nuclear magnetic resonance (nmr) – from basic to biomedical applications – from basic to...

Nuclear Magnetic Nuclear Magnetic Resonance Resonance (NMR)(NMR)

– – From Basic to Biomedical From Basic to Biomedical Applications Applications

黃太煌黃太煌中央研究院生物醫學科學研究所中央研究院生物醫學科學研究所

NCHU Physics - May 6, 2005NCHU Physics - May 6, 2005

History of NMR

Discovery - Physicists

1924 Pauli proposed the presence of nuclear magnetic moment to explain the hyperfine structure in atomic spectral lines

1930 Nuclear magnetic moment was detected using Stern-Gerlach

experiment by Estermann.1939 Rabi et al. first detected nuclear magnetic resonance pheno

menon by applying r.f. energy to a beam of hydrogen molecules.

1946 Purcell at Harvard reported nuclear resonance in paraffin wax and Bloch at Stanford found nuclear resonance in liquid water.

Development – Physicists and Chemists

1949 Chemical shift phenomenon was observed (Chemists)

1966 Ernst and Anderson first introduced the Fourier transform technique into NMR. (Chemists, chemists & chemists)

Late in the 1960s – 70s (NMR is dead !): High resolution solid state NMR was revived due to the effort of Wau

gh et al at MIT. Mutlidimensional NMR techniques were developed. Biological applications became feasible due to the introduction of

superconducting magnets (Biologists). NMR imaging was first demonstrated (Radiologist, MDs)

1980s Protein structure determination was achieved.MRI become a household term.

1990 - Protein structure determination and MRI become indispensable tools for biologists and radiologists, respectively

1950s NMR theories were developed (Physicists & Chemists)Applications to solid state physics

Felix Bloch 1952, Physics

Edward M. Purcell 1952, Physics

Kurt Wuthrich 2002, Chemistry

Richard R. Ernst 1992, Chemistry

Isador I. Rabi1944, Physics

Paul Lauterbur 2003, Medicine

Peter Mansfield 2003, Medicine

Huang’s group with Prof. Kurt Wüthrich, September 1, 2002

Introduction to NMR Spectroscopy

Bo

(Nuclei: 1H, 13C, 15N, 31P etc)

Resonance

Radio Wave

Nuclear spin(Bar magnet)

Energ

y

Bo= 0 Bo

E = h

Quantum mechanical view

Larmor Equation:

= Bo/ 2 = Larmor frequency; = nuclear gyric ratioBo = magnetic field strength (Telsla)

1nm 10 102 103 104 105 106 107

(the wave) X-ray UV/VIS Infrared Microwave Radio Frequency

(the transition) electronic Vibration Rotation Nuclear

(spectrometer) X-ray UV/VIS Infrared/Raman NMR

Fluorescence

NMR Spectroscopy

Where is it?

Basic Nuclear Spin Interactions

Nuclear Spin i Nuclear Spin j

Electrons

Phonons

3

1

Dominant interactions: H = HZ + HD + HS + HQ.

HZ = Zeeman Interaction HD = Dipolar Interactions HS = Chemical Shielding Interaction. HQ = Quadrupolar Interaction

6

HoHo

4

5

4

3

1 1

4

What can NMR do ?

1. Solid state Physics & Material Sciences.

2. Chemistry.

3. Biomedical Sciences:A. Magnetic Resonance Imaging (MRI).B. Protein structure and function.C. Drug design.D. Diagnostics.

NMR is probably the most versatile analytical technique.

What can NMR do ?

1. Material Sciences.

2. Chemistry.

3. Biomedical Sciences:A. Magnetic Resonance Imaging (MRI).B. Protein structure and function.C. Drug design.D. Diagnostics.

NMR is probably the most versatile analytical technique.

Biological systemBiological system

Gene Gene

Protein Protein

pathway pathway

Network Network

CellsCells

Organs Organs

IndividualsIndividuals

生命信息傳遞路徑 :

DNA 序列

蛋白質序列

蛋白質結構

Protein is the machine of life

What next ?What next ?

Determine all 3D structures !Determine all 3D structures ! - Proteins- Proteins - Nucleic acids (RNA)- Nucleic acids (RNA)

Structure determines functionStructure determines function

3D structure data base3D structure data base- Determine all folds- Determine all folds- Predict structure from sequence- Predict structure from sequence- Predict function- Predict function- Rational design of drugs- Rational design of drugs

((結構基因體學結構基因體學 ))

- Molecular design (protein, RNA)- Molecular design (protein, RNA)

NMR Sample (1 mM, 0.4 ml)2H, 13C, 15N-label

Obtain NMR spectra (3 weeks)

Assign resonances Automation ?

