Lecture #1 Juli Feigon

• Overview of course• Brief history of macromolecular NMR• Semi-classical description of NMR• 1D NMR experiment

1

Macromolecular NMR Spectroscopy

Course overview:Ø 5 weeks intro to macromolecular NMR spectroscopy.Ø Qualitative intro to modern NMR as applied to proteins and

nucleic acids.Ø You will be introduced to basics of NMR and where to go in

literature to learn things in more detail.Ø We will mostly use classical approach to look at spins.Ø Hopefully you will develop a feel for the field, with emphasis

on applications.Ø GOAL: Graduate course – get what you need out of itØ For people who go into field: tools to go on and learn more on your own

later.Ø For people outside the field: ability to follow seminars, critically

evaluate literature

Contact Info: Course material:

Prof. Juli Feigon basics of 1D NMRBoyer 241 nucleic acids and complexesfeigon@mbi.ucla.edu

Prof. Rob Clubb basics of 2D and 3D NMRBoyer 639 protein structurerclubb@mbi.ucla.edu

Dr. Robert Peterson NMR instrumentation & labMSB 1429peterson@mbi.ucla.edu

Office hours: After lecture and by appt.

Ø 16 lecturesØ Two OPTIONAL Tu lectures on product operatorsØ 4 problem sessions/discussion, usu ThØ [M230D: NMR lab last 4 weeks of quarter]Ø Problem sets

Ø due at START of problems sessionsØ You must turn in homework to attend the problem sessionØ Led by Dr. Peterson

Ø GradingØ Homework: 30%Ø Exam (2 hr during finals week) 70% Thurs March 23 9-11

Ø Handouts and ReadingØ Lectures will be posted on line (M230 web page)Ø Assigned readings posted on lineØ Relevant texts:

Ø Claridge: excellent on basics (except relaxation), but no applications to macromoleculeØ Evans: intro to 2D-3D NMR with basics and applications to both proteins and nucleic

acids. Good reference, but out of date.Ø Wuthrich: the original classic for macromolecular NMR. Ø Use lectures as a guide for what you need to know from reading.

Ø Other NMR books (handout)

M230B Structural Molecular Biology

Winter 2017 Prof. Robert Clubb Prof. Juli Feigon

with Dr. Robert Peterson

“Macromolecular NMR” Suggested reading: C=Claridge;

J=James Feb 15 W Intro and basics of 1D NMR JF C: 13-20; J: 1-12 (Evans: 1-13) Feb 16 Th NMR parameters & 1D spectra of proteins and nucleic acids JF C: 20-21; J:21-25 Feb 17 F Detection, acquisition, apodization, FT, phase JF C: 48-75 Feb

20

M

HOLIDAY

Feb 21 T Finish practical NMR JF Feb 22 W Relaxation, T1, T2 RC C: 21-44: J:12-21 TBD NMR lab #1: Overview of Instrumentation, NMR tour RP C: 45-48 (48-53; 59-63) Feb 23 Th Problem Session 1 RP Feb 24 F The NOE RC C: 277-296,301-306 Feb

27

M

Basics of 2DNMR: NOESY, COSY, TOCSY, HSQC

RC

C: 147-156

Feb 28 T Basics of 2DNMR: con’t RC Mar 1 W Sequential resonance assignment of small proteins JF Roberts (Chap. 4) NMR lab #2: tuning, matching, HSQC Mar 2 Th Problem Session 2 RP Mar 3 F Nucleic acids assignments and structure JF See course website Mar 6 M Conformational equilibria, chemical exchange, chemical

shift mapping JF See course website

Mar 7 T OPTIONAL lecture: Product Operators 1 RP Mar 8 W Heteronuclear NMR: bigger proteins RC See course website NMR lab #3: protein backbone assignment RP Mar 9 Th Problem Session 3 RP Mar 10 F Triple resonance NMR, 3D Structure determination RC See course website Mar 13 M Triple resonance NMR, 3D Structure determination RC See course website Mar 14 T OPTIONAL lecture: Product Operators 2 RP Mar 15 W Using NMR to study macromolecular dynamics RC See course website NMR lab #4: protein sidechain assignment & NOEs Mar 16 Th Drug binding and PRE methods to study macromolecular

interactions and structure RC See course website

Mar 17 F Problem Session 4 RP Mar 23 Th EXAM 9-11 am

M230 Rob Clubb and Juli Feigon

NMR BOOK LIST Some useful books both basic and advanced

Author(s) Title

Wüthrich, Kurt NMR of Proteins and Nucleic Acids

Bax, Ad Two-Dimensional Nuclear Magnetic Resonance in Liquids

Freeman, Ray A Handbook of Nuclear Magnetic Resonance

Claridge, Timothy D.W. High-Resolution NMR Techniques in Organic Chemistry

Derome, A.E. Modern NMR Techniques for Chemistry Research

Roberts, G.C.K., Editor NMR of Macromolecules: A Practical Approach

Evans, J.N.S. Biomolecular NMR Spectroscopy

Neuhaus, D. and Williamson, M. The Nuclear Overhauser Effecy in Structural and Conformational Analysis

Cavanagh, J., Fairbrother, W.J., Palmer A.G. III, Skelton, N.J.

