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Recognition, stability, and assembly in protein folding and protein-DNA interactions

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Title: Recognition, stability, and assembly in protein folding and protein-DNA interactions
Author(s): Robinson, Clifford Ralph
Doctoral Committee Chair(s): Sligar, Stephen G.
Department / Program: Biochemistry
Discipline: Biochemistry
Degree Granting Institution: University of Illinois at Urbana-Champaign
Degree: Ph.D.
Genre: Dissertation
Subject(s): Molecular Biology Biochemistry Biophysics
Abstract: A detailed understanding of the fundamental forces and mechanism which determine biomolecular structure and function is crucial to utilizing proteins and nucleic acids as therapeutic agents, as research tools, and in industrial and environmental applications. To elucidate the principles of macromolecular assembly and recognition, two systems were studied using genetic engineering, biochemical, and biophysical approaches.Specific aspects of the folding and stability of a four-helix-bundle protein, cytochrome b$\sb{562}$ were investigated. Contributions of electrostatic interactions between $\alpha$-helix macrodipoles to stability of antiparallel helices were evaluated. Thermodynamic double mutant cycles were constructed to determine that interactions between macrodipoles contribute approximately 2 kcal/mol to antiparallel helix packing. Additionally, calorimetric and hydrostatic pressure techniques were applied to characterize the "molten globule" conformation of apo-cytochrome b$\sb{562},$ and kinetic data was obtained to investigate the folding pathways of cytochrome b$\sb{562}$.Detailed studies were performed to test mechanisms of site specific protein-DNA interactions in restriction endonuclease systems. Using osmotic and hydrostatic pressure techniques, it was demonstrated that an unusual role is played by bound water molecules in mediating DNA sequence discrimination by the EcoRI endonuclease, providing the first molecular explanation for the well known phenomenon of restriction enzyme "star" activity. Kinetic assays revealed that approximately forty-five water molecules participate in the binding of EcoRI to canonical site DNA, while at least forty additional waters mediate recognition during catalytic steps. Finally, analogous roles were identified for waters in recognition by BamHI and PvuII endonucleases, and Hin recombinase.
Issue Date: 1994
Type: Text
Language: English
URI: http://hdl.handle.net/2142/19048
Rights Information: Copyright 1994 Robinson, Clifford Ralph
Date Available in IDEALS: 2011-05-07
Identifier in Online Catalog: AAI9512531
OCLC Identifier: (UMI)AAI9512531
 

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