ATOMIC STRUCTURES OF SINGLE-LAYER BETA-SHEET IN OSPA USING SURFACE MUTATIONS

这个子项目是众多研究子项目之一

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Outer surface protein A (OspA) from Borrelia burgdorferi contains a unique, single-layer beta-sheet that connects two globular domains. Although this beta-sheet segment is exposed to the solvent and thus it does not contain a hydrophobic core, it is highly stable. Therefore, OspA provides attractable platform for investigating the structure and energetics of nonglobular beta-sheets. The beta-sheet segment comprises two homologous beta-hairpins. We have previously demonstrated that the single-layer beta-sheet can be stably extended, in the context of OspA, by inserting copies of the beta-hairpin unit, reminiscent of the propagation of the cross-beta structure in peptide fibrils (cf. attached pdf file). Detailed molecular structures of the extended OspA would be very important for understanding the effects of the sheet extension and for further improving the design of single-layer beta-sheets. Our previous NMR characterization focused on the backbone structure of OspA+1bh, and it has been difficult to precisely define the structure of the single-layer beta-sheet region using NMR methods, because this region inherently has very few long-range interactions that are critical in NMR-based structure determination. Also NMR assignments of OspA+2bh has been technically challenging due to the presence of two 23-residue segments with an identical sequence. Thus the determination of precise crystal structures using APS synchrotron radiation is of a great interest. We have successfully crystallized OspA variants, each containing several copies of the hairpin inserts by optimizing initial conditions found in high-throughput screening at the Hauptman-Woodward institute and in-house screening.