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

2. 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. 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

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) 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 NMR) Practical aspects of NMR 1.instrumentation 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

7. 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 takes Reveals information about the conformation and structure. For diffraction, the limit of resolution is ½ wavelength!!

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. Protein Structures from an NMR Perspective Distance from Correct Structure NMR Data Analysis Correct structure X Not 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

11. Current PDB statistics (as of 3/27/2012) Exp.Met hod Proteins Nucleic Acids Protein/Nucleic Acid Complexes Total X-RAY658281346326070436 NMR81679751869335 ratio8.061.3817.53

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. MassChargeI Even I=0 EvenOdd I= integer OddI=half integer

17. How do we detect the NMR signal?

21. Practical aspects of NMR 1.instrumentation 2.Sample signal vs water signal 3.Sample preparation (very basic aspects & deal with specific labeling during the description of experiments) http://chem4823.usask.ca/nmr/magnet.html http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance

22. Practical aspects of NMR 1.instrumentation 2.Sample signal vs water signal 3.Sample preparation (very basic aspects & deal with specific labeling during the description of experiments) http://www.chemistry.nmsu.edu/Instrumentat ion/NMSU_NMR300_J.html

23. Illustrations of the Relationship Between MW,  c and T 2

24. Sample preparation using recombinant methods

25. Vinarov et al., Nature Methods - 1, 149 - 153 (2004) Cell-free protein production and labeling protocol for NMR-based structural proteomics

26. 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

27. 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!!

28. 15 N 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. 13 C HMQC spectrum of 50KDa protein complex (green) is a subset of the >250kDa multimeric protein complex (black) spectrum 2 H/ 15 N/ 12 C/ILVA( 1 H- 13 C 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: 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.

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 1 H- 15 N HSQC experiment correlates backbone amide 15 N through one-bond coupling to amide 1 H in principal, each amino acid in the protein sequence will exhibit one peak in the 1 H- 15 N HSQC spectra  also contains side-chain NH 2 s (ASN,GLN) and N  H (Trp)  position in HSQC depends on local structure and sequence  no peaks for proline (no NH) Side-chain NH 2

40. 3D NMR Experiments Consider a 3D experiment as a collection of 2D experiments  z-dimension is the 15 N chemical shift 1 H- 15 N 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 1 H, 15 N 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

41. 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. 2D 15 N-NH HSQC spectrum of the 30 kDa N-terminal domain of Enzyme I from the E. coli Effects of Deuterium Labeling only 15 N labeled 15 N, 2 H 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. 4.1Å 2.9Å NOE CHCHCHCH NH NH CHCHCHCH J 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 in environment - dihedral angle constraint Dipolar coupling constants (D) - bond vector orientation relative to magnetic field to magnetic field - alignment with bicelles or viruses D NMR Structure Determination

49. Protein Secondary Structure and Carbon Chemical Shifts 11 22 33 44 II  II  III  IV

50. NMR Structure Determination Protein Secondary Structure and 3J HN  Karplus relationship between  and 3J HN    =180o  3J HN   ~8-10 Hz   -strand   = -60o  3J HN  = ~3-4 Hz   -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 N H Energy landscape and dynamics high energy barriers = slow rate low energy barriers = fast rate

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 Leung et al, 2011 Virulence IFITs (1,2,3) zVP35/mVP35

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

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. 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: 625-633.

80. Phosphorylation Disrupts Autoinhibitory Interactions Amide resonances from N-terminal (Ac region) helix collapse to the center of the 1 H/ 15 N HSQC spectra and become extremely intense 13 C  and 13 C  assignments indicate that the N-terminus is random coil Aghazadeh, et al. Cell, 102: 625-633.

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 closed open 50:50 mixture Linearity of chemical shifts across multiple perturbations indicates a two-state equilibrium  obs = p o  o + (1-p o )  c

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

91. Rosen lab VavWASP/Cdc42 Nature.Nature. 1999 May 27;399(6734):379-83. Nature.Nature. 2000 Mar 9;404(6774):151-8. Science.Science. 1998 Jan 23;279(5350):509-14.

92. Final thoughts?

93. “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

94. Some Important Web Sites: RCSB Protein Data Bank (PDB)Database of NMR & X-ray Structures http://www.rcsb.org/pdb/ BMRB (BioMagResBank)Database of NMR resonance assignments http://www.bmrb.wisc.edu/ CATH Protein Structure ClassificationClassification of All Proteins in PDB http://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. http://scop.berkeley.edu DALICompares 3D-Stuctures of Proteins to http://www.ebi.ac.uk/dali/http://www.ebi.ac.uk/dali/ Determine Structural Similarities of New Structures NMR Information ServerNMR Groups, News, Links, Conferences, Jobs http://www.spincore.com/nmrinfo/ NMR Knowledge Base A lot of useful NMR links http://www.spectroscopynow.com/

95. 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