Obtain restrains(Distances, angles, Orientations etc) Calculate structures

Determine Protein Structure by NMR

NMR structures(Ensemble of 20 structures)

(111 amino acids)

1H Chemical Shift

15N Chemical Shift

1H Chemical Shift

13C

Chem

ical Sh

ift

15 N Shi

ft

Some challenges for Physicists

1. Pulse program development.

3. Protein structure determination. - Methods for determining the structure of large proteins and membrane proteins (Residual dipolar coupling (RDC) approach).

4. Protein dynamics: proteins are not rigid bodies. - Determine and simulate protein dynamics ? - Dynamics – functional relationship ?

5. Disordered proteins: Unfolded protein and proteins that are intrinsically disordered. - Protein folding mechanism and computer simulation.

2. Methods for faster data acquisition: - Multidimensional FT-NMR is slow & time consuming. - Reduced dimensionality approach.

90 90 90 90 90 180180180180

90 90 90 180180180

90 90180

t2/2 t2/2

t1/2 t1/2

t3

1H

15N

13C

13CO

Pulse program for 3D HNCA experiment

321321321332211),,(),,( dtdtdteeetttsF tititi

S(t1,t2,t3)

NMR spectrum = FT of time domain signal S(t1,t2,t3):

What kind of signal are we getting ?

90 90 90 90 90 180180180180

90 90 90 180180180

90 90180

t2/2 t2/2

t1/2 t1/2

t3

1H

15N

13C

13CO

Pulse program for 3D HNCA experiment

321321321332211),,(),,( dtdtdteeetttsF tititi

S(t1,t2,t3)

NMR spectrum = FT of time domain signal S(t1,t2,t3):

Time consuming !!!

Time it takes for obtain a 3D NMR by FT method:

If t1 = The time it takes to acquire a 1D spectrum N2 = Number of 1D traces in the 2nd dimension N3 = Number of 2D planes in the 3rd dimension Then, ttotal = t1 x N2 x N3

For a typical experiment:

t1 = 16 Sec; N2 = 256; N3 = 64 ttotal = 3 days Time consuming !!!

Can we acquire data faster than FT-NMR ?

1. 1. Chemical Shift :Chemical Shift : Difference in resonance frequency due Difference in resonance frequency due to chemical structure difference to chemical structure difference.. 2. 2. Resonance Intensity:Resonance Intensity: Determine number of spins.Determine number of spins...

3. 3. J-coupling:J-coupling: Resonance splitting due Resonance splitting due to through-bond spin coupling. to through-bond spin coupling.

4. 4. Nuclear Overhauser Effect (NOE):Nuclear Overhauser Effect (NOE): Energy transfer through dipolar coupling.Energy transfer through dipolar coupling. ..

5. 5. Residual dipolar coupling: Residual dipolar coupling: Non-vanishing dipolar coupling in oriented media.Non-vanishing dipolar coupling in oriented media. ..

6. 6. Relaxation rates (TRelaxation rates (T11, T, T2 2 etc):etc): Lost of magnetization due to dephasing (TLost of magnetization due to dephasing (T22) ) or energy transfer or energy transfer (T (T11) )

NMR ParametersNMR Parameters

R 1H1H

15N

1H

BO

I

t

LBD domain of BCKD CBD domain of BCKD

OnconaseE. Coli thioesterase IRC-RNase

Hath domain of HDGF (w/ heparin)

N248-365 of SARS CoV Telomere binding protein Mite allergen Blo t5

Gallery of structures determined in T.-h. Huang’s lab

The Solution Structure, Dynamics & Function The Solution Structure, Dynamics & Function of E. coli Thioesterase/Protease Iof E. coli Thioesterase/Protease I

An ubiquitous protein involved in:An ubiquitous protein involved in:

....

- Removal of acyl-chains from post-translational - Removal of acyl-chains from post-translational modified proteins such as Ras modified proteins such as Ras

- Fatty acid synthesis- Fatty acid synthesis

- Synthesis of polyketides, immunosupressants- Synthesis of polyketides, immunosupressants & peptide antibiotics & peptide antibiotics

- Bioluminescence- Bioluminescence

- Important industrial protein for synthesizing - Important industrial protein for synthesizing stereospecific acids, alcohols & esters etc stereospecific acids, alcohols & esters etc

- A new family of lipolytic proteins with no structure A new family of lipolytic proteins with no structure known.known.

Active siteOverlay of 20 structures

E coli thioesterase (20 kDa)

Getting much harder for larger proteins !







