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

Probe the Composition, Structure, Dynamics and Probe the Composition, Structure, Dynamics and Function of the Complete Range of Chemical Function of the Complete Range of Chemical Entities: from small organic molecules to large Entities: from small organic molecules to large molecular weight polymers and proteins. molecular weight polymers and proteins.

One of the One of the MOSTMOST Routinely used Analytical Routinely used Analytical Techniques Techniques

• Structural (chemical) elucidation

• Natural product chemistry.

• Synthetic organic chemistry. Analytical tool of choice of synthetic chemists.

• Study of dynamic processes

• Reaction kinetics.

• Study of equilibrium (chemical or structural).

• Structural (three-dimensional) studies

• Proteins.

• DNA. Protein/DNA complexes

• Polysaccharides

• Drug design

• Structure Activity Relationships by NMR

• Medicine - MRI

Common NMR UtilityCommon NMR Utility

2-phenyl-1,3-dioxep-5-ene2-phenyl-1,3-dioxep-5-ene

1313C NMR spectraC NMR spectra

11H NMR spectraH NMR spectra

NMRNMR: “fingerprint” of the compound’s chemical structure: “fingerprint” of the compound’s chemical structure

Protein Structures from NMRProtein Structures from NMR

2D NOESY Spectra at 900 MHz2D NOESY Spectra at 900 MHz Lysozyme Ribbon DiagramLysozyme Ribbon Diagram

1937 Rabi predicts and observes nuclear magnetic resonance1946 Bloch, Purcell first nuclear magnetic resonance of bulk sample1953 Overhauser NOE (nuclear Overhauser effect)1966 Ernst, Anderson Fourier transform NMR1975 Jeener, Ernst 2D NMR1985 Wüthrich first solution structure of a small protein (BPTI)

from NOE derived distance restraints1987 3D NMR + 13C, 15N isotope labeling of recombinant proteins

(resolution)1990 pulsed field gradients (artifact suppression)1996/7 new long range structural parameters:

- residual dipolar couplings from partial alignment in liquid crystalline media

- projection angle restraints from cross-correlated relaxation

TROSY (molecular weight > 100 kDa)Nobel prizes1944 Physics Rabi (Columbia)1952 Physics Bloch (Stanford), Purcell (Harvard)1991 Chemistry Ernst (ETH)2002 Chemistry Wüthrich (ETH)2003 Medicine Lauterbur (University of Illinois in Urbana ), Mansfield (University of Nottingham)

NMR HistoryNMR History

Some Suggested NMR ReferencesSome Suggested NMR References

“Basic One- and Two-Dimensional NMR Spectroscopy” Horst Friebolin

“Modern NMR Techniques for Chemistry Research” Andrew E. Derome

“NMR and Chemistry- an introduction to the fourier transform-multinuclear era” J. W. Akitt

“Nuclear Magnetic Resonance Spectroscopy” R. K Harris

“Protein NMR Spectroscopy: Principals and Practice” John Cavanagh, Arthur Palmer, Nicholas J. Skelton, Wayne Fairbrother

“NMR of Proteins and Nucleic Acids” Kurt Wuthrich

“Tables of Spectral Data for Structure Determination of Organic Compounds”Pretsch, Clerc, Seibl and Simon

“Spectrometric Identification of Organic Compounds” Silverstein, Bassler and Morrill

The Basics of NMR Hypertext based NMR course http://www.cis.rit.edu/htbooks/nmr/nmr-main.htm

Educational NMR Software All kinds of NMR softwarehttp://www.york.ac.uk/depts/chem/services/nmr/edusoft.html

NMR Knowledge Base A lot of useful NMR linkshttp://www.spectroscopynow.com/

NMR Information Server News, Links, Conferences, Jobshttp://www.spincore.com/nmrinfo/

Technical Tidbits Useful source for the art of shimminghttp://www.acornnmr.com/nmr_topics.htm

BMRB (BioMagResBank) Database of NMR resonance assignmentshttp://www.bmrb.wisc.edu/

