Theodore E. Carver

Distal pocket polarity in ligand binding to myoglobin: structural and functional characterization of a threonine68(E11) mutant.

Site-directed mutagenesis studies have confirmed that the distal histidine in myoglobin stabilizes bound O2 by hydrogen bonding and have suggested that it is the polar character of the imidazole side chain rather than its size that limits the rate of ligand entry into the protein. We constructed an isosteric Val68 to Thr replacement in pig myoglobin (i) to investigate whether the O2 affinity could be increased by the introduction of a second hydrogen-bonding group into the distal heme pocket and (ii) to examine the influence of polarity on the ligand binding rates more rigorously. The 1.9-A crystal structure of Thr68 aquometmyoglobin confirms that the mutant and wild-type proteins are essentially isostructural and reveals that the beta-OH group of Thr68 is in a position to form hydrogen-bonding interactions both with the coordinated water molecule and with the main chain greater than C=O of residue 64. The rate of azide binding to the ferric form of the Thr68 mutant was 60-fold lower than that for the wild-type protein, consistent with the proposed stabilization of the coordinated water molecule. However, bound O2 is destabilized in the ferrous form of the mutant protein. The observed 17-fold lowering of the O2 affinity may be a consequence of the hydrogen-bonding interaction made between the Thr68 beta-OH group and the carbonyl oxygen of residue 64. Overall association rate constants for O2, NO, and alkyl isocyanide binding to ferrous pig myoglobin were 3-10-fold lower for the mutant compared to the wild-type protein, whereas that for CO binding was little affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein-Protein and Protein-DNA Interactions at The Bacteriophage T4 DNA Replication Fork. Characterization of a Fluorescently Labeled DNA Polymerase Sliding Clamp

The T4 DNA polymerase holoenzyme is composed of the polymerase enzyme complexed to the sliding clamp (the 45 protein), which is loaded onto DNA by an ATP-dependent clamp loader (the 44/62 complex). This paper describes a new method to directly investigate the mechanism of holoenzyme assembly using a fluorescently labeled cysteine mutant of the 45 protein. This protein possessed unaltered function yet produced substantial changes in probe fluorescence intensity upon interacting with other components of the holoenzyme. These fluorescence changes provide insight into the role of ATP hydrolysis in holoenzyme assembly. Using either ATP or the non-hydrolyzable ATP analog, adenosine 5′-O-(3-thiophosphate), events in holoenzyme assembly were assigned as either dependent or independent of ATP hydrolysis. A holoenzyme assembly mechanism is proposed in which the 44/62 complex mediates the association of the 45 protein with DNA in an ATP-dependent manner not requiring ATP hydrolysis. Upon ATP hydrolysis, the 44/62 complex triggers a conformational change in the 45 protein that may be attributed to the clamp loading onto DNA.

Interactions among residues CD3, E7, E10, and E11 in myoglobins: attempts to simulate the ligand-binding properties of Aplysia myoglobin.

Site-directed mutations have been introduced singly and in combination at residues lysine/arginine45 (CD3), histidine64 (E7), threonine67 (E10), and valine68 (E11) in pig and sperm whale myoglobins. The mutations probe the roles of these key distal pocket residues and represent attempts to mimic the heme environment of Aplysia limacina myoglobin which achieves moderately high O2 affinity in the absence of a distal histidine. In the mollusc myoglobin, arginine-E10 is believed to swing into the heme pocket and provide a hydrogen bond to the bound O2. The association and dissociation rate constants for oxygen and carbon monoxide binding to H64V, T67A, T67V, T67E, T67R, V68I, V68T, H64V-T67R, H64V-V68T, H64V-V68I, and H64V-T67R-V68I pig myoglobin mutants and T67R, H64V-T67R, and R45D-H64V-T67R mutants of sperm whale myoglobin have been measured using stopped-flow rapid mixing and flash photolysis techniques. Replacement of histidine-E7 with valine in either pig or sperm whale myoglobin drastically lowers O2 affinity while increasing CO affinity. Two second-site mutations, T67R and V68T, increase O2 affinity in the H64V mutant, even though when introduced singly these mutations have no effect or lower KO2, respectively. However, the oxygen affinities of the H64V-T67R mutants are 5-10-fold lower than that of A. limacina myoglobin. The crystal structure of the pig H64V-T67R double mutant reveals that the valine-E7 side chain is approximately 1 A closer to the heme plane than in the mollusc protein which may restrict access of the arginine-E10 side chain into the heme pocket. The O2 affinity of the H64V-T67R double mutant is not altered by the R45D replacement but is reduced 10-fold by the V68I mutation. The interactive effects of the T67R, V68I, and V68T mutations with the H64V substitution are discussed in terms of O2, CO, and N3-binding and the crystal structures of the H64V-T67R, H64V-V68I, and H64V-V68T double-mutant proteins. In many instances, the effects of second-site mutations in the valine64 background are the opposite of those observed for the corresponding single mutations in the wild type background. These results can be understood in terms of the changes in the rate-determining steps for ligand association and dissociation and the loss of distal pocket water molecules which follow replacement of histidine64 by valine.

Internal fluorescent probes for studying irregular and metastable DNA structures

Time-resolved fluorescence anisotropy decay measurements are useful for characterizing differences in the local structural environment of probes attached to DNA molecules. We synthesized oligonucleotides with dansyl probes covalently attached to uridine near the center of a 17 base oligonucleotide. The fluorescence properties of these DNAs hybridized to complementary DNA were examined using time-correlated single photon counting. We present preliminary data indicating changes in the probe anisotropy in the presence of trifluoroethanol, divalent zinc cations, and under conditions promoting the formation of a hairpin junction at the site of probe incorporation. The results of these perturbations indicate that this approach will be useful for investigating site-specific and global structural phenomena in DNA.