Biomolecular NMR – A Brief...

Hercules 2020 NMR and Protein Dynamics

Martin Blackledge

Protein Dynamics and Flexibility by NMR

Institut de Biologie Structurale, Grenoble, France

Biomolecular NMR – A Brief Overview


Exploring the role of dynamics in protein function by NMR spectroscopy

Dynamics in microcrystalline

proteins Local dynamic

modes on multiple timescales....

Multidomain proteins with flexible linkers

Multidomain proteins with highly disordered

functional domains

Flexible domains in highly ordered

assemblies Intrinsically disordered

proteins

…and their role in functional complexes


Nuclear magnetic resonance (NMR) spectroscopy •  Placing a sample of nuclei with spin > 0 in a magnetic

field leads to a splitting of the nuclear spin energy levels

•  For nuclei of spin ½, two energy states are obtained

•  The populations of nuclei in the two states are given by the Boltzmann distribution

•  The energy splitting depends on the gyromagnetic ratio and the strength of the magnetic field

In NMR, we induce transitions between the two energy levels by applying a radiofrequency pulse:


Isotope labelling strategies for biomolecular NMR

Uniform 15N, 13C

Uniform 15N, 13C, 2D (partial or complete)

2D (uniform), Selective Methyl labelling 13CMet, 1HMet

Solution NMR sample conditions:

Concentration: > 50µM

Volume: > 100µL Isotope labelled Stable (days!)

Pure


Rotational diffusion times of

large folded proteins imposes

limits on NMR lineshape

Spectral quality is vastly improved if density of protons is decreased

As folded molecules get much larger selective

methyl labelling gives high quality spectra up to MDa

assemblies


Fingerprint of a protein: 1H-15N HSQC spectrum Folded protein 75 amino acids

Each NMR signal in the HSQC spectrum corresponds to a backbone or sidechain NH group

Chemical shifts depend on the electronic environment

of each nuclear spin

Information about structure and dynamics

•  Backbone 1H, 15N – domain interfaces, tertiary structure

•  Backbone 13C – secondary structure, location and propensity of α-helices, β-sheets, loops


Magnetization transfer

Dipole interaction (NOE) – Nuclear Overhauser Effect – Through space interaction – Distance dependent (1/r6) – NOESY -> distance restraints - < 6Å Scalar-coupling interaction (J coupling) – Through bond interactions – Chemical connectivities – Assignment – Dihedral angle dependence


From one- to two to three-dimensional NMR

h"p://www-‐keeler.ch.cam.ac.uk/lectures/

FT (t2)

2

2

1t1

t1

FT (t1)

t2

U

FT (t)

t

UI


Through-bond connectivities allow us to assign all nuclear spins throughout the protein

h"p://www.protein-‐nmr.org.uk


Classical Determination of Macromolecular

Structure in the Solution State

Interproton Distances from NOE Measurement

Conformational Ensemble



Inter-proton distances from NOESY

Conformational model

NOESY-based Protein Structure Determination




Interproton Distances from NOE Measurement

Conformational Ensemble



Inter-proton distances from NOESY

Conformational model

Tugarinov et al. PNAS 2005;102:3:622-627

NMR structure of the 723-residue (82-kDa) enzyme malate synthase G from Escherichia coli,

NOESY-based Protein Structure Determination


Determination of Kd CD2AP SH3" Ubiquitin" CD2AP SH3"

Ubiquitin"

Structures of protein complexes by NMR "Chemical Shift Mapping of Interfaces"


Free Ene

rgy

Protein Function Weighted average over all

populated states

Need for a detailed description of the energy

landscape

➡︎ Understanding the relationship between structure, dynamics and function

Proteins are plastic molecules exhibiting rich dynamics


Interconversion between natively folded protein and aggregation prone species

Conformational changes in proteins modulating functional activity

Trajectories of intrinsically disordered protein interactions with their partners

Dynamics are important for protein function and malfunction


Motional averaging up to the millisecond dictates interpretation of all NMR data"

ps ns µs ms s

Dipolar, scalar couplings, chemical shifts

Spin relaxation

Relaxation dispersion

Real time

Librational motion Rotational diffusion

Enzyme catalysis Signal transduction Ligand binding

Assembly

kex"δ

NMR and exchange!Conformational states kex << Δδ give rise to distinct peaks"Intermediate exchange broadens"peaks. ‘Fast’ exchange averages "to single resonance peak"

