The solution structure of a human immunodeficiency virus type-1 (HIV-1) Rev peptide bound to stem-loop IIB of the Rev response element (RRE) RNA was solved by nuclear magnetic resonance spectroscopy. The Rev peptide has an alpha-helical conformation and binds in the major groove of the RNA near a purine-rich internal loop. Several arginine side chains make base-specific contacts, and an asparagine residue contacts a G.A base pair. The phosphate backbone adjacent to a G.G base pair adopts an unusual structure that allows the peptide to access a widened major groove. The structure formed by the two purine-purine base pairs of the RRE creates a distinctive binding pocket that the peptide can use for specific recognition.

The near complete (>90%) NMR assignment of 15N, 13Cα, 13Cβ, and HN chemical shifts is presented for a 64 kDa trp repressor−operator complex consisting of two tandem dimers of 15N,13C,>90% 2H labeled trp repressor, unlabeled 22-base-pair DNA, and unlabeled corepressor, 5-methyltryptophan. The DNA sequence employed contains three copies of the palindromic sequence 5‘-CTAG-3‘, allowing two dimers of trp repressor to bind to each duplex operator DNA. Chemical shift data establish that each subunit within a given dimer in the complex is in a chemically distinct environment, and the pattern of chemical shift differences between subunits provides information regarding interdimer contacts. Because of the large size of the complex, a number of modifications were made to existing enhanced sensitivity triple-resonance correlation experiments which link 13Cβ, 15N, and HN chemical shifts; the pulse sequences which include these changes are presented. The experiments make use of constant-time chemical shift evolution of the carbon magnetization, resulting in significant improvements in spectral resolution compared to non-constant-time versions of the pulse schemes. An analysis of the utility of the enhanced sensitivity method for recording spectra of high molecular weight deuterated proteins indicates that this approach produces reasonable sensitivity gains for the 64 kDa trp repressor−operator complex studied here.

Abstract:Multidimensional heteronuclear NMR has been applied to the structural analysis of myotrophin, a novel protein identified from spontaneously hypertensive rat hearts and hypertrophic human hearts. Myotrophin has been shown to stimulate protein synthesis in myocytes and likely plays an important role in the initiation of cardiac hypertrophy, a major cause of mortality in humans. Recent cDNA cloning revealed that myotrophin has 118 amino acids containing 2.5 contiguous ANK repeats, a motif known to be involved in a wide range of macromolecular recognition. A series of two-and three-dimensional heteronuclear bond correlation NMR experiments have been performed on uniformly "N-labeled or uniformly '5N/'3C-labeled protein to obtain the 'H, "N, and I3C chemical shift assignments. The secondary structure of myotrophin has been determined by a combination of NOES, NH exchange data, 3JHNa coupling constants, and chemical shifts of 'Ha, "C,, and I3Cp. The protein has been found to consist of seven helices, all connected by turns or loops. Six of the seven helices (all but the C-terminal helix) form three separate helix-turn-helix motifs. The two full ANK repeats in myotrophin are characteristic of multiple turns followed by a helix-turn-helix motif. A hairpin-like turn involving L32-R36 in ANK repeat #1 exhibits slow conformational averaging on the NMR time scale and appears dynamically different from the corresponding region (D65-169) of ANK repeat #2.

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A concise, simple, and efficient method for remote C–H bond halogenation (Br and Cl) of 8‐aminoquinoline scaffolds at the geometrically difficult‐to‐access C5 position was explored with diverse substrate combinations in DMF. This protocol made use of inexpensive CuBr2 and CuCl2 as mediators and showed good to excellent yields for as many as 24 substrate combinations. The outstanding site selectivity of the reaction was reflected by the lack of formation of the undesired C7‐halogenated byproduct originating from the high nucleophilic reactivity of the 8‐aminoquinoline scaffold at the C7 position, even if excess amounts of the CuX2 salts were used.