Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR Export

@article{citeulike:3781080, abstract = {10.1073/pnas.0802286105 We present a structurally dynamic model for nucleotide- and actin-induced closure of the actin-binding cleft of myosin, based on site-directed spin labeling and electron paramagnetic resonance (EPR) in myosin II. The actin-binding cleft is a solvent-filled cavity that extends to the nucleotide-binding pocket and has been predicted to close upon strong actin binding. Single-cysteine labeling sites were engineered to probe mobility and accessibility within the cleft. Addition of ADP and vanadate, which traps the posthydrolysis biochemical state, influenced probe mobility and accessibility slightly, whereas actin binding caused more dramatic changes in accessibility, consistent with cleft closure. We engineered five pairs of cysteine labeling sites to straddle the cleft, each pair having one label on the upper 50-kDa domain and one on the lower 50-kDa domain. Distances between spin-labeled sites were determined from the resulting spin\^{a}€“spin interactions, as measured by continuous wave EPR for distances of 0.7\^{a}€“2 nm or pulsed EPR (double electron\^{a}€“electron resonance) for distances of 1.7\^{a}€“6 nm. Because of the high distance resolution of EPR, at least two distinct structural states of the cleft were resolved. Each of the biochemical states tested (prehydrolysis, posthydrolysis, and rigor), reflects a mixture of these structural states, indicating that the coupling between biochemical and structural states is not rigid. The resulting model is much more dynamic than previously envisioned, with both open and closed conformations of the cleft interconverting, even in the rigor actomyosin complex.}, author = {Klein, Jennifer C. and Burr, Adam R. and Svensson, Bengt and Kennedy, Daniel J. and Allingham, John and Titus, Margaret A. and Rayment, Ivan and Thomas, David D.}, citeulike-article-id = {3781080}, citeulike-linkout-0 = {http://dx.doi.org/10.1073/pnas.0802286105}, citeulike-linkout-1 = {http://www.pnas.org/content/105/35/12867.abstract}, citeulike-linkout-2 = {http://www.pnas.org/content/105/35/12867.full.pdf}, citeulike-linkout-3 = {http://view.ncbi.nlm.nih.gov/pubmed/18725645}, citeulike-linkout-4 = {http://www.hubmed.org/display.cgi?uids=18725645}, day = {2}, doi = {10.1073/pnas.0802286105}, journal = {Proceedings of the National Academy of Sciences}, keywords = {epr, experiment, motor, myosin, structural\_change}, month = {September}, number = {35}, pages = {12867--12872}, posted-at = {2008-12-12 10:03:15}, priority = {2}, title = {Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR}, url = {http://dx.doi.org/10.1073/pnas.0802286105}, volume = {105}, year = {2008} }

TY - JOUR ID - citeulike:3781080 L3 - citeulike-article-id:3781080 N2 - 10.1073/pnas.0802286105 We present a structurally dynamic model for nucleotide- and actin-induced closure of the actin-binding cleft of myosin, based on site-directed spin labeling and electron paramagnetic resonance (EPR) in myosin II. The actin-binding cleft is a solvent-filled cavity that extends to the nucleotide-binding pocket and has been predicted to close upon strong actin binding. Single-cysteine labeling sites were engineered to probe mobility and accessibility within the cleft. Addition of ADP and vanadate, which traps the posthydrolysis biochemical state, influenced probe mobility and accessibility slightly, whereas actin binding caused more dramatic changes in accessibility, consistent with cleft closure. We engineered five pairs of cysteine labeling sites to straddle the cleft, each pair having one label on the upper 50-kDa domain and one on the lower 50-kDa domain. Distances between spin-labeled sites were determined from the resulting spin–spin interactions, as measured by continuous wave EPR for distances of 0.7–2 nm or pulsed EPR (double electron–electron resonance) for distances of 1.7–6 nm. Because of the high distance resolution of EPR, at least two distinct structural states of the cleft were resolved. Each of the biochemical states tested (prehydrolysis, posthydrolysis, and rigor), reflects a mixture of these structural states, indicating that the coupling between biochemical and structural states is not rigid. The resulting model is much more dynamic than previously envisioned, with both open and closed conformations of the cleft interconverting, even in the rigor actomyosin complex. IS - 35 JF - Proceedings of the National Academy of Sciences EP - 12872 TI - Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR VL - 105 SP - 12867 KW - epr KW - experiment KW - motor KW - myosin KW - structural_change AU - Klein, Jennifer AU - Burr, Adam AU - Svensson, Bengt AU - Kennedy, Daniel AU - Allingham, John AU - Titus, Margaret AU - Rayment, Ivan AU - Thomas, David PY - 2008/09/02/ UR - http://dx.doi.org/10.1073/pnas.0802286105 ER -