ARTICLE R. Guzzi á A. Stirpe á M.P. Verbeet á L. Sportelli Structural heterogeneity of blue copper proteins: an EPR study of amicyanin and of wild-type and Cys3Ala/Cys26Ala mutant azurin Received: 3 August 2000 / Revised version: 25 January 2001 / Accepted: 25 January 2001 / Published online: 25 April 2001 Ó Springer-Verlag 2001 Abstract A comparative investigation of the eects of cooling rate and solvent physicochemical properties on the structural heterogeneity of wild-type and disul®de bond depleted azurin Cys3Ala/Cys26Ala) and of amicyanin has been performed by EPR spectroscopy and computer simulation. By describing the spectral features of the EPR spectra in terms of Gaussian dis- tributions of the components of the g $ and A $ tensors of the spin Hamiltonian, we have shown that either the cooling rate or the solvent composition aect the structural heterogeneity of the proteins. Such a hetero- geneity has been quanti®ed by the standard deviations rg || and rA || of the parallel components of the axially symmetric tensors. In particular, both parameters be- come smaller after the slow cooling cycle; such a re- duction is more signi®cant when glycerol is added as co- solvent to the protein solutions. The comparison of the rg || and rA || values found, for the copper proteins in- vestigated, highlights that the reduction is more marked in the azurins compared to amicyanin and that the Cy- s3Ala/Cys26Ala azurin mutant has a structural hetero- geneity lower than that shown by the wild-type protein. The remarkable similarity of the copper coordination sphere of the proteins suggests a more rigid structure of the azurin protein matrix in the absence of the disul®de bridge compared to wild-type azurin and of amicyanin with respect to both forms of azurin. The former result establishes an important role for the -SS- bond in modulating the ¯exibility of wild-type azurin. Keywords Blue copper proteins á Electron paramagnetic resonance á Cooling rate á Glycerol á Disul®de bridge Introduction To perform physiological functions, proteins fold natu- rally to their three-dimensional structure that is stabi- lized by the delicate equilibrium between the inter- and intramolecular interactions of the dierent residues and of these with the water molecules surrounding the pro- teins. The compactness of the structure is favored also by the presence of disul®de bridges. In fact, removal of one of these bridges entails a stability decrease of the native state in favor of the denatured one Cooper et al. 1992; Guzzi et al. 1999). Along with the eect on the thermodynamic stability, the role of the disul®de bridge on the dynamic properties of the proteins is also an interesting aspect to be addressed. Proteins are complex systems that allow a multiplicity of pathways for the folding process Wang et al. 1996), which have been justi®ed by admitting the existence of a great abundance of conformational substates CS). Ex- perimental evidence for the existence of the CS, for many years focused on heme proteins, has been recently provided also for the blue copper proteins, diering from the former mainly by the folding topology essen- tially b-strands instead of a-helices) and by the active site geometry Ehrenstein and Nienhaus 1992; Cannistraro et al. 1997; Guzzi et al. 1997). In these CS the protein molecules dier from each other by the structural ar- rangement of the amino acid side chains, by the position of the hydrogen bonds and by their energy Frauenfelder 1987; Frauenfelder et al. 1988). The CS number depends on the temperature and on the physicochemical prop- erties of the solvent in which the proteins are dissolved. At room temperature, ¯uctuations among CS occur continuously, whereas by decreasing the temperature below the glass transition temperature T g 180 K) the protein molecules freeze in a particular conformation Eur Biophys J 2001) 30: 171±178 DOI 10.1007/s002490100146 R. Guzzi á A. Stirpe á L. Sportelli &) Dipartimento di Fisica e UnitaÁ INFM, Laboratorio di Bio®sica Molecolare, UniversitaÁ dellaCalabria, 87030 Rende CS), Italy E-mail: sportelli@®s.unical.it Fax: +39-0984-493187 M.P. Verbeet Leiden Institute of Chemistry, Gorleaus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands