The effect of azide and thiocyanate on the structure and dynamics of wild type and disulfide bond depleted azurin and of amicyanin has been investigated by electron paramagnetic resonance (EPR)... Show moreThe effect of azide and thiocyanate on the structure and dynamics of wild type and disulfide bond depleted azurin and of amicyanin has been investigated by electron paramagnetic resonance (EPR) spectroscopy at low temperature. The analysis of the EPR spectra, which can be described in terms of Gaussian distributions of the components of the axial symmetric (g) over left right arrow and (A) over left right arrow tensors of the spin-Hamiltonian, has shown that the two small exogenous ligands, known as chaotropic agents, are effective in reducing the structural heterogeneity of the proteins, Such a reduction, quantified by the standard deviations sigma(gparallel to) and sigma(A\) and obtained by simulation of the experimental EPR spectra, depends on azide and thiocyanate concentration in solution. In particular, the comparison of the sigma(a\) and sigma(Aparallel to) values found for the protein samples investigated points out that the lower the protein to anion molar ratios (1:50; 1:100) are, the more marked the reduction in structural heterogeneity is. The thiocyanate effect is stronger than the azide one. Furthermore, the reduction in structural heterogeneity is more marked in the azurins than in amicyanin and the Cys3Ala/Cys26Ala azurin mutant is less flexible compared to the wild-type protein. The effect observed upon N-3(-) and SCN- addition in solution is very similar to that observed when glycerol is added to the solution, suggesting that such perturbing agents behave like cryoprotectors, affecting the protein-solvent interactions in such a way as to suppress the large amplitude motions, (C) 2002 Elsevier Science Inc. All rights reserved. Show less