Some NMR Web SitesSome NMR Web Sites

Basic NMR SpectrometerBasic NMR Spectrometer

Information in a NMR SpectraInformation in a NMR Spectra

1) Energy E = h

h is Planck constant is NMR resonance frequency 10-10 10-8 10-6 10-4 10-2 100 102

wavelength (cm)

-rays x-rays UV VIS IR -wave radio

ObservableObservable NameName QuantitativeQuantitative InformationInformation

Peak position Chemical shifts () (ppm) = obs –ref/ref (Hz) chemical (electronic)

environment of nucleus

Peak Splitting Coupling Constant (J) Hz peak separation neighboring nuclei (intensity ratios) (torsion angles)

Peak Intensity Integral unitless (ratio) nuclear count (ratio) relative height of integral curve T1 dependent

Peak Shape Line width = 1/T2 molecular motion peak half-height chemical exchange

uncertainty principaluncertainty in

energy

Source of the NMR SignalSource of the NMR Signal

From Quantum Theroy: Nuclear Spin (Think Electron Spin)

NMR “active” Nuclear Spin (I) = ½: 1H, 13C, 15N, 19F, 31P biological and chemical relevance Odd atomic mass

NMR “inactive” Nuclear Spin (I) = 0:12C, 16O Even atomic mass & number

Quadrupole Nuclei Nuclear Spin (I) > ½:

14N, 2H, 10B Even atomic mass & odd number

Zeeman Effect and Nuclear Spin Quantum NumberZeeman Effect and Nuclear Spin Quantum Number

I: hyperfine interaction associate with magnetization due to nuclear spin quantum transitions

Zeeman effect: splitting of energy levels in magnetic field

2I +1 possible energy levels

For I =1/2: m= -1/2 & 1/2

E= B

magnetogyric ratio (radians/Tesla) - unique value per nucleus 1H: 26.7519 x 107 rad T-1 s-1

Bo applied magnetic field - units:Tesla (Kg s-2 A-1)

NMR frequency:Bo

m: magnetic quantum number

NMR Spectra TerminologyNMR Spectra Terminology

Increasing field (Bo)Increasing frequency ()Increasing Increasing energy (E, consistent with UV/IR)

1H 13C 2H600 MHz 150 MHz 92 MHz

TMS

CHCl3

7.27 0 ppmincreasing decreasing low field high field down field up fieldhigh frequency () low frequencyde-shielding high shielding Paramagnetic diamagnetic

Another Viewpoint: Magnetic Moment (Nuclear Spin)Another Viewpoint: Magnetic Moment (Nuclear Spin)

magnetic moment()Ih

It is a vector quantity that gives the direction and magnitude (or strength) of the ‘nuclear magnet’

By convention:spin +1/2 => low energy statespin -1/2 =>

Analogous to current moving in a loop which induces a magnetic field (right-hand rule)

quantized by Planck’s constant (h)

Bo

= h / 4

Magnetic alignmentMagnetic alignment

In the absence of external field,each nuclei is energetically degenerate

Add a strong external field (Bo).and the nuclear magnetic moment: aligns with (low energy) against (high-energy)

NMR SensitivityNMR Sensitivity

Bo = 0

Bo > 0 E = h

N / N = e E / kTBoltzmman distribution:

The applied magnetic field causes an energy difference between aligned() and unaligned() nuclei

The population (N) difference can be determined from

The E for 1H at 400 MHz (Bo = 9.5 T) is 3.8 x 10-5 Kcal / mol

Very Small !Very Small !~64 excess spins ~64 excess spins per million in lower per million in lower statestate

Low energy gap

NMR SensitivityNMR Sensitivity

EhBo /2

NMR signal depends on:1) Number of Nuclei (N) (limited to field homogeneity and

filling factor)2) Gyromagnetic ratio (in practice 3)3) Inversely to temperature (T)4) External magnetic field (Bo

2/3, in practice, homogeneity)5) B1

2 exciting field strengthN / N = e E / kT

Increase energy gap -> Increase population difference -> Increase NMR signal

E ≡ Bo≡

- Intrinsic property of nucleus can not be changed.

