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Characterizing the dynamics of the leader-linker interaction in the glycine riboswitch with site-directed spin labeling.

ABSTRACT: Site-directed spin labeling with continuous wave electron paramagnetic resonance (EPR) spectroscopy was utilized to characterize dynamic features of the kink-turn motif formed through a leader-linker interaction in the Vibrio cholerae glycine riboswitch. Efficient incorporation of spin-labels into select sites within the phosphate backbone of the leader-linker region proceeded via splinted ligation of chemically synthesized spin-labeled oligonucleotides to in vitro transcribed larger RNA fragments. The resultant nitroxide EPR line shapes have spectral characteristics consistent with a kink-turn motif and reveal differential backbone dynamics that are modulated by the presence of magnesium, potassium, and glycine.

SUBMITTER: Esquiaqui JM

PROVIDER: S-EPMC4059530 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Characterizing the dynamics of the leader-linker interaction in the glycine riboswitch with site-directed spin labeling.

Esquiaqui Jackie M JM Sherman Eileen M EM Ionescu Sandra A SA Ye Jing-Dong JD Fanucci Gail E GE

Biochemistry 20140528 22

Site-directed spin labeling with continuous wave electron paramagnetic resonance (EPR) spectroscopy was utilized to characterize dynamic features of the kink-turn motif formed through a leader-linker interaction in the Vibrio cholerae glycine riboswitch. Efficient incorporation of spin-labels into select sites within the phosphate backbone of the leader-linker region proceeded via splinted ligation of chemically synthesized spin-labeled oligonucleotides to in vitro transcribed larger RNA fragmen ...[more]

PMID: 24849816

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Project description:Simulation studies have been performed to evaluate the utility of site-directed spin labeling for determining the structures of protein-ligand complexes, given a known protein structure. Two protein-ligand complexes were used as model systems for these studies: a 1.9-A-resolution x-ray structure of a dihydrofolate reductase mutant complexed with methotrexate, and a 1.5-A-resolution x-ray structure of the V-Src tyrosine kinase SH2 domain complexed with a five-residue phosphopeptide. Nitroxide spin labels were modeled at five dihydrofolate reductase residue positions and at four SH2 domain residue positions. For both systems, after energy minimization, conformational ensembles of the spin-labeled residues were generated by simulated annealing while holding the remainder of the protein-ligand complex fixed. Effective distances, simulating those that could be obtained from (1)H-NMR relaxation measurements, were calculated between ligand protons and the spin labels. These were converted to restraints with several different levels of precision. Restrained simulated annealing calculations were then performed with the aim of reproducing target ligand-binding modes. The effects of incorporating a few supplementary short-range (< or =5.0 A) distance restraints were also examined. For the dihydrofolate reductase-methotrexate complex, the ligand-binding mode was reproduced reasonably well using relatively tight spin-label restraints, but methotrexate was poorly localized using loose spin-label restraints. Short-range and spin-label restraints proved to be complementary. For the SH2 domain-phosphopeptide complex without the short-range restraints, the peptide did not localize to the correct depth in the binding groove; nevertheless, the orientation and internal conformation of the peptide was reproduced moderately well. Use of the spin-label restraints in conjunction with the short-range restraints resulted in relatively well defined structural ensembles. These results indicate that restraints derived from site-directed spin labeling can contribute significantly to defining the orientations and conformations of bound ligands. Accurate ligand localization appears to require either a few supplementary short-range distance restraints, or relatively tight spin-label restraints, with at least one spin label positioned so that some of the restraints draw the ligand into the binding pocket in the latter case.

Dataset Information

Characterizing the dynamics of the leader-linker interaction in the glycine riboswitch with site-directed spin labeling.

Publications

Characterizing the dynamics of the leader-linker interaction in the glycine riboswitch with site-directed spin labeling.

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