nmr in biology: structure, dynamics and energetics - bio 5068 · outline for bio 5068 december 8...

NMR in biology: Structure, dynamics and energetics

Gaya Amarasinghe, Ph.D.Department of Pathology and Immunology

[email protected] 7752

NMR?Nuclear Magnetic Resonance

Spectroscopy

Today, we will look at how NMR can provide insight into biological macromolecules. This information oftencompliment those obtained from other structuralmethods.

http://www.cryst.bbk.ac.uk/PPS2/projects/schirra/html/1dnmr.htm

NMR Spectra contains a lot of useful information: from small molecule to macromolecule.

http://www.nature.com/nature/journal/v418/n6894/fig_tab/nature00860_F1.html

• Few peaks• Sharper lines• Overall very easy to interpret

• Many peaks• Broader lines• Overall NOT very easy to interpret

• Structure determination by NMR

• NMR relaxation– how to look at molecular motion (dynamics by NMR)

• Ligand binding by NMR – Energetics

Outline for Bio 5068

December 8• Why study NMR (general discussion)

1.What is the NMR signal (some theory)2.What information can you get from NMR (structure, dynamics, and energeticfrom chemical shifts, coupling (spin and dipolar), relaxation)3.What are the differences between signal from NMR vs x-ray crystallography(we will come back to this after going through how to determine structures byNMR)

• Practical aspects of NMR1.instrumentation2.Sample signal vs water signal3.Sample preparation (very basic aspects & deal with specific labeling duringthe description of experiments)

• Assignments and structure determination1.2-D experiments2.3/4-D experiments3.Restraints and structure calculations4.Assessing quality of structures5.NMR structure quality assessment6.Comparison with x-ray

• Some examples of how NMR is used in biology

Nuclear transitions

Rotational transitions

Translational transitions

Electronic transitions

Diffractions

NMR works in the rf range‐after absorption of energy by nuclei,

dissipation of energy and the time it takesReveals information about the conformation and structure.

For diffraction, the limit of resolution is ½ wavelength!!

Protein Structures from an NMR PerspectiveBackground

– We are using NMR Information to “FOLD” the Protein.

– We need to know how this NMR data relates to a protein structure.

– We need to know the specific details of properly folded protein structures to verify the accuracy of our own structures.

– We need to know how to determine what NMR experiments are required.

– We need to know how to use the NMR data to calculate a protein structure.

– We need to know how to use the protein structure to understand biological function

Protein Structures from an NMR Perspective

Distance from

Correct Structure

NMR Data Analysis

Correct structure

XNot A Direct Path!

Interpreting NMR Data Requires Making Informed “Guesses” to Move Toward the “Correct” Fold

Initial rapid convergence to approximate correct fold

Iterative “guesses” allow “correct” fold to emerge

Analyzing NMR Data is a Non‐Trivial Task!there is an abundance of data that needs to be interpreted

Current PDB statistics (as of 3/27/2012)

Exp.Method Proteins Nucleic

AcidsProtein/Nucleic Acid Complexes Total

X‐RAY 65828 1346 3260 70436NMR 8167 975 186 9335

ratio 8.06 1.38 17.53

Nuclei are positively chargedmany have a spin associated with them.

Moving charge—produces a magnetic field that has a magnetic moment

Spin angular moment

Mass Charge I

Even Even I=0

Even Odd I= integer

Odd I=half integer

How do we detect the NMR signal?

•Practical aspects of NMR1.instrumentation2.Sample signal vs water signal3.Sample preparation (very basic aspects & deal withspecific labeling during the description of experiments)

http://chem4823.usask.ca/nmr/magnet.html

http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance

•Practical aspects of NMR1.instrumentation2.Sample signal vs water signal3.Sample preparation (very basic aspects & deal withspecific labeling during the description of experiments)

http://www.chemistry.nmsu.edu/Instrumentation/NMSU_NMR300_J.html

Illustrations of the Relationship Between MW, c and T2

Sample preparation using recombinant methods

Vinarov et al., Nature Methods ‐ 1, 149 ‐ 153 (2004)

Cell‐free protein production and labeling protocol for NMR‐based structural proteomics

Sample requirements and sensitivity

Methyl groups are more sensitive than isolated Ha spins

Source : www.chem.wisc.edu/~cic/nmr/Guides/Other/sensitivity‐NMR.pdf

Sample requirements and sensitivity

Cryoprobes are 3‐4 times better S/N than standard probes (2x in high salt)

Source : www.chem.wisc.edu/~cic/nmr/Guides/Other/sensitivity‐NMR.pdf

M not mM!!

