caa a22 4500 445325542 CHVBK 20180317142719.0 cr unu---uuuuu 170323e20110901xx s 000 0 eng 10.1007/s10858-011-9545-x doi (NATIONALLICENCE)springer-10.1007/s10858-011-9545-x A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data [Elektronische Daten] [Nicolas Fawzi, Mark Fleissner, Nicholas Anthis, Tamás Kálai, Kálmán Hideg, Wayne Hubbell, G. Clore] The measurement of 1H transverse paramagnetic relaxation enhancement (PRE) has been used in biomolecular systems to determine long-range distance restraints and to visualize sparsely-populated transient states. The intrinsic flexibility of most nitroxide and metal-chelating paramagnetic spin-labels, however, complicates the quantitative interpretation of PREs due to delocalization of the paramagnetic center. Here, we present a novel, disulfide-linked nitroxide spin label, R1p, as an alternative to these flexible labels for PRE studies. When introduced at solvent-exposed α-helical positions in two model proteins, calmodulin (CaM) and T4 lysozyme (T4L), EPR measurements show that the R1p side chain exhibits dramatically reduced internal motion compared to the commonly used R1 spin label (generated by reacting cysteine with the spin labeling compound often referred to as MTSL). Further, only a single nitroxide position is necessary to account for the PREs arising from CaM S17R1p, while an ensemble comprising multiple conformations is necessary for those observed for CaM S17R1. Together, these observations suggest that the nitroxide adopts a single, fixed position when R1p is placed at solvent-exposed α-helical positions, greatly simplifying the interpretation of PRE data by removing the need to account for the intrinsic flexibility of the spin label. Springer Science+Business Media B.V., 2011 Paramagnetic relaxation enhancement nationallicence Site-directed spin-labeling nationallicence Electron paramagnetic resonance nationallicence Fawzi Nicolas Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 20892-0520, Bethesda, MA, USA aut Fleissner Mark Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, 90095, Los Angeles, CA, USA aut Anthis Nicholas Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 20892-0520, Bethesda, MA, USA aut Kálai Tamás Institute of Organic and Medicinal Chemistry, University of Pécs, Szigeti str. 12, 7624, Pécs, Hungary aut Hideg Kálmán Institute of Organic and Medicinal Chemistry, University of Pécs, Szigeti str. 12, 7624, Pécs, Hungary aut Hubbell Wayne Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, 90095, Los Angeles, CA, USA aut Clore G. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 20892-0520, Bethesda, MA, USA aut Journal of Biomolecular NMR Springer Netherlands 51/1-2(2011-09-01), 105-114 0925-2738 51:1-2<105 2011 51 10858 https://doi.org/10.1007/s10858-011-9545-x text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-011-9545-x text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Fawzi Nicolas Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 20892-0520, Bethesda, MA, USA aut NATIONALLICENCE 700 1- Fleissner Mark Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, 90095, Los Angeles, CA, USA aut NATIONALLICENCE 700 1- Anthis Nicholas Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 20892-0520, Bethesda, MA, USA aut NATIONALLICENCE 700 1- Kálai Tamás Institute of Organic and Medicinal Chemistry, University of Pécs, Szigeti str. 12, 7624, Pécs, Hungary aut NATIONALLICENCE 700 1- Hideg Kálmán Institute of Organic and Medicinal Chemistry, University of Pécs, Szigeti str. 12, 7624, Pécs, Hungary aut NATIONALLICENCE 700 1- Hubbell Wayne Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, 90095, Los Angeles, CA, USA aut NATIONALLICENCE 700 1- Clore G. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 20892-0520, Bethesda, MA, USA aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 51/1-2(2011-09-01), 105-114 0925-2738 51:1-2<105 2011 51 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer