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

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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.