C)3 for 13C is 64xN)3

for 15N is 1000x

1H is ~ 64x as sensitive as 13C and 1000x as sensitive as 15N !

Consider that the natural abundance of 13C is 1.1% and 15N is 0.37%relative sensitivity increases to ~6,400x and ~2.7x105x !!

signal (s) 44BBoo22NBNB11g(g()/T)/T

NMR SensitivityNMR Sensitivity

Increase in Magnet Strength is a Major Means to Increase SensitivityBut at a significant cost!

~$800,000 ~$2,00,000 ~$4,500,000

E = h = Bo / 2

E = h Bo / 2

NMR Frequency Range (NMR Frequency Range (expensive radiosexpensive radios))

For 1H in normal magnets (2.35 - 18.6 T), this frequency isin the 100-800 MHz range.

10-10 10-8 10-6 10-4 10-2 100 102

wavelength (cm)

-rays x-rays UV VIS IR -wave radio

= 2 o = B (radians)Precession or Larmor frequency:

l

angular momentum (l)

Simply, the nuclei spins about itsaxis creating a magnetic moment

Classical View of NMR (Classical View of NMR (compared to Quantum viewcompared to Quantum view))

Maxwell: Magnetic field Moving charge≡

Bo

o

Apply a large external field (Bo)and will precess about Bo at its Larmor () frequency.

Important: This is the same frequency obtained from the Important: This is the same frequency obtained from the energy transition between quantum statesenergy transition between quantum states

Bulk magnetization Bulk magnetization (M(Moo))

Mo

y

x

z

x

y

z

Bo Bo

Now consider a real sample containing numerous nuclear spins:

Mo (N - N)

xiyjzk

Since is precessing in the xy-plane, Mo = ∑ zk – zk

is quantized ( or ), Mo has a continuous number of states, bulk property.

An NMR ExperimentAn NMR Experiment

Mo

y

x

z

x

y

z

Bo Bo

We have a net magnetization precessing about Bo at a frequency of o with a net population difference between aligned and unaligned spins.

Now What?

Perturbed the spin population or perform spin gymnasticsBasic principal of NMR experiments

Mo

z

x

i

B1

Transmitter coil (y)

yBo

An NMR ExperimentAn NMR Experiment

To perturbed the spin population need the system to absorb energy.

Two ways to look at the situation: (1) quantum – absorb energy equal to difference in spin states(2) classical - perturb Mo from an excited field B1

B1 off…

(or off-resonance)

Mo

z

x

B1

z

x

Mxy

y y1

1

Right-hand rule

resonant condition: frequency (1) of B1 matches Larmor frequency (o)energy is absorbed and population of and states are perturbed.

An NMR ExperimentAn NMR Experiment

And/Or:And/Or: Mo now precesses about B1

(similar to Bo) for as long as the B1 field is applied.

Again, keep in mind that individual spins flipped up or down(a single quanta), but Mo can have a continuous variation.

An NMR ExperimentAn NMR Experiment

What Happens Next?

The B1 field is turned off and Mxy continues to precess about Bo at frequency o.

z

x

Mxy

Receiver coil (x)

y

NMR signal

o

The oscillation of Mxy generates a fluctuating magnetic field which can be used to generate a current in a receiver coil to detect the NMR signal.

FID – Free Induction Decay

NMR Signal Detection - NMR Signal Detection - FIDFID

Mxy is precessing about z-axis in the x-y plane

Time (s)

y y y

The FID reflects the change in the magnitude of Mxy as the signal is changing relative to the receiver along the y-axis

Again, it is precessing at its Larmor Frequency (o).

NMR Signal Detection - Fourier NMR Signal Detection - Fourier TransformTransform

So, the NMR signal is collected in the Time - domain

But, we prefer the frequency domain.

