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Nanometer distance measurements in RNA using site-directed spin labeling.

ABSTRACT: The method of site-directed spin labeling (SDSL) utilizes a stable nitroxide radical to obtain structural and dynamic information on biomolecules. Measuring dipolar interactions between pairs of nitroxides yields internitroxide distances, from which quantitative structural information can be derived. This study evaluates SDSL distance measurements in RNA using a nitroxide probe, designated as R5, which is attached in an efficient and cost-effective manner to backbone phosphorothioate sites that are chemically substituted in arbitrary sequences. It is shown that R5 does not perturb the global structure of the A-form RNA helix. Six sets of internitroxide distances, ranging from 20 to 50 A, were measured on an RNA duplex with a known X-ray crystal structure. The measured distances strongly correlate (R(2) = 0.97) with those predicted using an efficient algorithm for determining the expected internitroxide distances from the parent RNA structure. The results enable future studies of global RNA structures for which high-resolution structural data are absent.

SUBMITTER: Cai Q

PROVIDER: S-EPMC1959523 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Nanometer distance measurements in RNA using site-directed spin labeling.

Cai Qi Q Kusnetzow Ana Karin AK Hideg Kálmán K Price Eric A EA Haworth Ian S IS Qin Peter Z PZ

Biophysical journal 20070525 6

The method of site-directed spin labeling (SDSL) utilizes a stable nitroxide radical to obtain structural and dynamic information on biomolecules. Measuring dipolar interactions between pairs of nitroxides yields internitroxide distances, from which quantitative structural information can be derived. This study evaluates SDSL distance measurements in RNA using a nitroxide probe, designated as R5, which is attached in an efficient and cost-effective manner to backbone phosphorothioate sites that ...[more]

PMID: 17526583

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Project description:Protein structures are not static but sample different conformations over a range of amplitudes and time scales. These fluctuations may involve relatively small changes in bond angles or quite large rearrangements in secondary structure and tertiary fold. The equilibrium between discrete structural substates on the microsecond to millisecond time scale is sometimes termed conformational exchange. Protein dynamics and conformational exchange are believed to provide the basis for many important activities, such as protein-protein and protein-ligand interactions, enzymatic activity and protein allostery; however, for many proteins, the dynamics and conformational exchange that lead to function are poorly defined. Spectroscopic methods, such as NMR, are among the most important methods to explore protein dynamics and conformational exchange; however, they are difficult to implement in some systems and with some types of exchange events. Site-directed spin labeling (SDSL) is an EPR based approach that is particularly well-suited to high molecular-weight systems such as membrane proteins. Because of the relatively fast time scale for EPR spectroscopy, it is an excellent method to examine exchange. Conformations that are in exchange are captured as distinct populations in the EPR spectrum, and this feature when combined with the use of methods that can shift the free energy of conformational substates allows one to identify regions of proteins that are in dynamic exchange. In addition, modern pulse EPR methods have the ability to examine conformational heterogeneity, resolve discrete protein states, and identify the substates in exchange. Protein crystallography has provided high-resolution models for a number of membrane proteins; but because of conformational exchange, these models do not always reflect the structures that are present when the protein is in a native bilayer environment. In the case of the Escherichia coli vitamin B12 transporter, BtuB, the energy coupling segment of this protein undergoes a substrate-dependent unfolding, which acts to couple this outer-membrane protein to the inner-membrane protein TonB. EPR spectroscopy demonstrates that the energy coupling segment is in equilibrium between ordered and disordered states, and that substrate binding shifts this equilibrium to favor an unfolded state. However, in crystal structures of BtuB, this segment is resolved and folded within the protein, and neither the presence of this equilibrium nor the substrate-induced change is revealed. This is a result of the solute environment and the crystal lattice, both of which act to stabilize one conformational substate of the transporter. Using SDSL, it can be shown that conformational exchange is present in other regions of BtuB and in other members of this transporter family. Conformational exchange has also been examined in systems such as the plasma membrane SNARE protein, syntaxin 1A, where dynamics are controlled by regulatory proteins such as munc18. Regulating the microsecond to millisecond time scale dynamics in the neuronal SNAREs is likely to be a key feature that regulates assembly of the SNAREs and neurotransmitter release.

Dataset Information

Nanometer distance measurements in RNA using site-directed spin labeling.

Publications

Nanometer distance measurements in RNA using site-directed spin labeling.

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