Biomacromolecule-biomacromolecule interactions as probed by NMR spectroscopy

Transcription

1 Biomacromolecule-biomacromolecule interactions as probed by MR spectroscopy Tzeng, S.-R. & Kalodimos,. G. Dynamic activation of an allosteric regulatory protein. ature 462, (2009). W. Milo Westler Bchm/hm 872 Selected Topics in Macromolecular and Biophysical hemistry Spring 2013

2 Protein Structure Determination Ubiquitin MQIFVKTLTGKTITLEVEPSDTIEVKAKIQDKEGIPPD QQRLIFAGKQLEDGRTLSDYIQKESTLLVLRLRGG With apologies to Staples

3 Ubiquitin MQIFVKTLTGKTITLEVEPSDTIEVKAKIQDKEGIPPD QQRLIFAGKQLEDGRTLSDYIQKESTLLVLRLRGG

4 Ubiquitin MQIFVKTLTGKTITLEVEPSDTIEVKAKIQDKEGIPPD QQRLIFAGKQLEDGRTLSDYIQKESTLLVLRLRGG

5 verview of structure determination Backbone assignments (,, α, β, and ) {we know the sequence!} Sidechain assignments rest of carbons and protons E constraints Glu-Asn-Val-Lys-Ala-Lys

6 verview of structure determination Backbone assignments (,, α, β, and ) Sidechain assignments rest of carbons and protons E constraints

7 verview of structure determination Backbone assignments (,, α, β, and ) Sidechain assignments rest of carbons and protons E constraints

8 onstraints for structure determination Proton-proton distances < 5Å Dihedral angle constraints chemical shifts (TALS+ and TARA) scalar coupling constants stereospecific assignments Residual dipolar couplings global relative orientation of bond vectors requires alignment in magnetic field Phage, bicells, stretched gels, etc. ydrogen bond constraints Deduced ross -bond scalar coupling

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10 Random starting structure with predicted secondary structure (from 13 chemical shifts) Red: helix Yellow: sheet Green: coil

11 Random structure + all experimental restraints (distances <5Å & dihedral angles)

12 Folded structure -network of distance restraints

13 Raw data Fourier transform: A linear transform

14 FT Raw data Linear??-practical solution-?? S-RSETTA (small proteins) In principle QM (age of universe computation) PIE Relaxation matrix analysis on-linear E constraints P-ard* Assignments *

15 FT Raw data Linear??-practical solution-?? S-RSETTA (small proteins) In principle QM (age of universe computation) PIE Relaxation matrix analysis on-linear E constraints P-ard* Assignments dynamics binding chemistry *

16 Vitamin D receptor- Ligand binding domain hemical shift perturbations observed in solution by MR (hormone vs 2MD) verlay of 6 crystal structures with different ligands 1. Singarapu, K. K. et al. Ligand-Specific Structural hanges in the Vitamin D Receptor in Solution. Biochem. 50, (2011).

17 Vitamin D receptor ligand binding domain MR spectral differences observed for 2 different ligands

18 Binding equilibria Unlabeled protein Labeled protein Dissociation equilibrium constant, K : d koff kon AB A+ B K on kon: usually diffusion controlled ~ 10 s to 10 s k : characteristic of the complex off koff = = k k = k + k : exchange rate ex on off A B AB d k off k on

19 Binding curves good S/ Low S/ weak S/ ~o S/ MR is esp. useful for K d >10-6 (K d <10-6 ok for bound state) and protein concentrations ~50µM-1mM

20 hemical shift and line width effects due to exchange (e.g. binding) Equal populations Unequal populations k ex >> δω k ex Fast exchange (averaged line position) δ=pa*δa+pb*δb Slow exchange (two lines) δa,δb k ex << δω 20

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23 ydrogen exchange

24 ydrogen exchange Mueller, G., Smith, M., hapman, M. D., Rule, G. S. & Benjamin, D.. ydrogen exchange nuclear magnetic resonance spectroscopy mapping of antibody epitopes on the house dust mite allergen Der p 2. The Journal of biological chemistry 276, (2001).

