Probing local mobility in carbonic anhydrase: EPR of spin-labelled SH groups introduced by site-directed mutagenesis

Abstract

Cloned human carbonic anhydrase, HCAII, and mutants thereof have been investigated by spin-probing methods during its unfolding caused by guanidine·HCl. The spin-probe, N-(2,2,5,5-tetramethyl-1-ylooxypyrrolidin-3-yl)iodoacetamide, has been regiospecifically introduced into cyst-eines by site-directed mutagenesis in various positions of the protein structure. Here we focus on EPR spectra of three different spin-labelled enzymes at various guanidine-HCl concentrations (at equilibrium). The following spin-labelled mutants are discussed: W16C/C206S, W97C/C206S and F176C/C206S. The EPR spectra of the three mutants differ markedly at low guanidine·HCl concentration (0–1 mol dm^–3) particularly within the series W97C/C206S and F176C/C206S, showing the characteristic anisotropic slow motional features. The W16C/C206S label position is much more mobile in the folded structures. The rotational correlation times reflect the local environment of the spin-probe in the folded enzyme: in W97C it is located in the core, near the active centre, in W16C and F176C at more peripheral positions. All samples gave EPR spectra characteristic of a ‘free’ unfolded protein chain at guanidine-HCl concentrations of ca. 3 mol dm^–3 and above, and could be characterised by using one component in lineshape simulations. The spectra of the W97C/C206S and F176C/C206S samples in low concentrations of guanidine·HCl (between ca. 0.1 and 2.0 mol dm^–3) could only be reproduced in simulations by introducing several components associated with rather different rotational correlation times. This seems to imply the co-existence of at least two dynamic structures in equilibrium during the intermediate stages of the unfolding process. It is compatible with earlier suggestions of a folding intermediate based on optical characterisation.