Role of proximal residues in controlling CO binding reactions in myoglobin
Chien, Ellen Yu-Lin Tsai
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Permalink
https://hdl.handle.net/2142/23844
Description
Title
Role of proximal residues in controlling CO binding reactions in myoglobin
Author(s)
Chien, Ellen Yu-Lin Tsai
Issue Date
1996
Doctoral Committee Chair(s)
Sligar, Stephen G.
Department of Study
Biology, Molecular
Chemistry, Biochemistry
Biophysics, General
Discipline
Biology, Molecular
Chemistry, Biochemistry
Biophysics, General
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Chemistry, Biochemistry
Biophysics, General
Language
eng
Abstract
Heme proteins are ubiquitous in biological systems and it is of interest to understand how the structure of various heme proteins determines their respective biological function. Using site-directed mutagenesis, the amino acids Leu89, Ser92, and His97 have been changed to understand why these residues are highly conserved in myoglobin across species, to elucidate the structural and functional roles played by these residues. These residues are shown to be participants in forming a hydrogen bonding network with the proximal histidine, His93. The effect of disrupting the hydrogen bonding interactions is examined via CO flash photolysis and resonance Raman spectroscopy. Furthermore, residues number 87 to 94 have been replaced by the amino acids in the respective locations in hemoglobin $\beta$-chain (residues number 86 to 93) to examine the importance of the F-helix in controlling ligand binding in myoglobin and hemoglobin $\beta$-chain. Based on the modeled structures of the mutants using CHARMM, and the evidence presented in this thesis, we propose that not only is the orientation of the proximal histidine important in controlling ligand binding to the heme, the dynamics of the F-helix also play an important role in controlling the inner barrier in myoglobin and hemoglobin.
In order to understand the structure-function relationships in proteins, it is important to have accurate structures. Proteins are not static entities. How does one determine if a given crystal structure is a good representation of the protein in solution? How does one obtain structural information when a protein does not lend itself to crystallization nor is it amenable to NMR structure determination? In this thesis, we have explored the possibility of using 4-fluorotryptophan as an electrostatic probe in myoglobin and hemoglobin to gain both electrostatic and structural information via $\sp{19}$F-NMR. We show that the combination of the fluorine's chemical shift determined from the $\sp{19}$F-NMR spectra of labeled proteins and the calculated chemical shielding using existing crystal structures can be used to gain structural and electrostatic information. This thesis demonstrates that this combination is a new avenue for structural analysis.
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