τ

Cang(τ)

Spin relaxation !Fast motional information "

1H

15N

First-order interactions!Chemical shifts, dipolar couplings"population-weighted average sampled up to millisecond

Three important timescales in NMR: •  Larmor timescale: τ=1/ω0

–  Efficiency of spins state transitions during the relaxation •  Spectral range: τ=1/Δν

–  Spectrum features: chemical shift range, couplings, ... –  Averaging of the interactions by motions at higher

frequencies –  Perturbation of spectral appearance by motions/processes

occurring around this timescale •  Equilibrium constant time T1 / Signal lifetime T2

–  NMR experiment ó perturbation of spins system –  Typical timescale for (macro)molecules in solution: 100 ms-s

(T1) / 10ms-s (T2) –  Determine the lowest frequency of motions that can be

characterized during one NMR experiment


Spin state equilibrium"

rf pulse"

Spin state excitation"1 transition every 3.1013 year"

NMR spin relaxation: the problem

B0


Spin state equilibrium"Spin state excitation"Spin state relaxation"

Molecular motion"

Local fields Beff"

The real experiment

Back to equilibrium "in ~1-10 s"

B0 rf pulse"

Only the photons generated by Beff which have the “right” energy are able to induce transitions - relaxation depends on motional timescale

and on magnetic field


Relaxation rates can be described in terms of the motion "of the relaxation-active interactions"

Dipole-dipole (DD)""

Spin I experiences"a distance and

orientation dependent local field due to the magnetic

moment of the nearby spin S"

"

15N relaxation (spin 1/2)

relaxation active mechanisms :"

Source of fluctuating fields"

DD I

S


Relaxation rates can be described in terms of the motion "of the relaxation-active interactions"

Dipole-dipole (DD)""

+"

Anisotropic "electronic"

environment -"chemical shift

anisotropy""

Assumed axially !symmetric and

coaxial with NH"Source of fluctuating fields"

CSA

DD

15N relaxation (spin 1/2)

relaxation active mechanisms :"


Spectral density function : J(ω) "

Describes the mobility of the inter-nuclear vector in terms of the distribution of

frequency components""

Fourier transform of the time-dependent auto-

correlation function c(τ) "

J(ω)"

ω


Spectral density function : J(ω) "

Describes the mobility of the inter-nuclear vector in terms of the distribution of frequency

components""

All spin relaxation rates can be described in terms of J(ω) at characteristic frequencies of

spin system " ""

J(ω)"

ω


R1(X)"R2(X)"

nOeH-X"

J(ω)"

ωx" ωH ωH-ωx"0" ωH+ωx"

ω =2πf - Larmor frequency, depends on static field strength"

80MHz" 800MHz" 18.8 Tesla"40MHz" 400MHz" 9.4 Tesla"

ω/2π =f"

Sampling of the spectral density function - Heteronuclear two spin system"


Interpretation in terms of dynamic amplitude and timescale"

J(ω)"

ω

τc

Amplitude of internal dynamics - S2"

Timescale of internal motion - τi"


Lipari-Szabo Modelfree Analysis : Fitting {S2, τi} to relaxation data from Calbindin

Apo form

Calcium bound

Ordered

Disordered


Relaxation in Methyl Groups!

Due to symmetry of rotation 13C relaxation in methyl groups reports on

reorientational"dynamics of the C-C

bond axis""

S2axis"


Aspartate transcarbamoylase 300 kDa

ClpP 300 kDa

Proteosome 670 kDa

Methyl-TROSY NMR of large molecular machines !

Kay and co-workers


NATURE Vol 445 8 February 2007

ps-ns methyl motion

conformational exchange (kex ~1500 s-1)


Tzeng & Kalodimos Nature 2012

Conformational entropy strongly influences binding

CAP variants have markedly different affinities for DNA, despite the CAP−DNA-binding interfaces being essentially identical in the various complexes – importance of internal dynamics on binding


Dynamics in microcrystalline

proteins

…and their role in functional complexes

Multidomain proteins with flexible linkers

Multidomain proteins with highly disordered

functional domains

Flexible domains in highly ordered

assemblies Intrinsically disordered

proteins

Local dynamic modes on multiple

timescales....

