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Abstract

We describe solid state NMR measurements on frozen solutions of the complex of the 24-residue HIV-1 gp120 V3 loop peptide RP135 with the Fab fragment of the anti-gp120 antibody 0.5beta, using rotational echo double resonance (REDOR). In order to probe possible hydrogen bonding between arginine side chains and glycine backbone carbonyls in the region of the conserved Gly-Pro-Gly-Arg (GPGR) motif of the V3 loop, RP135 samples were prepared with 15N labels at the eta nitrogen positions of arginine side chains and 13C labels at glycine carbonyl positions and 13C-detected 13C-15N REDOR measurements were performed on peptide/antibody complexes of these labeled samples. Such hydrogen bonding was previously observed in a crystal structure of the V3 loop peptide/antibody complex RP142/59.1 [Ghiara et al. (1994) Science, 264, 82-85], but is shown by the REDOR measurements to be absent in the RP135/0.5beta complex. These results confirm the antibody-dependent conformational differences in the GPGR motif suggested by previously reported solid state NMR measurements of phi and psi backbone dihedral angles in the RP135/0.53 complex. In addition, we describe REDOR measurements on the helical synthetic peptide MB(i+4)EK in frozen solution that establish our ability to detect 13C-15N dipole-dipole couplings in the distance range appropriate to these hydrogen bonding studies. We also report the results of molecular modeling calculations on the central portion RP135, using a combination of the solid state NMR restraints of Weliky et al. [Nat. Struct. Biol., 6, 141-145, 1999] and the liquid state NMR restraints of Tugarinov et al. (Nat. Struct. Biol., 6, 331-335, 1999]. The dynamics calculations demonstrate the mutual compatibility of the two sets of experimental structural restraints and reduce ambiguities in the solid state NMR restraints that result from symmetry and signal-to-noise considerations.

Magic angle spinning NMR of viruses

“…Another study from the Weliky group introduced an approach to examine lyophilized whole cells containing an expressed 154-residue gp41 fragment using MAS NMR, and detected helical structure at the C-terminal of this protein construct by REDOR experiments [193]. In addition to the studies on fusion peptides, MAS NMR was also applied to investigate the V3 loop in gp120 and its interaction with antibody fragments [194, 195]. …”

Glycosylation of HIV-1 gp120 V3 Loop: Towards the Rational Design of a Synthetic Carbohydrate Vaccine

“…7). A sequence comparison from different isolates shows that there are three conserved regions within V3loop: an Nterminal loop, a GPG crown (or crest), and a C-terminal -helix [20,[103][104][105]. Residues 306-320 (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) were designated V3 crown and residues 296 to 305 (1-10) as well as 321 to 330 (26)(27)(28)(29)(30)(31)(32)(33)(34)(35) were designated N and C-terminal strands of the V3 stem [106].…”

Structural and dynamic studies of proteins by high-resolution solid-state NMR

“…Rather than exploiting sample orientation, MAS experiments average orientation-dependent interactions as an alternative approach to achieve narrow resonances, thereby increasing resolution and sensitivity in the spectrum. Several groups are currently investigating selectively labeled membrane peptides and proteins by high-resolution MAS SSNMR [83][84][85][86][87][88][89][90][91][92][93][94][95][96], or investigate on general features of these proteins [97][98][99][100][101]. The ability of SSNMR to report on structural features of fully labeled ligands interacting with integral membrane proteins was demonstrated for several systems.…”