Fourier Transform is a mathematical procedure that transforms time domain data into frequency domain

z

x

Mxy

yBo

z

x

Mxy

yo

Laboratory Frame Rotating Frame

Laboratory Frame vs. Rotating FrameLaboratory Frame vs. Rotating Frame

To simplify analysis we convert to the rotating frame.

Simply, our axis now rotates at the Larmor Freguency (o). In the absent of any other factors, Mxy will stay on the x-axis

All further analysis will use the rotating frame.

Chemical Chemical ShiftShift

Up to this point, we have been treating nuclei in general terms.Simply comparing 1H, 13C, 15N etc.

If all 1H resonate at 500MHz at a field strength of 11.7T, NMR would not be very interesting

Beff = Bo - Bloc --- Beff = Bo( 1 - )

is the magnetic shielding of the nucleus

The chemical environment for each nuclei results in a unique local magnetic field (Bloc) for each nuclei:

Chemical Chemical ShiftShiftAgain, consider Maxwell’s theorem that an electric current in a loop

generates a magnetic field. Effectively, the electron distribution in the chemical will cause distinct local magnetic fields that will either add to or subtract from Bo

HO-CH2-CH3

Aromaticity, electronegativity and similar factors will contribute to chemical shift differences

Beff = Bo( 1 - )

de-shielding high shieldingShielding – local field opposes Bo

The NMR scale (The NMR scale (, ppm), ppm)

- ref

= ppm (parts per million) ref

Instead use a relative scale, and refer all signals () in the spectrum to the signal of a particular compound (ref).

Bo >> Bloc -- MHz compared to Hz

Comparing small changes in the context of a large number is cumbersome

Tetramethyl silane (TMS) is a common reference chemicalH3C Si CH3

CH3

CH3

IMPORTANT: absolute frequency is field dependent ( = Bo / 2)

The NMR scale (The NMR scale (, ppm), ppm)

Chemical shift is a relative scale so it is independent of Bo. Same chemical shift at 100 MHz vs. 900 MHz magnet

IMPORTANT: absolute frequency is field dependent ( = Bo / 2)

At higher magnetic fields an NMR spectra will exhibit the same chemical shifts but with higher resolution because of the higher frequency range.

Chemical Shift TrendsChemical Shift Trends

• For protons, ~ 15 ppm:

0TMS

ppm

210 7 515

Aliphatic

Alcohols, protons to ketones

Olefins

AromaticsAmidesAcids

Aldehydes

Chemical Shift TrendsChemical Shift Trends

• For carbon, ~ 220 ppm:

ppm

50150 100 80210

Aliphatic CH3,CH2, CH

Carbons adjacent toalcohols, ketones

Olefins

Aromatics,conjugated alkenes

C=O of Acids,aldehydes, esters

0TMS

C=O inketones

Predicting Chemical Shift AssignmentsPredicting Chemical Shift Assignments

Numerous Experimental NMR Data has been compiled and general trends identified

• Examples in Handout

• See also: “Tables of Spectral Data for Structure Determination of Organic Compounds” Pretsch, Clerc, Seibl and Simon

“Spectrometric Identification of Organic Compounds” Silverstein, Bassler and Morrill

• Spectral Databases: Aldrich/ACD Library of FT NMR Spectra Sadtler/Spectroscopy (UV/Vis, IR, MS, GC and NMR)

Predicting Chemical Shift AssignmentsPredicting Chemical Shift Assignments

Predict the chemical shifts of:

Benzene Shift NO2 effect NH2 effect TotalChange sign since table lists as downfield shifta 7.27 0.95 -0.75 7.47 ppmd 7.27 0.33 -0.75 6.85 ppmc 7.27 0.17 -0.24 7.20 ppmb 7.27 0.95 -0.63 7.59 ppm

From table 3-6-1 in handout:Substituent Shift relative to benzene (ppm)

ortho meta paraNO2 -0.95 -0.17 -0.33NH2 0.75 0.24 0.63

NH2

NO2

A

BC

D

Predicting Chemical Shift AssignmentsPredicting Chemical Shift Assignments

Predict the chemical shifts of:

