BCHM 313 – Physical Biochemistry

1. BCHM 313 – Physical Biochemistry Dr. Michael Nesheim (Coordinator) nesheimm@queensu.ca Rm. A210 Botterell Dr. Steven Smith (Co-coordinator) steven.smith@queensu.ca Rm. 615 Botterell Dr. Susan Yates yates@queensu.ca Rm. 623 Botterell

2. BCHM 313 – Physical Biochemistry • Topics • Protein NMR – Smith • Macromolecular Crystallography – Yates • Hydrodynamics – Nesheim • Equilibrium Binding – Nesheim • Enzyme Kinetics – Nesheim • Spectroscopy – Nesheim • Evaluation • Midterm test – 35% • Final exam – 65%

3. Protein NMR Spectroscopy Determining three-dimensional structures and monitoring molecular interactions (http://pldserver1.biochem.queensu.ca/~rlc/steve/313/)

4. Outline • N-dimensional NMR • Resonance assignment in proteins • NMR-based structure determination • Molecular interactions • Reference textbooks: • Lehninger • others available in my office.

5. Nuclear Magnetic Resonance (NMR) • MRI – Magnetic Resonance Imaging (water) • In-vivo spectroscopy (metabolites) • Soild-state NMR (large structures) • Solution NMR • Chemical structure elucidation • - Natural product chemistry • - Synthetic organic chemistry – analytical tool of choice for chemists • Biomolecular structural studies (3D structures) • Proteins • DNA and Protein/DNA complexes • Polysaccharides • Molecular interactions • Ligand binding and screening (Biotech and BioPharma) • Nobel prizes (3): Felix Bloch, 1952 (Physics); Richard • Ernest, 1991 (Chemistry); Kurt Wuthrich, 2002 (Chemistry)

6. Spectroscopy & Nuclear Spin • Absorption (or emission) spectroscopy (IR, UV, vis). Detects the • absorption of radiofrequencies (electro-magnetic radiation) by certain nuclei in a molecule - NMR • Unfortunately, some quantum mechanics are needed to understand it • Only nuclei with spin number (I)  0 can absorb/emit electro-magnetic radiation • Nuclei with even mass number & even number of protons (12C): • I = 0 • With even mass number & odd number of protons (14N): I = 1, 2, 3…. • With odd mass number (1H, 15N, 13C, 31P): I = 1/2, 3/2 ……. • Spin states of nucleus (m) are quantified: mI= (2I + 1) • Thus, for biologically relevant nuclei, two spin states exist: 1/2, -1/2

7. Spin ½ Nuclei Align in Magnetic Fields Efficiency factor- nucleus Energy Bo DE = h g Bo/2 Constants Strength of magnet • In ground state all nuclear spins are disordered – no energy difference degenerate • Since nuclei are small magnets, they orient when a strong magnetic field is applied – small excess aligned with field (lower energy)

8. Intrinsic Sensitivity of Nuclei Nucleusg % Natural Relative Abundance Sensitivity 1H 2.7 x 108 99.98 1.0 13C 6.7 x 107 1.11 0.004 15N -2.7 x 107 0.36 0.0004 31P 1.1 x 108 100. 0.5

9. H1 hn = DE 2. pump in energy Resonance: Perturb Equilibrium β 1. equilibrium DE Bo Efficiency factor- nucleus α DE = h g Bo/2 Constant Strength of magnet β α 3. non-equilibrium

10. Return to Equilibrium (Relax) β DE 3. Non-equilibrium α hn = DE 4. release energy (detect) β 5. equilibrium α

11. Nβ Nα -DE/kT = e Magnetic Resonance Sensitivity Sensitivity (S) ~ D(population) Efficiency factor- nucleus S ~ DN = DE = h g Bo/2 • E is small - @ r.t. ~1:105 • Thus, intrinsically low sensitivity • *Need lots of sample Constant Strength of magnet Increase sensitivity by increasing magnetic field strength

12. Energy/Frequency Relationship For a particular nucleus: DE = h  = B/2 DE = hB/2 Have to consider precession since: 1) nucleus has inherent spin and 2) application of a large external magnet generates a torque which results in precession Precession occurs around B at frequency, termed Larmor frequency ().  = B/2 =  wo m Bo Reason why 14.1 Telsa magnet often called 600 MHz magnet.

