Efficient solid-phase synthesis of Vpr from HIV-1 using low quantities of uniformly 13C-, 15N-labeled amino acids for NMR structural studies.

Abstract

The 96-amino acid protein Vpr functions as a regulator of cellular processes involved in the human immunodeficiency virus, type 1 (HIV-1) life cycle, including cell-cycle arrest at the G2/M check point, promotion of the HIV-1 preintegration complex for nuclear transport, induction of apoptosis and transcriptional activation of a variety of viral and cellular promoters. Preliminary 1H NMR experiments performed on Vpr fragments showed the presence of several helical regions. However, the assignment of many protons in the amide region of the complete sequence of Vpr proved to be impossible due to the overlap of multiple NOE cross peaks. Moreover, because of its cytotoxicity, it is difficult to produce large quantities of 15N- and 13C-labeled Vpr using molecular biology approaches. Therefore, the solid-phase peptide synthesis of (1-96)Vpr, labeled at 22 selected positions, using recently commercially available uniformly 13C-, 15N-labeled fmoc amino acids, has been optimized to produce large quantities (104 mg, 15% yield) of pure compound, while minimizing the quantity of labeled amino acids used for each coupling. As expected two-dimensional heteronuclear NMR experiments performed with this protein allowed the unequivocal assignments of all the proton signals. This study shows that introduction of few labeled 13C/15N labeled amino acids in selected positions facilitates the determination of structure solution of small protein accessible by solid-phase peptide synthesis, and could allow dynamic studies of their conformational behavior to be carried out.