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(Bio)-applications of high-field NMR

(Bio)-applications of high-field NMR. Aims. To give an overview of the capability of NMR to answer biological questions To make aware of limitations To give a basic idea about structure determination by NMR To make aware of NMR sample requirements

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(Bio)-applications of high-field NMR

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  1. (Bio)-applications of high-field NMR

  2. Aims • To give an overview of the capability of NMR to answer biological questions • To make aware of limitations • To give a basic idea about structure determination by NMR • To make aware of NMR sample requirements • To enable you to decide whether NMR would be a useful method in your research

  3. Outline • Introduction to biological applications of NMR • Basics of solution structure determination of proteins • Heteronuclear NMR • NMR of nucleic acids • NMR and dynamic phenomena • (some more applications)

  4. What can NMR do for biology ? • 3D Structure determination of proteins and nucleic acids • Assess stability and folding of proteins • Binding studies (Proteins, DNA, Ligands) • Protein dynamics and “reactions”: possible to look at timescales between ps and days • Elucidation of structure of biomarkers, metabolites, and synthetic pathways • NMR of bio-fluids and tissues • In vivo NMR • Magnetic Resonance Imaging

  5. 3D Structure determinations Express and purify protein (or isolate from natural source) • Initial characterisation • - Identity, composition • Concentration • Stability (buffers, salt, pH, temperature) GSDIIDEFGTLDDSATICRVCQKPGDLVMCNQCEFCFHLDCHLPALQDVPGEEWSCSLCHVLPDLKEEDVDLQACKLN Protein sequence Acquire NMR spectra Evaluation: Sequential Assignment Extraction of distance restraints and other structural data 3D structure

  6. 3D Structure determinations 1. The sample In solution: • ca. 0.2-1 mM protein solution (ca. 200-500 mL) • Smaller than 35 kDa • Preferentially in native form, not aggregated.... • Usually nothing paramagnetic (e.g. Cu(II), Fe(II) or Fe(III), … • Recombinant expression necessary for proteins > 8kDa (for isotopic labelling with 13C and 15N)

  7. 3D Structure determinations • The spectra

  8. Fourier Transform pulse sequences • The simplest 1D experiment: 1. Radiofrequency pulse with high power 2. Recording of the free induction decay (FID) Acquisition Repeat - but need to make sure that excitation from previous scan has completely vanished  relaxation delay

  9. 0.10 0.20 0.30 0.40 0.50 0.60 0.70 1D NMR “Free induction decay” (FID) Time domain (s) Fourier Transformation Frequency domain 1D NMR spectrum (s-1)

  10. Typical 1H NMR spectrum of a small molecule Recorded in 90% H2O/10% D2O 8H aliphatic 16H (aromatic) H2O High field Low field 4H 1 10 9 8 7 6 5 4 3 2 1 d1H (ppm) Aromatic protons are affected by electron cloud (“ring current”) of aromatic ring (deshielded – the field experienced by aromatic protons is weaker than B0, consequently the resonance frequency is lower

  11. H O H N C C C H 2 H N N H H 1H NMR spectrum of a 55 amino acid protein C225H356N70O80S9 a NH aliphatic side-chain Backbone b CH(a) d Side-chain H2O e NH and aromatic 10 9 8 7 6 5 4 3 2 1 0 10 9 8 7 6 5 4 3 2 1 0 d1H (ppm)

  12. 10 10 9 9 8 8 7 7 6 6 5 5 4 4 3 3 2 2 1 1 0 0 NMR spectrum of a 66 kD protein:Size limitation • Heavy overlap • Broad lines d1H (ppm)

  13. Relaxation • Relaxation is the process that brings the excited system (e.g. after the rf pulse) back to its equilibrium state • Transversal (T2): “spin-spin” • Longitudinal (T1): “spin-lattice” • Line-width of signal is reciprocally related to T2: fast relaxation  broad lines • Both T1 and T2 are dependent on molecular motions, e.g. for proteins molecular “tumbling” (correlation time tc (1/tumbling rate: large molecules have long tc) and backbone dynamics/conformational fluctuations

  14. Factors influencing the quality of NMR spectra: pH • Backbone amide protons very important for structure determination • But: Can dissociate and hence exchange with bulk protons (from water) • Exchange leads to loss of signal intensity • Exchange rates usually most favourable at pH 3-5

  15. Factors influencing the quality of NMR spectra: ions • salt and buffer • proteins usually require the presence of buffers and/or salt • but: salt and buffer ions add to spectral noise  loss of signal intensity • Usually not more than 50-100 mM total • NB: Buffer must not contain non-exchangeable protons (otherwise need deuterated compound) e.g.: O D O H D C H C 2 2 D O C D C N D H O C H C N H 2 2 2 2 D C H C 2 2 O D O H Tris Tris-d11