No limitation of rotational correlation of the protein – simplifies analysis

Extends range: should be sensitive to a broader range of timescales

>100000 protein crystal structures : little information about associated dynamics

Development of approaches for insoluble systems

Cole & Torchia Chem Phys (1991), Mack et al Biopolymers (2000), Giraud et al JACS (2004), Giraud, et al JACS . (2005), Lorieau & McDermott JACS (2006), Chevelkov et al JACS (2007), Agarwal et al JACS (2007), Yang et al JACS (2009), Lewandowski et al JACS (2010) Schanda et al JACS (2010) , Lewandowski et al JACS (2011)…..

Exploring the role of dynamics in protein function using NMR


Solution R1 C’ R1 Cα R1 Cβ

Dynamic studies of crystalline proteins by solid state NMR relaxation

Lewandowski, Sein, Sass, Blackledge, Emsley (2010) J Am Chem Soc 132 1246-1248

Lewandowski, Sein, Sass, Grzesiek, Blackledge, Emsley (2011) J Am Chem Soc 133 16762-16765 Mollica, Baias, Lewandowski…, Rienstra, Emsley, Blackledge. (2012) J. Phys. Chem. Lett., 3, 3657–3662

Józef Lewandowski | [email protected] | Asilomar 2011

distribution of 15N R1 and R1! in GB1

Q56

M1

avg. R1! 18 kHz 2.7 ± 2 s-1

10 20 30 40 500

4

8

16

R1! (

1/s

)

residue

10 20 30 400.0

0.1

0.27

0.35

R1 (

1/s

)

50

1GHz, 60 kHz MAS, 24ºC

! common features between R1 and R1! profiles

R1! (1/s)

Wednesday, April 6, 2011

Probing slower motions with rotating frame relaxation (R1ρ)

Solid state relaxation probes compared to solution RDC studies

crystal

solution

Intrinsic Dynamics in Crystalline Proteins

Development of multiple probes to sample fast and slow motions on protein

backbone and sidechains


Atomic Resolution Structural Dynamics in Crystalline Proteins from NMR and Molecular Dynamics Simulation

Mollica et al J. Phys Chem Lett, (2012)

250ns MD simulation 32 explicit copies -

8 unit cells,

Principal component of motion resembles RDC-derived slow

motion found in solution

2GI9

Av MD

Central copy + neighbours

Central copy

Snapshots

Understanding averaging properties of NMR observables in crystalline proteins

32*56 angular correlation functions

Chemical shifts

Nuclear spin relaxation

Intrinsic Dynamics in Crystalline Proteins


Neutron spectroscopy

X-ray crystallography

Proteins transition from inert to functional molecules, as they awake from their deep freeze

But when, and how?

Solution state NMR

Mössbauer spectroscopy

Dielectric relaxation

Frauenfelder et al., P. N. A. S. (2009) Doster, Cusack, Petry, Nature (1989) Parak, Formanek, Acta Crystallogr. A (1971) Knab, Chen, He, Markelz, P. Ieee (2007) Jansson, BergmanSwenson, J. P. C. (2011) Weik, Colletier, Acta Cryst,. D (2010) Vitkup, Ringe, Petsko, M. Karplus, NSB (2000) Tarek, D. J. Tobias. Phys Rev Lett (2002) Doster, Eur. Biophys. J. Biophy. (2008) Fenimore et al., Chem. Phys., (2013) Lee & Wand, Nature (2001)

TeraHertz

spectroscopy

Temperature Dependence of Protein Dynamics

backboneR1: 15N,13C’

R1ρ &R2’: 15N , 13C’, 13Cα

hydration water+

side chain

side chain

R1,CP : 1H

R1:13CH3, 15Nζ R1ρ &R2’:13CH3, 15Nζ

αα

bulk water R1, 1H: 1H

hydrated protein crystals

fast (ps-ns): R1slow (ns-ms): R1ρ & R2’ bulk w

R1, 1H, :

NMR can provide unique insight into the molecular origin of the Protein dynamical transition


Multiprobe relaxation measurements reporting on

solvent, sidechains and backbone from 105-280K

Temperature dependence of protein motions – Hierarchy and activation energies

τ = τ 0 expEa

RT!

