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dc.contributor.advisor Olson, John S.
dc.creatorCarver, Theodore Edward, Jr
dc.date.accessioned 2009-06-04T00:41:18Z
dc.date.available 2009-06-04T00:41:18Z
dc.date.issued 1993
dc.identifier.urihttps://hdl.handle.net/1911/16606
dc.description.abstract Picosecond, nanosecond, and bimolecular reactions of ligands with myoglobins containing distal pocket mutations at positions 29, 45, 64, and 68 were examined at 20$\sp\circ$C, in 0.1 M potassium phosphate pH 7.0. The Val$\sp{68}{\to}$Ile mutation hindered intramolecular iron-ligand bond formation, slowing recombination of NO on picosecond time scales and O$\sb2$ on nanosecond time scales. Picosecond NO recombination was enhanced by increasing the size of residue 29. The rates for the major picosecond rebinding phase were 1.8, 2.5, 29, and $\ge$100 ns$\sp{-1}$ for Ala$\sp{29}$, Val$\sp{29}$, Leu$\sp{29}$(native), and Phe$\sp{29}$ myoglobin. In contrast to this trend, the Leu$\sp{29}{\to}$Phe mutation caused a 10-fold decrease in the rate of nanosecond NO recombination. These effects were interpreted in terms of a model for picosecond and nanosecond ligands based on diffusion of ligands within the protein matrix. On picosecond time scales, the photodissociated ligand appears to reside in a space bordered by His$\sp{64}$ and Leu$\sp{29}$ close to the iron atom. On nanosecond time scales, unbound ligands have diffused farther away into a space adjacent to Ile$\sp{107}$. The Leu$\sp{29}{\to}$Phe mutation caused the distal pocket to become more compartmentalized, enhancing picosecond recombination and hindering nanosecond recombination. The Leu$\sp{29}{\to}$Val and Leu$\sp{29}{\to}$Ala substitutions caused picosecond and nanosecond intermediates to become less distinct. The Val$\sp{68}{\to}$Phe mutation enhanced nanosecond O$\sb2$ recombination by reducing the volume available to the unbound ligand. Thus, distal pocket structure plays a key role in the internal kinetic barriers to ligand recombination. Replacement of His$\sp{64}$ with apolar residues facilitated ligand entry into the protein, due to loss of the distal water molecule in deoxymyoglobin. The Val$\sp{68}{\to}$Thr mutation produced the opposite effect, hindering ligand entry by stabilizing the distal H$\sb2$O. No definite information was gained about pathways of ligand entry and escape, but there appears to be a global protein barrier to ligand exit that causes the rate of ligand escape to be $\sim$1 $\times$ 10$\sp7$s$\sp{-1}$, regardless of mutations in the distal pocket.
dc.format.extent 192 p.
dc.format.mimetype application/pdf
dc.language.iso eng
dc.subjectBiochemistry
Biophysics
dc.title Kinetic barriers to ligand binding in myoglobin
dc.type.genre Thesis
dc.type.material Text
thesis.degree.department Chemistry
thesis.degree.discipline Natural Sciences
thesis.degree.grantor Rice University
thesis.degree.level Doctoral
thesis.degree.name Doctor of Philosophy
dc.identifier.citation Carver, Theodore Edward, Jr. "Kinetic barriers to ligand binding in myoglobin." (1993) Diss., Rice University. https://hdl.handle.net/1911/16606.


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