Emag=-μB

E:electric field

B:magnetic field

μ:magnetic momentB B

μ

μ

Low energy(-) High energy(+)

μinduced = μ0-1VΧB

μ0=awkward constant

V=volume of object

Χ=magnetic susceptibility

Random directed spin polarization

Spin precession

dj/dt=μxB0

ω = γBon resonance : ωo-ωr = 0

off resonance : ωo-ωr = 0

(ωr = B1 에 의한 각속도 )

Excitation using RF pulse

θ = ω1 tp = 2πγB1tp

- tp 를 변화시켜 precession 의 각도를 변화시킬 수 있음

- B1 이 클 수록 짧은 tp 를 사용가능

Spin-lattice Relaxation

Transverse Relaxation

B = Bo (1-σ) ω = γB

δ = (ω - ωREF) x106 / ωREF

Chemical shift

Deshielding effect in benzen molecule

NMR signal and Fourier Transform

The 90-FID Sequence The Spin-Echo Sequence

The Inversion Recovery Sequence

Copyright © 1997-99 J.P. Hornak.All Rights Reserved

Simple pulse sequences

The 2D NMR Spectrum

Pulse Sequence

Spectrum

Coupled spinsBefore mixing

After mixing

t1 t2

The Power of 2D NMR:Resolving Overlapping Signals

1D

2D

2 signalsoverlapped

2 cross peaksresolved

COrrelation SpectroscopY (COSY)

Two 90 degree pulses are applied insequence.

The time t1 is the time between the firstand second pulse.

The signal will vary with the time between the pulses (t1).

In a 2D COSY spectrum, cross-peakswill exist where there is spin-spincoupling between nuclei.

TOCSY(Total COrrelation SpectroscopY)

NOE Spectroscopy

The first two pulses are an inversion for allprotons. We label everything with chemical shifts and couplings.

The magnetization component that ends up in the -Z axis evolves duringthe mixing time tm and dipolar coupled spins will undergo NOE.

The 2D spectrum will have chemical shifts in f1 and f2.

The cross peaks are for nuclei that are dipolar coupled.

~500 resonances

Resolve resonances by multi-dimensional experiments•Proteins have thousands of signals

•Assign the specific signal for each atom

•Thousands of interactions between atoms- also need to be assigned

•Need to transform representation from spectrum through interactions

between atoms to spatial coordinates

Proteins Have Too Many Signals! 1H NMR Spectrum of Ubiquitin

3D- detect signals 3 times

Same as 1D experiment

t2t1

t33D NMR Pulse Sequence

(t3)

Experiments are composites acronyms are composites

Using this simplified approach we can understand the design of more complex NMR experiments as not much more than putting predefined blocks together, just like Lego.

Higher Dimensional NMR:Built on the 2D Principle

90º pulse

Higher Dimensional NMR:Built on the 2D Principle

There are four such building blocks:1. the preparation period which leads to the production of magnetization

to start the experiment can be as simple as a single 90o pulse. 2. the evolution period allows the magnetization to precess or evolve to

create one of the frequency axes in our multidimensional experiment. 3. the mixing period is designed to transfer magnetization or coherence

from one spin to another. This is the most complicated of all building blocks. The simple period between two 90o pulses to allow time for dipolar interactions to exchange magnetization and the spin lock in the TOCSY experiment to develop J-couplings. All they are doing is getting magnetization from one spin to another one.

4. The final building block is the detection period.

Strategies for Sequential Assignment

Beginning from the residue in the middle, i, we can start to collect intraresidual cross peaks between amide proton and / protons.

Moving to the NOESY (red) we are thus able to identify the proton of the preceding amino acid, i-1.

The intraresidue cross peak of amide and protons for residue i-1 can then be identified by moving horizontally until the appropriate TOCSY cross peak is identified.

Assignment Strategies for Secondary structure

NMR sample preparation from E.coli system

Media using NMR group

1. LB(Luria-Bertani) media : rich media (Bacto-trypton, Bacto-yeast extract and NaCl)

2. M9 media : minimal media (essential metals, NaCl and nutrients for synthesis of proteins which you

need)Usage of M9 media

M9

15N labeled protein

13C-15N labeled protein

2H-13C-15N labeled protein

Selective labeled protein

1.Essential Metals and NaCl

2. 12C-glucose

13. 5NH4Cl

4. H2O

1. Essential Metals and NaCl

2. 13C-glucose

3. 15NH4Cl

4. H2O

1. Essential Metals and NaCl

2. 13C,D-glucose

3. 15NH4Cl

4. D2O or H2O and D2O

- As purpose, % of D2O can be regulated.

1. Essential Metals and NaCl

2. individulal amino acid and 13C-15N or 13C-15N labeled Amino acid for selective label

3. H2O

NMR sample preparation from E.coli system

Expression system His-tagging and GST fusion system

M 1 2 3 4 5

116.3

97.4

66.3 55.4

36.531

21.514.4

6

3.52.5

His tag-fusion protein (17.4kDa)

TEV (27KDa)

DNA binding domain

(13.8KDa)

M 1 2 3 4

116.3

97.4

66.3 55.4

36.531

21.514.4

6

3.52.5

GST-fusion protein (37.5kDa)

GST (26KDa)

DNA binding domain

(11.5KDa)

M 1 2 3 4 5 6

116.3

97.4

66.3 55.4 36.531

21.5

14.4

6

His tagging DNA binding domain of AtB

AT

(18KDa)

Concepts of NMR Structure calculation

Concepts of NMR Structure calculation

1) 2)

3)

4)5)

