Karsten W. Theis
Assistant Professor
Department: Biochemistry and Molecular Biology
Voice Phone Number: (413) 577-2890
Fax Phone Number: (413) 545-3291
E-mail Address: theis@biochem.umass.edu
Postal Address:
Room 1003
Lederle Graduate Research Center
University of Massachusetts Amherst
Amherst, MA 01003
Graduate Program Affiliations: Chemistry, Molecular and Cellular Biochemistry
Participant (and P.I.) of the BMB Teaching Initiative
Welcome to the Theis lab
Located in the Department for Biochemistry and Molecular Biology at the University of Massachusetts Amherst, our lab is interested in the structural biology of DNA repair and molecular motors. We use X-ray crystallography to solve the atomic structures of biological macromolecules, making use of the newly established X-ray diffraction facility housed in the department right across the lab.
Lab members
Molecular motors and DNA repair
Molecular motors are a class of proteins that use the free energy of ATP hydrolysis to move in some way or another. Often, this movement is on a substrate like an actin fiber (for instance myosin, the protein that is instrumental in contracting your muscles) or DNA (for instance helicases, proteins that separate the two strands of duplex DNA). The motor proteins are either processive, going through multiple rounds of ATP hydrolysis, dissociation and binding to move along the substrate, or non-processive, jumping to another substrate after a single ATP hydrolysis event. A large subgroup of the molecular motors acting on DNA have a common domain structure: two loosely connected domains with an ATPase active site at their interface. ATP hydrolysis is thought to cause domain movements, which act on the DNA via DNA binding sites found on both domains. The cartoon below depicts how two DNA motor proteins might work.
On the left is a helicase translocating along one strand of DNA. In the process (I'm just showing half of the cycle), the other strand of DNA is stripped off. The mechanism of the helicase has been likened to the way inchworms move. Inchworms have a long flexible body and alternate between holding on to the ground with their front and back feet, streching and contracting their bodies to move ahead. Dale Wigley and coworkers extensively studied the helicase PcrA to unravel the details of the inchworm mechanism (reviewed for instance in Singleton and Wigley (2002), J. Bact. 184:1819-1826).
On the right is a gyrase changing the topology of DNA. The mechanism is very similar, but the gyrase in this cartoon holds on to the DNA with both domains, causing the DNA to be scrunched when the two domains move towards each other. Daniela Stock and coworkers solved the structure of a reverse gyrase that led them to propose the mechanism of action shown in the cartoon (Rodriguez and Stock (2002) ,EMBO J. 21, 418-426).
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X-ray crystallography
This picture shows three stages in solving a biological structure by X-ray crystallography. (It is a larger version of the picture you will find on the Biochemistry and Molecular Biology Department web site next to my research interests.) The background is a diffraction image, which we can observe on a X-ray sensitive detector when illuminating a crystal with X-rays. The spots of varying intensity carry the structural information we are interested in. The forground is electron density (cyan) and the interpretation of it, the atomic model (shown as space-filling model). X-rays interact with the electrons of a molecule, not with the nuclei. When we infer the structure from the X-ray diffraction data, we first obtain an electron density, which is then interpreted by building an atomic model. For clarity, I moved the electron density a bit towards the viewer so that it is not covered by the atomic model.
For this picture, I combined the diffraction image from one project with the electron density and model of a different project. The molecule is alloxanthine, a nucleotide analog used as a drug to treat gout. Results from both projects have been published:
1) José A. Carrodeguas, Karsten Theis, Daniel F. Bogenhagen, and Caroline Kisker. (2001) Crystal Structure and Deletion Analysis Show that the Accessory Subunit of Mammalian DNA Polymerase, PolgB, Functions as a Homodimer. Molecular Cell 7: 43-54.
2) James J. Truglio, Karsten Theis, Silke Leimkuehler, Roberto Rappa, KV Rajagopalan and Caroline Kisker. (2002) Crystal structures of the active and alloxanthine-inhibited forms of xanthine dehydrogenase from Rhodobacter capsulatus. Structure 10:115-125.
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Conformational change
Conformational change lies at the heart of molecular motors. However, it is also important in many other biological processes. We recently published a paper in collaboration with Craig Martin's lab on conformational change in T7 RNA polymerase titled "Topological and conformational analysis of the initiation and elongation complex of t7 RNA polymerase suggests a new twist." [Pubmed]. According to the model we suggest in the paper, the growing DNA/RNA hybrid (shown as blue/red spheres below) pushes away the promoter binding site (which comprises the rigid domain shown in purple and the specificity loop shown in orange) until the specificity loop is streched to its limit; the promoter DNA then dissociates and the specificity loop disengages from the rigid domain; as the last step, the rigid domain rotates by 220 degrees (allowing the green loop attached to it to refold and interact with the specificity loop to form the RNA exit tunnel). While the model is hypothetical except for the initial (PDB 1QLN) and final (PDB 1MSW) structures, which were solved by the Steitz lab (Yale University), the hypotheses concerning the intermediates are testable because the model describes a defined sequence of events of disrupting interactions and forming new ones.
The following is a collection of animations illustrating conformational change, as envisioned in models based on structures showing the start and end point of the conformational changes. To view these animations, you have to install the chime viewer.
T7 RNA polymerase transition from initiation to elongation
Comparison of domain orientation in UvrB from Bacillus caldotenax and Thermus thermophilus
Inhibition of Trypsin by Serpin Part I, Part II, Part III
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Courses
Fall 2008
General Biochemistry (Biochemistry 523)
Structural Bioinformatics (Biochemistry 697E)
Spring 2008
Sophomore Colloquium (Biochemistry HO1)
Analytical Chemistry Laboratory (Chemistry 312)
Hands-on crystallography (Biochemistry 697N)
Crystallography journal club (Biochemistry 695A)
Fall 2007
no courses, on parental leave
Spring 2007
Hands-on crystallography (Biochemistry 697N)
Crystallography journal club (Biochemistry 695A)
Fall 2006
General Biochemistry (Biochemistry 523)
Molecular motor proteins (Biochemistry 697E)
Spring 2006
Advanced Biochemistry (MCB 623)
Hands-on crystallography (Biochemistry 697N)
Crystallography journal club (Biochemistry 695A)
Fall 2005
General Biochemistry (Biochemistry 523)
Spring 2005
Advanced Biochemistry (MCB 623)
Fall 2004
General Biochemistry (Biochemistry 523)
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