{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T11:28:37Z","timestamp":1789558117171,"version":"build-2803163510"},"reference-count":59,"publisher":"National Academy of Sciences","issue":"39","content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2011,9,27]]},"abstract":"<jats:p>\n                    A disulfide-linked nitroxide side chain (R1) is the most widely used spin label for determining protein topology, mapping structural changes, and characterizing nanosecond backbone motions by site-directed spin labeling. Although the internal motion of R1 and the number of preferred rotamers are limited, translating interspin distance measurements and spatial orientation information into structural constraints is challenging. Here, we introduce a highly constrained nitroxide side chain designated RX as an alternative to R1 for these applications. RX is formed by a facile cross-linking reaction of a bifunctional methanethiosulfonate reagent with pairs of cysteine residues at\n                    <jats:italic>i<\/jats:italic>\n                    and\n                    <jats:italic>i<\/jats:italic>\n                    \u00a0+\u00a03 or\n                    <jats:italic>i<\/jats:italic>\n                    and\n                    <jats:italic>i<\/jats:italic>\n                    \u00a0+\u00a04 in an \u03b1-helix, at\n                    <jats:italic>i<\/jats:italic>\n                    and\n                    <jats:italic>i<\/jats:italic>\n                    \u00a0+\u00a02 in a \u03b2-strand, or with cysteine residues in adjacent strands in a \u03b2-sheet. Analysis of EPR spectra, a crystal structure of RX in T4 lysozyme, and pulsed electron-electron double resonance (ELDOR) spectroscopy on an immobilized protein containing RX all reveal a highly constrained internal motion of the side chain. Consistent with the constrained geometry, interspin distance distributions between pairs of RX side chains are narrower than those from analogous R1 pairs. As an important consequence of the constrained internal motion of RX, spectral diffusion detected with ELDOR reveals microsecond internal motions of the protein. Collectively, the data suggest that the RX side chain will be useful for distance mapping by EPR spectroscopy, determining spatial orientation of helical segments in oriented specimens, and measuring structural fluctuations on the microsecond time scale.\n                  <\/jats:p>","DOI":"10.1073\/pnas.1111420108","type":"journal-article","created":{"date-parts":[[2011,9,13]],"date-time":"2011-09-13T02:44:44Z","timestamp":1315881884000},"page":"16241-16246","update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":159,"title":["Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy"],"prefix":"10.1073","volume":"108","author":[{"given":"Mark R.","family":"Fleissner","sequence":"first","affiliation":[{"name":"Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-7008;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael D.","family":"Bridges","sequence":"additional","affiliation":[{"name":"Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-7008;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Evan K.","family":"Brooks","sequence":"additional","affiliation":[{"name":"Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-7008;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Duilio","family":"Cascio","sequence":"additional","affiliation":[{"name":"University of California, Los Angeles\u2013Department of Energy Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095-1570; and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tam\u00e1s","family":"K\u00e1lai","sequence":"additional","affiliation":[{"name":"Institute of Organic and Medicinal Chemistry, University of P\u00e9cs, H-7624 P\u00e9cs, Szigeti Strasse 12, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"K\u00e1lm\u00e1n","family":"Hideg","sequence":"additional","affiliation":[{"name":"Institute of Organic and Medicinal Chemistry, University of P\u00e9cs, H-7624 P\u00e9cs, Szigeti Strasse 12, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wayne L.","family":"Hubbell","sequence":"additional","affiliation":[{"name":"Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-7008;"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2011,9,12]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"crossref","first-page":"294","DOI":"10.1002\/prot.340060312","article-title":"Site-directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation","volume":"6","author":"Todd A","year":"1989","unstructured":"A Todd, V Crozel, F Levinthal, C Levinthal, WL Hubbell, Site-directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation. 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