Resolving fast and slow motions in the internal loop containing stem-loop 1 of HIV-1 that are modulated by Mg(2+) binding: role in the kissing–duplex structural transition

Stem loop 1 (SL1) is a highly conserved hairpin in the 5′-leader of the human immunodeficiency virus type I that forms a metastable kissing dimer that is converted during viral maturation into a stable duplex with the aid of the nucleocapsid (NC) protein. SL1 contains a highly conserved internal loop that promotes the kissing–duplex transition by a mechanism that remains poorly understood. Using NMR, we characterized internal motions induced by the internal loop in an SL1 monomer that may promote the kissing–duplex transition. This includes micro-to-millisecond secondary structural transitions that cause partial melting of three base-pairs above the internal loop making them key nucleation sites for exchanging strands and nanosecond rigid-body stem motions that can help bring strands into spatial register. We show that while Mg(2+) binds to the internal loop and arrests these internal motions, it preserves and/or activates local mobility at internal loop residues G272 and G273 which are implicated in NC binding. By stabilizing SL1 without compromising the accessibility of G272 and G273 for NC binding, Mg(2+) may increase the dependence of the kissing–duplex transition on NC binding thus preventing spontaneous transitions from taking place and ensuring that viral RNA and protein maturation occur in concert

Review NMR studies of RNA dynamics and structural plasticity using NMR residual dipolar couplings

An increasing number of RNAs are being discovered that perform their functions by undergoing large changes in conformation in response to a variety of cellular signals, including recognition of proteins and small molecular targets, changes in temperature, and RNA synthesis itself. The measurement of NMR residual dipolar couplings (RDCs) in partially aligned systems is providing new insights into the structural plasticity of RNA through combined characterization of large-amplitude collective helix motions and local flexibility in noncanonical regions over a wide window of biologically relevant timescales (<milliseconds). Here, we review RDC methodology for studying RNA structural dynamics and survey what has been learnt thus far from application of these methods. Future methodological challenges are also identified. © 2007 Wiley Periodicals, Inc. Biopolymers 86: 384–402, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected] Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56044/1/20765_ftp.pd

Direct detection of N-H[...]N hydrogen bonds in biomolecules by NMR spectroscopy.

International audienceA nuclear magnetic resonance (NMR) experiment is described for the direct detection of N-H[...]N hydrogen bonds (H-bonds) in 15N isotope-labeled biomolecules. This quantitative HNN-COSY (correlation spectroscopy) experiment detects and quantifies electron-mediated scalar couplings across the H-bond (H-bond scalar couplings), which connect magnetically active (15)N nuclei of the H-bond donor and acceptor. Detectable H-bonds comprise the imino H-bonds in canonical Watson-Crick base pairs, many H-bonds in unusual nucleic acid base pairs and H-bonds between protein backbone or side-chain N-H donor and N acceptor moieties. Unlike other NMR observables, which provide only indirect evidence of the presence of H-bonds, the H-bond scalar couplings identify all partners of the H-bond, the donor, the donor proton and the acceptor in a single experiment. The size of the scalar couplings can be related to H-bond geometries and as a time average to H-bond dynamics. The time required to detect the H-bonds is typically less than 1 d at millimolar concentrations for samples of molecular weight < or = approximately 25 kDa. A C15N/13C-labeled potato spindle tuber viroid T1 RNA domain is used as an example to illustrate this procedure

Macromolecular NMR spectroscopy for the non-spectroscopist

NMR spectroscopy is a powerful tool for studying the structure, function and dynamics of biological macromolecules. However, non-spectroscopists often find NMR theory daunting and data interpretation nontrivial. As the first of two back-to-back reviews on NMR spectroscopy aimed at non-spectroscopists, the present review first provides an introduction to the basics of macromolecular NMR spectroscopy, including a discussion of typical sample requirements and what information can be obtained from simple NMR experiments. We then review the use of NMR spectroscopy for determining the 3D structures of macromolecules and examine how to judge the quality of NMR-derived structures