Phosphorylation-induced changes in backbone dynamics of the dematin headpiece C-terminal domain
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Dematin is an actin-binding protein abundant in red blood cells and other tissues. It contains a villin-type ‘headpiece’ F-actin-binding domain at its extreme C-terminus. The isolated dematin headpiece domain (DHP) undergoes a significant conformational change upon phosphorylation. The mutation of Ser74 to Glu closely mimics the phosphorylation of DHP. We investigated motions in the backbone of DHP and its mutant DHPS74E using several complementary NMR relaxation techniques: laboratory frame 15N NMR relaxation, which is sensitive primarily to the ps–ns time scale, cross-correlated chemical shift modulation NMR relaxation detecting correlated μs–ms time scale motions of neighboring 13C′ and 15N nuclei, and cross-correlated relaxation of two 15N–1H dipole–dipole interactions detecting slow motions of backbone NH vectors in successive amino acid residues. The results indicate a reduction in mobility upon the mutation in several regions of the protein. The additional salt bridge formed in DHPS74E that links the N- and C-terminal subdomains is likely to be responsible for these changes.
KeywordsDematin headpiece NMR relaxation Model-free Cross-correlation Slow motions Backbone dynamics
We thank Zhenghui G. Jiang and Benjamin S. Frank for preparation of the protein samples. We are grateful to Prof. Arthur Palmer for a critical reading of the manuscript, Prof. Geoffrey Bodenhausen’s laboratory for the assistance with the pulse sequences and to Prof. Milo Westler for technical assistance. The study made use of the National Magnetic Resonance Facility at Madison, which is supported by NIH grants P41RR02301 (BRTP/NCRR) and P41GM66326 (NIGMS). Additional equipment was purchased with funds from the University of Wisconsin, the NIH (RR02781, RR08438), the NSF (DMB-8415048, OIA-9977486, BIR-9214394), and the USDA. This work has been supported by the University of Alaska Anchorage fund number 104110-11970 to L.V. and National Institute of Health grant number GM26335 to C.J.M.
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