Protein NMR Spectroscopy: Principles and Practice (2nd edition)

Freeman, Ray Spin Choreography: Basic Steps in High Resolution NMR

Keeler, James Understanding NMR Spectroscopy

Pochapsky, Thomas and Susan Sondej NMR for Physical and Biological Scientists

Tang, Quincy Structural Biology: Practical NMR Application

NMR: structures, dynamics, interactions

Mueller & Feigon EMBO (2003) Ubl domain of HHR23A in complex with S5A-UIM2 of the proteosome

Theimer, Blois, & Feigon, Mol Cell (2005)Structure of a conserved pseudoknot from human telomerase RNA

Wu, Henras, Chanfreau, & Feigon PNAS (2004)Structure of the yeast RNAse III dsRBD in complex with dsRNA

Hud, Schultze, Sklenar & Feigon JMB (1999) Binding sites and dynamics of ammonium ions in a telomere repeat DNA quadruplex

Dynamics in solution: protein folding, enzyme catalysis, and ligand recognition

Example: Naik, et al & Clubb, JBC (2006) : Dynamics of Calcium modulated loop closure of Sortase A. Calcium binding quenches picosecond time-scale motions near the binding pocket.

Integrative structural biology of Tetrahymena telomerase: NMR and crystal structures of protein and RNA domains fit into 9Å resolution cryoEM map

Jiang, Chan et al Feigon, Science, 2015

p65 xRRM:TER stem 4 complex determined by NMR and crystallographyp65 N-terminal

domain is intrinsically disordered (IDP), determined by NMR

TER pseudoknot determined by NMR

p19 structure determined by crystallography;2° structure by NMR

p19:p45 complex and interface detected by NMR

p45C determined by crystallography

Domains connected by flexible linkers, invisible in EM map

Dynamics of flexible domains can be studied by NMR!

Teb2C structure determined by NMR

Integrative structural biology of S. aureus Iron-regulated surface determinant (IsdH) protein and recognition of human hemoglobin: interdomain orientations from RDC, dihedral angle, NOE, PRE, and SAXS data

Sjodt et al Clubb, JMB, 2015

NMR is a “new” technique….. a little magnet history1932 Stern and Gerlach experimentally detect nuclear magnetic moment in a molecular beam of silver atoms (its quantized).

1945 Nuclear magnetic resonance detected in solution (H2O by Bloch et al.) and in a solid (parafin by Purcell et al.). Bloch and Purcell received the Nobel Prize in Physics in 1952.

~1964 First superconducting magnet for NMR (200 MHz). Dramatically increases sensitivity and resolution.

1966 Ernst and Anderson introduce pulse Fourier transform NMR. Increases sensitivity ~100 fold.

1958 First protein structure determined by crystallography (Myoglobin by Kendrew)

A little history: developments that made NMR useful for thestudy of macromolecules in the past 40 years

a) Resolution increases with increasing magnetic fieldRecall: chemical shift in ppm will be the same

at all fields, but Hz will vary.

1976-1st 300MHz

NOTE: ω=2πυChemical shift ⇒ δ = (υsample - υref)106

υrefυ in Hz

example: 2 peaks separated by 0.2 ppm at 100 MHz → 20 Hz500 MHz → 100 Hz

Installed Feb 2004 at UCLA: ~$2.2 million ⇒ 800 MHz →Highest field spectrometer currently ⇒ 1 GHz →

commercially available (23.5 Tesla)Installed in Lyon, FR 2009

1.2 GHz NMR to be installed in Frankfurt in2017

in ppm

1) Development of high field spectrometers

increasing B0

B01

B02no B0

ΔE

b) Sensitivity ↑ as field ↑

Spins 1/2 line up with or againstfield with a separation of energy ΔE

##

= e-ΔE/kT = β= α

NOTE: Population difference is small. ∴ NMR is inherently insensitive

∴ with higher fields, can look at lower concentrations of macromolecules

Typically: 0.5 - 2 mM in 200 to 500 µl

with cryoprobes à µM!