C |C – C – C – C – C – C 2

Chemical shift is determined by sum of carbon types.From Table 3.2 in handout:

=Bs + ∑ DmAsm +SN3 +sN4 - empirical formula

S – number of directly bonded carbonsDm – number of directly bonded carbons having M attached carbonsNp – number of carbons P bonds away

2 = B2 + [1xA23+ 1xA21 ] + [1x2] + [1x2]

2 = 15.34 + [1X16.70 +1x0] + [1x-2.69] +[1x0.25] = 29.60 ppm

Coupling ConstantsCoupling Constants

Energy level of a nuclei are affected by covalently-bonded neighbors spin-states

13C

1H 1H 1H

one-bond

three-bond

I SS

S

I

I

J (Hz)

Spin-States of covalently-bonded nuclei want to be aligned.

The magnitude of the separation is called coupling constant (J) and has units of Hz.

+J/4

-J/4

+J/4

Coupling ConstantsCoupling Constants

IMPORTANT: Coupling constant pattern allow for the identification of bonded nuclei.

Multiplets consist of 2nI + 1 lines I is the nuclear spin quantum number (usually 1/2) and

n is the number of neighboring spins.

The ratios between the signal intensities within multiplets are governed by the numbers of Pascals triangle.

Configuration Peak Ratios

A 1

AX 1:1

AX2 1:2:1

AX3 1:3:3:1

AX4 1:4:6:4:1

Coupling ConstantsCoupling Constants

NMR RelaxationNMR Relaxation

After the B1 field (pulse) is removed the system needs to “relax” back to equilibrium

Mz = M0(1-exp(-t/T1))

T1 is the spin-lattice (or longitudinal) relaxation time constant.

Think of T1 as bulk energy/magnetization exchange with the “solvent”.

Please Note: General practice is to wait 5xT1 for the system to have fully relaxed.

NMR RelaxationNMR Relaxation

Mx = My = M0 exp(-t/T2)

T2 is the spin-spin (or transverse) relaxation time constant.In general: T1 T2

Think of T2 as the “randomization” of spins in the x,y-plane

Related to line-shape

Please Note: Line shape is also affected by the magnetic fields homogeneity

(derived from Hisenberg uncertainty principal)

NMR Time ScaleNMR Time Scale

Time Scale Chem. Shift ( Coupling Const. (J) T2 relaxationSlow k << A- B k << JA- JB k << 1/ T2,A- 1/ T2,B

Intermediate k = A - B k = JA- JB k = 1/ T2,A- 1/ T2,B

Fast k >> A - B k >> JA- JB k >> 1/ T2,A- 1/ T2,B

Range (Sec-1) 0 – 1000 0 –12 1 - 20

NMR time-scale refers to the chemical shift timescale.

k = (he-ho)

Exchange Rates from NMR DataExchange Rates from NMR Data

k = (o2 -  e

2)1/2/21/2

k = o / 21/2

k = o2 /2(he - ho)

h – peak-width at half-height – peak frequencye – with exchangeo – no exchangef – mole fraction – chemical shift

obs = f11 + f22

f1 +f2 =1

Continuous Wave (CW) vs. Pulse/Fourier TransformContinuous Wave (CW) vs. Pulse/Fourier Transform

NMR Sensitivity Issue

A frequency sweep (CW) to identify resonance is very slow (1-10 min.)Step through each individual frequency.

Pulsed/FT collect all frequencies at once in time domain, fast (N x 1-10 sec)

Increase signal-to-noise (S/N) by collecting multiple copies of FID and averaging signal.

S/N number of scans

* =tp

NMR PulseNMR Pulse

FT

A radiofrequency pulse is a combination of a wave (cosine) of frequency o and a step function

Pulse length (time, tp)

The fourier transform indicates the pulse covers a range of frequencies

Hisenberg Uncertainty principal again: .t ~ 1/2Shorter pulse length – larger frequency envelopeLonger pulse length – selective/smaller frequency envelope

Sweep Width f ~ 1/t

NMR PulseNMR Pulse

z

x

Mxy

y

z

x

y

Mo

B1

ttp

t = * tp * B1

NMR pulse length or Tip angle (tp)

The length of time the B1 field is on => torque on bulk magnetization (B1)

A measured quantity – instrument dependent.