13. z x y Bulk Magnetization Two spins All spins Sum z x z z y Bo Mo b x x y y a

14. z x z y x y Power of Fourier Transform z t 90 RF pulse x y  = B B A t f Fourier Transform  Variation of signal at X axis vs. time NMR frequency

15. Pulse Fourier Transform NMR z z z z 90 RF pulse t x x x x y y y y 1 = B 2 = B A t f 2 1 Fourier Transform NMR time domain  Variation in amplitude vs. time NMR frequency domain  Spectrum of frequencies

16. Fourier Transform Data Analysis Time domain (t) Pulse FT NMR Experiment 90º pulse Experiment acquisition (t) equilibration equilibration detection of signals FID

17. NMR terminology • Magnetic field (B) felt by each nucleus affected by its local electronic environment - big difference between B (MHz) and Blocal (hundreds of Hz) ie. parts per million (ppm, ) • Use relative scale and refer all signals in spectrum to a signal from a reference compound (DSS)  - ref  = ref

18. z x z y x y Summary All spins Sum z z Bo b Mo } x x y y a DE = hBo/2  = B =  DE = h z FID – time domain t 90 RF x y

19. Summary (cont) FID – time domain frequency domain 10 0 ppm Chemical shift – relative scale

20. NMR terminology Scalar and Dipolar Coupling Through Bonds Through Space Coupling of nuclei gives information on structure

21. OH CH2 CH3 Resonance Assignment CH3-CH2-OH Which signal from which H atoms? The key attribute: Use scalar and dipolar couplings to match the set of signals with the molecular structure

22. Proteins Have Too Many Signals! 1H 1D NMR Spectrum of Ubiquitin ~500 resonances 1H (ppm) Resolve resonances by multi-dimensional experiments

23. Examples of Amino Acids

24. NMR experiment 90º pulse 1D acquisition (t) equilibration equilibration detection of signals 2D 2D detect signals twice (before/after couple) 90º pulse evolution (t1) mixing acquisition (t2) preparation Transfers between coupled spins Same as 1D experiment

25. 2D NMR: Coupling is the Key 2D detect signals twice (before/after couple) 90º pulse evolution (t1) mixing acquisition (t2) preparation Transfers between coupled spins Same as 1D experiment t1 t1 t2 t2

26. 2D NMR Spectrum excitation acquisition (t2) Pulse sequence evolution (t1) mixing preparation Either: Spectrum HB t1 t2 Before mixing Coupled spins or: HA t1 t2 After mixing

27. The Power of 2D NMR Resolving Overlapping Signals 1D 2 signals overlapped 2D 2 cross peaks resolved

28. Multi-Dimensional NMR Built on the 2D Principle 3D detects signals 3 times excitation 90º pulse acquisition (t3) mixing evolution (t1) mixing evolution (t2) preparation t2 t1 Same as 1D experiment t3

29. Protein NMR: Practical Issues Hardware: • Magnet: homogeneous, high field - $$$$ • Electronics: stable, tunable • Environment: temperature, pressure, humidity, stray fields Sample Preparation: • Recombinant protein expression (E. coli, Pichia pastoris etc) • Volume: 300 L – 600 L • Concentration: 1D ~ 50 M, nD ~ 1mM ie. @ 20 kDa, 1mM = 10 mg • Purity: > 95%, buffers • Sensitivity (): isotope enrichment (15N, 13C)

30. Protein NMR: Practical Issues (cont.) Solution Conditions: • Variables: buffer, ionic strength, pH, temperature • Binding studies: co-factors, ligands • No crystals! Molecular Weight: • up to 30 – 40 kDa for 3D structure determination • > 100 kDa: uniform deuteration, residue and site-specific, atom-specific labeling • Symmetry reduces complexity: 2 x10 kDa  20 kDa

31. NMR Spectrum to 3D structure? | | 1H (ppm) 0 12

32. Critical Features of Protein NMR Spectra • The nuclei are not mutually coupled • Regions of the spectrum correspond to different parts of the amino acid • Tertiary structure leads to increased dispersion of resonances • chemical shifts associated with each nucleus influenced by local chemical environment – nearby nuclei Each amino acid gives rise to an independent NMR sub-spectrum, which is much simpler than the complete protein spectrum