2) Development of a variety of multiple pulse NMR techniques whichhave greatly expanded the info. we can get - 2D and 3D NMR!!

1st 2D experiment proposed by Jeener1st published 2D experiment by Ernst1st applications to proteins (Ernst and Wüthrich labs) ⇒ 1st revolution2D NOESY experiment invented. Can be used to measure interproton distances1st 2D NMR of DNA (Feigon, et al., Kearns)]1st 3D protein structure determination (Wüthrich)1st 3D NMR experiment (Oschkinat, Griesinger, Ernst, Gronenborn, Clore)Triple resonance 3D NMR: 13C, 15N, 1H (Bax) ⇒ 2nd revolutionHeteronuclear relaxation experiments for protein backbone dynamics! (Bax & Kay)Application of RDC (residual dipolar couplings) (Bax and Prestegard groups)TROSY (transverse relaxation optimized spectroscopy; Pervushin & Wüthrich)plus many more recent developments.

1971:1975:1976-7:1979:[1982:1985:1988:1990:1992:1990’s:1997:

2002: Nobel prizeKurt Wüthrich

1991: Nobel prizeRichard Ernst

3) SamplesØ Need mgs of samples (like crystallography)Ø proteins ⇒ cloning, overexpression; cell-free expression.Ø DNA, RNA ⇒ chemical and enzymatic synthesisØ 15N, 13C, 2D labeling methodsØ for larger proteins selective aa and methyl labeling

4) Development of computers & computational methods

Ø handle large 2D, 3D, 4D data setsØ ability to process and analyze NMR dataØ structure calculations and visualization

(like crystallography)

~0.5 ml of protein or nadissolved in bufferedwater.

Why do (solution state) NMR vs crystallography?

Ø can’t always get crystal.Ø can look at >1 conformation and equilibrium between them.Ø can get information on dynamics and mobility.Ø can vary conditions (pH, ionic strength, etc.).Ø can look at partially structured and intrinsically disordered proteins (IDPs).Ø can study protein and nucleic acid folding.Ø can identify weak ligand interactions.Ø occasionally more biologically relevant.Ø can quickly determine (< 2hrs) if protein or na is folded.Ø v. easy to map protein-protein and protein-na interfaces, even for weak binding.Ø can “see” small populated or invisible states of proteins (NEW!).Ø NMR data can help with designing constructs for xtallography…

⇒ X-ray Crystallography and NMR are COMPLEMENTARY techniques

Ø size 1986 10 kD 2D proton NMR (Wuthrich)1992 18 kD heteronuclear 3D, 4D NMR

interleukin II (Clore & Gronenborn)1996 ~30 kD deuterium labeling also2002 ~60 kD (and 3D folds up to 100 kD)

Wüthrich assigned resonances in 900 kD GroEl-GroES complex Nature 418 207 (2002)

2005 82 kD aa specific methyl labeling;malate synthase G fold (Lewis E Kay)

2008 refined using combined NMR/SAXS

today 128 kD Hybrid approach using RDCs and SAXS for dynamic macromolecular assemblies; example bacterial Enzyme I (Clore) [reviewed in Chemical Reviews 2015]

Ø solubility and aggregation problemsØ time

Limitations (for getting complete 3D structure using solution NMR)

NMR gives information on a wide range of timescales

ps ns µs ms sinternalmotions τc

2° structuralelement movement

domain movement

chemical exchange

exchange spectroscopyR2, R1ρ, CPMGR1, R2het NOE

RDCs

StructureDynamicsInteractions

Most nuclei have an intrinsic quantum mechanical property called “nuclear spin”

This is a quantized value described by (I), the spin magnetic quantum number.

General features:

Spin magnetic quantum number

I = ½ for 1H, 13C, 15N, 31PI = 0 for 12C I = 1 for 2H (deuterium) ( 1 neutron, 1 proton), 14N

Even Mass nuclei that have Even number of neutrons have I=0 (not interesting from the NMR point of view) Even Mass nuclei that have Odd number of neutrons have an integer spin quantum numberOdd Mass nuclei have half integer Spin quantum number

Solution state NMR work mostly makes use of “spin ½” nuclei (I = ½)

1H, 13C, 15N, 31P

Qualitatively,the dipole moment arisesfrom motion ofspinning particle

γ: magnetogyric ratio; constant for a given nucleus[γ is a proportionality constant between J and µ]

In the presence of a magnetic field (Bo), the energy of the dipole is:

applied field(also written Ho)

