Structure determination and characterization of Saccharomyces cerevisiae profilin

Janina C. Eads, Nicole M. Mahoney, Sergey Vorobiev, Anne R. Bresnick, Kuo Kuang Wen, Peter A. Rubenstein, Brian K. Haarer, Steven C. Almo

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Abstract

The structure of profilin from the budding yeast Saccharomyces cerevisiae has been determined by X-ray crystallography at 2.3 Å resolution. The overall fold of yeast profilin is similar to the fold observed for other profilin structures. The interactions of yeast and human platelet profilins with rabbit skeletal muscle actin were characterized by titration microcalorimetry, fluorescence titrations, and nucleotide exchange kinetics. The affinity of yeast profilin for rabbit actin (2.9 μM) is approximately 30-fold weaker than the affinity of human platelet profilin for rabbit actin (0.1 μM), and the relative contributions of entropic and enthalpic terms to the overall free energy of binding are different for the two profilins. The titration of pyrene-labeled rabbit skeletal actin with human profilin yielded a K(d) of 2.8 μM, similar to the K(d) of 2.0 μM for the interaction between yeast profilin and pyrene-labeled yeast actin. The binding data are discussed in the context of the known crystal structures of profilin and actin, and the residues present at the actin-profilin interface. The affinity of yeast profilin for poly-L-proline was determined from fluorescence measurements and is similar tO the reported affinity of Acanthamoeba profilin for poly-L- proline. Yeast profilin was shown to catalyze adenine nucleotide exchange from yeast actin almost 2 orders of magnitude less efficiently than human profilin and rabbit skeletal muscle actin. The in vivo and in vitro properties of yeast profilin mutants with altered poly-L-proline and actin binding sites are discussed in the context of the crystal structure.

Original languageEnglish (US)
Pages (from-to)11171-11181
Number of pages11
JournalBiochemistry
Volume37
Issue number32
DOIs
StatePublished - Aug 11 1998

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Eads, J. C., Mahoney, N. M., Vorobiev, S., Bresnick, A. R., Wen, K. K., Rubenstein, P. A., Haarer, B. K., & Almo, S. C. (1998). Structure determination and characterization of Saccharomyces cerevisiae profilin. Biochemistry, 37(32), 11171-11181. https://doi.org/10.1021/bi9720033