NMR PulseNMR Pulse

z

x

Mxy

y

z

x

y

Mo / 2

Some useful common pulses

90o

Maximizes signal in x,y-planewhere NMR signal detected

z

x

-Moy

z

x

y

Mo

180o

90o pulse

180o pulse

Inverts the spin-population.No NMR signal detected

Can generate just about any pulse width desired.

NMR Data AcquisitionNMR Data Acquisition

Collect Digital Data ADC – analog to digital converter

0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00t1 sec

SR = 1 / (2 * SW)

The Nyquist Theorem says that we have to sample at least twice as fast as the fastest (higher frequency) signal.

Sample Rate

- Correct rate, correct frequency-½ correct rate, ½ correct frequency Folded peaks!Wrong phase!

SR – sampling rate

carrier

234 233 232 231 230 229 228 227 226 225 224 223f1 ppm

Quadrature detectionQuadrature detection

Frequency of B1 (carrier) is set to center of the spectra.

• small pulse length to excite entire spectrum• minimizes folded noise

How to differentiate between peaks upfield and downfield from carrier?

carrier

If carrier is at edge of spectra, then peaks are all positive or negative relative to carrier. But excite twice as much including noise

(B1)

B

F

B

F

PH = 0

PH

= 9

0PH = 0

PH = 90

F

F

S

S

Quadrature Quadrature detectiondetection

Use two detectors 90o out of phase.

Phase of Peaksare different.

Receiver GainReceiver Gain

The NMR-signal received from the resonant circuit in the probehead needs to be amplified to a certain level before it can be handled by the computer.

The detected NMR-signals vary over a great range due to differences in the inherent sensitivity of the nucleus and the concentration of the sample.

Data Processing – Window FunctionsData Processing – Window Functions

0 0.10 0.20 0.30 0.40 0.50t1 sec

Good stuff Mostly noise

The NMR signal Mxy is decaying by T2 as the FID is collected.

Emphasize the signal and decrease the noise by applying a mathematical function to the FID

F(t) = 1 * e - ( LB * t ) – line broadening Effectively adds LB in Hz to peak

Line-widths

Sensitivity Resolution

0 0.10 0.20 0.30 0.40 0.50t1 sec

1080 1060 1040 1020 1000 980 960 940 920 900f1 ppm

0 0.10 0.20 0.30 0.40 0.50t1 sec0 0.10 0.20 0.30 0.40 0.50

t1 sec

1080 1060 1040 1020 1000 980 960 940 920 900f1 ppm

FT FT

LB = -1.0 HzLB = 5.0 Hz

Can either increase S/N or Resolution Not Both!

Increase Sensitivity Increase Resolution

NMR Data sizeNMR Data size

digital resolution (DR) as the number of Hz per point in the FID for a given spectral width.

DR = SW / SI SW - spectral width (Hz)SI - data size (points)

Remember: SR = 1 / (2 * SW)

Also: SW = 1/2DW

Dwell time DW

TD

A Number of Interdependent Values (calculated automatically)

AQ = TD * DW= TD/2SWH

Total Data Acquisition Time:

Should be long enough to allow complete delay of FID

Higher Digital Resolution requires longer acquisition times

231.40 231.39 231.38 231.37 231.36 231.35 231.34 231.33 231.32 231.31 231.30 231.29 231.28 231.27 231.26 231.25 231.24

f1 ppm

231.42 231.40 231.38 231.36 231.34 231.32 231.30 231.28 231.26 231.24 231.22 231.20f1 ppm

0 0.20 0.40 0.60 0.80 1.00 1.2 1.4 1.6 1.8 2.0 2.2t1 sec

8K data 8K zero-fill

8K FID 16K FID

Zero FillingZero Filling

Improve digital resolution by adding zero data points at end of FID

No zero-filling 8K zero-filling

MultiDimensional NMRMultiDimensional NMR

1D NMR

Up to now, we have been talking about the basic or 1D NMR experiments

More complex NMR experiments will use multiple “time-dimensions” to obtaindata and simplify the analysis.

