instructor: tai-huang huang ( 黃太煌 ) 中央研究院生物醫學科學研究所 tel....
DESCRIPTION
NMR 3- Pulse sequence and NMR experiments. Instructor: Tai-huang Huang ( 黃太煌 ) 中央研究院生物醫學科學研究所 Tel. (886)-2-2652-3036; E. mail: [email protected] Web site: www.nmr.ibms.sinica.edu.tw/~thh/biophysics/NMR-2.ppt Reference: - PowerPoint PPT PresentationTRANSCRIPT
Instructor: Tai-huang Huang ( 黃太煌 )中央研究院生物醫學科學研究所
Tel. (886)-2-2652-3036; E. mail: [email protected]
Web site: www.nmr.ibms.sinica.edu.tw/~thh/biophysics/NMR-2.ppt
Reference:
Cavanagh, J. et al., “Protein NMR Spectroscopy-Principles and Practice”, Academic Press, 1996.
Term paper:
Find a NMR paper and write a report on the subject related to the paper.
NMR 3- Pulse sequence and NMR experiments
液態樣品 取得NMR圖譜 圖譜分析結構計算
( hours/days to weeks) ( weeks to months)( days to weeks)
Steps involved in determining protein structures by NMR
NMR II- Pulse sequence and NMR experiments
Collecting NMR signals NMR signal is detected on the xy plane. The oscillation of Mxy generate a current in a coil , which is the NMR signal. Due to the “relaxation process”, signal decay with time. This time dependent signal is called “free induction decay” (FID)
Mxy
time
(if there’s no relaxation ) (the real case with T1 &T2)
•The Bloch Equations: dM/dt = M x B + relaxation terms
dMx(t) / dt = [ My(t) * Bz - Mz(t) * By ] - Mx(t) / T2 --------- (1) dMy(t) / dt = [ Mz(t) * Bx - Mx(t) * Bz ] - My(t) / T2 --------- (2)dMz(t) / dt = [ Mx(t) * By - My(t) * Bx ] - ( Mz(t) - Mo ) / T1 ------ (3)
Rotating frame:
Let [dM(t)/dt]rot = [dM(t)/dt]lab+M(t) x = M(t) x [γB(t) + ]
Let Beff = B(t) + /γ ------------------- (4)Thus, if B(t) + /γ= 0, or B(t) = - , Beff = 0 dM(t)/dt = 0, M(t) is time independent.
In the absence of RF field and B(t) = Bo or B(t) = -Bo = - o = Larmor frequency.In a frame rotating at Larmor frequency the magnetization is static. The Bloch equations become:
dMz(t) / dt = [ Mo - Mz(t)/ T1 -------------- (5)dMx(t) / dt = - Mx(t) / T2 -------------- (6)dMy(t) / dt = - My(t)/T2 -------------- (7)
Bo= Bo - o/
X
Y
Z
Bo
Solutions: Mz = Mo – [Mo –Mz(0)]exp(-t/T1) -------------- (8) Mx = Mx(0)exp(-t/T2); -------------- (9) My = My(0)exp(-t/T2); -------------- (10)
T1 relaxation in the Z-direction and T2 relaxation on the xy-plane If we obsere the spins in a frame which rotate at exactly the Larmor frequency then we see the spin state stationary (Static). What if we observe the spin at a frequency which is from the Larmor frequency ? Both Mx and My will rotate at Hz.
Experimentally what is the rotating frame ?
Transmitter Probe
Receiver Digitizer
Computero
o - o
Signal is in rotating frame (kHz)
106 – 109 Hz
Effect of RF-field:dMz(t)/dt = [Mx(t)Br
y(t) – My(t)Brx(t)] – [Mz(t) – Mo]/T1
dMx(t)/dt = - My(t) – Mz(t)Bry(t) – Mx(t)/T2 ----------- (11)
dMy(t)/dt = Mx(t) – Mz(t)Brx(t) – My(t)/T2
where Brx(t) = Br
ocos and Bry(t) = Br
osin
= -γΔBo - rf = o - rf is the offset.
