NMR structural analysis of a peptide mimic of the bridging sheet of HIV-1 gp120 in methanol and water

Abstract

gp120 is a subunit of the Env (viral envelope protein) of HIV-1. The protein consists of inner and outer domains linked by a bridging sheet. Several gp120 residues that bind the neutralizing antibody 17b as well as the cellular co-receptor CCR5 (CC chemokine receptor 5), are located in the bridging sheet. Peptides that mimic the 17b-binding regions of gp120 would be useful potential immunogens for the generation of neutralizing antibodies against HIV-1. Towards this end, a 26-residue, four-stranded beta-sheet peptide was designed on the basis of the structure of the bridging sheet, and its structure was characterized in methanol by NMR. In methanol, amide and alpha-proton resonances were well resolved and dispersed. A number of interstrand NOEs (nuclear Overhauser effects) were observed, providing good evidence for multiple turn beta-hairpin structure. NOEs also provided good evidence for all Xxx-D-Pro bonds in the trans configuration and all three turns formed by a two residue D-Pro-Gly segment to be of type II' turn. The structure conforms well to the designed four-stranded beta-sheet structure. Approx. 20% of the peptide was estimated to adopt a folded conformation in water, as evidenced by CD spectroscopy. This was consistent with smaller, but still significant, downfield shifts of C(alpha)H protons relative to random-coil values. A second peptide was designed with two disulphide bonds to further constrain the peptide backbone. While structured in methanol, this peptide, like the previous one, also exhibits only partial structure formation in water, as evidenced by CD spectroscopy.

Conformational analysis and design of cross‐strand disulfides in antiparallel β‐sheets

“…Such engineered disulfides may provide a general method for stabilizing β‐rich proteins. Engineered cross‐strand disulfides may also be a useful strategy to rigidify β‐strands in peptide models of antiparallel β‐sheets 62, 63. In addition, we have rationalized the preference of cross‐strand disulfides for NHB relative to HB positions.…”

A systematic analysis of the beta hairpin motif in the Protein Data Bank

“…Beta strands are known to participate in protein-protein interactions that are often facilitated by specific amino acid orientations 1 and beta hairpin motifs are no different. [2][3][4] Indeed, these motifs are a core feature in a diverse array of bioactive molecules, from large beta barrel proteins that transport cargo through cellular membranes [5][6][7] to substantially smaller antimicrobial peptides and peptide derivatives. [8][9][10] Whether through self-aggregation, 11,12 target binding, 13 or amphipathic structure formation, 6,14 beta hairpin motifs facilitate a range of different biological functions.…”

Engineering a β‐Helical d,l‐Peptide for Folding in Polar Media

“…[57] We also assessed the possible influence of solvent by computing a second set of Dd values using the RC shifts obtained by Kessler and colleagues in 1:1 chloroform/methanol ( Figure 5 b). [58][59][60] In both cases, all non-turn residues of 1 m are downfield-shifted from random-coil values, suggesting a b-sheet-like conformation ( Figure 5 a and b). To our knowledge, these results constitute the first explicit demonstration that the NMR spectroscopic chemical shifts of b helices behave similarly to those of other b-sheet-like structures.…”

“…[50][51][52][54][55][56] To test this hypothesis, we plotted differences in the [57] b) Dd plotted for 1 m by using the RC values obtained by Kessler and co-workers in 1:1 CHCl 3 /MeOH. [58][59][60] c) Dd plotted for 1 w by using the aqueous RC values of Dobson. [57] d) Differences in the 1 H NMR CdH chemical shifts of 1 m with respect to 1 w (Dd = d 1 m Àd 1 w ).…”