15N TROSY spectrum of 50KDa protein complex (green) is a subset of the >250kDa multimeric protein complex (black), but most peaks in the multimeric

complex disappear

13C HMQC spectrum of 50KDa protein complex (green) is a subset of the >250kDa multimeric protein complex (black) spectrum

2H/15N/12C/ILVA(1H-13C methyl) in 400 mM NaCl buffer

Why use NMR ?

Some proteins do not crystallize (unstructured, multidomain) crystals do not diffract well can not solve the phase problem

Functional differences in crystal vs in solution

can get information about dynamics


Overview of Some Basic Structural Principals:

a) Primary Structure: the amino acid sequence arranged from the amino (N) terminus to the carboxyl (C) terminus polypeptide chain

b) Secondary Structure: regular arrangements of the backbone of the polypeptide chain without reference to the side chain types or conformation

c) Tertiary Structure: the three‐dimensional folding of the polypeptide chain to assemble the different secondary structure elements in a particular arrangement in space.

d) Quaternary Structure: Complexes of 2 or more polypeptide chains held together by noncovalent forces but in precise ratios and with a precise three‐dimensional configuration.

Protein Structure Determination by NMR

•Stage I—Sequence specific resonance assignment

•State II – Conformational restraints

•Stage III – Calculate and refine structure

Resonance assignment strategies by NMR

NMR Assignments 3D NMR Experiments

• 2D 1H‐15N HSQC experiment• correlates backbone amide 15N through one‐bond coupling to amide 1H• in principal, each amino acid in the protein sequence will exhibit one peak in the 1H‐15N HSQC spectra

also contains side‐chain NH2s (ASN,GLN) and NH (Trp) position in HSQC depends on local structure and sequence no peaks for proline (no NH)

Side‐chain NH2

3D NMR Experiments• Consider a 3D experiment as a collection of 2D experiments

z‐dimension is the 15N chemical shift• 1H‐15N HSQC spectra is modulated to include correlation through coupling to a another backbone atom

• All the 3D triple resonance experiments are then related by the common 1H,15N chemical shifts of the HSQC spectra • The backbone assignments are then obtained by piecing together all the “jigsaw” puzzles pieces from the various NMR experiments to reassemble the backbone

NMR Assignments

Ni-1

H

Ci-1

H

Ci-1

O

Ni

H

Ci

Ci-1

H

Ci

Ci

O

NMR Assignments

3D NMR Experiments• Amide Strip

3D cube 2D plane amide strip

Strips can then be arranged in backbone sequential order to visual confirm assignments

NMR Assignments 4D NMR Experiments

• Consider a 4D NMR experiment as a collection of 3D NMR experiments

still some ambiguities present when correlating multiple 3D triple‐resonance experiments 4D NMR experiments make definitive sequential correlations

increase in spectral resolution– Overlap is unlikely

loss of digital resolution– need to collect less data points for the 3D experiment– If 3D experiment took 2.5 days, then each 4D time point would be a multiple of 2.5 days i.e. 32 complex points in A‐dimension would require an 80 day experiment

loss of sensitivity– an additional transfer step is required– relaxation takes place during each transfer

Get less data that is less ambiguous?

NMR Assignments

Why use deuteration?

• What are the advantages?

• What are the disadvantages?

2D 15N‐NH HSQC spectrum of the 30kDa N‐terminal domain of Enzyme I from the E. coli

Effects of Deuterium Labeling

only 15N labeled 15N, 2H labeled

Current Opinion in Structural Biology 1999, 9:594–601

NMR Structure Determination

With The NMR Assignments and Molecular Modeling Tools in Hand:• All we need are the experimental constraints

Distance constraints between atoms is the primary structure determination factor. Dihedral angles are also an important structural constraint

What Structural Information is available from an NMR spectra?

How is it Obtained?

How is it Interpreted?