  16. Water suppression • Proteins are usually studied in aqueous solution: 90% H2O/10% D2O. • D2O required for “lock”: ensures stable field • Typically, protein concentration a few mM • Ca. 100 M protons from water (i.e. a 100000-fold excess) • Various ways for “getting rid” of water signal: • “Presaturation”: Irradiation of water resonance at low power before high-power rf pulse (during the relaxation delay) • “Watergate”: Selective pulse flanked by gradient pulses • DPFGSE (Double Pulsed Field Gradient Spin Echo) or “Excitation sculpting” (AJ Shaka)

  17. Principles of 2D NMR • 2D NMR experiments are composed of a series of 1D experiments • Involves • Irradiation of a nucleus (as in 1D) • “Incremented delay” (different for each 1D experiment) (also called “evolution”) • Magnetisation transfer to other nucleus that is “coupled” to irradiated nucleus Signal detection (as in 1D) • Results in information on correlations between nuclei

  18. Principles of 2D NMR 1D NMR: acquisition preparation e.g. relaxation delay and rf pulse t2 2D NMR are a series of 1D experiments: acquisition evolution preparation mixing t1 t2 What is detected depends on what happens during mixing time (spin coupling) This time period changes between the various individual 1D experiments  gives a second time domain

  19. Principles of 2D NMR Generated from FID as in 1D Repeated several hundred times with different evolution times t1 (also called incremented delay) 1st dimension: Last FID; incremented delay = 0.5 s (e.g.) time (s) time (s) Etc.... 2nd dimension: 2nd FID; incremented delay = 10 us 1st FID; incremented delay = 0 Frequency (Hz or ppm) Fourier Transformation of the second dimension gives the second frequency axis

  20. Principles of 2D NMR: Fully FT transformed spectrum 1st dimension (F2) (the third dimension is the peak intensity) 2nd dimension (F1) The “FID” for the second dimension is generated by the “incremented delay”

  21. The mixing time • Correlation between nuclei happens during the mixing time • Reciprocal relationship to observed coupling • large couplings - short mixing time • Difficult to detect small couplings, as mixing takes too long, and at end of mixing time no magnetisation left (due to relaxation) • If coupling through space: Long range - long mixing time

  22. Homonuclear 2D NMR • Typical experiments: • DQF-COSY (double-quantum-filtered correlation spectroscopy: up to 3-bond coupling • TOCSY (total correlation spectroscopy): entire residues • NOESY (nuclear Overhauser enhancement spectroscopy): through space • COSY and TOCSY are based on scalar coupling (through bonds), NOESY on dipolar coupling

  23. Identification of spinsystems E.g. Valine: 0 ppm • Protons have characteristic shifts • Tabulated • Each amino acid has a characteristic “pattern” in the various 2D spectra F1 C H H C H 3 3 C O C H C N H Expected TOCSY spectrum 10 10 0 ppm F2

  24. 2D NMR techniques: TOCSY and COSY in proteins 0 ppm Ala10 H(b) H(a) O H TOCSY N C C H C H F1 3 H(a) H(b) TOCSY and COSY help identifying the type of residue COSY amide 10 10 0 ppm F2

  25. Regions in 2D spectra NH-to-sidechain crosspeaks aromatic H(a)-to-H(b) H(a)-to methyl (Ala, Thr,Leu, Val, Ile) H(b)-to-methyl (Leu,Val, Ile) TOCSY spectrum of a decapeptide (Luteinising hormone releasing hormone)

  26. COSY TOCSY Intra-residue Cys11 NOESY Intra-residue Ala10 Inter-residue, sequential Sequential assignment NOESY connects residues that are adjacent to each other 0 ppm O O H H H N C C N C C H C H 3 C11 H C H A10 F1 S C d 10 10 10 0 ppm F2

  27. Overlay of TOCSY with NOESY Sequential assignment H(a) Amide H

  28. Break

  29. Recognising secondary structure:Chemical shift index • Shifts of backbone atoms are sensitive towards secondary structure (a helix, b sheet etc) • Comparison of experimental shifts with tabulated “random coil” shifts (one for each amino acid) • Quick and robust method, 95% accuracy • Can utilise H(a) protons (13C backbone shifts also useful) • Each residue with a shift larger than expected gets an index of 1 • Each residue with a shift smaller than expected gets an index of -1 • Residues within random coil shift get a 0

  30. 3 5 7 9 53 55 15 33 43 45 47 49 51 11 17 21 23 25 27 29 35 37 39 41 31 13 19 Chemical shift index: Example No recognisable secondary structure b strands a helix MTKKIKCAYHLCKKDVEESKAIERMLHFMHGILSKDEPRKYCSEACAEKDQMAHEL -----HHHEE---------HHHHHHHHH--------------HHHHHHHHHH---- (secondary structure prediction by jpred) C N

  31. Secondary Structure Can also Be Characterised by Regular Patterns of NOEs H(a) of residue 47 NH of residue 50 NH of residue 51 Strong NOEs between NH’s of adjacent residues NOE between Ha(i) and NH(i+3) a helix