"#

$

%&

Protein dynamics Master equation#

Data fit to simple Arrhenius relationships

J(ω) = Ck,amplitudeτ k

1+ω 2τ k2

k=1

n

∑

Lewandowski et al Science 348 578 (2015)


R1 100ps-100ns R1r,2 10ns-µs

Most relaxation rates are reproduced

by two or three contributions


Activation energies converge at high

temperatures

Estimation of activation energies/

timescales associated with

protein motions

Direct observation of hierarchical protein dynamics




-168°C -113°C 0°C

PROTEIN

DYNAMICS

Backbone

Solvent

Sidechain

NM

R R

ela

xa

tio

n R

1

•  Temperature-dependent NMR relaxation allows direct

visualisation of distinct

structural/dynamic

contributions in the same

experimental system

•  Activation energies of dominant

modes of backbone, sidechain

and solvent motions

•  Reconciles different transition

temperatures observed using

diverse physical techniques

Temperature dependence of protein motions – Hierarchy and activation energies



Dynamic timescales and NMR!

ps ns µs ms s


Spin relaxation


Real time



Assembly

kex"δ


τ

Cang(τ) Spin relaxation !Fast motional information "Quenched by rotational diffusion"

1H

15N

First-order interactions!

Chemical shifts, dipolar couplings"

population-weighted average sampled up to

millisecond

[email protected]!


Studying low populated and transient protein forms Free Ene

rgy

Minor States

Protein folding

Enzyme catalysis

Molecular recognition

Allosteric regulation

Aggregation

…

P ≈ 0.5 %

NMR exchange techniques see beyond the ground state


kex/Δω≪1

kex/Δω<1

kex/Δω≈1

kex/Δω>1

kex/Δω≫1

NMR exchange techniques see beyond the ground state


τexch=20ms

Rex=f{pA, Δω, kex, νCPMG}

CPMG Relaxation Dispersion – Probing the origin of exchange line-broadening

CPMG Relaxation Dispersion:

Atomic resolution characterisation of exchange equilibria Structure, Kinetics, Thermodynamics


Korzhnev et al. Science, 329, 1312-1316 (2010)

FF domain Observing a

folding intermediate using CPMG dispersion

2.8%

kex = 1800 s-1


FF Domain Korzhnev et al. Science, 329, 1312-1316 (2010)

SH3 Domain Neudecker et al. Science, 336, 362-366 (2012)

T4 Lysozyme L99A Bouvignies et al. Nature, 477, 111-114 (2011)

CPMG structures of weakly populated (invisible) states


Signal intensity

A (99 %)

B (1 %)

15N (ppm) B1 field

CEST profile depends on kex, Δω, R1 and R2

Sensitive to exchange 10 s-1 < kex < 400 s-1

Chemical exchange saturation transfer (CEST)

Forsen & Hoffman, 1963, Vallurupalli et al. 2012

⎯⎯→⎯ →bak

⎯⎯⎯←→abk

A B

abbaex kkk →→ +=


Dij = −γ iγ jµ0h8π3

P2 cosθ t( )( )rij3

Dipole-dipole interaction between two magnetic

moments

θ B0

r

Coupling averaged to zero

Isotropic liquid

Incomplete orientational sampling -

residual dipolar coupling

Anisotropic liquid

Dipolar Coupling


Resonance Frequency " 1H"

Residual Dipolar Couplings "

15N"

I"

II"

III"

I"

II"

III"

II"

III"

I"


φ

θ

Azz

Ayy

Axx

Alignment frame (tensor) defined by 5 independent parameters - (Aa,Ar,α,β,γ)"

"

Residual dipolar couplings report on the orientation of

internuclear vectors relative to

molecular alignment tensor"

"

Azz

Ayy

Axx

Structural Information from Residual Dipolar Couplings"


Alignment frame (tensor) defined by 5 independent parameters - (Aa,Ar,α,β,γ)"

"

Structural Information from Residual Dipolar Couplings"

Available orientations of

internuclear bond for measured RDC"

Dmax

Dmin

Azz

Ayy

Axx

Dmax

Dmin

Azz

Ayy

Axx

Dmax"

Dmin"

Azz

Ayy

Axx


Steric Alignment""

Bicelles"Alcohol mixtures"

Strained Gels"…..""""""

