Judith Klein-Seetharaman Department of Structural Biology NMR Spectroscopy Judith Klein-Seetharaman Department of Structural Biology jks33@pitt.edu

Objectives of this Lecture and Practicum Resources Physical principle Sample requirements Parameters that are measured by NMR Dynamics by NMR Limitations Practical aspects Setup of NMR experiments (downstairs) 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Resources Websites http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf http://www.bmrb.wisc.edu/ http://www.biochem.ucl.ac.uk/bsm/nmr/ubq/ http://nobelprize.org/nobel_prizes/chemistry/laureates/2002/wutrich-lecture.pdf http://www.cis.rit.edu/htbooks/nmr/ http://www.ch.ic.ac.uk/local/organic/nmr.html http://www.spectroscopynow.com/ http://www.chem.queensu.ca/FACILITIES/NMR/nmr/webcourse/ http://spincore.com/nmrinfo/ http://www.chembio.uoguelph.ca/driguana/NMR/TOC.HTM http://www.embl-heidelberg.de/nmr/sattler/embo/handouts/griesinger_lecture_pof.pdf http://dupont.molbio.ku.dk/teach/course/introNMR.pdf http://www.infochembio.ethz.ch/links/en/spectrosc_nmr_lehr.html 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Resources Books NMR Books: Protein NMR Techniques (Methods in Molecular Biology) by A. Kristina Downing (Editor) Protein NMR Spectroscopy: Principles and Practice by John Cavanagh, Wayne J. Fairbrother, III, Arthur G. Palmer, Nicholas J. Skelton, Mark Rance Spin Dynamics: Basics of Nuclear Magnetic Resonance by Malcolm H. Levitt Principles of Nuclear Magnetic Resonance in One and Two Dimensions by Richard R. Ernst, Geoffrey Bodenhausen, Alexander Wokaun 200 and More NMR Experiments: A Practical Course by Stefan Berger, Siegmar Braun Basic One- and Two-Dimensional NMR Spectroscopy by Horst Friebolin NMR Spectroscopy: Basic Principles, Concepts, and Applications in Chemistry by Harald Günther NMR Data Processing by Hoch NMR: The Toolkit by P. J. Hore, J. A. Jones, S. Wimperis Nuclear Magnetic Resonance by P. J. Hore NMR for Physical and Biological Scientists by Thoma Pochapsky Understanding NMR Spectroscopy by James Keeler NMR of Proteins (Topics on Molecular and Structural Biology) by G. M. Clore, A. M. Gronenborn The Nuclear Overhauser Effect in Structural and Conformational Analysis by David Neuhaus, Michael P. Williamson Biophysics Books with chapters on NMR: Biophysical Chemistry: Part II: Techniques for the Study of Biological Structure and Function by Charles R Cantor, Paul R Schimmel Principles of Physical Biochemistry by Kensal E van Holde, Curtis Johnson, Pui Shing Ho 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Objectives of this Lecture and Practicum Resources Physical principle Sample requirements Parameters that are measured by NMR Dynamics by NMR Limitations Practical aspects Setup of NMR experiments (downstairs) 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Nuclei in a magnetic field http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Energy Difference http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Macroscopic View http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Experiment: Recycle delay dependent on T1 relaxation http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

The NMR signal Analogy: conducting loop rotating in a magnetic field: http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Analogy: conducting loop rotating in a magnetic field: 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Fourier Transform http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Soft pulses vs. hard pulses http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Obtaining a spectrum http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Product Operator Formalism http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

HSQC Experiment http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

HSQC TOCXY http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Signal Intensity Boltzmann distribution http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Objectives of this Lecture and Practicum Resources Physical principle Sample requirements Parameters that are measured by NMR Dynamics by NMR Limitations Practical aspects Setup of NMR experiments (downstairs) 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Sample requirements: Sources Think of the requirements that we may need to fulfil! 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Comparison of expression systems for rhodopsin Example Comparison of expression systems for rhodopsin spacebio.net/modules/ mb_teare.html genetics.med.harvard.edu/ ~winston/ http://www.icr.ac.uk/structbi/baculovirus/img/infectedsf9.jpg http://www.gla.ac.uk/ibls/BMB/mdh/images/conrd1-cos-golgi.gif http://www.wjgnet.com/images/english/V11/2576-2a.jpg ___________ ___________ ___________ ___________ ___________ ___________ What are the advantages and disadvantages of each expression system? 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Where can we get these molecules from? 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Sources of biomolecules Summary Native sources Best quality (correct fold, posttranslational modifications etc.) Not always best quantity Limitations in labeling No mutants Chemical synthesis Good for small molecules Not good for large proteins Biosynthesis A variety of expression systems exist, all with their advantages and disadvantages. Required for isotope labeling for NMR 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Objectives of this Lecture and Practicum Resources Physical principle Sample requirements Parameters that are measured by NMR Dynamics by NMR Limitations Practical aspects Setup of NMR experiments (downstairs) 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

