Recent developments in multidimensional, multinuclear
Our interest in developing further methodology for arginine side-chain assignment was peaked during structural NMR methods have greatly increased the scope of proteins and nucleic acids that can be studied by this technology (1). studies of an HIV-1 Rev peptide in complex with stemloop IIB of the Rev response element (RRE) RNA (4). One particularly promising area of investigation relates to structural studies of protein-RNA interactions and a number of Eleven of the 23 residues in the Rev peptide are arginines and many make specific contacts with bases on the RNA or NMR-derived structures of such complexes have been reported in the past few years (2-4). Accurate structures of these mo-are in position to establish electrostatic or hydrogen-bonding interactions with the phosphate backbone of the nucleic acid lecular complexes depend on the assignment of intermolecular NOEs correlating proximal protein and nucleic acid spins. A (4). Not surprisingly, in some cases it has been difficult to link H e protons with the aliphatic part of the side chain on key residue in stabilizing protein-nucleic acid interactions is arginine (4, 5). In fact, statistical analyses of protein structures the basis of experiments which correlate H d and H e chemical shifts ( 13). The difficulty arises in cases where the H d shifts establish that arginine is one of the three most abundant amino acids at molecular interfaces (6). Furthermore, arginines play of one residue overlap with those of another, or where cross peaks are simply absent. With this in mind, we present a a critical role in RNA recognition, especially in proteins with arginine-rich motifs such as TAT and REV from HIV, the N number of experiments which correlate the guanidino H e proton with either arginine aliphatic side-chain-carbon or proteins from lambdoid phages, and many viral and ribosomal -proton chemical shifts. The large numbers of potential aliproteins (7, 8). Additionally, site-specific substitutions of arginine for lysine in a number of proteins have been shown to enhance stability, suggesting that arginine residues might serve as stabilizing elements in proteins (9).
Given the importance of arginine residues, it is clearly necessary that methods be developed for the unambiguous assignment of arginine side-chain chemical shifts, with focus on the guanadino group in particular, since many of the molecular interactions involve contacts with this moiety. Several years ago, Yamazaki et al. developed experiments for correlating arginine H e , N e , and C z chemical shifts employing exclusively magnetization transfer via scalar connectivities (10). Subsequently, Wittekind and co-workers demonstrated that it is possible to observe (H e , N e , H d ) correlations in the HNHA-gly experiment (11) and Boelens et al. developed a scheme for correlating guanidino H e , N h chemical shifts (12). Finally, Yamazaki and co-workers FIG. 1. Portion of the 1 H-15 N HSQC spectrum of the 1:1 complex of have recently developed a family of experiments for sea 1.5 mM sample of 15 N, 13 C-labeled HIV-1 Rev peptide-RRE RNA illus- quence-specific assignment of arginine guanidino 15 N and trating the excellent dispersion that is often present in the (N e , H e ) region of correlation maps recorded for RNA-peptide complexes.