the three conformations in the region 0-4 M Gu-HCI were obtained by least squares fitting of the simulated spectra to the experimental ones. The folding intermediate was found to have a maximum population of 39 +/- 4% at approximately 0.7 M Gu-HCI.






HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 1995 by the Biophysical Society.