Characterizing protein interactions using NMR
Group Leader page
Structures of individual macromolecules can now often be determined very quickly. However, in order to understand a macromolecule’s function one generally needs also to characterize at a structural level its interactions with other molecules, and this is one area where NMR spectroscopy can often make a key contribution. Experiments for solution studies of protein interactions with other proteins, peptides, DNA, RNA and other ligands are already widely used, but there are several areas where real difficulties exist, especially for relatively weak complexes that are often of great biological relevance.
Current methods mainly detect either atom-specific nuclear Overhauser effects (NOEs) across the interface, or else chemical shift changes during titration of the protein with a binding partner. However, interfacial resonances are commonly rather broad, which can greatly reduce the sensitivity of experiments designed to detect intermolecular NOEs selectively, while chemical shift changes during a titration can indicate either direct interaction or secondary conformational changes resulting from binding. To address these problems we propose to develop new experiments and extend existing ones in order to give a more robust approach to characterizing interfaces using NMR.There are a variety of target systems at this laboratory to which such methodology would be applicable. The main targets that we are already working on are some protein complexes involved in DNA repair,1,2,3 as well as protein interactions involved in vesicle trafficking (with David Owen, CIMR), intracellular immunity (with Leo James), chromatin targeting (with Douglas Higgs [Oxford]),4 and mRNA splicing (with Kiyoshi Nagai).5,6
Potential students will gain experience in many techniques. These include primarily NMR spectroscopy and computation, as well as protein expression and purification. A wide range of biochemical and molecular biological techniques used to characterize proteins and their interactions will be used as necessary.
- Eustermann, S., Wu, W.F., Langelier, M.F., Yang, J.C., Easton, L.E., Riccio, A.A., Pascal, J.M. and Neuhaus, D. (2015)
Structural Basis of Detection and Signaling of DNA Single-Strand Breaks by Human PARP-1.
Mol. Cell, 60, 742-754.
- Eustermann, S., Videler, H., Yang, J.C. , Cole, P.T., Gruszka, D., Verprintsev, D. and Neuhaus, D. (2011)
The DNA-binding domain of human PARP-1 interacts with DNA single-strand breaks as a monomer through its second zinc finger. .
J. Mol. Biol., 407, 149-170.
- Eustermann, S., Brockmann, C., Mehrotra, P.V., Yang, J.C. , Loakes, D., West, S.C., Ahel, I. and Neuhaus, D. (2010).
Solution structures of the two PBZ domains from human APLF and their interaction with poly(ADP-ribose).
Nature Struct. Molec. Biol., 17, 241-243.
- Eustermann, S., Yang, J.C. , Law, M.J., Amos, R., Chapman, L., Jelinska, C., Garrick, D., Clynes, D., Gibbons, R.J., Rhodes, D., Higgs, D.R. and Neuhaus, D. (2011)
Combinatorial readout of histone H3 modifications specifies localization of ATRX to heterochromatin.
Nature Struct. Molec. Biol., 18, 777-782.
- van Roon, A.M., Loening, N.M., Obayashi, E.,Yang, J.C. , Newman, A.J., Hernandez, H., Nagai, K. and Neuhaus, D. (2008)
Solution structure of the U2 snRNP protein Rds3p reveals a knotted zinc-finger motif.
P.N.A.S., 105, 9621-9626.
- van Roon, A.M., Yang, J.C., Mathieu, D., Bermel, W., Nagai, K. and Neuhaus, D. (2015)
113Cd NMR Experiments reveal an unusual metal cluster in the solution structure of the yeast splicing protein Bud31p.
Angewandte Chemie, International Edition, 54, 4861-4864.