The NMR phenomenon* NOTE: I will use only vector formalism; no product operators

1) Certain nuclei have magnetic moments (‘spin’)u Nuclear angular momentum ; I = nuclear spin quantum number

For protons, I = 1/2 ⇒ spin 1/2 nuclei in units of ħu Nuclear magnetic dipole moment

For spin 1/2 nuclei in B0:m = -1/2

m = 1/2 α

βFor spin 1/2, nuclei line up withor against field; have 2 differentenergy levels

ΔE=6.6x10-26 joules

at 500 MHz

small

2) Nuclei precess around applied magnetic field(due to angular momentum and torque) at the Larmor frequency.

cycles per sec (Hz)radians per sec

From classical mechanics:torque on µ at angle θ to µ.

z

y

x

B0

individualspin

classical picture

y

x

Bo

individual spin

y

x

zzensemble of spins

more upthan down

no phase coherence(not really a classical concept)

y

x

z

Classical (vector sum)

net magnetization

Mz = Mo = equilibrium magnetization

Mx, My = 0

Larmor equation

3) Transitions are induced between 2 states by application of arotating field (B1) in the xy plane at the Larmor frequency

y

x

B0z

Resonance condition: υo = υrf

frequency ofB1 field in xy plane

B1[Note: B1 is actually a linear field oscillating along

x-axis → equivalent to 2 counter rotating vectors]

4) The rotating frame

In order to follow the bulk magnetization, it is easiest to view motion of spins (vector sum) in the rotating frame, i.e. make our reference frame rotate at ω0.

y’

x’

z

Bo

B1

B1 now lies along x’.

(unless otherwise explicitly stated, the reference framewill always be the rotating frame from now on)

Relative magnitude of Bo and B1 at 400 MHz with 10 µs 90o pulse:If γH = (2π X42.58 X106) = 2.6754 X108 rad s-1T-1,

then Bo =9.34 T;For 90o pulse, t = π/2 γH B1; B1 = 5.87 X 10-4 T

5) The pulse!In our FT experiments, an RF pulse at the Larmor frequency is used

to knock the magnetization into the xy plane

y’

x’

z

M0B1

B0

y’

x’

z90º pulseapplied along x’:

by turning onB1 for

a time t[M X B1]

net magnetization My along y’; phase coherentno phase coherence in xy plane, Mxy = 0

(ie. phase of precession is random)

x

M = -M0or πx pulse no net magnetization

in xy plane

The time that the rf pulse is on determines the angle that the bulk magnetization is tipped. Only puts all magnetization as My.

⇒ Multiple pulse NMR is combination of various π and π/2 pulses which affect spins in different ways.

If spins have frequencies other than υo(i.e. slightly different because of different electronic environments), then

3 spins with differentchemical shifts A, B, C

Y’

x’

z

υC < υB

υB (υ0)υA > υB

all spins will initially be along y, but will precess at different frequencies than the rotating frame (slightly different),so will precess around z-axis at a frequency equal to chemical shift difference from υ0

individual spins of given type, not bulk magnetization

But of course, spins reestablish equilibrium after the rf pulse is turned off.Lose transverse (i.e. xy plane) magnetization as a result of relaxation processes (next week)Spins dephase, lose phase coherence in xy plane ⇒ T2 relaxation

y’

x’

y’

x’

time

right after90° pulse

⇐ observesignal along y

6) The FID (free induction decay)Signal is observed as voltage induced in rf coil - observed along y’

Imaginary case with no relaxation:

t →

ifω0 ≠ ωi

ifω0 = ωi

t →

Note: ∴ For a 180° pulse, nothing along y, ∴ no detected signal

Result is an FID which decays exponentially

7) So, a normal 1D FT experiment goes like this:

pulse (90° pulse) ← actually can be < 90 °

delaytime

to allow systemto come backto equilibrium

T1, T2

acquire signalfor a time t1 until

it has decayed

n ← to signal average for greater S/N

⇓ FFT (Cooley-Tukey algorithim)

1D FT NMR spectrumƒ(t) → ƒ(ω)

FT

Note: T1 ≠ t1T2 ≠ t2

Ø1D NMR experiments are described using the Bloch equations (1946) which predict the behavior of nuclear spins when they are treated as “Bulk” magnetization. [Felix Bloch (Swiss-born American physicist (1905-1983)]

ω0

ω0 = ωi

ω0 ≠ ωi

2 spinsωi , ωj

ω0

ω0ωi

ωi ωj

time → frequency →FT

Fourier transformation (FT)of the FID gives rise to theabsorption (NMR) spectrum

Relative chemical shift determinedby Larmor frequency of nuclei

∆υ1/2 (linewidth at half height)