1. Protein structures cannot be described by static stick and ball models.

2. How to characterize protein structures ?

Nuclear Magnetic Resonance

(NMR)

Molecular dynamic simulation of E. coli thioesterase/protease I

NMR Relaxation & Protein DynamicsNMR Relaxation & Protein Dynamics

R1 =1/T1 = (d2/4)[J(H - N) + 3J(N) + 6J(H + N)] + c2J(N) --------- (1)

R2 =1/T2 = (d2/8)[4J(0) + J(H - N) + 3J(N) + 6J(H) + 6J(H + N)]

+ (c2/6)[4J(0) + 3J(N)] + Rex ------------------------------- (2)

where d = (ohN H/82)(rNH-3), c = N(σ‖- σ)/3.

o : permeability constant of free space; h: Planck constant;

i : magnetogyric ratio of spin i; i: Larmor frequency of spin i;

rNH = 1.02 Å: length of the NH bond vector; Rex: exchange rate;

σ‖- σ = -170 ppm (size of the CSA tensor of 15NH)

NMR Relaxation & Protein DynamicsNMR Relaxation & Protein Dynamics

The relaxation rates are dominated by dipolar interaction and chemical shift anisotropic interaction, and is related to the correlation time, J(), by the following equations:

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

Relaxation Data

Obtained in two fields:

: 500 MHz

: 600 MHz

Determine T1, T2 & NOE

of every residue

Backbone dynamics of TEP-I

(a) S2 (fast motion)

(b) Rex (Slow motion)

Huang, Y.T. et al.* (2001) J. Mol. Biol. 307, 1075-1090.

National Research Program for Genomic Medicine High-filed NMR core Facility

Structure of SARS-CoV Nucleocapsid (N) Protein

Tai-huang HuangTai-huang HuangInstitute of Biomedical SciencesInstitute of Biomedical Sciences

Academia SinicaAcademia Sinica

IBMS,IBMS, January 14, 2005January 14, 2005

http://www.nmr.sinica.edu.tw/

Structural proteins:

S: Spike protein

E: Envelop protein

M: Membrane protein

N: Nucleocapsid protein

N(45-181)

N(248-365)

N(45-365) (70 kDa)

Dissecting the domain structure of the SARS CoV N protein

=

+N(45-365) (70 kD)

(321 a.a.)

1 45 181 248 365 422

RBD OGD

Overall structural organization of N protein

Structured (136 a.a.) RNA binding domain (RBD)

Structured (117 a.a.)(Oligomerization Domain, OGD)

Disordered N-terminus(44 a.a.)

Disordered

Linker (67 a.a.)

DisorderedC-terminus(57 a.a.)

? It forms dimer.

Structure of the dimer.(Solved by Abbott group, May 2004)

N

C C

1-44(44 a.a.)

366-422(57 a.a.)

RBG

N

182-248(67a.a.)

RBG

OGDOGD

366-422(57 a.a.)

45-181

248-365

1-44(44 a.a.)

SARS Project

First isolated from human hepatoma-derived Huh7 cells (Nakamura, 1994).

Mitogenic activities toward various cell lines, including fibroblasts, endothelial cells and smooth muscle cells. (Gastric cancer)

Involved in the development of vascular tissue, kidney and liver, and in lung remodeling after injury. Treatment of restenosis.

Human Hepatoma-derived Growth Factor (hHDGF)

The first member of a new family of heparin binding growth factors of important functions.

NLS

1 100 240

PWWP/HATH domain C-terminal domain

Structure hHDGF

PWWP

Nuclear localization signalHMG-like (PXXP(PKRP,PSEP)

Ordered

PWWP/HATH

Disordered

Internalization DNA synthesis

1 100 240

Order-disorder Prediction with PONDR (Predictors Of Natural Disordered Region)Dunker et. al. (2002). Biochemistry 41, 6573-6582.

HATH

Ordered Disordered

All HRPs consist of a structuredN-terminal HATH domain and

a disordered C-terminal domain

NMR Structure of the hath domain (N-100)

(A) (B)

(C)

Proposal: Domain-swapping Model of HATH-dimer(Strands 1 & 2 of chain 1 pair with strands 2,3 & 4 of chain 2 and vise versa)

C1 -

- C2

|

N1 |

N2

Loop 23(Chain 1)

Strands 1 & 2

Loop 23(Chain 2)

Proposed Model of HATH-Dimer

Heparin binding site

Domain-swapped dimer

周靜怡

800 MHz Magnet

www.nmr.sinica.edu.tw

Thank you !

nuclear magnetic resonance (nmr) – from basic to biomedical applications – from basic to...

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