In a 1D NMR experiment the FID acquisition time is the time domain (t1)

Multidimensional NMR experiments may also observe multiple nuclei (13C,15N) in addition to 1H.But usually detect 1H.

2D COSY (Correlated SpectroscopY): Correlate J-coupled NMR resonances

MultiDimensional NMRMultiDimensional NMR

A series of FIDs are collected where the delay between 90o pulses (t1) is incremented. t2 is the normal acquisition time.

MultiDimensional NMRMultiDimensional NMR

During the t1 time period, peak intensities are modulated at a frequency corresponding to the chemical shift of its coupled partner.

Solid line connects diagonal peaks(normal 1D spectra). The off-diagonalor cross-peaks indicate a correlationbetween the two diagonal peaks – J-coupled.

Karplus Equation – Coupling Constants Karplus Equation – Coupling Constants

Relates coupling constant toTorsional angle.

Used to solve Structures!

J = const. + 10Cos

Karplus Equation – Coupling Constants Karplus Equation – Coupling Constants

For Protein Backbones

Nuclear Overhauser Effect (NOE)Nuclear Overhauser Effect (NOE)

Interaction between nuclear spins mediated through empty space (5Ă) (like ordinary bar magnets). Important: Effect is Time-Averaged!

Give rise to dipolar relaxation (T1 and T2) and specially to cross-relaxation and the NOE effect.

the 13C signals are enhanced by a factor1 + = 1 + 1/2 . (1H)/(13C) ~ max. of 2

Perturb 1H spin populationaffects 13C spin population NOE effect

DEPT ExperimentDEPT Experiment: Distortionless Enhancement by Polarization Transfer: Distortionless Enhancement by Polarization Transfer

13C spectra is perturbed basedOn the number of attached 1H

Takes advantage of differentpatterns of polarization transfer1H-13C NOE

2D NOESY (Nuclear Overhauser Effect)2D NOESY (Nuclear Overhauser Effect)

Diagonal peaks are correlated by through-spaceDipole-dipole interaction.

NOE is a relaxation factor that builds-up duringThe “mixing-time (m)

The relative magnitude of the cross-peak is Related to the distance (1/r6) between the Protons (≥ 5Ă).

Basis for solving a Structure!

Protein NMRProtein NMR

Number of atoms in a protein makes NMR spectra complex

Resonance overlap

Isotope label protein with 13C and 15Nand spread spectra out in 3D and 4D

Protein NMRProtein NMR

How do you assign aprotein NMR spectra?

A collection of “COSY”-likeexperiments that sequentiallywalk down the proteins’ backbone

3D-NMR experiments thatRequire 13C and 15N labeledProtein sample

Detect couplings to NHDetect couplings to NH

Protein NMRProtein NMR

Assignment strategy

We know the primary sequence of the protein.

Connect the overlapping correlation between NMR experiments

Protein NMRProtein NMR

Molecular-weight Problem

Higher molecular-weight –> more atoms –> more NMR resonance overlap

More dramatic:NMR spectra deteriorate with increasingmolecular-weight.

MW increases -> correlation time increases-> T2 decreases -> line-width increases

NMR lines broaden to the point of not being detected!

With broad lines, correlations (J, NOE) become less-efficient

Protein NMRProtein NMR

How to Solve the Molecular-weight Problem?

1) Deuterium label the protein.• replace 1H with 2H and remove efficient relaxation paths• NMR resonances sharpen• problem: no hydrogens -> no NOEs -> no structure• actually get exchangeable (NH –NH) noes can augment with specific 1H labeling

2) TROSY• line-width is field dependent