In a common experimental situation in pulse NMR, B1 is applied for a time p << T1, T2 and neither B1 nor is time dependent. Thus, during the time when B1 is on eq. 11 becomes:dMz(t)/dt = Mx(t)Br
y(t) – My(t)Brx(t)
dMx(t)/dt = - My(t) – Mz(t)Bry(t) ----------- (12)
dMy(t)/dt = Mx(t) – Mz(t)Brx(t)
The solution of eq. 12 is a series of rotations about the axis perpendicular to the applied B1 field. The signal can be described as:
Mx(t) = Mosincos(t)exp(-t/T2) My(t) = Mosinsin(t)exp(-t/T2)
B1
Bo
Br
Bloch Equations (Phenomenological equations):
dMx/dt = (M x H)x – Mx/T2 -------------------- (1)
dMy/dt = (M x H)y – My/T2 -------------------- (2)
dMz/dt = (M x H)z – (Mo – Mz)/T1 ----------- (1)
For H1 along the x-axis and H1 0 and in steady statei.e. dM/dt = 0 we can solve the above simultaneous Equations to get:
Mx = o(oT2) -------- (3)
(Lorenzian lineshape, absorption)
My = o(oT2) -------- (4)
(Dispersion)
22
212
)(1
)(
T
HT
o
o
22
21
)(1 T
H
o
My
Mx
Fourier transformation (FT)
FT
FT
FT
Function at
exponential LorenzianAt zero Hz
Absorption: Mx = Mo/[1 + ( - )2T22]
Dispersion signal: My = Mo(-)/[1 + ( - )2T22]
Lorenzian at
MMx My
= 1/T2
Pulsed NMR spectroscopy (only signal on X-Y plan is observable)
90o-pulse: Iz Iy Sees a strong signal
180o-pulse: Iz -Iz Sees no signal.
0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00t1 sec
90x
180x
0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00t1 sec
180x
90x
FT
FT234 233 232 231 230 229 228 227 226 225 224 223
f1 ppm
X X
X X
Y Y
Y Y
Pulsed NMR spectroscopy (only signal on X-Y plan is observable)
-90o-pulse: Iz Iy Sees a strong negative signal
-180o-pulse: Iz -Iz Sees no signal.
0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00t1 sec
90x
180x
0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00t1 sec
-180x
-90x (same as 270x)
FT
FT234 233 232 231 230 229 228 227 226 225 224 223
f1 ppm
Y Y
Y Y
X X
X X
Spin-echo pulse: 90o--180o--detection1. Refocus chemical shift. 2. Decouple of heteronuclear J-coupleing
0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00t1 sec
0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00t1 sec
90x
FT234 233 232 231 230 229 228 227 226 225 224 223
f1 ppm
Y
X
180x
00.100.200.300.400.500.600.700.800.901.00t1sec
Detection
90x
180x
(Inversion)
XX
Y Y
Y
X
Y
X
(Dephasing)
(Refocusing)
(Excitation)
00.100.200.300.400.500.600.700.800.901.00t1sec
(Detection)
Pulse of finite length
1. Long weak pulse:
Square waver SINC function (sinx/x) If is very short then one will excite a broad spectral region. Long pulse excite only finite region of the spectrum.
2. Shape pulse: SINC function (sinx/x) Square wave
Gaussian Gaussian
tB1
0
1/
FT
Power
Sinx/x
1/
Power
1D one pulse 1H
Aliphatic Aromatic & Amide
Types of NMR
Experiments
Homo Nuclear: Detect proton.
Heteronuclear – Other nuclei, 13C, 15N, 31P etc.
Huge Water signal(110 M compare to 1 mM for normal protein sample)
Water suppression is an important issueDynamic range problem.
3. 1-1 pulse: = 0
to
1/to
1/to
4. 1331 pulse: Similar to 11 pulse but more complicated
5. Gradient enhanced pulse sequence (Watergate):
Receiver on(/2)-X
(/2)X
(/2)-Y (/2)-Y
GZ
1H
Gradient causes
Homo Nuclear 2D NMR – Need two variable times
Basic 1D Experiment
Basic 2D Experiment
Homo Nuclear 2D NMR – Need two variable times
1. Needs two time variables t1 and t2 for chemical shift to evolve.
2. Needs to decide what interaction do you wish to observe ? J-coupling – short and long range coupling. Take place on x-y plane only. NOE – Take place when magnetization is in Z-direction.
3. In heterouclear NMR one needs a way to transfer magnetization between nuclei. J-coupling (the larger the easier to transfer magnetization). Need to adjust the time duration of the coupling (Maximum when coupling time = 1/2J. If J = 100 Hz, = 5 ms)
J-coupling
•Nuclei which are bonded to one another could cause an influence on each other's effective magnetic field. This is called spin-spin coupling or J coupling.
