Date of Award
multi-domain proteins, SH3 domains, Crk-II protein
The past few decades, considerable progress has been made in understanding the biophysical properties of proteins using small modular domains such as SH3 domains. However, there is a surprising lack of knowledge regarding how these properties are affected when the domain is placed back within its full-length multi-domain protein. Using a combination of expressed protein ligation (EPL) and in vivo amino acid replacement of tryptophans with tryptophan (Trp) analogues, we have developed an integrated approach that allows the domain-specific incorporation of optical probes into large recombinant proteins. The Src homology 3 (SH3) domain from the c-Crk-l adaptor protein has been labeled with a Trp analogue, 7-azatryptophan (7AW), using E.coli Trp auxotrophs. Biophysical analysis shows that incorporation of 7AW does not significantly perturb the structure or function of the isolated domain. Ligation of 7AW labeled SH3 domain to the c-Crk-l Src homology 2 (SH2) domain, via EPL, generated the multi-domain protein, c-Crk-l, with a domain specific label. Studies on this labeled protein show that the biochemical and thermodynamic properties of the SH3 domain do not change within the context of a larger multi-domain protein. We have also utilized this technique and segmental isotopic labeling to study the Cterminal SH3 (cSH3) domain of the signaling adaptor protein, Crk-ll. Several studies suggest that the cSH3 domain plays an important regulatory role in the protein. However, no structural information is available on this domain and relatively little is known about its binding partners. We have solved the solution NMR structure of the C-terminal SH3 domain and it adopts the standard SH3 fold comprising a fivestranded p-barrel. Thermodynamic and kinetic studies show that the domain folds in a reversible two-state manner and that the stability of the fold is similar to that observed for other SH3 domains. Studies on the cSH3 domain specifically labeled within Crk-ll have provided the strongest evidence yet that the cSH3 domain interacts in an intramolecular fashion to regulate Crk-ll. The techniques developed here should be applicable to many other multi-domain proteins. This sets the stage for a better understanding of the biophysical properties of domains within these complex systems.
Muralidharan, Vasant, "Exploring the Biophysical Properties of Domains Within Multi-Domain Proteins" (2006). Student Theses and Dissertations. 58.