1. 3-hour Open book exam. No discussion among yourselves.

Transcription

1 Lecture 13 Review 1. 3-hour Open book exam. No discussion among yourselves. 2. Simple calculations. 3. Terminologies. 4. Decriptive questions. 5. Analyze a pulse program using density matrix approach (omonuclear 2D). 6. Analyze a pulse program using product operator approach (eteronuclear 2D).

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13 Lecture 13 Protein Structure determination by NMR Nuclear Spin Interactions ( 核子自旋交互作用 ) 6 Electrons Nuclear Spin i 2 Nuclear Spin j 1 o 5 o 4 4 Phonons 4 Dominant interactions: = Z + D + S + Q. Z = Zeeman Interaction D = Dipolar Interactions S = Chemical Shielding Interaction. Q = Quadrupolar Interaction

14 NMR spectra of samples in different physical states Small molecule in solution 1 Porphorin 1 Macromolecule in solution 6 kz Myoglobin Gel 1 Fat 31 P Solid sample 22 kz 3 -CMP Single crystal 13 C δ Chemical shift is orientation dependent

15 NMR Parameters ( 參數 ) (Measurable quantities) 1. Chemical Shift : Difference in resonance frequency due to chemical structure difference (in ppm). 2. Resonance Intensity: Determine number of spins.. 3. J-coupling: Resonance splitting due to through-bond spin coupling Nuclear Overhauser Effect (NOE): Energy transfer through dipolar coupling. 1 R Residual dipolar coupling: Non-vanishing dipolar coupling in oriented media.. 6. Relaxation rates (T 1, T 2 etc): Lost of magnetization due to dephasing (T 2 ) or energy dissipation (T 1 ) M 15 N t B O θ 1 15 N- 1 vector orientation

16 1. Chemical Shift NMR Parameters The chemical shift of a nucleus is the difference between the resonance frequency of the nucleus and a standard, relative to the standard. This quantity is reported in ppm and is given by the symbol δ, δ (ω - ω REF ) x10 6 /ω REF Where ω REF is the reference frequency of the standard compound, i.e. the methyl resonance of tetramethylsilane (TMS) or 2,2-dimethyl-2-silapentane-5- sulfonate (DSS). In this relative scale, the δ value is independent of magnet field used. (i.e same in 100 Mz magnet (2.35 T) or in a 600 Mz magnet (14.1 T). Deshielded (low field) 15 Acids Aldehydes Aromatics Amides Olefins Alcohols, protons α to ketones 2 Aliphatic 0 TMS ppm Shielded (upfield)

17 Chemical Shift Referencing: The 1 chemical shift was referenced to 2,2-dimethyl-2-Silapentane-5-sulfonate dmethy (DSS) at 0 ppm. The 15 N and 13 C chemical shift values were referenced using the consensus ratio of Ξ of and for 15 N/ 1 and 13 C/ 1, respectively (Wishart and Case, Method. Enzymol. 338, (2001)) Ξ ratio (Nucleus-specific frequency ratio: Determine the precise 1 resonance frequency of DSS then multiply this frequency by Ξ of a particular nucleus one obtains the exact resonance frequency reference at 0 ppm of that nucleus.

18 Proton chemical shift in some diamagnetic structures (12 ppm) Aliphatic 2 O 2 (4.75 ppm) Aromatic Amide group Chemical shift (ppm from DSS)

19 13 C chemical shift in some diamagnetic structures 200 ppm range (large dispersion, better resolution) Functional group Chemical shifts Aliphatic Chemical shift (ppm from DSS)

20 Chemical shift ranges of 15 N (800 ppm) In biomacromolecular NMR one observe mostly amide nitrogen (- 15 N-) and side chain amino nitrogens (Arg and Lys) (- 15 N 3 or - 15 N 2 ). Amide nitrogen resonates at ~ ppm range and 80 ppm for N 2. Notice, amide nitrogen shift spans ~ 40 ppm.

