Andrew P Capaldi

Andrew P Capaldi

Associate Professor, Molecular and Cellular Biology
Associate Professor, Genetics - GIDP
Associate Professor, BIO5 Institute
Member of the General Faculty
Member of the Graduate Faculty
Primary Department
Contact
(520) 626-9376

Research Interest

Andrew Capaldi, PhD, researches the signaling pathways and transcription factors in a cell that are organized into circuits. They allow cells to process information and make decisions. For Dr. Capaldi, the work arises in understanding both how these circuits are built from their components, and how they function and malfunction. To address these questions, he is working to reverse engineer the circuitry that controls cell growth in budding yeast using a combination of genomic, proteomic and computational methods. http://capaldilab.mcb.arizona.edu

Publications

Shah, A. M., Conn, D. A., Li, S. -., Capaldi, A., Jäger, J., & Sweasy, J. B. (2001). A DNA polymerase β mutator mutant with reduced nucleotide discrimination and increased protein stability. Biochemistry, 40(38), 11372-11381.

PMID: 11560485;Abstract:

DNA polymerase β (pol β) offers a simple system to examine the role of polymerase structure in the fidelity of DNA synthesis. In this study, the M282L variant of pol β (M282Lβ) was identified using an in vivo genetic screen. Met282, which does not contact the DNA template or the incoming deoxynucleoside triphosphate (dNTP) substrate, is located on α-helix N of pol β. This mutant enzyme demonstrates increased mutagenesis in both in vivo and in vitro assays. M282Lβ has a 7.5-fold higher mutation frequency than wild-type pol β; M282Lβ commits a variety of base substitution and frameshift errors. Transient-state kinetic methods were used to investigate the mechanism of intrinsic mutator activity of M282Lβ. Results show an 11-fold decrease in dNTP substrate discrimination at the level of ground-state binding. However, during the protein conformational change and/or phosphodiester bond formation, the nucleotide discrimination is improved. X-ray crystallography was utilized to gain insights into the structural basis of the decreased DNA synthesis fidelity. Most of the structural changes are localized to site 282 and the surrounding region in the C-terminal part of the 31-kDa domain. Repositioning of mostly hydrophobic amino acid residues in the core of the C-terminal portion generates a protein with enhanced stability. The combination of structural and equilibrium unfolding data suggests that the mechanism of nucleotide discrimination is possibly affected by the compacting of the hydrophobic core around residue Leu282. Subsequent movement of an adjacent surface residue, Arg283, produces a slight increase in volume of the pocket that may accommodate the incoming correct base pair. The structural changes of M282Lβ ultimately lead to an overall reduction in polymerase fidelity.

Capaldi, A. P., Ferguson, S. J., & Radford, S. E. (1999). The Greek key protein apo-pseudoazurin folds through an obligate on-pathway intermediate. Journal of Molecular Biology, 286(5), 1621-1632.

PMID: 10064719;Abstract:

Folding of the 123 amino acid residue Greek key protein apo-pseudoazurin from Thiosphaera pantotropha has been examined using stopped-flow circular dichroism in 0.5 M Na2SO4 at pH 7.0 and 15°C. The data show that the protein folds from the unfolded state with all eight proline residues in their native isomers (seven trans and one cis) to an intermediate within the dead-time of the stopped-flow mixing (50 ms). The urea dependence of the rates of folding and unfolding of the protein were also determined. The ratio of the folding rate to the unfolding rate (extrapolated into water) is several orders of magnitude too small to account for the equilibrium stability of the protein, consistent with the population of an intermediate. Despite this, the logarithm of the rate of folding versus denaturant concentration is linear. These data can be rationalised by the population of an intermediate under all refolding conditions. Accordingly, kinetic and equilibrium measurements were combined to fit the chevron plot to an on-pathway model (U⇆I⇆N). The fit shows that apo-pseudoazurin rapidly forms a compact species that is stabilised by 25 kJ/mol before folding to the native state at a rate of 2 s-1. Although the data can also be fitted to an off-pathway model (I⇆U⇆N), the resulting kinetic parameters indicate that the protein would have to fold to the native state at a rate of 86,000 s-1 (a time constant of only 12 μs). Similarly, models in which this intermediate is bypassed also lead to unreasonably fast refolding rates. Thus, the intermediate populated during the refolding of apo-pseudoazurin appears to be obligate and on the folding pathway. We suggest, based on this study and others, that some intermediates play a critical role in limiting the search to the native state.