NMR in biology: Structure, dynamics and energetics

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. 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

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 11 • 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—next class) • 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

8. 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.

9. 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

10. Protein Structures from an NMR Perspective X Not A Direct Path! Distance from Correct Structure Correct structure NMR Data Analysis Analyzing NMR Data is a Non-Trivial Task! there is an abundance of data that needs to be interpreted Initial rapid convergence to approximate correct fold Iterative “guesses” allow “correct” fold to emerge Interpreting NMR Data Requires Making Informed “Guesses” to Move Toward the “Correct” Fold

12. Current PDB statistics (as of 3/27/2012)

14. 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

18. How do we detect the NMR signal?

21. Next time—pick up on chemical shifts

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) http://chem4823.usask.ca/nmr/magnet.html http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance

23. 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

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, 149 - 153 (2004)

26. Segment labeling can simplify NMR spectra Native chemical ligation Expressed protein ligation Muir et al. Curr Opin Biotechnol. 2002 Aug;13(4):297-303.

27. 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

28. 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

29. 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

30. 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.

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

34. Resonance assignment strategies by NMR

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

43. 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

44. 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

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

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

47. NMR Assignments • 3D NMR Experiments • 3DHN(CA)CO Experiment • correlates NHi to COi • relays the transfer through Cai without chemical shift evolution • uses stronger one-bond coupling • contains only intra correlation • provides a means to sequential connect NH and CO chemical shifts • match NHi-COi (HN(CA)CO with NHi-COi-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 1JNCa 1JCaC’ 1JNH

48. NMR Assignments • 3D NMR Experiments • 3DHN(CA)CO Experiment HNCO and HN(CA)CO pair for one residues NH Connects HNi-COi with HNi-COi-1 Amide “Strips” from the 3D HNCO and HN(CA)CO experiments arranged in sequential order Journal of Biomolecular NMR, 9 (1997) 11–24

49. 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?

50. NMR Assignments