Analysis of Protein Structures

NMR in biology: Structure, dynamics
and energetics
Gaya Amarasinghe, Ph.D.
Department of Pathology and Immunology
CSRB 7752
Nuclear Magnetic Resonance
Today, we will look at how NMR can provide insight in
to biological macromolecules. This information often
compliment those obtained from other structural
NMR Spectra contains a lot of useful information:
from small molecule to macromolecule.
• 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
• 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 energetic
from 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 by
• Practical aspects of NMR
2.Sample signal vs water signal
3.Sample preparation (very basic aspects & deal with specific labeling during
the description of experiments)
• Assignments and structure determination
1.2-D experiments
2.3/4-D experiments
3.Restraints and structure calculations
4.Assessing quality of structures
5.NMR structure quality assessment
6.Comparison with x-ray
Some examples of how NMR is used in biology
For diffraction, the limit
of resolution is ½
Electronic transitions
Translational transitions
Rotational transitions
Nuclear transitions
NMR works in the rf rangeafter absorption of energy by nuclei,
dissipation of energy and the time it takes
Reveals information about the conformation
and structure.
Protein Structures from an NMR Perspective
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
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
Analyzing NMR Data is a Non-Trivial Task!
there is an abundance of data that needs to be interpreted
Not A Direct Path!
Interpreting NMR Data Requires
Making Informed “Guesses” to
Move Toward the “Correct” Fold
Distance from Correct Structure
Initial rapid convergence to
approximate correct fold
Correct structure
NMR Data Analysis
Iterative “guesses” allow
“correct” fold to emerge
Current PDB statistics (as of 3/27/2012)
Nucleic Protein/Nucleic
Acids Acid Complexes
Nuclei are positively charged
many have a spin associated with them.
Moving charge—produces a magnetic field that has a magnetic moment
Spin angular moment
I= integer
I=half integer
How do we detect the NMR signal?
•Practical aspects of NMR
2.Sample signal vs water signal
3.Sample preparation (very basic aspects & deal with
specific labeling during the description of experiments)
•Practical aspects of NMR
2.Sample signal vs water signal
3.Sample preparation (very basic aspects & deal with
specific labeling during the description of experiments)
Illustrations of the Relationship Between MW, tc and T2
Sample preparation using recombinant methods
Cell-free protein production and labeling protocol for NMR-based structural proteomics
Vinarov et al., Nature Methods - 1, 149 - 153 (2004)
Sample requirements and sensitivity
Methyl groups are more sensitive than isolated Ha spins
Source :
Sample requirements and sensitivity
mM not mM!!
Cryoprobes are 3-4 times better S/N than standard probes (2x in high salt)
Source :
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
HMQC spectrum of 50KDa protein complex (green) is a subset of the
>250kDa multimeric protein complex (black) spectrum
methyl) in 400 mM NaCl buffer
Why use NMR ?
 Some proteins do not crystallize (unstructured,
 crystals do not diffract well
 can not solve the phase problem
 Functional differences in crystal vs in solution
 can get information about dynamics
Protein Structures from an NMR Perspective
Overview of Some Basic Structural Principals:
Primary Structure: the amino acid sequence arranged from the amino (N)
terminus to the carboxyl (C) terminus  polypeptide chain
Secondary Structure: regular arrangements of the backbone of the
polypeptide chain without reference to the side chain types or conformation
Tertiary Structure: the three-dimensional folding of the polypeptide chain to
assemble the different secondary structure elements in a particular
arrangement in space.
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
•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 NeH (Trp)
 position in HSQC depends on local structure and sequence
 no peaks for proline (no NH)
Side-chain NH2
NMR Assignments
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
Ci-1 O
Ci-1 Ci-1
• 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
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
 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
– relaxation takes place during each
Get less data that is less ambiguous?
NMR Assignments
Why use deuteration?
• What are the advantages?
• What are the disadvantages?
Effects of Deuterium Labeling
only 15N labeled
2D 15N-NH HSQC spectrum of the 30
kDa N-terminal domain of Enzyme I
from the E. coli
15N, 2H
Current Opinion in Structural Biology 1999, 9:594–601
Protein Structure Determination
by NMR
•Stage I—Sequence specific resonance
•State II – Conformational restraints
•Stage III – Calculate and refine structure
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?
NMR Structure Determination
- 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
NMR Structure Determination
Protein Secondary Structure and Carbon Chemical Shifts
3 I
NMR Structure Determination
Protein Secondary Structure and 3JHN
• Karplus relationship between f and 3JHN
f =180o  3JHN = ~8-10 Hz  -strand
 f = -60o  3JHN = ~3-4 Hz  -helix
Vuister & Bax (1993) J. Am.Chem. Soc. 115:7772
Protein Structure Determination
by NMR
•Stage I—Sequence specific resonance
•State II – Conformational restraints
•Stage III – Calculate and refine structure
Protein Structures from an NMR Perspective
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
10 restraints/residue
13 restraints/residue
16 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
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
 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
Energy landscape and dynamics
high energy barriers = slow rate
low 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)
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
•regions that interact with solvent or binding
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
Non-self dsRNA recognition is inhibited by filoviral VP35
at multiple steps in the IFN production pathway
IFITs (1,2,3)
Leung et al, 2011 Virulence
VP35 IID structure revealed two functionally important
conserved basic patches
viral replication
“first” basic patch
IFN inhibition
“central” basic patch
All VP35 binders contain a common pyrrollidinone
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 dataset
Currently, the efficacy and PD/PK characteristics are being tested.
Autoinhibited Multi-Domain Proteins are Critical in
Many Signal Transduction Pathways
• Numerous multi-domain
proteins transmit signals
from 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
Aghazadeh, et al. Cell, 102: 625-633.
• Y3 is buried in the interface
Phosphorylation Disrupts Autoinhibitory Interactions
Aghazadeh, et al. Cell, 102: 625-633.
• Amide resonances from N-terminal (Ac region) helix collapse to the center of
the 1H/15N HSQC spectra and become extremely intense
• 13C and 13C assignments indicate that the N-terminus is random coil
How is Y3 Accessed by Kinases?
A general problem in autoinhibition/allostery: activators must contact buried sites
Chemical Shift Can Report on Population
• Linearity of chemical
shifts across multiple
perturbations indicates
a two-state equilibrium
wobs = powo + (1-po)wc
Mutants Sample a Range of Population Distributions
• Linearity strongly suggests an
equilibrium between Y3bound and Y3-unbound states
wobs = powo + (1-po)wc
Conformational equilibrium controls Vav activation by Src family kinases
Science. 1998 Jan 23;279(5350):509-14.
Nature. 2000 Mar 9;404(6774):151-8.
Nature. 1999 May 27;399(6734):379-83.
Rosen lab
Final thoughts?
Some Other Recommended Resources
“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 Important Web Sites:
RCSB Protein Data Bank (PDB)
Database of NMR & X-ray Structures
BMRB (BioMagResBank)
Database of NMR resonance assignments
CATH Protein Structure Classification Classification of All Proteins in PDB
SCOP: Structural Classification of Proteins Classification of All Structures into
Families, Super Families etc.
Compares 3D-Stuctures of Proteins to
Determine Structural Similarities of New
NMR Information Server
NMR Groups, News, Links, Conferences, Jobs
NMR Knowledge Base
A lot of useful NMR links
Many slides have been either taken directly or adapted from the following sources:
David Cistola (Wash U)
Kevin Gardner/Carlos Amzcua (UTSW)
Or as cited