Protein NMR spectroscopy

Transcription

1 Protein NMR spectroscopy Perttu Permi National Biological NMR Center, Institute of Biotechnology, University of elsinki Protein NMR spectroscopy course, 19th January 2009

2 1 spectrum of 20 kda Ca-binding protein, calerythrin

3 2gx3 1zk6 2aav 1wm4 1nya 1szl 2rol 2hd7 2jzv 1vex 2bbx 2k2m 2v6z 2k7p 2k3t 2kdg 2alb 2k7q

4 NMR studies of biological macromolecules why bother? No crystals needed, native like conditions: solution state, in cell NMR, nonnative & unstructured states, prions, amyloids Molecular interactions in solution, binding epitope mapping at residue level, NMR signal is very sensitive to changes in chemical environment low affinity interactions Characterization of dynamics and mobility in various timescales (ps to days) - folding, kinetics, enzymatic reactions, domain movements January 2008, in PDB 6053 (38181) protein, 800 (1015) nucleic acid and 137 (1762) protein/nucleic acid complex structures determined by NMR (Xray) NMR suitable for proteins < kda complementary to X-ray crystallography

5 Proteins suitable for NMR studies Modular proteins Membrane proteins Partially unfolded or instrisically unfolded protein

6 Solution Structure of the Integral uman Membrane Protein VDAC-1 in Detergent Micelles iller et al., Science, 2008 NMR solution structure of the integral membrane enzyme DsbB Zhou et al., Mol. Cell, 2008

7 Spring 2007: 40 NMR spectrometers Cell-free protein synthesis 2500 proteins solved (~1200 by NMR)

8 What can be studied by NMR? Structural characterization of various molecules, small organic compounds, natural products, oligosaccharides, proteins, RNA, DNA Molecular dynamics in various timescales Molecular interactions (Kd range from nm to mm) ydrogen exchange Residue specific pka values, reaction dynamics, enzymatic activity, folding, thermostability Drug molecule screening/ligand optimization NMR inside a living cell (In-cell NMR) Metabo(n/l)omics data

9 Structural characterization of various molecules Small proteins Protein ligand complexes Integral membrane proteins Protein-DNA complexes Protein-RNA complexes C 3 N C 3 N C 3 N C3 3C N C3 C3 C 2 N N C3 3 C N 3 C 3C N 2 N C 3 C3 C3 C 3 C 3 C3 C3 C 3 C 3 C3 C 3 N C3 N2 Large proteins Strychnine Lipopolysaccharides Intrinsically unfolded proteins

10 NMR in structural biology Case study: Structural characterization of ADF- domains

11 CLP/Coactosins Low sequence homology (~15%) to ADF/Cofilin family members Biological function unclear Binds solely F-actin with low affinity Coactosin is the fourth member of ADF- domain family Saccaromyces cerevisiea Cofilin Mus musculus Coactosin ellman et al., FEBS Lett., 2004 ellman et al., J. Biomol NMR, 2004

12 Structural differences to ADF/cofilins L36 Y Y64 3 L Y64 Y101 3 Stabilization of long a3-helix, which is important for the G- actin binding, is obtained with different amino acids 1 Y31 T94 3 F Y F59 T94 Could there be any other explanation for missing G-actin binding? ellman et al., FEBS Lett., 2004 ellman et al., J. Biomol NMR, 2004

13 Twinfilin Composed of tandem repeat of ADF- domains (twf-n and twf-c), connected with a 35-residue linker and followed by an extended C-terminal tail Low sequence homology, 15 and 25%, to other ADF/Cofilin family members Binds ADP-G actin; Kd s ~ 0.05, 0.5 and 0.05 M for the full-length twinfilin, twf-n, and twf-c, respectively rientation of 3-4 extension in twf-n makes a steric hindrance, preventing F-actin binding Two domain architecture obsolote, even an evolutionary defect C-terminal tail binds the heterodimeric barbed-end capping protein S. cerevisiea Cofilin