4.1Å

2.9Å

NOE

CH

NH

NH

CHJ

NOE- a through space correlation (<5Å)- distance constraint

Coupling Constant (J)- through bond correlation- dihedral angle constraint

Chemical Shift- very sensitive to local changes

in environment- dihedral angle constraint

Dipolar coupling constants (D)- bond vector orientation relative

to magnetic field- alignment with bicelles or viruses

D

NMR Structure Determination

NMR Structure Determination Protein Secondary Structure and Carbon Chemical Shifts

1

2 3

4

I

II

IIIIV

NMR Structure Determination Protein Secondary Structure and 3JHN

• Karplus relationship between and 3JHN =180o 3JHN ~8‐10 Hz ‐strand = ‐60o 3JHN = ~3‐4 Hz ‐helix

Vuister & Bax (1993) J. Am.Chem. Soc. 115:7772


What Information Do We Know at the Start of Determining A Protein Structure By NMR?

Effectively Everything We have Discussed to this Point!

The primary amino acid sequence of the protein of interest.► All the known properties and geometry associated with each amino acid and peptide bond within the protein.► General NMR data and trends for the unstructured (random coiled) amino acids in the protein.

The number and location of disulphide bonds.► Not Necessary can be deduced from structure.

7 restraints/residue




Wüthrich et al. , J. Virol. February 15, 2009; 83:1823‐1836

Analysis of the Quality of NMR Protein Structures With A Structure Calculated From Your NMR Data, How Do You Determine the Accuracy and Quality of the Structure?

• Consistency with Known Protein Structural Parameters bond lengths, bond angles, dihedral angles, VDW interactions, etc

all the structural details discussed at length in the beginning• Consistency with the Experimental DATA

distance constraints, dihedral constraints, RDCs, chemical shifts, coupling constants all the data used to calculate the structure

• Consistency Between Multiple Structures Calculated with the Same Experimental DATA

Overlay of 30 NMR Structures

Analysis of the Quality of NMR Protein Structures

As We have seen before, the Quality of X‐ray Structures can be monitored by an R‐factor

• No comparable function for NMR • Requires a more exhaustive analysis of NMR structures

Analysis of the Quality of NMR Protein Structures Root‐Mean Square Distance (RMSD) Analysis of Protein Structures

• A very common approach to asses the quality of NMR structures and to determine the relative difference between structures is to calculate an rmsd

an rmsd is a measure of the distance separation between equivalent atoms

two identical structures will have an rmsd of 0Å the larger the rmsd the more dissimilar the structures

0.43 ± 0.06 Å for the backbone atoms 0.81 ± 0.09 Å for all atoms

Analysis of the Quality of NMR Protein Structures Is the “Average” NMR Structure a Real Structure?

• No‐it is a distorted structure level of distortions depends on the similarity between the structures in the ensemble provides a means to measure the variability in atom positions between an ensemble of structures

Expanded View of an “Average” Structure

Some very long, stretched bonds

Position of atoms are so scrambled the graphics program does not know which atoms to draw bonds between Some regions of the structure

can appear relatively normal

Timescales of Protein Motion

N

HEnergy landscape and dynamicshigh energy barriers = slow ratelow energy barriers = fast rate

Why do proteins move?• Broad, shallow energy potential

– Thermal energy is sufficient for the protein to sample many different conformations• Change in conditions

– Interaction with a small molecule or binding partner, change in temperature, ion concentration, etc.

– Now a different conformation is lower in energy• Sequence encodes both protein structure and protein flexibility

– Non‐bonded interactions determine the lowest energy conformation(s)

Sequence

StabilityFlexibility

Function Function requires•Stability: the right chemical and spatial features in the right place to bind ligand, catalyze a chemical reaction, etc.•Flexibility: the ability to move in order to control access in and out of the active site and to provide energy for chemical reactions

Summary ‐‐‐ NMR relaxation/dynamics

• High sensitivity and site specific information

• may need isotopic labeling

•May require assignment of resonances

• Can help narrow construct space and identify interfaces

•regions that interact with solvent or binding partners

NMR Analysis of Protein-Ligand Interactions

NMR Monitors the Different Physical Properties That Exist Between a Protein and a Ligand

Summary ‐‐‐ NMR ligand binding• High sensitivity and site specific information

• may need isotopic labeling

•May require assignment of resonances

• Affinity measurements are only valid for low affinity interactions

• Complex structures can be determined for high affinity interactions

Some examples of how NMR can provide information about biological

systems

Non-self dsRNA recognition is inhibited by filoviral VP35 at multiple steps in the IFN production pathway

