Protein Labeling Strategies for Improving the Efficiency of Structure Determination by NMR

LuxU;residual dipolar coupling;Protein expression;NMR;calbindin D28K;isotopic labeling

Rogers, Constance Ann ; John Cavanagh, Committee Chair,William Miller, Committee Member,Michael Goshe, Committee Member,Rogers, Constance Ann ; John Cavanagh ; Committee Chair ; William Miller ; Committee Member ; Michael Goshe ; Committee Member

University:North Carolina State University

关键词: LuxU;    residual dipolar coupling;    Protein expression;    NMR;    calbindin D28K;    isotopic labeling;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/588/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

Rapid and efficient methods for preparing isotopically labeled recombinant proteins and refining solved structures via NMR are presented. The former approach was developed for 2H⁄13C⁄15N isotopic labeling and specific protonation of the methyl groups of isoleucine, leucine, and valine (ILV) residues of rat brain calbindin D28K, a calcium sensor and buffer. This protocol produces cell mass using unlabeled rich media followed by exchange into labeled media at high cell density. Allowing for a short period for growth recovery and unlabeled metabolite clearance, the cells were induced. Additionally, the solution structure of LuxU, a subunit of the quorum sensing circuit of Vibrio harveyi, has been refined using residual dipolar coupling (RDC). In slightly anisotropic environments, large one-bond internuclear dipolar interactions no longer average to zero, and therefore can provide information on the average orientation of the corresponding vectors relative to the magnetic field. This ordering was induced by introducing Pf1 filamentous bacteriophage into a solution of 13C15N isotopically labeled LuxU.