title: Domain-specific incorporation of noninvasive optical probes into recombinant proteins.
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.
authors.affiliation: Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, New York 10021, USA.
authors.forename: Vasant
authors.initials: V
authors.lastname: Muralidharan
authors.forename: Jaehyun
authors.initials: J
authors.lastname: Cho
authors.forename: Michelle
authors.initials: M
authors.lastname: Trester-Zedlitz
authors.forename: Lukasz
authors.initials: L
authors.lastname: Kowalik
authors.forename: Brian T
authors.initials: BT
authors.lastname: Chait
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Raleigh
authors.forename: Tom W
authors.initials: TW
authors.lastname: Muir
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D008024:Ligands; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C006657:7-azatryptophan; D014364:Tryptophan