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제108회 대한화학회 학술발표회, 총회 및 기기전시회 안내 Structural and dynamic studies of single mutant adenylate kinase from mycobacterium tuberculosis

2011년 8월 5일 16시 51분 42초
Ⅰ-BIO.P-236 이곳을 클릭하시면 발표코드에 대한 설명을 보실 수 있습니다.
목 <발표Ⅰ>
저자 및
권애진, 원호식
한양대학교 응용화학과, Korea
Tuberculosis disease is still today a serious threat to life worldwide. So far it is well known that adenylate kinase from mycobacterium tuberculosis (AKmt), a protein of 181 residues play an essential role for the bacterial survival. Crystal and NMR solution structural studies shown that AKmt enzyme have an ATP binding P-loop (7-13 residues).This loop is considered very active towards ATP binding due to its high flexibility. Presently we done mutation in p-loop by site directed mutagenesis exchange alanine residue with serine at position 11 (11 Ala 11 Ser). AKmtA11S enzyme was cloned, expressed in Escherichia coli and purified as a 15N labeled protein for NMR studies. Wild and mutant (A11S) AKmt enzymes were studied in free and complex forms with binding ligands (ATP, AMP & MgCl2) by using 1H-15N HSQC titration and relaxation NMR experiments. Dissociation constant (Kd) calculations for ATP binds binary complex of mutant and wild type AKmt shows that upon mutation the enzyme activity increase by 10-folds. Analysis of the 15N relaxation data enables us to obtain global rotational correlation time (τm) for both wild and mutant type’s enzymes in free and complex forms. Further relaxation data were fitted by using modelfree analysis, calculating order parameter (S2) and internal correlation time (te) of amide vectors along the polypeptide sequence. In support with NMR data at 10ns MD simulation was also run to closely explain the dynamic motion, calculated RMSD, RMSF and principal component analysis.