Leung et al, 2011 Virulence

IFITs (1,2,3)

zVP35/mVP35

“first” basic patch “central” basic patch

VP35 IID structure revealed two functionally importantconserved basic patches

viral replication IFN inhibition

All VP35 binders contain a common pyrrollidinonescaffold

NMR-based studies reveal quantitative structure/activity relationships (QSAR)

NMR provides a medium throughput quantitation of ligand binding

A subset of residues important for VP35-NP binding are also important for inhibitor binding

Crystal structure(s) of Zaire ebolavirus IID-GA228 complex reveals key protein-small molecule contacts

~30 different small molecule-VP35 IID structures provides a comprehensive SAR datasetCurrently, the efficacy and PD/PK characteristics are being tested.

Autoinhibited Multi‐Domain Proteins are Critical in Many Signal Transduction Pathways

• Numerous multi‐domainproteins transmit signalsfrom the T‐cell receptor

Rosen lab

Vav proto‐oncoprotein is a key GEF that regulates Rho family GTPases

• A member of the Dbl family of guanine nucleotide exchange factors (GEF) for the Rho family of GTP binding proteins.

• Important in hematopoiesis, playing a role in T‐cell and B‐cell development and activation.

• DH domain is inhibited by contacts with the Acidic (Ac) region and is relieved by phosphorylation of the Ac region tyrosines

A Helix From the Ac Domain Binds in the DH Active Site: Autoinhibition by Occlusion

• Y3 is buried in the interfaceAghazadeh, et al. Cell, 102: 625‐633.

Phosphorylation Disrupts Autoinhibitory Interactions

• Amide resonances from N‐terminal (Ac region) helix collapse to the center ofthe 1H/15N HSQC spectra and become extremely intense

• 13C and 13C assignments indicate that the N‐terminus is random coil

Aghazadeh, et al. Cell, 102: 625‐633.

How is Y3 Accessed by Kinases?A general problem in autoinhibition/allostery: activators must contact buried sites

?

Chemical Shift Can Report on Population Distribution

closed

open

50:50mixture

• Linearity of chemicalshifts across multipleperturbations indicates a two‐state equilibrium

obs = poo + (1-po)c

Mutants Sample a Range of Population Distributions

obs = poo + (1-po)c

Conformational equilibrium controls Vav activation by Src family kinases

Open

Closed

• Linearity strongly suggests anequilibrium between Y3‐bound and Y3‐unbound states

Rosen lab

Vav WASP/Cdc42

Nature. 1999 May 27;399(6734):379‐83.

Nature. 2000 Mar 9;404(6774):151‐8.

Science. 1998 Jan 23;279(5350):509‐14.

Final thoughts?

“NMR of Proteins and Nucleic Acids” Kurt Wuthrich

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

“Principles of Protein Structure” G. E. Schulz & R. H. Schirmer

“Introduction to Protein Structure” C. Branden & J. Tooze

“Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis” R. Copeland

“Biophysical Chemistry” Parts I to III, C. Cantor & P. Schimmel

“Principles of Nuclei Acid Structure” W. Saenger

Some Other Recommended Resources

Some Important Web Sites:RCSB Protein Data Bank (PDB) Database of NMR & X‐ray Structureshttp://www.rcsb.org/pdb/

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

CATH Protein Structure Classification Classification of All Proteins in PDBhttp://www.biochem.ucl.ac.uk/bsm/cath/

SCOP: Structural Classification of Proteins Classification of All Structures into http://scop.berkeley.edu Families, Super Families etc.

DALI Compares 3D‐Stuctures of Proteins to http://www.ebi.ac.uk/dali/ Determine Structural Similarities of New

Structures

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

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

Many slides have been either taken directly or adapted from the following sources:

http://www.bionmr.com/forum/educational‐web‐pages‐16/lectures‐nmr‐spectroscopy‐protein‐structures‐university‐nebraska‐lincoln‐324/

David Cistola (Wash U)

Kevin Gardner/Carlos Amzcua (UTSW)

Or as cited

nmr in biology: structure, dynamics and energetics - bio 5068 · outline for bio 5068 december 8...

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