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Abstract
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 15N-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 Phe78 mutants. The extent of DNA cleavage in vitro corresponds well to the rate of chromophore release. The results provide the first clear-cut indication of how toxin release can be controlled by a specific side chain of a carrier protein.
Highlights
Parameswaran Hariharan‡§, Wenchuan Liang¶, Shan-Ho Chou§, and Der-Hang Chin‡1 From the ‡Department of Chemistry, National Chung Hsing University, Taichung 40227, Taiwan, the §Institute of Biochemistry, National Chung Hsing University, Taichung 40227, Taiwan, and the ¶Department of Biochemistry, Beckman Center, Stanford University School of Medicine, Stanford, California 94305. Antitumor antibiotic chromoproteins such as neocarzinostatin involve a labile toxin that is tightly bound by a protective protein with very high affinity but must be freed to exert its function
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
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 Phe78 mutants
Summary
Physiological release of a noncovalently bound ligand from a protein complex is often triggered by a specific contact with some trans-acting agents in a cell Such contact, if present, is more likely to occur on the solvent-exposed surface than the interior. Dynamics and relaxation of NCS reveal internal motion and localized flexibility in the loop regions around the binding cleft [21, 22] We speculate that these loop residues (residues 75– 80 in Loop 1 and residues 98 –102 in Loop 2) may play a pivotal role and set up mutagenesis studies to explore the effect of these side chains on chromophore release. Such concern could not be overlooked because our study highly depends on the quality of the folded conformation
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