14 Solution structure of C-terminal domain of Twinfilin Table 1 NMR and refinement statistics for the solution structure of the C-twinfilin C-twinfilin NMR distance constraints Distance constraints Total NE 3112 Intra-residue 755 Inter-residue Sequential ( i-j = 1) 821 Medium-range ( i-j 4) 591 Long-range ( i-j 5) 945 Structure Statistics Violations (mean and s.d.) Distance constraints (Å) Max. distance constraint violation (Å) Deviations from idealized geometry Bond lengths (Å) s.d Bond angles (º) s.d Average pairwise r.m.s.d.* (Å) eavy 0.74 Backbone 0.31 * Pairwise r.m.s.d. was calculated among 15 refined structures for residues Paavilainen, ellman et al. Proc. Natl. Acad. Sci. USA, 2007

15 Structural and functional differences among ADF-s S. cerevisiea Cofilin M. musculus twf-c M. musculus twf-n The structure of the C-terminal domain of mouse twinfilin is very similar to yeast cofilin Further functional studies revealed that twf-c is also functionally similar to cofilin i.e. it binds the ADP-G actin but also co-sediments with the F-actin in a cofilin-like manner Two-domain architecture is required for capping actin filament barbed ends, neither domain is able to cap the barbed end alone Domains can be swapped without loosing the capping activitity Paavilainen, ellman et al. Proc. Natl. Acad. Sci. USA, 2007

16 Structural characterization of natively unfolded proteins 10% of proteins has been predicted to be fully disordered 40-50% of eukaryotic proteins have natively unstructured regions longer than fifty residues The proportion of intrinsically unstructured proteins in genome increases along with the complexity of organism Molecular recognition: transition between disordered ordered states Associated to several diseases e.g., Alzheimer, Parkinson, cancer, diabetes Attractive targets for NMR owing to slow transverse relaxation

17 Structural characterization of natively unfolded proteins

18 Studies of protein dynamics

19 Dynamics in NMR: molecular motions manifest themselves as differential relaxation Fast loop reorientation Sidechain rotations Librational dynamics Slow loop reorientation Ligand binding, Enzymatic kinetics Folding 1 ps 1 ns 1 s 1 ms 1 s T 1, T 2, 15 N-noe T 1, T 2 Chemical shift T 1, T 2 relaxation, CCR CPMG Residual dipolar couplings (RDCs) EXSY /D exchange

20 Model-free approach defines molecular motions in folded proteins eikkinen et al., BMC Struct Biol, 2009

21 Spectral density mapping more approriate describing molecular motion in IUPs

22 Dynamics in ADF- domains C CACTSIN-LIKE PRTEIN C-TERMINAL DMAIN F TWINFILIN -extension C -extension N N C C b N N 200 T -Relaxation time mouse CLP 1WM T -Relaxation time mouse TwfC 2D

23 Differences in dynamics between coactosin and ADF/cofilins Extension formed by 3-4 strands, earlier shown to be critical for the F-actin binding, is highly flexible 10-residue long C-terminal tail is completely unstructured ellman et al., FEBS Lett., 2004 ellman et al., J. Biomol NMR, 2004

24 Studies of molecular interactions

25 Studies of molecular interactions Magnitude of the chemical shift changes upon binding: Length of vector = bound free 2 N ppm N

26 Structure activity relationship by NMR 15 N labelled protein 15 N- 1 correlation spectrum (reference) verlay the reference and the spectra with different compounds + compound 1 2 3

27 Structure activity relationship by NMR Shuker et al., Science, 1996

28 ydrogen/deuterium exchange C N A C N B D 2 Canet et al. Nature Struct. Biol. 2002

29 ydrogen/deuterium exchange Canet et al. Nature Struct. Biol. 2002

30 Determination of residue specific pka values Crystal structure of protein G (PDB ID 1pga)(28) showing all lysine side chains in blue and all aspartate and glutamate side chains in green. The residues involved in salt bridges are circled. Similar interactions are found in other structures (PDB IDs 1pgb,(28) 2qmt,(36) 1mi0,(37) 2on8, and 2onq(38)). verlay of 2CN spectra of lysine N3 groups at p 8.0 (purple), 10.0 (red), and (blue). Change in lysine N chemical shift with p fit to eq 1 for K12 (cyan), K18 (black), K21 (green), K36 (blue), K39 (yellow), and K58 (red). Tomlinson et al., J. Am. Chem. Soc., 2009

31 NMR inside living cells (In-Cell NMR)