1. NMR in biology: Structure, dynamics and energetics
Gaya Amarasinghe, Ph.D.
Department of Pathology and Immunology
CSRB 7752

2. Nuclear Magnetic Resonance Spectroscopy
NMR?
Nuclear Magnetic Resonance Spectroscopy
Today, we will look at how NMR can provide insight in to biological macromolecules. This information often compliment those obtained from other structural methods.

3. 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

4. Structure determination by NMR
NMR relaxation– how to look at molecular motion (dynamics by NMR)
Ligand binding by NMR – Energetics

5. Outline for Bio 5068 December 8 Why study NMR (general discussion)
What is the NMR signal (some theory)
What information can you get from NMR (structure, dynamics, and energetic from chemical shifts, coupling (spin and dipolar), relaxation)
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 NMR)
Practical aspects of NMR
instrumentation
Sample signal vs water signal
Sample preparation (very basic aspects & deal with specific labeling during the description of experiments)
Assignments and structure determination
2-D experiments
3/4-D experiments
Restraints and structure calculations
Assessing quality of structures
NMR structure quality assessment
Comparison with x-ray
Some examples of how NMR is used in biology

7. For diffraction, the limit of resolution is ½ wavelength!!
Diffractions
Electronic transitions
Translational transitions
Rotational transitions
Nuclear transitions
NMR works in the rf range-
after absorption of energy by nuclei, dissipation of energy and the time it takes
Reveals information about the conformation and structure.

8. Protein Structures from an NMR Perspective
Background
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

9. X 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 X
Not A Direct Path!
Initial rapid convergence to approximate correct fold
Distance from Correct Structure
NMR Data Analysis
Correct structure
Iterative “guesses” allow “correct” fold to emerge
Interpreting NMR Data Requires Making Informed “Guesses” to Move Toward the “Correct” Fold

11. Protein/Nucleic Acid Complexes
Current PDB statistics (as of 3/27/2012)
Exp.Method
Proteins
Nucleic Acids
Protein/Nucleic Acid Complexes
Total
X-RAY
65828
1346
3260
70436
NMR
8167
975
186
9335
ratio
8.06
1.38
17.53

13. 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

14. Mass
Charge I
Even
I=0
Odd
I= integer
I=half integer

17. How do we detect the NMR signal?

21. Practical aspects of NMR
instrumentation
Sample signal vs water signal
Sample preparation (very basic aspects & deal with specific labeling during the description of experiments)

22. Practical aspects of NMR
instrumentation
Sample signal vs water signal
Sample preparation (very basic aspects & deal with specific labeling during the description of experiments)

23. Illustrations of the Relationship Between MW, tc and T2

24. Sample preparation using recombinant methods

25. Cell-free protein production and labeling protocol for NMR-based structural proteomics
Vinarov et al., Nature Methods - 1, (2004)

26. Sample requirements and sensitivity
Methyl groups are more sensitive than isolated Ha spins
Source :

27. Sample requirements and sensitivity
mM not mM!!
Cryoprobes are 3-4 times better S/N than standard probes (2x in high salt)
Source :

28. 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

29. 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

30. 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

31. 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.

34. Protein Structure Determination by NMR
Stage I—Sequence specific resonance assignment
State II – Conformational restraints
Stage III – Calculate and refine structure

35. Resonance assignment strategies by NMR

39. 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

40. 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
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

41. 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

42. 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?

43. NMR Assignments

44. Why use deuteration? What are the advantages?
What are the disadvantages?

45. Effects of Deuterium Labeling
2D 15N-NH HSQC spectrum of the 30 kDa N-terminal domain of Enzyme I from the E. coli
only 15N labeled
15N, 2H labeled
Current Opinion in Structural Biology 1999, 9:594–601

46. Protein Structure Determination by NMR
Stage I—Sequence specific resonance assignment
State II – Conformational restraints
Stage III – Calculate and refine structure

47. 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?

48. NMR Structure Determination
4.1Å
2.9Å
NOE
CaH NH J
- 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
Dipolar coupling constants (D)
- bond vector orientation relative
to magnetic field
- alignment with bicelles or viruses D

49. NMR Structure Determination
Protein Secondary Structure and Carbon Chemical Shifts a1 a2 a3 a4 bI
bII
bIII
bIV

50. NMR Structure Determination
Protein Secondary Structure and 3JHNa
Karplus relationship between f and 3JHNa
f =180o  3JHNa = ~8-10 Hz  b-strand
f = -60o  3JHNa = ~3-4 Hz  a-helix
Vuister & Bax (1993) J. Am.Chem. Soc. 115:7772

51. Protein Structure Determination by NMR
Stage I—Sequence specific resonance assignment
State II – Conformational restraints
Stage III – Calculate and refine structure

53. 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.

57. 7 restraints/residue
10 restraints/residue
13 restraints/residue
16 restraints/residue

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

59. 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

60. 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

61. 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

62. 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

63. Timescales of Protein Motion
Energy landscape and dynamics high energy barriers = slow rate
low energy barriers = fast rate H N

64. 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
Stability
Flexibility
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

65. 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

66. NMR Analysis of Protein-Ligand Interactions
NMR Monitors the Different Physical Properties That Exist Between a Protein and a Ligand

67. 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

68. Some examples of how NMR can provide information about biological systems

69. Non-self dsRNA recognition is inhibited by filoviral VP35 at multiple steps in the IFN production pathway
zVP35/mVP35
IFITs (1,2,3)
Leung et al, 2011 Virulence

70. VP35 IID structure revealed two functionally important
conserved basic patches
viral replication
IFN inhibition
“first” basic patch
“central” basic patch

71. All VP35 binders contain a common pyrrollidinone scaffold

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

73. NMR provides a medium throughput quantitation of ligand binding

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

75. Currently, the efficacy and PD/PK characteristics are being tested.
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.

77. Autoinhibited Multi-Domain Proteins are Critical in Many Signal Transduction Pathways
Numerous multi-domain
proteins transmit signals
from the T-cell receptor
Rosen lab

78. 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

79. A Helix From the Ac Domain Binds in the DH Active Site: Autoinhibition by Occlusion
Y3 is buried in the interface
Aghazadeh, et al. Cell, 102:

80. Phosphorylation Disrupts Autoinhibitory Interactions
Aghazadeh, et al. Cell, 102:
Amide resonances from N-terminal (Ac region) helix collapse to the center of
the 1H/15N HSQC spectra and become extremely intense
13Ca and 13Cb assignments indicate that the N-terminus is random coil

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

82. Chemical Shift Can Report on Population Distribution
Linearity of chemical
shifts across multiple
perturbations indicates
a two-state equilibrium
closed
50:50
mixture
open
wobs = powo + (1-po)wc

83. Mutants Sample a Range of Population Distributions
Open
Closed
Linearity strongly suggests an
equilibrium between Y3-
bound and Y3-unbound states
wobs = powo + (1-po)wc
Conformational equilibrium controls Vav activation by Src family kinases

91. Vav WASP/Cdc42 Rosen lab Science. 1998 Jan 23;279(5350):509-14.
Nature Mar 9;404(6774):151-8.
Nature May 27;399(6734):
Rosen lab

92. Final thoughts?

93. 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

94. 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.
DALI Compares 3D-Stuctures of Proteins to
Determine Structural Similarities of New
Structures
NMR Information Server NMR Groups, News, Links, Conferences, Jobs
NMR Knowledge Base A lot of useful NMR links

95. 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