JOHNSON, KENNETH

Kenneth Johnson

Professor
Molecular Biosciences

Roger J. Williams Centennial Professor in Biochemistry


kajohnson@mail.utexas.edu

Phone: 512-471-0434

Office Location
MBB 3.122

Postal Address
The University of Texas at Austin
Molecular Biosciences, College of Natural Sciences
2506 Speedway
Austin, TX 78712

Ph.D., Molecular Biology, University of Wisconsin (1975)
B.S., Chemistry, University of Iowa (1971)

Research In The Johnson Lab

Research in the Johnson Lab is currently focused on four topics. In each we apply state-of-the art kinetic methods developed in the Johnson lab to answer mechanistic questions of biological importance

Effectiveness versus toxicity of nucleoside analogs used to treat HIV infections and the mechanistic basis for the evolution of resistance. In our prior work we were the first to establish a quantitative link between the toxicity of nucleoside analogs and the kinetics of incorporation by the human mitochondrial DNA polymerase. We also were the first to quantify the kinetics of incorporation of normal nucleotides and nucleoside analogs by HIV reverse transcriptase. This enabled us to compute an enzymatic toxicity index defining the effectiveness versus toxicity of nucleoside analogs based upon their kinetics of incorporation by HIV RT versus the human mitochondrial DNA polymerase, respectively. We are continuing this work by developing new methods to measure the steps leading to the incorporation of nucleotides on the millisecond time scale and to apply these methods to understand the evolution of resistance to nucleoside analogs.


Role of protein conformational dynamics in enzyme specificity. The role of induced-fit in enzyme specificity has long been debated in the enzymology field. In our recent work on HIV RT and on other DNA polymerases we have established the role of substrate-induced changes in enzyme structure in enzyme specificity. Binding of the substrate in an initial weak-binding state triggers a large conformational change with movements up to 25 Å, to hold the substrate tightly and bring catalytic residues into proper alignment to facilitate catalysis. In contrast, binding of an incorrect substrate leads to weak binding and misalignment of catalytic residues. Thus the large conformational change, occurring on a millisecond time scale, determines enzyme specificity. Further studies to understand the kinetic and thermodynamic basis for enzyme specificity are based several upon novel approaches including single molecule kinetic methods to monitor events occurring at the active sites of individual enzymes, and molecular dynamics simulations to predict the pathway and energetics of the conformation change.

Structure and mechanism of the human mitochondrial DNA helicase. Replication of the human mitochondrial DNA requires the cooperative action of the DNA helicase to unwind the DNA and the DNA polymerase to replicate each single strand. The DNA helicase forms a hexameric ring around single stranded DNA and, in a reaction coupled to ATP hydrolysis, walks along the single stranded DNA to displace the duplex. We are currently working to reconstitute the replication complex and study the role of ATP binding and hydrolysis in DNA unwinding.

Correlating mutations in the human mitochondrial DNA polymerase and helicase with heritable diseases. There are numerous mutations in the humane genes for the mitochondrial DNA polymerase and helicase that are linked to heritable mitochondrial disorders. We are using our sophisticated structural and kinetic methods to understand the molecular basis for these defects. Understanding the physiological effects resulting from biochemical defects is challenging and requires the most accurate measurements and comprehensive analysis. To approach this problem and many others, we have developed new computer methods for rigorously fitting kinetic and equilibrium binding data without the standard simplifying assumptions employed in other labs.

Representative Publications

Kellinger, M. W. and Johnson, K. A. (2010) Nucleotide-dependent conformational change governs specificity of HIV reverse transcriptase. PNAS 107, 7734-7739.

Batabyal, D., McKenzie, J. L., and Johnson, K. A. (2010) Role of histidine-932 of the human mitochondrial DNA polymerase in nucleotide discrimination and inherited disease. J. Biol. Chem. 285, 34191-201

Jin, Z., Johnson, K. A. (2011) Role of a GAG hinge in the nucleotide-induced conformational change governing nucleotide specificity by T7 DNA polymerase. J. Biol. Chem. 286,1312-22

Jin, Z., Deval, J., Johnson, K. A., and Swinney, D. C. (2011) Assembly and transient kinetic analysis of the dengue polymerase elongation complex. J. Biol. Chem. 286, 2067-77

Kellinger, M. W., and Johnson, K. A. (2011) Role of induced-fit in limiting discrimination against AZT by HIV reverse transcriptase. Biochemistry 50, 5008-5015.

Estep, P. A., and Johnson, K. A. (2011) Effect of the Y955C mutation on mitochondrial DNA polymerase nucleotide incorporation efficiency and fidelity. Biochemistry 50, 6376-86