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Sligar, Stephen G. Robinson, Clifford Ralph 2011-05-07T11:55:21Z 2011-05-07T11:55:21Z 10000-01-01 1994 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." Made available in DSpace on 2011-05-07T11:55:21Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9512531.pdf: 6851053 bytes, checksum: f592c2d0300fb05f40d6384bcd6659b3 (MD5) Previous issue date: 1994 Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:34:18Z Item is restricted indefinitely. Restriction data tranferred 2014-07-01T11:13:04-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission ETDs are only available to UIUC Users without author permission U of I Only AAI9512531 (UMI)AAI9512531 http://hdl.handle.net/2142/19048 eng Copyright 1994 Robinson, Clifford Ralph Molecular Biology Biochemistry Biophysics Recognition, stability, and assembly in protein folding and protein-DNA interactions text Biochemistry Biochemistry University of Illinois at Urbana-Champaign Dissertation Ph.D.