Domain-specific incorporation of noninvasive optical probes into recombinant proteins

Vasant Muralidharan, Jaehyun Cho, Michelle Trester-Zedlitz, Lukasz Kowalik, Brian T. Chait, Daniel P. Raleigh, Tom W. Muir

Research output: Contribution to journalArticle

24 Scopus citations

Abstract

An integrated approach is described that allows the domain-specific incorporation of optical probes into large recombinant proteins. The strategy is the combination of two existing techniques, expressed protein ligation (EPL) and in vivo amino acid replacement of tryptophans with tryptophan (Trp) analogues. The Src homology 3 (SH3) domain from the c-Crk-I adaptor protein has been labeled with a Trp analogue, 7-azatryptophan (7AW), using Escherichia coli Trp auxotrophs. Structural, biochemical, and thermodynamic studies show that incorporation of 7AW does not significantly perturb the structure or function of the isolated domain. Ligation of the 7AW-labeled SH3 domain to the c-Crk-I Src homology 2 (SH2) domain, via EPL, generated the multidomain protein, c-Crk-I, with a domain-specific label. Studies of this labeled protein show that the biochemical and thermodynamic properties of the SH3 domain do not change within the context of a larger multidomain protein. The technology described here is likely to be a useful tool in enhancing our understanding of the behavior of modular domains in their natural context, within multidomain proteins.

Original languageEnglish (US)
Pages (from-to)14004-14012
Number of pages9
JournalJournal of the American Chemical Society
Volume126
Issue number43
DOIs
StatePublished - Nov 3 2004
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Chemistry(all)

Fingerprint Dive into the research topics of 'Domain-specific incorporation of noninvasive optical probes into recombinant proteins'. Together they form a unique fingerprint.

  • Cite this

    Muralidharan, V., Cho, J., Trester-Zedlitz, M., Kowalik, L., Chait, B. T., Raleigh, D. P., & Muir, T. W. (2004). Domain-specific incorporation of noninvasive optical probes into recombinant proteins. Journal of the American Chemical Society, 126(43), 14004-14012. https://doi.org/10.1021/ja0466199