Date of Award

1996

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Mary D. Barkley

Abstract

Sequence specific protein-DNA interaction involves changes in protein conformation and dynamics. Research work involved in this thesis is the study of structure and dynamics of EcoRI restriction endonuclease N-termini. The N-termini of EcoRI endonuclease has been shown to be essential for DNA cleavage and also stabilize EcoRI-DNA complex. But they are not resolved in the X-ray crystal structure. An Asn3Cys mutation was made at the N-termini by site-directed mutagenesis and the mutant was subjected to cysteine crosslinking, pyrene labeling and fluorescence studies. Chemical crosslinking of Asn3Cys mutant and steady-state fluorescence studies of pyrene-labeled mutant indicated that the N-termini are in close proximity and probably involved in the dimer interface. Time resolved fluorescence measurements revealed dynamics of the N-termini by examining the dissociation and reformation of pyrene excimers as well as the monomer spectral shifts caused by N-terminal segment movements. Fluorescence anisotropy decay analysis indicated the N-termini are on the protein surface and not totally disoriented in solution. Substrate DNA binding, however, causes the N-termini to be more mobile without affecting the proximity relationship. Fluorescence energy transfer experiments were carried out using a double mutant Asn3CysTrp104Tyr. The through space distance between the fluorescent labels, MIANS or 1,5-IAEDANS, and Trp246 is around 30 A. Substrate DNA binding decreased the distance to 26 A but cofactor Mg$\sp{2+}$ ion did not cause any distance change. Single Trp246 fluorescence in the double mutant also revealed possible conformational changes upon substrate DNA or cofactor binding. Experimental evidence indicates that the N-termini are located at the dimer interface but distal to the DNA binding site. The findings provide further insight into the function of the N-termini of EcoRI endonuclease.

ISBN

9780591288681

Pages

127

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