  32. Very strong sequential NOEs (from H(a) to NH of next residue) • Also information on tertiary structure: Strong NOEs between neighbouring strands b sheet

  33. Recognising the fold: Analysis of backbone NOEs Backbone trace C b hairpin Residue number a helix N Antiparallel b sheet Residue number (Predicted by homology modelling, consistent with CSI and fold analysis)

  34. Distance restraints from NOESY • The NOE is a dipolar interaction: Through space • A cross peak between two nuclei means that magnetisation transfer through dipolar interactions between two neighbouring spins must have taken place during the mixing time. This means that the two nuclei are close together in space. • The cross peak intensity is defined as follows: • I = k g12g22r-6 S J(w)

  35. Real-world example: 100 ms 2D NOESY of a 55 aa protein • 356 protons • Ca. 2000 peaks • Intra-residue • Sequential • Long-range

  36. NMR restraints • evaluated ca 1000 1H peaks • 600 peaks unambiguously assigned • extracted about 300 relevant distance restraints (3-5 Å)

  37. B A X X Use of coupling constants to gain structural information • 3J-scalar coupling constants (extracted from dedicated NMR spectra) are dependent on dihedral (or torsional) angles B B : A A Dihedral angle dihedron

  38. Karplus relationship Coupling constant 3J Dihedral angle 3J = a cos2a- b cosa+ c; a, b, c are empirical parameters - tabulated for various combinations of nuclei

  39. Structure calculations • A number of programs available, most popular: XPlor, Cyana, CNS... • Randomised starting structures • Use distance restraints (+ various other experimental data) together with generic atom masses, chirality, electric charges, Van der Waals radii, covalent bond lengths and angles, peptide geometry… (constraints) • Several methods: 1) Distance geometry (DG): calculation of distance constraint matrices of for each pair of atoms (older method) 2) Restrained Molecular Dynamics (MD): Simulate molecular motions (e.g. torsions around bonds) 3) Simulated Annealing (SA): heat to a high temperature (e.g. 3000 °K) followed by slow cooling steps • Methods 2 and 3 work towards the energetically favourable final structure under the influence of a force field derived from the restraints and constraints

  40. There is always more than one solution to the parameter set: The results of an NMR structure determination are presented as an “ensemble of conformers” 20, structures, all atoms

  41. The ensemble (20 structures) Backbone traces

  42. Average structure Ensembles are awkward to handle, if one wants to inspect the structure, therefore calculation of anaverage structure is useful. “Sausage”: Backbone representation of average structure; thickness of tube indicates deviations between individual conformers

  43. Final average structure Initial average structure is only mean between positions of individual atoms in different conformers - bonds and angles strongly distorted - need to do force-field based energy minimisation. Newer approach: Select representative conformer

  44. Validation • Structural statistics: • Violation of restraints • root-mean-square deviations between individual conformers and the mean structure • Back-calculations: does the structural model give rise to a NOESY spectrum that resembles the experimental data ? • Is the structure physically reasonable ?  Comparison of the resulting structure with empirical parameters: • E.g. Whatcheck and Procheck : Look at bond lengths, angles, dihedrals, van-der-Waals contacts, stereochemistry.....

  45. Heteronuclear NMR • Common nuclei: 15N, 13C • Usually requires uniform labelling expression of protein in cells that live on 15NH4Cl as single nitrogen source, and (e.g.) 13C-glucose as single carbon source • Other nuclei: • 31P (the only stable isotope) : useful for DNA • 113Cd or 111Cd : Cd has eight stable isotopes - needs enrichment

  46. Labelling strategies • Uniform • Selective, e.g. all histidine residues • Chain selective (for hetero-oligomers) • Partial • e.g. deuterate only aliphatic protons • For solid-state NMR: Use only x % isotopically labelled nitrogen or carbon source: “dilute spins” • Or: Mix uniformly-labelled with unlabelled protein • Or: use differentially labelled 13C sources • Differential labelling (mixture of 2 compounds, observe signals of only one): Useful for protein/protein or protein/DNA interactions

  47. 15N • Natural abundance: 0.368% • Spin ½ • Receptivity relative to 1H: 0.00000384 • Need isotopic labelling • Recombinant protein expression in minimal medium with 15NH4Cl as single nitrogen source • Relatively cheap: ca. 15£/l culture (which can be enough for one NMR sample)

  48. O H N C C H C H 3 1H,15N correlation (HSQC) d15N 105 110 115 120 125 130 135 9.0 7.0 d 1H

  49. Advantages of 15N labelling:Quick way to explore folding well folded Unfolded/random coil HSQC spectra taken from NMR pages of the Max-Planck-Institut für Biochemie, Martinsried.

  50. Advantages of labelled proteins Isotope editing 15N 1H 1H 3D [1H,15N,1H] HSQC-TOCSY and HSQC-NOESY

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