University of Illinois Urbana-Champaign

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

Robinson, Clifford Ralph

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Description

Title
Recognition, stability, and assembly in protein folding and protein-DNA interactions
Author(s)
Robinson, Clifford Ralph
Issue Date
1994
Doctoral Committee Chair(s)
Sligar, Stephen G.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
  • Molecular Biology
  • Biochemistry
  • Biophysics
Language
eng
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."
Type of Resource
text
Permalink
http://hdl.handle.net/2142/19048
Copyright and License Information
Copyright 1994 Robinson, Clifford Ralph

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Graduate Dissertations and Theses at Illinois PRIMARY
Graduate Theses and Dissertations at Illinois
Dissertations and Theses - Biochemistry