25 ydrogen exchange

26 ross-saturation

27 ross-saturation Takahashi,., akanishi, T., Kami, K., Arata, Y. & Shimada, I. A novel MR method for determining the interfaces of large protein-protein complexes. ature structural biology 7, (2000).

28 ross-saturation

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30 PRE- Paramagnetic relaxation enhancement

31 PRE- Paramagnetic relaxation enhancement Berardi, M. J., Shih, W. M., arrison, S.. & hou, J. J. Mitochondrial uncoupling protein 2 structure determined by MR molecular fragment searching. ature 476, (2011).

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33 Paramagnetic pseudocontact shift

34 Paramagnetic pseudocontact shift Ubbink, M., Ejdebäck, M., Karlsson, B. G. & Bendall, D. S. The structure of the complex of plastocyanin and cytochrome f, determined by paramagnetic MR and restrained rigid-body molecular dynamics. Structure (London, England : 1993) 6, (1998).

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36 Residual dipolar couplings (RD)

37 Residual dipolar couplings (RD) rtega-roldan, J. L. et al. Accurate characterization of weak macromolecular interactions by titration of MR residual dipolar couplings: application to the D2AP S3-:ubiquitin complex. ucleic acids research 37, e70 (2009).

38 Protein/ membrane interactions urr pin Struct Biol August; 20(4): on-micellar systems for solution MR spectroscopy of membrane proteins.thomas Raschle, Sebastian iller, Manuel Etzkorn, and Gerhard Wagner J. Am. hem. Soc., 2009, 131 (49), pp Structural and Functional haracterization of the Integral Membrane Protein VDA-1 in Lipid Bilayer anodiscs. Thomas Raschle, Sebastian iller, Tsyr-Yan Yu, Amanda J. Rice, Thomas Walz, and Gerhard Wagner Science 29 August 2008: Vol. 321 no pp Solution Structure of the Integral uman Membrane Protein VDA-1 in Detergent Micelles. Sebastian iller, Robert G. Garces,* Thomas J. Malia, Vladislav Y. rekhov, Marco olombini, Gerhard Wagner Biochim Biophys Acta Jun;1818(6): doi: /j.bbamem Epub 2011 ov 15. Solution MR spectroscopic characterization of human VDA-2 in detergent micelles and lipid bilayer nanodiscs. Yu TY, Raschle T, iller S, Wagner G.

39 MR study of the proteosome Tugarinov, V. & Kay, L. E. Methyl groups as probes of structure and dynamics in MR studies of high-molecular-weight proteins. hembiochem : a European journal of chemical biology 6, (2005). Religa, T. L., Sprangers, R. & Kay, L. E. Dynamic regulation of archaeal proteasome gate opening as studied by TRSY MR. Science (ew York,.Y.) 328, (2010). t / t /j 7317/f ll/ t l Ruschak, A. M., Religa, T. L., Breuer, S., Witt, S. & Kay, L. E. The proteasome antechamber maintains substrates in an unfolded state. ature 467, (2010). ansen, D. F. & Kay, L. E. Determining valine side-chain rotamer conformations in proteins from methyl 13 chemical shifts: application to the 360 kda half-proteasome. Journal of the American hemical Society 133, (2011).

40 Various techniques to study macromolecular interactions Wang, X., Lee,.-W., Liu, Y. & Prestegard, J.. Structural MR of protein oligomers using hybrid methods. Journal of structural biology 173, (2011). Wang, X., Watson,., Sharp, J. S., andel, T. M. & Prestegard, J.. ligomeric structure of the chemokine L5/RATES from MR, MS, and SAXS data. Structure (London, England : 1993) 19, (2011). Lee,.-W. et al. Three-dimensional structure of the weakly associated protein homodimer SeR13 using RDs and paramagnetic surface mapping. Protein science : a publication of the Protein Society 19, (2010). Jain,. U., Wyckoff, T. J.., Raetz,. R.. & Prestegard, J.. Rapid analysis of large protein-protein complexes using MR-derived orientational constraints: the 95 kda complex of LpxA with acyl carrier protein. Journal of molecular biology 343, (2004).