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A novel 5 displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA.

ABSTRACT: An RNA spin-labeling technique was developed using the well-characterized interaction between the HIV Rev peptide and the Rev response element (RRE) RNA as a model system. Spin-labeled RNA molecules were prepared by incorporating guanosine monophosphorothioate (GMPS) at the 5' end using T7 RNA polymerase and then covalently attaching a thiol-specific nitroxide spin label. Three different constructs of the RRE RNA were made by strategically displacing the 5' end within the native three-dimensional structure. Nitroxide-to-nitroxide distance measurements were made between the specifically bound RNA and peptide using electron paramagnetic resonance (EPR) spectroscopy. The dipolar EPR method can reliably measure distances up to 25 A, the calculation of which is derived from the 1/r3 dependence of the broadening of EPR lines in motionally frozen samples. This RNA-labeling technique, dubbed 5' displacement spin labeling, extends the usefulness of the dipolar EPR method developed for analysis of protein structure. The advantage of this technique is that it is applicable to large RNA systems such as the ribosome, which are difficult to study by other structural methods.

SUBMITTER: Macosko JC

PROVIDER: S-EPMC1369839 | biostudies-other | 1999 Sep

REPOSITORIES: biostudies-other

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A novel 5 displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA.

Macosko J C JC Pio M S MS Tinoco I I Shin Y K YK

RNA (New York, N.Y.) 19990901 9

An RNA spin-labeling technique was developed using the well-characterized interaction between the HIV Rev peptide and the Rev response element (RRE) RNA as a model system. Spin-labeled RNA molecules were prepared by incorporating guanosine monophosphorothioate (GMPS) at the 5' end using T7 RNA polymerase and then covalently attaching a thiol-specific nitroxide spin label. Three different constructs of the RRE RNA were made by strategically displacing the 5' end within the native three-dimensiona ...[more]

PMID: 10496217

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Project description:KCNE1 is a single transmembrane protein that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in the genes encoding either protein can result in diseases such as congenital deafness, long QT syndrome, ventricular tachyarrhythmia, syncope, and sudden cardiac death. Despite the biological significance of KCNE1, the structure and dynamic properties of its physiologically relevant native membrane-bound state are not fully understood. In this study, the structural dynamics and topology of KCNE1 in bilayered lipid vesicles was investigated using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A 53-residue nitroxide EPR scan of the KCNE1 protein sequence including all 27 residues of the transmembrane domain (45-71) and 26 residues of the N- and C-termini of KCNE1 in lipid bilayered vesicles was analyzed in terms of nitroxide side-chain motion. Continuous wave-EPR spectral line shape analysis indicated the nitroxide spin label side-chains located in the KCNE1 TMD are less mobile when compared to the extracellular region of KCNE1. The EPR data also revealed that the C-terminus of KCNE1 is more mobile when compared to the N-terminus. EPR power saturation experiments were performed on 41 sites including 18 residues previously proposed to reside in the transmembrane domain (TMD) and 23 residues of the N- and C-termini to determine the topology of KCNE1 with respect to the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) lipid bilayers. The results indicated that the transmembrane domain is indeed buried within the membrane, spanning the width of the lipid bilayer. Power saturation data also revealed that the extracellular region of KCNE1 is solvent-exposed with some of the portions partially or weakly interacting with the membrane surface. These results are consistent with the previously published solution NMR structure of KCNE1 in micelles.

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A novel 5 displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA.

Publications

A novel 5 displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA.

Similar Datasets

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