Lectures for CCB 538 James Aramini, PhD.-Structure Activity Relationships by NMR 5) Solid State NMR 6) Medicine - MRI - Metabolomics MRI images of the Human Brain NMR Structure

J.A.! 04/21/14!

Nuclear Magnetic Resonance

Lectures for CCB 538

James Aramini, PhD.

CABM 014A [email protected]

"

"

Outline""

1.  Introduction / Spectroscopy Overview!

2.  NMR Spectroscopy – Theory and Practice!

3.  Protein NMR & Resonance Assignment!

4.  Structure Determination &! !Biological NMR Applications !

April 21!!!!April 23!!April 28!

Introduction""

“The Tree of Knowledge of Science”!!! !Richard Ernst !!(Nobel Prize in Chemistry 1991)!

!

Physics!

Chemistry!

Biology!

Medicine!

Richard Ernst!Father of modern NMR!Nobel Prize 1991 !!

Biological Spectroscopy""! Spectroscopy is the study of matter and its

properties by investigating light, sound, or particles that are emitted, absorbed or scattered by the matter under investigation. !!! !! E = hν"

Biological Spectroscopy""

X-ray Crystallography!

NMR Spectroscopy!

Circular !Dichroism!UV Spectroscopy!

λ = 280nm proteins!λ = 260nm nucleic acids!

Mass !Spectrometry !

16421.13!

Actual:!16447.5!

+ H/D exchange!

Infrared!

(for 2o structure)!

NMR: Historical Timeline 1946: Bloch, Purcell: first 1H NMR spectrum"1950: Proctor, Yu: 14N NMR spectrum of NH4NO3"

"" "" Dickinson: 19F NMR of fluorinated compounds"" " -> concept of “chemical shift”"

1953: “Overhauser effect” -> NOE"" " -> internuclear distance information"

1966: Ernst: Fourier Transform (“FT”) NMR"1971: Jeener: 2D FT NMR"1985: Wüthrich: first protein NMR solution structure

" BPTI (6.5 kDa; 58 a.a.); homonuclear (1H)"≈1990: Bax, Kay, Wagner, others: 3D NMR + 13C,15N "" " isotopic labeling protein NMR assignment/structure"

To date: Explosion of technical and experimental advances""""""

""

1958: First X-ray crystal structure of protein (Kendrew et al) Myoglobin (17 kDa; 153 a.a.)

1913: First X-ray Structure (Bragg;

Diamond)

NMR: Historical Timeline 1950: “Frequency shift” (chemical shift)"Dickinson: 19F NMR in 1950"""

""""

""

δ = 117 ppm"

1 Tesla = 10,000 Gauss"-> 0.7 Tesla field"-> 30 MHz instrument"

Consider:"600 MHz spectrometer"-> 14.1 Tesla"ΔField"

Literature (19F vs. CFCl3):"SbF3: 85 ppm"BeF2: -20 ppm"

NMR: Historical Timeline 1966: Continuous Wave (CW) vs. Fourier Transform (FT)"""

""""

""

CW: field/frequency “sweep” (“OLD”)"FT: simultaneous excitation (“modern NMR”)"

"- signal averaging (S/N) - higher resolution - PW excitation SW""- pulse sequence programming - line shape distortions (vs. CW)"

CW!

FT!

Protein Structure Determination:Xray vs. NMR

X-ray " " " " "NMR"• diffracting crystals • observe “heavy” atoms 1920s 1st structure of protein: 1950s Practical Aspects

•  Producing enough protein for trials; SeMet labeling

•  Crystallization time and effort •  Crystal quality / stability •  Synchotron beam lines

Kurt Wüthrich Nobel Prize 2002!• solution or solid state

• observe distances between H’s 1940s 1st structure of protein: 1985 Practical Aspects

• Producing enough (2H)13C/15N labeled protein for collection

•  Sample “conditioning” •  Size of protein •  Spectral analysis is slow and

error prone

John Kendrew!Nobel Prize 1962!