13C
1H 1H 1H
one-bondthree-bond
•Each spin now seems to has two energy ‘sub-levels’ depending on the state of the spin it is coupled to:
The magnitude of the separation is called coupling constant (J) and has units of Hz.
aa
ab ba
bb
I SS
S
I
IJ (Hz)
ψ Ψ
Cα
Cβ
Cγ
ωN
χ1
χ2
C’
N
H
H
H H
O
C’
Cα
94
1115
2J(13C15N) = 9
35
55
140
35
15
94
11
J-coupling of backbone nuclei (Hz)3J(HN-CA) = 4 – 11 Hz depends on secondary structure.
< 6 Hz -helix > 8 Hz -stand
Heteronuclear 2D NMR (HETCOR) – (Need ways to couple different
nuclei)
t1
t11
t21
t31
t41
FT (t2)
FT (t1) Transpose (t2)
2
1
2D-NMR Spectrum – stack plot
2D spectrum (Countour plot)
Determining Macromolecular Structures
(1)Prepare
NMR samples2H, 13C and/or 15N-
Labeled
(3)Assign NMR
resonances
(2)Obtain
NMR spectra -( 1D, 2D, 3D & 4D)
(4)Obtain
NMR restraintsdistances,
dihedral angles bond orientations
(5)Structure
Calculation and
refinement
Determining Macromolecular Structures
(3)Assign NMR
resonances
1. Assign all resonances to a specific amino acid.2. Assign to a specific nucleus.3. Proton resonances are most important for structure determination.4. Homonuclear 2D NMR for small proteins (< 10kDa).5. Heteronuclar NMR are required for larger proteins (> 10 kDa)6. Deuteration is needed for protein > 30 kDa.
Homonuclear NMR – small protein
1000 protons to assign.1D clear is unable to do the job.
Determination of the Structure of RC-RNase
1. A pyridine-Guanine specific Ribonuclease found only in the oocyte of bullfrog (Rana catesbeiana).
2. It is also a lectin with cytotoxic and antitumor activity.
3. A single chain poplypeptide with 111 amino acids and four disulfide bonds.
4. The structure of RC-RNase has not been determined.
Reference: 1. Chen et al., 1996, J. Biomol. NMR 8 331-344. 2. Chang et al., 1998, J. Mol. Biol. 283 231-244.
Assignment of Protein NMR Resonances
1. Spin system (amino acid) identification:
- Rely on J-coupling (2-D COSY & TOCSY) COSY: Cross peaks observed for Nearest neighbors only (e.g. NH
to Hα only) TOCSY: All coupled spins are potentially observable (e.g. NH to
Hα, Hβ, Hγ…etc). - Chemical shifts of the observed COSY and TOCSY cross peaks.
2. Sequential resonance assignment:
- Assign resonances to a specific amino acid (e.g. Gly-10 etc). - NOESY (NH- Hα, Hβ etc). - Heteronuclear 3-D NMR expts. (15N-13Cα, CO).
(COrrelated SpectroscopY)Through bond J-coupling Assign adjacent resonances
(Nuclear Overhauser Effect SpectroscopY) Through space dipolar effect Determine NOE Measuring distance Assign resonances
(TOtal Correlated SpectroscopY) (TOC SY)
Through bond relayed J-coupling Assign full spin system (residues type)
(Homonuclear HAtman-HAhn spectroscopY)
(Multiple Quantum Filtered COrrelated SpectroscopY)Through bond J-coupling similar to COSY Assign adjacent resonances More sensitive
COSY: (MQF-COSY; DQF-COSY) 1. Off-diagonal resonances due to 1JNHC one bond J-coupling.
2. Assign adjacent resonances.3. One can select a magnetization transfer pathway (efficiency) by varying the evolution time.
TOCSY: ( HOHAHA)
1. Off-diagonal resonances due to relayed J-coupling.2. Magnetization transfer thru Hartmann-Hahn cross polarization.3. Assign long range correlated resonances (Whole a.a. system).
NOESY: 1. Off-diagonal resonances due to NOE.2. Magnetization transfer thru energy transfer due to thru space dipolar effect.
I R-6 Determine distances.3. Sequential resonance assignments.
