1H, 15N, 13C and 13CO resonance assignments and secondary structure of villin 14T, a domain conserved among actin-severing proteins.
Sequence-specific assignments have been made for the 1H, 15N, 13C and 13CO resonances of 14T, the 126-residue amino-terminal domain of the actin-severing protein villin. Villin is a member of a family of proteins that regulate cytoskeletal actin by severing, capping and nucleating actin filaments. Actin binding is dependent on calcium and disrupted by phosphatidyl inositol 4,5-bisphosphate. Actin-severing proteins are built from three or six repeats of a conserved domain, represented by 14T. Expression in Escherichia coli facilitated incorporation of 15N and 13C isotopes and application of triple-resonance, backbone-directed strategies for the sequential assignments. Elements of regular secondary structure have been identified by characteristic patterns of NOE cross peaks and values of vicinal 3JHNH alpha coupling constants. Amide protons that exchange slowly (rates less than 1.0 x 10(-4) per min) are concentrated in the central beta-sheet and the second and third alpha-helices, suggesting that these elements of secondary structure form very stable hydrogen bonds. Assignments for the amide nitrogens and protons have been examined as a function of pH and calcium concentration. Based on the conservation of chemical shifts in the core of the domain, villin 14T maintains the same overall fold in the pH range from 4.15 to 6.91 and the calcium range from 0 to 50 mM. The calcium data indicate the presence of two calcium-binding sites and suggest their locations.
Markus
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MA
Committee on Higher Degrees in Biophysics, Harvard University, Cambridge, MA 02138.
Nakayama
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T
Matsudaira
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Wagner
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D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002247:Carbon Isotopes; D002352:Carrier Proteins / Q000737:chemistry*; D016969:Desulfovibrio vulgaris / Q000737:chemistry; D005418:Flavodoxin / Q000737:chemistry; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy; D008840:Microfilament Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D017434:Protein Structure, Tertiary
D000199:Actins; D002135:Calcium-Binding Proteins; D002247:Carbon Isotopes; D002352:Carrier Proteins; D005418:Flavodoxin; D008840:Microfilament Proteins; D009587:Nitrogen Isotopes; C026615:villin; D006859:Hydrogen; D002118:Calcium