Physical Biochemistry

David Cowburn

Much of the machinery of the cell -- enzymes, transport factors, signaling complex, transcriptional and translational devices -- involve proteins’ interaction with other proteins, with other bio-macromolecules and with low molecular weight ligands. The large scale systems analysis of these interactions is highly complex. Using and developing the tools of structural biology, molecular dynamics simulation, molecular biology and protein engineering, we are attempting to provide such analysis in leading edge systems of practical biological interest.

In many proteins the role of ‘unstructured' regions can be assessed using improved NMR and scattering measurements probing how multiple weak interactions can give rise to specificity and selectivity of biological activity. These interactions can also be probed in cell using direct observation of multiple expressed protein systems by NMR, for a direct study of protein-protein interactions. A new area of application is using these methods to understand the dynamic structures of the FG-rich Nuclear Pore proteins, and their interactions with carriers and cargo. Novel functional roles of ‘unfoldable’ protein regions are being discovered. The nuclear pore transport mechanism contains potential significant targets for next generation therapeutics in cancer and infectious disease, which are being targeted for validation.

The mechanism of the intein reaction, internal splicing of proteins, is of general interest for protein engineering and as a model for several posttranslational modification mechanisms involving thioesterification. NMR is being used as an essential tool for probing this unusual reaction involving breaking and making peptide bonds, with substantial flexibility of the coordinating entities. All these studies also involve developing new analytical applied mathematical methods.

Protein kinases are critical mediators in development, differentiation, and homeostasis. Genetic and phenotypic modifications of their activities and of related phosphatases are commonly associated with many diseases states including infections, cancers, autoimmunity and developmental disorders. Recent advances have introduced selective inhibitors to these enzymes, and the potential for understanding the chemical biology of their interactions and for therapeutics are significant. A substantial part of their regulation and substrate interactions involve major molecular movements (“dynamics”) which are probed using NMR and other methods adding to static structural information from NMR or crystallography.

Selected References

163. Hayama, R., Sparks, S., Dutta, K., Hecht, L., Cabana, C., Karp, J., Rout, M.P., and Cowburn, D. (2018). Thermodynamic characterization of the multivalent interactions underlying selective translocation through the Nuclear Pore Complex. The Journal of biological chemistry 10.1074/jbc.AC117.001649 Editors’ pick. F1000 citation, PMC5868264

162. Sparks, S., Temel, D., Rout, M., and Cowburn, D. (2018). Deciphering the 'fuzzy' interaction of FG nucleoporins and transport factors using SANS. Structure 26, 477-484 e474, PMC5929991

161. Warren C, Matsui T, Karp JM, Chen H-Y, Onikubo T, Cahill S, Cowburn D, Brenowitz M, Girvin M, Shechter D. Dynamic disorder of the Histone chaperone Nucleoplasmin regulates histone binding and release. Nature Communications. (2017) 8 PMC5738438

160. Stevens AJ, Sekar G, Shah NH, Mostafavi AZ, Cowburn D, Muir TW. A promiscuous split intein with expanded protein engineering applications. Proceedings of the National Academy of Sciences of the United States of America. 2017;114 (32):8538-43. PMC5559002 P

159. Upla, P., Kim, S.J., Sampathkumar, P., Dutta, K., Cahill, S.M., Chemmama, I.E., Williams, R., Bonanno, J.B., Rice, W.J., Stokes, D.L., Cowburn, D., et al. (2017). Molecular Architecture of the Major Membrane Ring Component of the Nuclear Pore Complex. Structure 25, 434-445 PMC5342941 P

158. Karp, J.M., Sparks, S., and Cowburn, D. (2017). Effects of FGFR2 kinase activation loop dynamics on catalytic activity. PLoS Comput Biol 13, e1005360 PMC5313233

157. Liu D, Cowburn D. Segmental isotopic labeling of proteins for nmr study using intein technology (2017) Methods in molecular biology. 2017;1495:131-45

156. Khoo Y, Singer A, Cowburn D. Bias Correction in Saupe Tensor Estimation. arXiv preprint arXiv:160606975. 2016

155. Khoo Y, Singer A, Cowburn D. Integrating nOe and RDC using sum-of-squares relaxation for protein structure determination. J Biomol NMR. 2017;68 (3):163-85

154. Liu D, Yaun Y, Xu R, Cowburn D. Domain interactions of C-terminal Src Kinase determined through NMR spectroscopy with segmental isotope labeling. Protein and Cell. 2017; 8 , 67-71

153. Liu D, Cowburn D. Combining biophysical methods to analyze the disulfide bond in SH2 domain of C-terminal Src kinase. Biophysics Reports. 2016;10.1007/s41048-016-0025-4:1-11 10.1007/s41048-016-0025-4

152. Raveh, B., Karp, J. M., Sparks, S., Rout, M., Sali, A., and Cowburn, D. (2016). Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex. Proceedings of the National Academy of Sciences. 2016;113:E2489-E97 10.1073/pnas.1522663113.P

151. Stevens, A.J., Brown, Z.Z., Shah, N.H., Sekar, G., Cowburn, D., and Muir, T.W. (2016). Design of a Split Intein with Exceptional Protein Splicing Activity. Journal of the American Chemical Society 138, 2162-2165 P

150. Ferrage, F., Dutta, K., and Cowburn, D. (2015). Identification of Hydrophobic Interfaces in Protein-Ligand Complexes by Selective Saturation Transfer NMR Spectroscopy. Molecules 20, 21992-21999

149. Xia, Y., Eryilmaz, E., Zhang, Q., Cowburn, D., and Putterman, C. (2016). Anti-DNA antibody mediated catalysis is isotype dependent. Molecular immunology 69, 33-43 P

148. Xia, Y., Eryilmaz, E., Der, E., Pawar, R.D., Guo, X., Cowburn, D., and Putterman, C. (2016). A peptide mimic blocks the cross-reaction of anti-DNA antibodies with glomerular antigens. Clin Exp Immunol 183, 369-379

147. Hough LE, Dutta K, Sparks S, Temel DB, Kamal A, Tetenbaum-Novatt J, Rout MP, Cowburn D. “The molecular mechanism of nuclear transport revealed by atomic-scale measurements”. eLife. 2015;4 10.7554/eLife.10027P