Electrostatic "Alignment"

"Bacteriophage"

Charged bicelles"Purple membranes"

…."

Dmax

Dmin

Azz

Ayy

Axx

Dmax

Dmin

Azz

Ayy

Axx


Residual dipolar couplings as constraints for structure refinement"

RDC-refined NMR ensemble calculated using 2-stage

restrained molecular dynamics with floating

alignment tensor

Aa = (9.28±0.11)10-4Ar = (1.12±0.14)10-4 RDC-Refined nOe/J-coupling-only

D(exp)

30

30-40-40

σ=(3.5±1.7)Å σ=(1.5±0.2)Å

D(calc)

Provide long-range order complementary to local distances"


Long-range Conformational restraints

from RDCs""

Orientation of structural domains relative to a common reference

frame"""

Example - ""•  Measurement of RDC in modules of known structure"•  Determine alignment axes"•  Superpose axes"


E.g.: Millet, et al. (2003) Proc. Natl. Acad. Sci. USA 100, 12700-12705

Structural changes associated with domain reorientation in MBP

Orientation of structural domains using Dipolar Couplings"


Orientation of Interaction Partners in Molecular Complexes"

E.g. : Clore & Schwieters (2003) J. Am. Chem. Soc. 125, 2902

In combination with chemical shift mapping, intermolecular nOe, paramagnetic effects


Bondensgard et al. (2002) Biochemistry 41, 11532

Orientation of structural domains using Dipolar Couplings"

The Global Conformation of the Hammerhead Ribozyme Determined Using Residual Dipolar Couplings


Dynamic timescales and NMR!

ps ns µs ms s


Spin relaxation


Real time



Assembly

kex"δ


τ

Cang(τ) Spin relaxation !Fast motional information "Quenched by rotational diffusion"

1H

15N

First-order interactions!

Chemical shifts, dipolar couplings"

population-weighted average sampled up to

millisecond

How can we access these timescales by

simulation?

[email protected]!


Residual Dipolar Couplings : Quantitative description of protein dynamics in solution"

φ

θ

Azz

Ayy

Axx

Dij

Atomic resolution description "of protein flexibility on physiologically "

important timescales"

Dipolar couplings provide direct probes of all conformational sub-states sampled up to the millisecond "

Intrinsic Protein Dynamics and Molecular Recognition


Multiple timescale motions in CD2AP SH3C from RDCs and relaxation

• 15 different alignment media

• 1912 RDCs 1DNH, 2DC’HN , 1DC’Ca

Relaxation Ensemble averaged simulation

S2NH

S2NH

Relaxation ps-ns RDC ps-ms

RDC-only average structure

Intrinsic Protein Dynamics and Molecular Recognition

αCi αCi-1

NH

O

N C’

3D Gaussian Axial Fluctuation Model

Accelerated MD

Rfree : 10% of data left out of 3DGAF/AMD analysis

Cross validation of independent data

High resolution structure and dynamics on ps to ms

timescales

Dependence of fast motions on nature of slowly

interconverting substates forming the ensemble

Statistical Ensemble


Describing multiple timescale motions in proteins by NMR "

SH3C

Ubi

GB3

Relaxation ps-ns RDC ps-ms

S2NH

S2NH

S2NH



Incorporation of

SAXS

potential into the

CNS hybrid potential

Characterisation of

structure

refinement

protocols

Combination of Residual Dipolar Couplings and Small Angle Scattering"






Lapinaite, Gabel, Carlomagno et al. Nature, 1-5 (2013)

Catalytic structure of the box C/D ribonucleoprotein bound to substrate RNA

Combination of solution-state NMR and small-angle X-ray and neutron scattering

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Biomolecular NMR – A Brief...

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