The conformations of the protein and nucleic acid backbones in the filamentous viruses fd and Pf1 are characterized by one- and two-dimensional solid-state NMR experiments on oriented virus solutions. Striking differences are observed between fd and Pf1 in both their protein and DNA structures. The coat proteins of fd and Pf1 are almost entirely alpha helical and in both viruses most of the helix is oriented parallel to the filament axis. fd coat protein is one stretch of alpha helix that is slightly slued about the filament axis. In Pf1 coat protein two distinct sections of alpha helix are present, the smaller of which is tilted with respect to the filament axis by about 20 degrees. The DNA backbone structure of fd is completely disordered. By contrast, the DNA backbone of Pf1 is uniformly oriented such that all of the phosphodiester groups have the O-P-O plane of the nonesterified oxygens approximately perpendicular to the filament axis.

The major coat protein of filamentous bacteriophage adopts its membrane-bound conformation in detergent micelles. High-resolution 1H and 15N NMR experiments are used to characterize the structure and dynamics of residues 30-40 in the hydrophobic midsection of Pf1 coat protein in sodium dodecyl sulfate micelles. Uniform and specific-site 15N labels enable the immobile backbone sites to be identified by their 1H/15N heteronuclear nuclear Overhauser effect and allow the assignment of 1H and 15N resonances. About one-third of the amide N-H protons in the protein undergo very slow exchange with solvent deuterons, which is indicative of sites in highly structured environments. The combination of results from 1H/15N heteronuclear correlation, 1H homonuclear correlation, and 1H homonuclear Overhauser effect experiments assigns the resonances to specific residues and demonstrates that residues 30-40 of the coat protein have a helical secondary structure.

NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to produce segmentally labeled proteins is beneficial. Here we show that the S. aureus transpeptidase sortase A can be used to catalyze the ligation of two separately expressed domains of the same protein, MecA (B. subtilis). The yield of purified, segmentally labeled MecA protein conjugate is ∼40%. The resultant HSQC spectrum obtained from this domain-labeled conjugate demonstrates successful application of sortase A for segmental labeling of multi-domain proteins for solution NMR study.

A critical step in the HIV-1 lifecycle involves reverse transcription of the viral genomic RNA (gRNA). Human tRNA Lys3 serves as a primer for transcription initiation and is selectively enriched in virus particles. Human lysyl-tRNA synthetase (hLysRS) is also packaged into virions. Recently, a tRNA-like element (TLE) within the HIV-1 gRNA was shown to mimic the global tRNA fold and bind competitively to hLysRS, suggesting a mechanism of tRNA targeting to the primer binding site (PBS) and release from the synthetase. Here, we use NMR to investigate hLysRS anticodon-binding domain (ACB) binding to six RNA oligonucleotides, including a hairpin derived from the HIV-1 gRNA TLE. We show that ACB interacts with submicromolar affinity to U-rich RNA oligonucleotides-the tRNA Lys3 anticodon stem-loop (ACSL), the WT TLE, and a nonanucleotide, U9. In contrast, the ACB bound only weakly to two TLE loop mutants and a C9 nonanucleotide. NMR chemical shift perturbations induced by each RNA indicate that the ACSL and the WT TLE both interact with the ACB in a strikingly similar manner. Taken together, these findings support the conclusion that tRNA mimicry by the HIV-1 genome leads to a highly specific protein-RNA interaction that facilitates efficient primer release from hLysRS prior to reverse transcription.

The dynamics and conformation of the peptide antigen MHKDFLEKIGGL bound to the Fab' fragment of the monoclonal antipeptide antibody B13A2, raised against a peptide from myohemerythrin, have been investigated by isotope-edited NMR techniques. The peptides were labeled with 15N (98%) or 13C (99%) at the backbone of individual amino acid residues. Well-resolved amide proton and nitrogen backbone resonances were obtained and assigned for eight of the 12 residues of this bound peptide. Significant resonance line width and chemical shift differences were observed. The 15N and 1H line width variations are attributed to differential backbone mobilities among the bound peptide residues which are consistent with the previously mapped epitope of this peptide antigen. Local structural information was obtained from isotope-directed NOE studies. The approximate distances associated with the experimental NOEs were estimated on the basis of a theoretical NOE analysis involving the relative integrated intensities of the NOE and source peaks. In this way, the sequential NH-NH NOEs obtained for seven of the Fab'-bound peptide residues were shown to correspond to interproton separations of approximately 3 A or less. Such short distances indicate that the backbone dihedral angles of these residues are in the alpha rather than the beta region of phi,psi conformational space; the peptide most likely adopts a helical conformation from F5 to G11 within the antibody combining site. The significance of these results with respect to the type and extent of conformational information obtainable from studies of high molecular weight systems is discussed.