21 Example of 1D : 1 spectra, 13C spectra of Codeine C NO 3, MW= C

22 2. J-coupling (More than one spins) Nuclei which are connected by chemical bonds form a coupled system and cause splitting on the energy level, thus cause resonance splitting This is called spin-spin coupling or J coupling C one-bond three-bond Energy diagram of two spin system: Each spin now seems to has two energy sub-levels depending on the state of the spin it is coupled to: αβ 2 ββ αα βα I S J (z) The magnitude of the separation is called coupling constant units of z. (J) and has

23 Number of lines N neighboring spins: split into N + 1 lines 1. Single spin: 2. One neighboring spins: -C C - 3. Two neighboring spins: -C 2 C -

24 Use of J-coupling for structure determination (Dihedral angle) One neighboring spins: -C α N - 3 J Nα 3 J Nα From coupling constant (J) one can determine the dihedral angles from the following Karplus equations, where 3 J Nα is the coupling constant between Cα N. χ 2 3 J N α 3 3 J J αβ1 αβ = 6.4 cos 2 ( φ 60) 1.4 cos( φ 60) = 9.5cos 2 ( χ 120) 1.6 cos( χ 120) = 9.5cos χ cos χ C χ 1 ψ N Cβ Cα Ψ C N ω C α 3 J Nα = 4 11 z depends ds on secondary structure. t 3 J Nα < 6 z α-helix; 3 J Nα > 8 z β-stand

25 For through-bond 3D NMR (Magnetization transfer) - J-coupling of backbone nuclei (z) C γ χ 2 C β 35 χ 1 2 J( 13 C α N) = 9 C ψ 15 N 11 Ψ C α ω 55 C 15 N 11 C α 94 O

26 3. Nuclear Overhauser Effect (NOE) RF I r S XNOE = 1 + (d 2 /4)(γ / γ N )[6J(ω + ω N ) J(ω - ω N )] T 1 where d = (μ o hγ N γ /8π 2 )(r N -3 ), J(ω) is the spectral density function 1. Distance info: XNOE r -6 ; 2. Dynamics: XNOE J (ω )

27 4. Residual dipolar coupling in partially oriented media Bicells Phage

28 Dipolar interactions of peptide plane nuclei 1 B Z o Dipolar pairs 1-15 N 13 C α - 15 N 13 CO - 15 N 13 CO - 13 C α 13 C α 15 N 13 C O 1-13 C α 1 13 C α Y X

29 S Dipolar Field I D Residual Dipolar Coupling: S: Scaling factor = 0 in non-viscous liquid media γ: Nuclear gyric ratios μohsγ Iγ S ( θ, ϕ ) = ( ){(3cos θ 1) Asin θ cos2 ϕ } 3 16π R 2 IS + R: Distance between spins I and S

30 Protein structure Stick & ball representation (Cartesian space) Peptide plane representation (Angular space) Strings of Peptide planes

31 4. NMR Relaxation

32 Applications I. Structure: - Protein structure up to 60 kda has been reported (easier for < 20 kda) - Can observe good protein signal up to 800 kda. II. Dynamics (Motion): - Characterize molecular l motion (4th dimension) III. Drug screening: - igh throughput h t (1000 samples per day) - Atomic details - Lead discovery. IV. Magnetic Resonance Imaging (MRI): V. Metabolomics (Small molecule identification): n):

33 Determine Protein Structure by NMR NMR Sample (1 mm, 0.4 ml) 2, 13C, 15N-label Obtain NMR spectra (3 weeks) Assign resonances Automation? NMR structures (Ensemble of 20 structures) Calculate structures Obtain restrains (Distances, angles, Orientations etc)

34 Resonance Assignment Sidechain N & COO

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36 2D-NMR Spectrum - Diagonal resonances same as in 1D spectrum Diagonal resonances same as in 1D spectrum - off-diagonal cross peaks are due to interactions such as NOE or J-coupling

37 1 1 NOESY of RC-RNase S I

38 omonuclear 2D NMR experiments (Nuclear Overhauser Effect SpectroscopY) Through space dipolar effect Determine NOE Measuring distance Assign resonances (COrrelated SpectroscopY) Through bond J-coupling Assign adjacent resonances (Multiple Quantum Filtered COrrelated SpectroscopY) Through bond J-coupling similar to COSY Assign adjacent resonances More sensitive (TOtal Correlated SpectroscopY) (TOC SY) TOSY Through bond relayed J-coupling Assign full spin system (residues type)

39 COSY v.s. TOCSY spectra (Fingerprint region) COSY TOCSY δ γ β α N C3 C2 C 2 C CO ical Shi ift (ppm m) c Chem 1 Aliphatic γ β γ β α α N Chemical Shift (ppm)

