Andrew P Capaldi
Associate Professor, Genetics - GIDP
Associate Professor, Molecular and Cellular Biology
Associate Professor, BIO5 Institute
Primary Department
(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

Buchan, J. R., Capaldi, A. P., & Parker, R. (2012). TOR-tured yeast find a new way to stand the heat. Molecular cell, 47(2), 155-7.
BIO5 Collaborators
Ross Buchan, Andrew P Capaldi

In this issue, Takahara and Maeda (2012) discover that together, Pbp1 and sequestration of the TORC1 complex in cytoplasmic mRNP stress granules provides a negative regulatory mechanism for TORC1 signaling during stress.

Gorski, S. A., Capaldi, A. P., Kleanthous, C., & Radford, S. E. (2001). Acidic conditions stabilise intermediates populated during the folding of Im7 and Im9. Journal of Molecular Biology, 312(4), 849-863.

PMID: 11575937;Abstract:

The helical bacterial immunity proteins Im7 and Im9 have been shown to fold via kinetic mechanisms of differing complexity, despite having 60% sequence identity. At pH 7.0 and 10°C, Im7 folds in a three-state mechanism involving an on-pathway intermediate, while Im9 folds in an apparent two-state transition. In order to examine the folding mechanisms of these proteins in more detail, the folding kinetics of both Im7 and Im9 (at 10°C in 0.4 M sodium sulphate) have been examined as a function of pH. Kinetic modelling of the folding and unfolding data for Im7 between pH 5.0 and 8.0 shows that the on-pathway intermediate is stabilised by more acidic conditions, whilst the native state is destabilised. The opposing effect of pH on the stability of these states results in a significant population of the intermediate at equilibrium at pH 6.0 and below. At pH 7.0, the folding and unfolding kinetics for Im9 can be fitted adequately by a two-state model, in accord with previous results. However, under acidic conditions there is a clear change of slope in the plot of the logarithm of the folding rate constant versus denaturant concentration, consistent with the population of one or more intermediate(s) early during folding. The kinetic data for Im9 at these pH values can be fitted to a three-state model, where the intermediate ensemble is stabilised and the native state destabilised as the pH is reduced, rationalising previous results that showed that an intermediate is not observed experimentally at pH 7.0. The data suggest that intermediate formation is a general step in immunity protein folding and demonstrate that it is necessary to explore a wide range of refolding conditions in order to show that intermediates do not form in the folding of other small, single-domain proteins. © 2001 Academic Press.

Capaldi, A. P., C., M., Kleanthous, C., Roder, H., & Radford, S. E. (2001). Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate. Nature Structural Biology, 8(1), 68-72.

PMID: 11135674;Abstract:

Many proteins populate partially organized structures during folding. Since these intermediates often accumulate within the dead time (2-5 ms) of conventional stopped-flow and quench-flow devices, it has been difficult to determine their role in the formation of the native state. Here we use a microcapillary mixing apparatus, with a time resolution of ∼150 μs, to directly follow the formation of an intermediate in the folding of a four-helix protein, Im7. Quantitative kinetic modeling of folding and unfolding data acquired over a wide range of urea concentrations demonstrate that this intermediate ensemble lies on a direct path from the unfolded to the native state.

Worley, J., Sullivan, A., Luo, X., Kaplan, M. E., & Capaldi, A. P. (2015). Genome-Wide Analysis of the TORC1 and Osmotic Stress Signaling Network in Saccharomyces cerevisiae. G3 (Bethesda, Md.).

The Target of Rapamycin kinase Complex I (TORC1) is a master regulator of cell growth and metabolism in eukaryotes. Studies in yeast and human cells have shown that nitrogen/amino acid starvation signals act through Npr2/3 and the small GTPases Gtr1/2 (Rags in humans) to inhibit TORC1. However, it is unclear how other stress and starvation stimuli inhibit TORC1, and/or act in parallel with the TORC1 pathway, to control cell growth. To help answer these questions, we developed a novel automated pipeline and used it to measure the expression of a TORC1 dependent ribosome biogenesis gene (NSR1) during osmotic stress in 4700 Saccharomyces cerevisiae strains from the yeast knock-out collection. This led to the identification of 440 strains with significant and reproducible defects in NSR1 repression. The cell growth control and stress response proteins deleted in these strains form a highly connected network, including; 56 proteins involved in vesicle trafficking and vacuolar function; 53 proteins that act downstream of TORC1 according to a rapamycin assay--including components of the HDAC Rpd3L, Elongator, and the INO80, CAF-1 and SWI/SNF chromatin remodeling complexes; over 100 proteins involved in signaling and metabolism; and 17 proteins that directly interact with TORC1. These data provide an important resource for labs studying cell growth control and stress signaling, and demonstrate the utility of our new, and easily adaptable, method for mapping gene regulatory networks.

Jones, S., Reader, J. S., Healy, M., Capaldi, A. P., Ashcroft, A. E., Kalverda, A. P., Smith, D. A., & Radford, S. E. (2000). Partially unfolded species populated during equilibrium denaturation of the β-sheet protein Y74W apo-pseudoazurin. Biochemistry, 39(19), 5672-5682.

PMID: 10801317;Abstract:

Apo-pseudoazurin is a single domain cupredoxin. We have engineered a mutant in which a unique tryptophan replaces the tyrosine residue found in the tyrosine comer of this Greek key protein, a region that has been proposed to have an important role in folding. Equilibrium denaturation of Y74W apo- pseudoazurin demonstrated multistate unfolding in urea (pH 7.0, 0.5 M Na2SO4 at 15 °C), in which one or more partially folded species are populated in 4.3 M urea. Using a variety of biophysical techniques, we show that these species, on average, have lost a substantial portion of the native secondary structure, lack fixed tertiary packing involving tryptophan and tyrosine residues, are less compact than the native state as determined by fluorescence lifetimes and time-resolved anisotropy, but retain significant residual structure involving the trytophan residue. Peptides ranging in length from 11 to 30 residues encompassing this region, however, did not contain detectable nonrandom structure, suggesting that long-range interactions are important for stabilizing the equilibrium partially unfolded species in the intact protein. On the basis of these results, we suggest that the equilibrium denaturation of Y74W apo-pseudoazurin generates one or more partially unfolded species that are globally collapsed and retain elements of the native structure involving the newly introduced tryptophan residue. We speculate on the role of such intermediates in the generation of the complex Greek key fold.