RC-RNaseDQF-COSY (Fingerprint region)
1. NH-Hα only (Intra residue)
同一胺基酸
2. Splitting 3JHNα
δ1/ppm
TOCSY (Spin System Identification) RC-RNase
1. J-Coupling: HN→Hα→Hβ…….2. Identify Spin System(a.a. type)
1H – 1H NOESY of RC-RNase
r
RF
Nuclear Overhauser Effect (NOE)
XNOE = 1 + (d2/4)(H/ N)[6J(H + N) – J(H - N)] T1
where d = (ohN H/82)(rNH-3),
XNOE r-6
I S
1. Larger proteins(> 10 kDa)1. Need to label the protein with 13C and 15N, and may be 2H.
2. Need to do heteronuclear multidiemnsional NMR (3D or 4D)
3. Heteronculear has larger chemical shift dispersion, thus better resolution. (13C ~ 200 ppm; 15N ~ 300 ppm)
4. Energy transfer between heteronuclei by J-coupling.
ψ Ψ
Cα
Cβ
Cγ
ωN
χ1
χ2
C’
N
H
H
H H
O
C’
Cα
94
1115
2J(13C15N) = 9
35
55
140
35
15
94
11
J-coupling of backbone nuclei (Hz)3J(HN-CA) = 4 – 11 Hz depends on secondary structure.
< 6 Hz -helix > 8 Hz -stand
1H Chemical Shift
13C
Chem
ical Sh
ift
15 N Shi
ft
Advantages of heteronuclear NMR:
1. Large chemical shift dispersion Increased resolution.2. Large coupling constant (Easy to transfer magnetization.3. Thru bond connectivity Easy assignments.4. Permit easier analysis of protein dynamics.5. Permit determining the structure of larger proteins (> 100 kDa).
Disadvantages of heteronuclear NMR:
1. Must label the protein with 13C and/or 15N.a). Expensive.b). Time consuming.
2. Technically much more complicated.3. More demanding on spectrometers.4. Much larger data size.
二維核磁共振基本原理 (HETCOR)
Homonuclear: 同核 (1H); Heteronuclear: 異核 (1H, 13C, 15N etc)
Decoupling
1H
15N t1
t2
2D 15N-1H Heteronuclear Single Quantum Correlation Spectroscopy) (15N-HSQC)
Efficientcy sin(2J)Maximum transfer when 2J = /2. or = 1/4J = 1/4x94 = 2.5 ms
Magnetization transfer from 1H to 15N
15N chemical shift evolution
Magnetization transfer from 15N to 1H
1H detection
90x 90x 90x180x 180x 180x
180x180x 90x
Amide Proton Resonance Assignments of Thioesterase I
Decoupling
1H
15N t2
t3
3D NOESY-HSQC
NOESY 15N-HSQC
90x 90x 90x180x 180x 180x
180x180x 90x
NOE
90x90x
Dec
t1
ψ Ψ
Cα
Cβ
Cγ
ωN
χ1
χ2
C’
N
H
H
H H
O
C’
Cα
94
1115
2J(13C15N) = 9
35
55
140
35
15
94
11
J-coupling of backbone nuclei (Hz)3J(HN-CA) = 4 – 11 Hz depends on secondary structure.
< 6 Hz -helix > 8 Hz -stand
Decoupling
1H
15N t1
t3
3D HNCA
90x 90x 90x180x 180x 180x
180x180x 90x
Decoupling13Ct2
180x 90x
90x 180x
180x
13CO Decoupling
90x
90x
= 1/4JN-CA = 1/4x10 = 25 ms for optimal detection= 1/4JH-N = 1/4x94 = 2.5 ms
Detect: 1HN, 15N and 13C
Heteronuclear multidimensional NMR experiments for resonance assignments
Magnetization transfer pathway:
1H 15N 13C 15N
1H 1H Detection
Detect 1H, 13C, 15N resonances
Permit sequential correlation of backbone 1H-13C-15N resonances !!!
N NC CCO CO11Hz
9Hz
1. In HNCA experiment the stronger cross peak belongs to its own CA and the weaker one belongs to precedent amino acid.
2. Combine HNCA with HN(CO)CA one can assign the CA resonances unambiguously.
3. Use several sets of thru-bond 3D experiment one can assign all Backbone resonances.
4. Side chain resonances: HCCH-TOCSY, TOCSY-HSQC or NOESY-HSQC.
Side-Chain assignments
Resonance Assignments
I. Homonuclear: 1. Use 2D NMR (COSY, TOCSY, NOESY) to assign spin system (a.a. type). 2. Use NOESY to do sequential assignments.