33. Regions of a protein 1H NMR Spectrum What would an unfolded protein look like?

34. Solutions to the Challenges • Increase dimensionality of spectra to better resolve signals: 1  2  3  4 • Detect signals from heteronuclei (13C, 15N) • Better resolution of signals/chemical shifts not correlated nuclei • More information to identify signals • Lower sensitivity to MW of protein

35. 1D Protein 1H NMR Spectrum

36. Resolve Peaks by Multi-D NMR A BONUSregions in 2D spectra provide protein fingerprints If 2D cross peaks overlap go to 3D

37. T L R S G G S Basic Strategy to Assign Resonances in Protein • Assign resonances for each amino acid • Put amino acids in order • Sequential assignment (R-G-S, T-L-G-S) • Sequence-specific assignment 1 2 3 4 5 6 7 R - G - S - T - L - G - S

38. Acronyms for Basic Experiments Differ Only in the Nature of Mixing Homonuclear Heteronuclear HSQC Heteronuclear COSY COrrelation SpectroscopY Scalar Coupling (thru-bond) Hetero-TOCSY TOCSY TOtal Correlation SpectroscopY Dipolar Coupling NOESY Nuclear Overhauser Effect (Enhancement) SpectroscopY NOESY-HSQC (thru-space)

39. Homonuclear 1H Assignment Strategy • For proteins up to ~ 10 kDa • Scalar couplings to identify resonances/spin systems/amino acids, dipolar couplings to place in sequence • Based on backbone HN (unique region in 1H spectrum, greatest dispersion of resonances, least overlap) • Concept: Build out from the backbone to identify the side-chain resonances (unique spin systems) • 2nd dimension resolves overlap, 3D rare

40. COSY (3-bond) Homonuclear 1H Assignment Strategy Step 1: Identify Spin System TOCSY dCH3 H – H – H – C – N – C – C – – = aH O H H Alanine HN

41. COSY (3-bond) TOCSY CH3 H3C – – C – H – H – C – H – N – C – C – – = H O H Leucine Homonuclear 1H Assignment Strategy Step 1: Identify Spin System d’CH3 dCH3 gH b’H bH aH HN

42. dCH3 CH3 H3C – – H – C – H H – H – – C – H – C – H – N – C – C – aH – = N – C – C – O – H = H O H Alanine - open circles Leucine - closed circles HN Homonuclear 1H Assignment Strategy Step 1: Identify Spin System COSY (3-bond) d’CH3 dCH3 TOCSY gH b’H bH – aH H HN

43. A B C D • • • • Z Homonuclear 1H Assignment Strategy Step 2: Fit residues in sequence Minor Flaw: All NOEs mixed together! Use only these to make sequential assignments Long Range Sequential Intraresidue • Sequential NOEs •  HN-HN (i, i + 1) •  H-HN (i, i + 1) Medium-range (helices: H-HN (i, i + 3,4)))

44. Homonuclear 1H Assignment Strategy Step 2: Fit residues in sequence d’CH3 NOESY = dCH3 CH3 H3C – – bCH3 COSY/TOCSY + C – H gH H – – b’H H – H – H – C – H C bH – – – N – C – C N – C – C aH – – – = – = aH O H H H O H HN HN

45. Extended Homonuclear 1H Strategy • For proteins up to ~ 15 kDa • Same basic idea as 1H strategy: based on backbone HN • Concept: When backbone 1Hoverlaps  disperse with backbone 15N • Use heteronuclear 3D experiments to increase signal resolution • 1H 1H 15N

46. Solutions to the Challenges • Increase dimensionally of spectra to better resolve signals: 1  2  3  4 • Detect signals from heteronuclei (13C, 15N) • Labeling with NMR-observable 13C, 15N isotopes • Better resolution of signals/chemical shifts not correlated nuclei • More information to identify signals • Lower sensitivity to MW of protein

47. Isotopic Labeling • Require uniform 15N/13C labeling ie. Every carbon and nitrogen isotopically labeled • How? • Grow bacteria on minimal media (salts) supplemented with 15N-NH4Cl and 13C-glucose as soles sources of nitrogen and carbon • lower yields than protein expression than on enriched media, therefore need very good recombinant expression system

48. Double Resonance Experiments Increases Resolution/Information Content

49. H R 15N – C – C – 15N – C O R H Heteronuclear NMR: 15N-Edited Experiments Increases Resolution/Information Content

50. 3D Heteronuclear NMR: 15N-Edited Experiments +