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

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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.

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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

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

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Outline for Bio 5068 December 10 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 Assessing quality of structures 1.NMR structure quality assessment 2.Comparison with x-ray

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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 wavelength!!

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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

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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

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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

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Nuclei are positively charged many have a spin associated with them. Moving chargeproduces a magnetic field that has a magnetic moment Spin angular moment

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

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How do we detect the NMR signal?

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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

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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

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Sample preparation using recombinant methods

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

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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

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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!!

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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

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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.

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Protein Structure Determination by NMR Stage ISequence specific resonance assignment State II Conformational restraints Stage III Calculate and refine structure

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Resonance assignment strategies by NMR

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Illustrations of the Relationship Between MW, c and T 2

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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

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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

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3D NMR Experiments Amide Strip 3D cube 2D plane amide strip Strips can then be arranged in backbone sequential order to visual confirm assignments

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NMR Assignments 3D NMR Experiments 3D HNCO Experiment common nomenclature letters indicate the coupled backbone atoms correlates NH i to C i-1 (carbonyl carbon, CO or C) no peaks for proline (no NH) Like the 2D 1 H- 15 N HSQC spectra, each amino acid should display a single peak in the 3D HNCO experiment identifies potential overlap in 2D 1 H- 15 N HSQC spectra, especially for larger MW proteins most sensitive 3D triple resonsnce experiment may observe side-chain correlations 1 J NC 1 J NH

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NMR Assignments 3D NMR Experiments 3D HN(CA)CO Experiment correlates NH i to C i relays the transfer through C i without chemical shift evolution uses stronger one-bond coupling contains only intra correlation provides a means to sequential connect NH and C chemical shifts match NH i -CO i (HN(CA)CO with NH i -CO i-1 (HNCO) not sufficient to complete backbone assignments because of overlap and missing information every possible correlation is not observed need 2-3 connecting inter and intra correlations for unambiguous assignments no peaks for proline (no NH) breaks assignment chain but can identify residues i-1to prolines 1 J C C 1 J NH 1 J NC

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NMR Assignments 3D NMR Experiments 3D HN(CA)CO Experiment Amide Strips from the 3D HNCO and HN(CA)CO experiments arranged in sequential order HNCO and HN(CA)CO pair for one residues NH Connects HN i -CO i with HN i -CO i-1 Journal of Biomolecular NMR, 9 (1997) 1124

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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?

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NMR Assignments

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

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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, 9:594601

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Protein Structure Determination by NMR Stage ISequence specific resonance assignment State II Conformational restraints Stage III Calculate and refine structure

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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?

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4.1 2.9 NOE CHCHCHCH NH NH CHCHCHCH J NOE - a through space correlation (