Proteins with a long flexible polymeric tail attached at their N- or C-terminus are studied using molecular dynamics (MD) simulations of a coarse-grained model for protein folding where the temperature is regulated by either the Berendsen or the Langevin thermostat. These thermostats show different abilities to regulate the temperature of these systems that include flexible and more rigid regions. In the simulations with the Berendsen thermostat, the flexible tail is significantly hotter than the protein, both in its folded and unfolded states. Upon weakening the strength of the Berendsen thermostat, the temperature gradient between the fast and the slow degrees of freedom is significantly decreased, yet linkage between the temperatures of the flexible tail and the protein remains. The Langevin thermostat is proven to regulate the temperature of these inhomogenous systems reliably, without discriminating between the slow and fast degrees of freedom. The Langevin thermostat is less sensitive than is the Berendsen thermostat to the strength of the coupling between the protein system and the thermal bath. Our study calls for special care in choosing the thermostat for MD simulations of systems with inhomogenous degrees of freedom. Using the Berendsen thermostat with strong coupling would result in mistaken thermodynamic descriptions of such systems.

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.

The V3 region of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1 (HIV-1) is a potential target for an anti-HIV-1 vaccine. Peptides corresponding to V3 form three variations of a beta-hairpin conformation when bound to anti-V3 HIV-1 neutralizing antibodies. The conformation of a V3(IIIB) peptide bound to the 0.5beta antibody, generated against an X4 gp120, has been postulated to represent the V3 conformation of X4 viruses while the conformations of a V3(MN) and a V3(CONSENSUS) peptide bound to the 447-52D human monoclonal antibody were postulated to represent the R5A and R5B V3 conformations of R5 viruses, respectively. To constrain the conformation of synthetic V3 peptides to these X4, R5A, and R5B conformations, we formed disulfide bonds between Cys residues whose location in a peptide template representing the entire V3(CONSENSUS) epitope recognized by the broadly neutralizing 447-52D antibody was changed systematically. In a previous study [Mor, A., et al. (2009) Biochemistry 48, 3288-3303] we showed that these constrained peptides adopted conformations resembling the three antibody-bound V3 conformations according to the location of the disulfide bonds. Here we show that these constrained peptides, with the exception of peptides in which the disulfide bond flanks the GPGR segment, retain high-affinity binding to the 447-52D antibody. Compared with peptides designed to mimic the X4 conformation, peptides designed to mimic either the R5A or R5B conformation had higher affinity to 447-52D. It is possible that constrained peptides which mimic the R5A and R5B conformations of the V3 and retain high-affinity binding to 447-52D are good candidates for eliciting a broad neutralizing antibody response similar to that of 447-52D.

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