40 COSY (Fingerprint region) δ N δ (0.62) γ C3 C2 C-C 3 C (1.32) (1.03) β γ (1.76) (0.71) α (8.75) γ β γ α (4.85) CO I N Chemical Shift (ppm) ical Shif ft (ppm) α Chem 1 Isoleucine See only N and α correlation

41 TOCSY (Spin System Identification) RC-RNase 1. J-Coupling: N α β. 2. Identify Spin System(a.a. type) δ N δ γ β α δ C3 γ (0.62?) C2 (1.32?) γ (1.03?) C-C 3 γ γ C (4.85) β (2.60) (0.71?) (8.75) γ β γ α Isoleucine CO α I12 δ 1 /ppm γ 2.60 β 4.85 α

42 2D 15 N- 1 eteronuclear Single Quantum Correlation Spectroscopy ( 15 N-SQC) 1 90 x 90 x 90 x 180 x 180 x 180 x τ τ τ t x 90 x 180 x 15 N t 1 Decoupling Magnetization 15 N chemical shift 1 detection transfer evolution from 1 to 15 N Magnetization transfer from 15 N to 1 Observe 15 N spectrum in t 1 and 1 spectrum in t 2 dimension Excellent resolution Each peak codes for one amide group ( 15 N- 1 ), i.e. one amino acid. Detect 15 N at 1 sensitivity.

43 15 N-eteronculear Single Quantum Spectroscopy ( 15- N SQC) Q81

44 23 kda protein (191 a.a.) Cannot be assigned using 2D NMR alone 3 or higher dimensional NMR

45 Magnetization transfer thru bonds J-coupling of backbone nuclei (z) C γ χ 2 C β 35 χ 1 2 J( 13 C α N) = 9 C ψ 15 N 11 Ψ C α ω 55 C 15 N 11 C α 94 O

46 Magnetization transfer thru bonds

47 3D NCA 1 90 x x 90 x x x x x x 180 x τ τ τ t 3 15 N t x 90 x 180 x δ δ 180 x Decoupling 180 x 90 x 90 x 13 C α t 2 Decoupling 13 CO Decoupling Detect: 1 N, 15 N and 13 C α δ = 1/4J N-CA = 1/4x1010 = 25 ms for optimal detection τ= 1/4J -N = 1/4x94 = 2.5 ms

48 13 C Che emical Shift 1 Chemical Shift

49 eteronuclear multidimensional NMR experiments for resonance assignments Magnetization transfer pathway: 1 15 N 13 C 15 N 1 1 Detection Detect 1, 13 C, 15 N resonances Permit sequential correlation of Permit sequential correlation of backbone 1-13 C- 15 N resonances!!!

50 13 C 15 N Select a 1 frequency 15 N 1 Select a particular 13 C frequency Select a 15 N frequency 13 C 15 N 13 C 1 1

51 N Chemical shift (ppm) ft (ppm) mical shif 3 C Chem Chemical shift (ppm)

52 15 N 13 C 1 Select a 15 N frequency 1 Chemical shift (ppm) 13 C Chem mical shift (ppm)

53 N Chemical shift (ppm) ft (ppm) mical shif 3 C Chem Chemical shift (ppm)

54 R n-1 R n N C α CO N C α CO 9.0 z 11.0 z 1. In NCA experiment the stronger cross peak belongs to its own CA and the weaker one belongs to precedent amino acid. 2. Combine NCA with N(CO)CA one can assign the CA resonances unambiguously. NCA N C α CO N C α CO N(CO)CA 3. Use several sets of thru-bond 3D experiment one can assign all Backbone resonances. 4. Side chain resonances: CC-TOCSY, TOCSY-SQC or NOESY-SQC.

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56 Side-Chain assignments 3D C(C)-TOCSY ( 1-13 C- 1 ) Use 13 C to resolve the proton overlaps. Use 1-1 coupling as in 2D TOCSY to detect side chain connectivity. N δ γ β α C3 C2 C 2 C CO

57 15 N-eteronculear Single Quantum Spectroscopy ( 15-N SQC)

58 Assignment Table

59 p-dependent of proton exchange rates Backbone N exchange rates

60 Nucleotide N exchange rates