The oxidized blue copper proteins azurin and stellacyanin have been investigated through 1H NMR at 800 MHz and the results compared with those for plastocyanin (Bertini, I.; Ciurli, S.; Dikiy, A.; Gasanov, R.; Luchinat, C.; Martini, G.; Safarov, N. J. Am. Chem. Soc. 1999, 121, 2037). By exploiting saturation transfer between the oxidized and the reduced forms, all the hyperfine shifted signals can be assigned, including the β-CH2 protons of the coordinated cysteines, which are so broad not to be detected under direct observation. Both hyperfine shifts and line widths of the latter signals differ dramatically from one protein to another:  average hyperfine shifts of about 850, 600, and 400 ppm and average line widths of 1.2, 0.45, and 0.25 MHz are observed for azurin, plastocyanin, and stellacyanin, in that order. The observation of a nuclear line width of 1.2 MHz is unprecedented in high-resolution NMR in solution. These data are interpreted as a measure of the out-of-plane displacement of the copper ion, which increases on passing from azurin to plastocyanin to stellacyanin. The present approach seems general for the investigation of blue copper proteins.

Protein-protein association is often accompanied by changes in receptor and ligand structure. This interplay between protein flexibility and protein-protein recognition is currently the largest obstacle both to our understanding of and to the reliable prediction of protein complexes. We performed two sets of molecular dynamics simulations for the unbound receptor and ligand structures of 17 protein complexes and applied shape-driven rigid body docking to all combinations of representative snapshots. The crossdocking of structure ensembles increased the likelihood of finding near-native solutions. The free ensembles appeared to contain multiple complementary conformations. These were in general not related to the bound structure. We suggest that protein-protein binding follows a three-step mechanism of diffusion, free conformer selection, and refolding. This model combines previously conflicting ideas and is in better agreement with the current data on interaction forces, time scales, and kinetics.

Experimental data for the unfolding of cytochrome c and azurin by guanidinium chloride (GuHCl) are used to construct free-energy diagrams for the folding of the oxidized and reduced proteins. With cytochrome c, the driving force for folding the reduced protein is larger than that for the oxidized form. Both the oxidized and the reduced folded forms of yeast cytochrome c are less stable than the corresponding states of the horse protein. Due to the covalent attachment of the heme and its fixed tetragonal coordination geometry, cytochrome c folding can be described by a two-state model. A thermodynamic cycle leads to an expression for the difference in self-exchange reorganization energies for the folded and unfolded proteins. The reorganization energy for electron exchange in the folded protein is approximately 0.5 eV smaller than that for a heme in aqueous solution. The finding that reduced azurin unfolds at lower GuHCl concentrations than the oxidized protein suggests that the coordination structure of copper is different in oxidized and reduced unfolded states: it is likely that the geometry of Cu I in the unfolded protein is linear or trigonal, whereas Cu II prefers to be tetragonal. The evidence indicates that protein folding lowers the azurin reorganization energy by roughly 1.7 eV relative to an aqueous Cu(1, 10-phenanthroline) 2 2؉͞؉ reference system.The folding of a protein to its native three-dimensional structure is a spontaneous process, driven by the tendency of the peptide chain to assume the conformation of minimum free energy. As first clearly enunciated by Lumry and Eyring in 1954 (1, 2), the universal minimum for a given protein (i.e., for a specific amino acid sequence) may be reached at the expense of some local energy maximum. They further suggested that evolution has availed itself of this so-called rack phenomenon to create strain and distortion in prosthetic groups or coenzymes, thereby tuning the electronic properties by the mechanical force. This idea also led to a visualization of evolutionary fine tuning of active-site properties in protein superfamilies by small variations in amino acid sequences. The idea of conformationally induced strain in protein active sites was further developed both by Lumry himself (3) and by other authors. Vallee and Williams (4) stressed, in particular, how strain in the active site of the ground state of a catalytic metalloenzyme (e.g., a blue copper protein) can poise the metal ion for its reaction with substrate. The unique properties of blue copper were first described in 1960 (5), and they were attributed to a rack mechanism by one of us in 1964 (6). The first attempt to estimate the rack energy for blue copper, based on ligand-field considerations, was published in 1983 (7), and recently, Brill (8) has developed a model to calculate the mechanical energy associated with stress and strain and applied it to one specific blue protein, azurin. Interestingly, electronic structure calculations (9, 10) and spectroscopic experiments (9) have suggested...

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To better understand the interplay between protein-protein binding and protein dynamics, we analyzed molecular dynamics simulations of 17 protein-protein complexes and their unbound components. Complex formation does not restrict the conformational freedom of the partner proteins as a whole, but, rather, it leads to a redistribution of dynamics. We calculate the change in conformational entropy for seven complexes with quasiharmonic analysis. We see significant loss, but also increased or unchanged conformational entropy. Where comparison is possible, the results are consistent with experimental data. However, stringent error estimates based on multiple independent simulations reveal large uncertainties that are usually overlooked. We observe substantial gains of pseudo entropy in individual partner proteins, and we observe that all complexes retain residual stabilizing intermolecular motions. Consequently, protein flexibility has an important influence on the thermodynamics of binding and may disfavor as well as favor association. These results support a recently proposed unified model for flexible protein-protein association.

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