Protein nano-crystallogenesis

We demonstrate the feasibility of growing crystals of protein in volumes as small as 1 nanoliter, Advances in the handling of very small volumes (i.e. through ink-jet and other technologies) open the way towards fully automated system,,. The rationale for these experiments is the desire to develop a system that speeds up the structure determination of proteins by crystallographic techniques, where most of the precious protein sample is wasted for the identification of the ideal crystallisation conditions, An additional potential benefit of crystallisation in very small volumes is the potential improvement of the crystal quality through reduced convection during crystal growth. Furthermore, in such small volumes even very highly supersaturated conditions can be stable for prolonged periods, allowing additional regions of phase-space to be prospected for elusive crystallisation conditions. A massive improvement in the efficiency of protein crystallogenesis will cause a paradigm shift in the biomolecular sciences and will have a major impact in product development in (for example) the pharmaceutical industry

Crystal structure of the C-terminal SH2 domain of the p85 alpha regulatory subunit of phosphoinositide 3-kinase: An SH2 domain mimicking its own substrate

The binding properties of Src homology-2 (SH2) domains to phosphotyrosine (pY)-containing peptides have been studied in recent years with the elucidation of a large number of crystal and solution structures. Taken together, these structures suggest a general mode of binding of pY-containing peptides, explain the specificities of different SH2 domains, and may be used to design inhibitors of pY binding by SH2 domain-containing proteins. We now report the crystal structure to 1.8 Angstrom resolution of the C-terminal SH2 domain (C-SH2) of the P85 alpha regulatory subunit of phosphoinositide S-kinase (PI3 K). Surprisingly, the carboxylate group of Asp2 from a neighbouring molecule occupies the phosphotyrosine binding site and interacts with Arg18 (alpha A2) and Arg36 (beta B5), in a similar manner to the phosphotyrosine-protein interactions seen in structures of other SH2 domains complexed with pY peptides. It is the first example of a non-phosphate-containing, non-aromatic mimetic of phosphotyrosine binding to SH2 domains, and this could have implications for the design of substrate analogues and inhibitors. Overall, the crystal structure closely resembles the solution structure, but a number of loops which demonstrate mobility in solution are well defined by the crystal packing. C-SH2 has adopted a binding conformation reminiscent of the ligand bound N-terminal SH2 domain of PI3K, apparently induced by the substrate mimicking of a neighbouring molecule in the crystal