NMR Spectroscopy: A Short Course"

Typical Applications of NMR: 1) Structural (chemical) elucidation - Natural product chemistry - Synthetic organic chemistry - analytical tool of choice of synthetic chemists - used in conjunction with MS and IR 2) Study of dynamic processes - reaction kinetics - study of equilibrium (chemical or structural) 3) Structural (three-dimensional) studies - Proteins, Protein-ligand complexes - DNA, RNA, Protein/DNA complexes - Polysaccharides 4) Drug Design

- Structure Activity Relationships by NMR 5) Solid State NMR 6) Medicine

- MRI - Metabolomics

MRI images of the Human Brain

NMR Structure of MMP-13 complexed to a ligand

O

O

O

O

OH

OO

O

HO

NH

OH

OO

O

O

Taxol


Bo = 0 Bo > 0 Randomly oriented Highly oriented

Bo

Nuclear Spin"

N S

like bar magnets

Bo = 0

Bo > 0 ΔE = h ν

α

β

Nuclear Spin"

Nuclear spin µ = γ I h

µ - magnetic moment

γ - gyromagnetic ratio I - spin quantum number

h - Planck’s constant mI – magnetic quantum

number

µ

I is a property of the nucleus!

Mass # Atomic # I!!Odd Even or odd 1/2, 3/2, 5/2…!!Even Even 0!!Even Odd 1, 2, 3…..!

“NMR INACTIVE”"

I = 0!

“NMR ACTIVE”"! I = ½ ! I > ½ !

! “quadrupolar”!

Every element has at least 1 NMR active isotope

http://www.webelements.com/nmr.html!

Nuclear Spin Energy Levels"

2I + 1 nuclear energy levels => 2I transitions !!!

I = ½ " " " " " I > ½

ΔE = γhBo = hνL

I = 1! I = 3/2!

NMR Sensitivity and Magnet Technology

Increase in magnet strength is a major means of increasing Sensitivity

ΔE = h ν α B0

$0.8M ! $1.5M ! $4.0M!14.1 T 18.8 T 21.2 T!!1 Tesla = 10,000 G!Earth’s magnetic field: ≈ 0.6G!

S/N α (B0)3/2

NMR “Radio”"

MHz! MHz!0 200 400 600 800!

1H!

Isotope Net Spin γ / MHz T-1 Abundance / % 1H 1/2 42.58 99.98 2H 1 6.54 0.015 3H 1/2 45.41 0.0 31P 1/2 17.25 100.0 23Na 3/2 11.27 100.0 14N 1 3.08 99.63 15N 1/2 4.31 0.37 13C 1/2 10.71 1.108 19F 1/2 40.08 100.0

13C!15N! 31P!

ΔE = h νo ΔE = γ h Bo / 2π

“gyromagnetic ratio”!

B1 on

Mo

z

x B1

z

x

Mxy y y

ω1

ω1 ω1 = γB1

90o pulse


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

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

Free Induction"Decay (FID)"

Spectrum"

FT"

Let’s do a simple ! experiment:!

[Time (s)]"

[Frequency (Hz)]"

Bo Bo


Observable Name Quantitative Information Peak position Chemical shift (δ) δ(ppm) = [νobs – νref / νref ] x 106

chemical (electronic) (ppm) environment of nucleus Peak Splitting Coupling Constant (J) peak separation neighboring nuclei (Hz) (intensity ratios) (torsion angles) Peak Intensity Integral unitless (ratio) nuclear count (ratio) relative height of integral curve T1 dependent Peak Shape Line width Δυ = 1/πT2 molecular motion

(Hz) peak half-height chemical exchange “dynamics”

NMR Vocabulary"

δ


Chemical Shift"

ΔE = hνL = h (1 – σ) γBo / 2π “shielding constant”!(local fields at nucleus)!

electrons"

electrons in B0 => current => tiny field opposing B0 !

e- withdrawing ! “inductive effect” " !e- donating! !!


Chemical Shift"! ! ! “ring current effect” " !!

Deshielding! δ!

Shielding! δ!

Definition: " " "1H νref (= 0 ppm)""

" " " "organic: TMS!!

