Albany 2013: Book of Abstracts
June 11-15 2013
©Adenine Press (2012)
Conformational analysis of processivity clamps in solution demonstrates that protein structure does not correlate with dynamics
Processivity clamps, critical for efficient DNA replication in all organisms, are dimers or trimers that possess six similar domains organized in the shape of a ring. Each domain is highly conserved in its tertiary structure even though the amino acid sequences are divergent. We previously characterized the beta processivity clamp from Escherichia coli using hydrogen-deuterium exchange mass spectrometry (HXMS), which probes the solvent accessibility and hydrogen bonding of each backbone amide hydrogen, except those in prolines and at the first residue (Fang, 2011). We found that the three different domains within each monomer displayed different dynamics and that Domain I, which dissociates from Domain III to open the clamp, underwent partially cooperative local unfolding with a half-life of ~4 h. To determine how general our observations of a highly dynamic clamp protein were, we carried out a similar analysis using HXMS to characterize the dynamics of clamps from bacteriophage T4, yeast, archaeon, plant, and human. The different clamps show a wide range of dynamic behavior despite highly similar three-dimensional structures. Several of the other clamps undergo local unfolding events with half-lives that correlate with their reported stabilities on DNA. Bacteriophage T4 gp45 shows high deuterium uptake and undergoes widespread local unfolding events with half-lives of approximately 5 min. In human PCNA, local unfolding is observed at the trimer interface with a half-life of about 1 h. Furthermore, the C-terminal domain of proliferating cellular nuclear antigen (PCNA) was generally more dynamic than the N-terminal domain, indicating asymmetry in the dynamics of these proteins despite the obvious symmetry in the tertiary structure. The clamp proteins from bacteriophage T4, Arabidopsis thaliana, and human incorporate the most deuterium and are therefore the most dynamic. We find a striking range of dynamic properties of the clamp proteins despite their highly conserved tertiary structures.
This research has been supported by NSF CAREER Award MCB-0845033, Research Corporation for Science Advancement, American Cancer Society Research Scholar Grant RSG-12-161-01-DMC, NIH grant R01-GM086507, and a research collaboration with the Waters Corporation.
J. Fang, J.R. Engen, & P.J. Beuning (2011). Escherichia coli processivity clamp beta from DNA polymerase III is dynamic in solution. Biochemistry. 50, 5958-5968.
Penny J. Beuning 1
1Department of Chemistry