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1 Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, USA
2 Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA
(RECEIVED August 15, 2005; FINAL REVISION September 27, 2005; ACCEPTED October 5, 2005)
The ability of proteins to regulate their own enzymatic activity can be facilitated by changes in structure or protein dynamics in response to external regulators. Because many proteins contain SH2 and SH3 domains, transmission of information between the domains is a potential method of allosteric regulation. To determine if ligand binding to one modular domain may alter structural dynamics in an adjacent domain, allowing potential transmission of information through the protein, we used hydrogen exchange and mass spectrometry to measure changes in protein dynamics in the SH3 and SH2 domains of hematopoietic cell kinase (Hck). Ligand binding to either domain had little or no effect on hydrogen exchange in the adjacent domain, suggesting that changes in protein structure or dynamics are not a means of SH2/SH3 crosstalk. Furthermore, ligands of varying affinity covalently attached to SH3/SH2 altered dynamics only in the domain to which they bind. Such results demonstrate that ligand binding may not structurally alter adjacent SH3/SH2 domains and implies that other aspects of protein architecture contribute to the multiple levels of regulation in proteins containing SH3 and SH2 domains.
Keywords: protein dynamics; Src-homology 3; Src-homology 2; domains; Hck; ligand binding
Abbreviations: SH3, Src homology domain 3 • SH2, Src homology domain 2 • SH32, a joint construct of Hck SH3 and SH2 • SH32L, a construct of Hck containing SH3, SH2, and the natural Hck kinase linker • SH32HAL, same as SH32L but containing two point mutations in the linker • Hck, hematopoietic cell kinase • MS, mass spectrometry • HX, hydrogen exchange • YEEI, high-affinity SH2 binding peptide from hamster polyomavirus middle T antigen • PPII, polyproline class II
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051782206.
Reprint requests to: John R. Engen, Clark Hall 242, MSC03-2060, Department of Chemistry, University of New Mexico, Albuquerque, NM 87131-0001, USA; e-mail: engen{at}unm.edu; fax: (505) 277-2609.
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