! ! ! !aqueous: DSS ""


Chemical Shift Dispersion: 1H < 13C (!)"

1H!

13C!


Spin-Spin Coupling (a.k.a. Scalar Coupling; J-Coupling):"-  Through-bond interaction between nuclei via electrons"-  Multiplet = 2nI + 1 (n = # equivalent neighboring nuclei)"

2-4 bonds!CH3CHCl2!


Spin-Spin Coupling in Biological NMR:"I.  Information on torsional angles"

"=> structural information"

J related to torsional angle between coupled nuclei - Karplus "

C! C!H!

H!H!

H! θ

J(φ) = Acos2φ + Bcosφ + C Sugar Puckering:!

Phi in Proteins:!

HN"

Hα

3JHNHα

H2ʼ"

H1ʼ"

H2ʼ"

H1ʼ"

“S”"

“N”"

3JH1ʼH2ʼ"""" 7-8 Hz""""""" ≈ 1 Hz "


Spin-Spin Coupling in biological NMR:"II.  1H-X coupling (X = 13C, 15N, 31P)"

" "1J(1H-13C) ≈ 120-150 Hz"" " "1J(1H-15N) ≈ 90-95 Hz"" ""

"i. Extremely important in " ii. Residual Dipolar Couplings"multi-dimensional NMR"=> experimental set-up""

“INEPT”"

“isotropic”" “aligned”"

15N"

1H"


Nuclear Relaxation: The “Core” of NMR"• Relaxation: magnetization returning to equilibrium state"

" "after rf pulse"" " described by 2 time constants"

! ! ! T1 " " T2 " “spin-lattice” " “spin-spin”"

" " “longitudinal” "“transverse” "" " α sampling rate " α 1/Δν1/2 !

inversion recovery “spin-echo” / “CPMG”

!T2!

T2 ≤ T1 "


T1 and T2 are intimately related to molecular motion/dynamics"Many Nuclear Relaxation Mechanisms:"

" "I = ½ " " " " " ""Dipole-Dipole: " " " " " ""

! !α r-6 " " " "Chemical Shift Anisotropy (CSA):"

" "α B02!

Scalar Coupling:"" "X-Y (Y = I > ½)!

Others…."""! !I > ½"

Quadrupolar:""extremely efficient!

"

Principle relaxation mechanism for:""1H, 13C-1H, 15N-1H"


T1 and T2 are intimately related to molecular motion/dynamics"" " " " " ""

Dipole-Dipole Relaxation and Molecular Motion (τc): "" " " ""

broad signals

narrow signals

An NMR Spectrometer"Pulse generator

Fr equency synthesizer

switch amplifier

transmitter magnet

Pr obe

sample

RF am plifierdetectorAF amplifierADC

Computer

SPH 020"

NMR Probes and Samples"NMR Probes" NMR Samples"

NMR Robotics"

≈ 40 μL!≈ 400 μL!

≈ 270 μL!

“RT” vs. “CRP”!


Sample Preparation: Biological Solution NMR""I.  Concentration: ca. 0.1 to 1 mM range (ALOT!)""II.  Volume: 1.7-mm tube: 40 μL (sample limited)"

5-mm tube: 270 - 300 μL (Shigemi)""III.  pH: typically < 7 (avoid NH exchange)"IV.   Buffer: "

"i. 1H NMR: try to avoid big buffer peaks; deuteration """ii. isotope edited: not a big issue""

V.  Salt: increases pulse lengths and bad for CRP; < 500 mM"VI.  T, η: anything that τc is generally good ( Δν1/2)"

"=> η and T (if your protein can withstand it)""VII.  D2O: 5-10% D2O for a “lock”; 100% also used"VIII.  Labeling: 15N, 13C/15N, 2H/13C/15N….. "

Peptide NH exchange"vs. pH"

Documents

Lectures for CCB 538 James Aramini, PhD.-Structure Activity Relationships by NMR 5) Solid State NMR 6) Medicine - MRI - Metabolomics MRI images of the Human Brain NMR Structure