II. Heteronuclear: 1. Use backbone correlated heteronuclear 3D NMR to do sequential resonance assignments of all heteronuclei. (Need seveal sets) 2. Use HCCH-TOCSY, TOCSY-HSQC or NOESY-HSQC to assign side chain resonances.
III. New developments: Chemical shift information may be crucial for easy resonance assignents.
Chemical shift table
Possible term paper topics –IInstruction: 1. Paper submission and topic selection approval all by e. mail to [email protected]. Send me a title of the term paper from the list below or your choice for approval by April 15.3. Team paper due date: May 15, 2003.4. Format: Use Microsoft word file format (or other text format).5. Content:
I. Introduction: Describe the biological background and the problems to be solved. II. NMR techniques employed: Describe succinctly what type of NMR techniques are applied
and give some description of the NMR techniques.III. Results.IV. Discussion.
Some possible topics:
1. Strategies in assigning protein NMR resonances with examples. Ref. Lin, T. H., C. P. Chen, et al. (1998). " Multinuclear NMR resonance assignments and the secondary structure of Esc
herichia coli thioesterase/protease I: A member of a new subclass of lipolytic enzymes. J. Biomol. NMR, 11, 363-380." J. Biomol. NMR 11: 363-380.
2. Strategies in protein structure determination by NMR with examples. Ref. Chang, C.-F., H.-T. Chou, et al. (2002). "Solution Structure and Dynamics of the Lipoic Acid-bearing Domain of Human Mitochondrial Branched-chain alpha -Keto Acid Dehydrogenase Complex." J. Biol. Chem. 277(18): 15865-15873.
3. NMR and protein dynamics. Ref. Huang, Y. T., Y. C. Liaw, et al. (2001). "Backbone dynamics of Escherichia coli thioesterase/protease I: Evidence o
f a flexible active-site environment for a serine protease." J. Mol. Biol. 307: 1075-1090.
4. Applications of NMR in studying protein folding. Ref. Fersht, A. R. and V. Daggett (2002). "Protein folding and unfolding at atomic resolution." Cell 108(4): 573- 582.
Possible term paper topics - continue
5. Applications of NMR in drug discovery. Ref. Peng, J. W., C. A. Lepre, et al. (2001). "Nuclear Magnetic Resonance-based Approaches for lead generation in drug discovery." Method. Enzymology 338: 202-230.
6. Applications of NMR in enzyme catalysis. Ref. Xiao, B., C. Jing, et al. (2003). "Structure and catalytic mechanism of the human histone methyltransferase SET7/9." Nature 421(6923): 652-656.
7. Strategies in determining the structures of DNA and RNA by NMR. Ref. Allen, M., L. Varani, et al. (2001). " Nuclear magnetic resonance methods to study structure and dynamics of RNA-protein complexes." Method. Enzymology 339: 357-376.
8. Strategies in determining the structure of large proteins by NMR. Ref. Fiaux, J., E. B. Bertelsen, et al. (2002). "NMR snalysis of a 900 kDa GroEl-GroES complex." Nature 418(11): 207-21
1. Riek, R., J. Fiaux, et al. (2002). "Solution NMR Techniques for Large Molecular and Supramolecular Structures." J. A
m. Chem. Soc. 124(41): 12144-12153.
9. Use of residual dipolar coupling in NMR structure determination and refinement. Ref. Prestegard, J. H. (2000). "NMR structure of biomolecules using field oriented media and residual dipolar couplin
gs." Q. Rev. Biophys. 33(4): 371-424.
10. NMR in structural genomics. Ref. Yee, A., X. Chang, et al. (2002). "An NMR approach to structural proteomics." PNAS 99(4): 1825-1830.
11. NMR in determining membrane protein structure. Ref. Fernandez, C., C. Hilty, et al. (2002). " Lipid-protein interactions in DHPC micelles containing the integral membrane protein OmpX investigated by NMR spectroscopy." Proc. Natl . Acad. Sci. 99(21): 13533-13537. Fernandez, C., C. Hilty, et al. (2001). "Solution NMR studies of the integral membrane proteins OmpX and OmpA from Escherichia coli." FEBS Lett. 504(3): 173-178.
12. Functional MRI: Ref. Ugurbil K, Toth L, Kim DS.Related Articles, Links How accurate is magnetic resonance imaging of brain function?
Trends Neurosci. 2003 Feb;26(2):108-14.