NMR structure of a complex containing the TFIIF subunit RAP74 and the RNA polymerase II carboxyl-terminal domain phosphatase FCP1

PNAS May 13, 2003 vol. 100 no. 10 5688–5693

- Protein expression : cter RAP74 : GST fusion , cterFCP1 : double mutagenesis (E956A/L957A), GST fusion

-NMR sample :

20mM NaPO4(pH6.5) ans 1mM EDTA, Uniform double labele

-Bindibg assay:

GST coulmn, western

-NMR spectroscopy:

Varian Inova Unity 500, 600 and 800MHz, 299K, conventional 3D NMR spectroscopy,

2D 13C(F1) filtered, (F2) edited NOESY, 3D 15N/13C (F1)-filtered, (F3)-edited NOESY

-Structure calculation

Torsion Angle Molecular Dynamics protocol of CNS

PROCHECK-NMR, MOLMOL and MOLscript

Detection

3D 15N/13C (F1)-filtered, (F3)-edited NOESYIn D2O

<INEPT> <Reverse INEPT>

Alignment by liquid crystals bicelle phase : consisting of a mixture of long-chain phospholipids and detergent these adopt -lamellar phase of highly porous bilayers that cooperatively order in the magnetic field, with the bilayer normal orthogonal to the magnetic field

Recent Topic 1 : Residual Dipolar Coupling

Application : Structure refinement

Ribbon representation of three crystal structures of Ca2+/CaM in complex with cCaMKKp (1.8 Å, PDB entry 1IQ5), smMLCKp (2.2 Å, PDB entry 1CDL), and CaMKIIp (2.0 Å, PDB entry 1CDM). These kinase-derived peptides are drawn in magenta; the N- and C-terminal domains of CaM are shown in yellow and green, respectively, and the linker region and Ca2+are in gray.

Tapas.K.M.,Biochemistry, Vol. 41, No. 43, 2002

Structural alignment of the seven lowest energy structures (of 20 calculated) of the tandem WW domains ofPrp40 refined with and without RDC restraints.

Wiesner.S., et.al.,J. Mol. Biol. (2002) 324, 807–822

Parkin binds the Rpn10 subunit of 26S proteasomes through its ubiquitin-like domain

EMBO reports 4, 3, 301–306 (2003)

- NMR experiment

2D experiment : HSQC, constant time 1H-13C HSQC

3D experiment : HNCA, HN(CO)CA, HNCO, CBCA(CO)NH and CBCANH

Proton assignment : HBHA(CO)NH, HBHANH, 15N-edited TOCSY,

15N-edited NOESY,13C-edited TOCSY, HCACO and

HCCH-TOCSY

Rasidual dipolar coupling: cetyltri-ammonium bromide (CTAB)-

doped bicelle

Dihedral angle : HNHA

- Structure calculation

program : CNS ver1.1 and PROCHECK

Initial structure : NOE-derived inter proton distance restrains

Angle : TALOS, HNHA

Hydrogen bond : 1H-2H exchange experiment

Fig. 1 | The solution structure of the ubiquitin-like (Ubl) domain of parkin. (A) Stereo view of ten converged structures of the parkin Ubl domain. (B) Ribbon representation of the average structure.β-strands and α-helices are coloured yellow and pink, respectively, in (A) and (B).Numbers in (A) and (B) indicate aminoacid positions in the Ubl domain sequence.

Fig. 2 | Identification of the binding site for Rpn10196–306 in the parkin ubiquitin-like (Ubl) domain. (A) 1H-15N heteronuclear single-quantum coherence (HSQC)spectrum of the parkin Ubl domain in the presence (red) and absence (black) of equimolar quantities of Rpn10196–306. The peaks labelled with L-2, G-1 and S0 originate from the amino-terminal tag. (B) NMR chemical-shift-perturbation data for the parkin Ubl domain. The data are displayed for each residue according to the equation (0.2 δN 2 + δH 2)1/2,where δN and δH represent the change in nitrogen and proton chemical shifts on addition of Rpn10196–306.Asterisks indicate residues the peaks of which became undetectable due to broadening. Secondary structure elements for the parkin Ubl are shown below the graph. (C) Mapping of the perturbed residues of the Ubl domains of parkin and PLIC2 (Walters et al., 2002) on binding to Rpn10.Residues showing a chemical-shift-perturbation are coloured in red,with the colour gradient indicating the strength of the perturbation.Residues the peaks of which became undetectable on binding to Rpn10 are shown in purple.

Recent topic 2 : Paramagnetic Labelling of Proteins for Biomolecular NMR1. Protein complex 에 대한 Paramagnetic 현상의 이용

metalloprotein 에서 binding partner 에 대한 구조연구 방법의 새로운 대안

1) amide proton exchange experiment

2) HN 의 PCS(pseudocontact shift)

first : asymmetric isotope labeling and filtered NOESY experiments

Second :define a target function describing contributions from intra and intermolecular NOEs and to computationally obtain the proper orientation of the monomeric subunits within the dimer

The size of protein dimer is large

Deuteration is necessary

NOE measurement

Paramagnetic anisotropic susceptibility (PAS)

Paramagnetic probe is incorporated by modification of a unique cysteine residue with thiol-reactive EDTA

Paramagnetic metal

obtain long-range structural information specific to each monomeric component of a symmetric dimer using paramagnetic probes

15N TROSY spectrum collected at 900 MHz on 2H, 15N, 13C enriched ILV labeled sample of STAT4NT-EDTA-Co2+.

Examples of CH3 PCSs observed in a 13C CT-HSQC experiment

The distribution of measurable PCSs in ILV- STAT4NT

(1.6 per residue)