Please use this identifier to cite or link to this item:
|標題:||A new model for ligand release - Role of side chain in gating the enediyne antibiotic||作者:||Hariharan, P.
|關鍵字:||non-protein chromophore;neocarzinostatin chromophore;apo-neocarzinostatin;crystal-structure;deoxyribonucleic-acid;escherichia-coli;binding;apoprotein;chromoprotein;complex||Project:||Journal of Biological Chemistry||期刊/報告no：:||Journal of Biological Chemistry, Volume 281, Issue 23, Page(s) 16025-16033.||摘要:||
Antitumor antibiotic chromoproteins such as neocarzinostatin involve a labile toxin that is tightly bound by a protective protein with very high affinity but must also be freed to exert its function. Contrary to the prevalent concept of ligand release, we established that toxin release from neocarzinostatin requires no major backbone conformational changes. We report, herein, that subtle changes in the side chains of specific amino acid residues are adequate to gate the release of chromophore. A recombinant wild type aponeocarzinostatin and its variants mutated around the opening of the chromophore binding cleft are employed to identify specific side chains likely to affect chromophore release. Preliminary, biophysical characterization of mutant apoproteins by circular dichroism and thermal denaturation indicate that the fundamental structural characteristics of wild type protein are conserved in these mutants. The chromophore reconstitution studies further show that all mutants are able to bind chromophore efficiently with similar complex structures. NMR studies on N-15-labeled mutants also suggest the intactness of binding pocket structure. Kinetic studies of chromophore release monitored by time course fluorescence and quantitative high pressure liquid chromatography analyses show that the ligand release rate is significantly enhanced only in Phe(78) mutants. The extent of DNA cleavage in vitro corresponds well to the rate of chromophore release. The results provide the first clearcut indication of how toxin release can be controlled by a specific side chain of a carrier protein.
|Appears in Collections:||化學系所|
Show full item record
TAIR Related Article
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.