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

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.

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 http://www.cryst.bbk.ac.uk/PPS2/projects/schirra/html/1dnmr.htm http://www.nature.com/nature/journal/v418/n6894/fig_tab/nature00860_F1.html

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) 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

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.

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

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

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  

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

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

How do we detect the NMR signal?

Practical aspects of NMR instrumentation Sample signal vs water signal 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

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

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

Sample requirements and sensitivity mM not mM!! 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

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

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 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 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 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 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?

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

Protein Structure Determination by NMR Stage I—Sequence specific resonance assignment 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 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

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

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

Protein Structure Determination by NMR Stage I—Sequence specific resonance assignment 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 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 Energy landscape and dynamics high energy barriers = slow rate low energy barriers = fast rate H N

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

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

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

All VP35 binders contain a common pyrrollidinone scaffold

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

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.

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 Y3 is buried in the interface Aghazadeh, et al. Cell, 102: 625-633.

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 13Ca and 13Cb 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 Distribution Linearity of chemical shifts across multiple perturbations indicates a two-state equilibrium closed 50:50 mixture open wobs = powo + (1-po)wc

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

Vav WASP/Cdc42 Rosen lab 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 http://www.rcsb.org/pdb/ BMRB (BioMagResBank) Database of NMR resonance assignments http://www.bmrb.wisc.edu/ CATH Protein Structure Classification Classification 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. 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, Jobs http://www.spincore.com/nmrinfo/ NMR Knowledge Base A lot of useful NMR links http://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