The association of two proteins is preceded by a mutual diffusional search in solution. The role of translational and rotational diffusion in this process has been studied theoretically for many years. However, systematic experimental verification of theoretical results is still lacking. We report here measurements of association rates of the proteins beta-lactamase (TEM) and beta-lactamase inhibitor protein (BLIP) in solutions of glycerol and poly(ethylene glycol) of increasing viscosity. We also measured translational and rotational diffusion in the same solutions, using fluorescence correlation spectroscopy and fluorescence anisotropy, respectively. It is found that in glycerol both translational and rotational diffusion rates are inversely dependent on viscosity, as predicted by the classical Stokes-Einstein relations, while the association rate depends nonlinearly on viscosity. In contrast, the association rate depends only weakly on the viscosity of the polymer solutions, which results in a similar weak dependence of k(on) on viscosity. The data are modeled using the theory of diffusion-limited association. Deviations from the theory are explained by a short-range solute-induced repulsion between the proteins in glycerol solution and an attractive depletion interaction generated by the polymers. These results open the way to the creation of a unified framework for all nonspecific effects involved in the protein association process, as well as to better theoretical understanding of these effects. Further, they reflect on the complex factors controlling protein association within the crowded environment of cells and suggest that a high concentration of macromolecules does not significantly impede protein association.

The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 is a target for virus neutralizing antibodies. The V3 sequence determines whether the virus will manifest R5 or ×4 phenotypes and use the CCR5 or C×CR4 chemokine co-receptor, respectively. Previous NMR studies revealed that both R5- and ×4-V3 peptides bound to antibodies 0.5β and 447-52D form β-hairpin conformations with the GPGR segment at the turn. In contrast, in their free form, linear V3 peptides and a cyclic peptide consisting of the entire 35-residue V3 loop were highly unstructured in aqueous solution. Herein we evaluated a series of synthetic disulfide constrained V3-peptides in which the position of the disulfide bonds, and therefore the ring size, was systematically varied. NMR structures determined for singly and doubly disulfide constrained V3-peptides in aqueous solution were compared with those found for unconstrained V3JRFL and V3IIIB peptides bound to 447-52D and to 0.5β, respectively. Our study indicated that cyclic V3 peptides manifested significantly reduced conformational space compared to their linear homologues and that in all cases cyclic peptides exhibited cross strand interactions suggestive of β-hairpin like structures. Nevertheless, the singly constrained V3-peptides retained significant flexibility and did not form an idealized p-hairpin. Incorporation of a second disulfide bond results in significant overall rigidity and in one case, a structure close to that of V3MN peptide bound to 447-52D Fab was assumed and in another case a structure close to that formed by the linear V3IIIB peptide bound to antibody 0.5β was assumed.