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

The 96-amino acid protein Vpr functions as a regulator of cellular processe s involved in the human immunodeficiency virus, type 1 (HIV-1) life cycle, including cell-cycle arrest at the G(2)/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. P reliminary H-1 NMR experiments performed on Vpr fragments showed the presen ce of several helical regions. However, the assignment of many protons in t he 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 cytot oxicity, it is difficult to produce large quantities of N-15- and C-13-labe led Vpr using molecular biology approaches. Therefore, the solid-phase pept ide synthesis of (1-96)Vpr, labeled at 22 selected positions, using recentl y commercially available uniformly C-13-, N-15-labeled fmoc amino acids, ha s been optimized to produce large quantities (104 mg, 15% yield) of pure co mpound, while minimizing the quantity of labeled amino acids used for each coupling. As expected two-dimensional heteronuclear NMR experiments perform ed with this protein allowed the unequivocal assignments of all the proton signals. This study shows that introduction of few labeled C-13/N-15 labele d amino acids in selected positions facilitates the determination of struct ure solution of small protein accessible by solid-phase peptide synthesis, and could allow dynamic studies of their conformational behavior to be carr ied out.