Markus M A, Nakayama T, Matsudaira P, Wagner G
Committee on Higher Degrees in Biophysics, Harvard University, Cambridge, MA 02138.
J Biomol NMR. 1994 Jul;4(4):553-74. doi: 10.1007/BF00156620.
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.
已对肌动蛋白切断蛋白绒毛蛋白126个残基的氨基末端结构域14T的1H、15N、13C和13CO共振进行了序列特异性归属。绒毛蛋白是一类通过切断、封端和成核肌动蛋白丝来调节细胞骨架肌动蛋白的蛋白质家族成员。肌动蛋白结合依赖于钙，并被磷脂酰肌醇4,5 -二磷酸破坏。肌动蛋白切断蛋白由一个保守结构域的三个或六个重复序列组成，以14T为代表。在大肠杆菌中的表达促进了15N和13C同位素的掺入以及用于序列归属的三共振、基于主链的策略的应用。通过NOE交叉峰的特征模式和邻位3JHNHα耦合常数的值确定了规则二级结构的元素。交换缓慢（速率小于每分钟1.0×10(-4)）的酰胺质子集中在中央β折叠以及第二和第三α螺旋中，这表明这些二级结构元素形成了非常稳定的氢键。已研究了酰胺氮和质子的归属随pH和钙浓度的变化。基于结构域核心化学位移的保守性，绒毛蛋白14T在pH范围4.15至6.91以及钙浓度范围0至50 mM内保持相同的整体折叠。钙数据表明存在两个钙结合位点并暗示了它们的位置。