NMR parameters Chemical Shift See handout http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf See handout 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Chemical shift perturbation Figure 2 in “Cap-free structure of eIF4E suggests a basis for conformational regulation by its ligands Laurent Volpon, Michael J Osborne, Ivan Topisirovic, Nadeem Siddiqui and Katherine LB Borden The EMBO Journal (2006) 25, 5138–5149 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

The Nuclear Overhauser Effect NMR parameters The Nuclear Overhauser Effect http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Measuring NOE’s http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

NMR Parameters Dipolar Couplings http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

NMR Structure of Bcl-XL Bound to BH3 Peptide Structure was solved with a homolog of BH3 helix. Protein-protein interaction groove was identified on anti-apoptotic Bcl-XL. PDB ID:1G5J Drug design approach: SAR by NMR Identify a drug that binds in the BH3 pocket of Bcl-XL, inhibit binding to BID -> normal apoptosis. 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

NMR parameters chemical shifts NOE Dipolar coupling coupling constants HetNOE longitudinal relaxation rates (R1) transverse relaxation rates (R2) 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Objectives of this Lecture and Practicum Resources Physical principle Sample requirements Parameters that are measured by NMR Dynamics by NMR Limitations Practical aspects Setup of NMR experiments (downstairs) 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Theory -NMR Relaxation mechanism NMR Dynamics on Different Time Scales time scale type ns-ps fast internal motions us-ms slow internal motions ms-days proton exchange Proteins are dynamic molecules, they often undergo conformational changes while performing thir specific functions. NMR can be used to monitor the dynamic behavior on a broad range of timescales. Typically, 3 types, fast internal motions, slow internal motions and proton exchange. These motions are reported by measurement parameters. • rotational diffusion (τc) • translational diffusion (D) • internal dynamics of backbone and sidechains (τi) • degree of order for backbone and sidechains (S2) • conformational exchange (Rex) • interactions with other molecules (kon,koff) Protein are dynamic molecules http://www.bioc.aecom.yu.edu/labs/girvlab/nmr/course/relaxdyn NMR dynamics can be used on a broad range of timescales. 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Relaxation Longitudinal relaxation (T1): return of longitudinal (z-component) to its equilibrium value Transverse relaxation (T2): decay of transverse (x,y-component) 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

T1 Relaxation http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Experiment: Recycle delay dependent on T1 relaxation http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

T2 Relaxation http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Mechanisms of Relaxation http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Dipolar interaction 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Mechanisms of Relaxation http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Chemical shift anisotropy Scalar relaxation (chemical exchange, rapid T1 relaxation) Quadrupolar relaxation Spin rotation relaxation Interaction with unpaired electrons 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Quantification of motion - strategies to obtain dynamic information from NMR relaxation experiment Measure R1, R2, heteronuclear NOE “model free” approach Get order parameter S2 ,τe, τm This is the strategies to get dynamic information from experiment. First, then… 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Lipari-Szabo Model Free Approach http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Lipari Szabo Order parameters S2 http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Order parameters S2 τe, effective correlation function time for internal motions τm, overall tumbling correlation time for global motions 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Lipari–Szabo “model-free” approach - don’t depend on a specific physical model Estimate (τm) from R2/R1 for a selected subset of the residues fits to the observed relaxation data using various regression variables model-selection criteria are used to decide which choice is appropriate for each residue Reoptimize using the selected models. Uncertainties in the optimized parameters were obtained by Monte Carlo simulation. Model free approach is the most popular analytical methods, the name “model free” means it doesn’t depend on a specific physical model. Assuming that the global and internal motions are separable. It is a good approximation if the internal motions are sufficiently small. J(w); spectral density, is the fourier transform of the correlation function. (the function of correlation time) is a measure of amount of the motion present at different frequencies, ω, to cause the transitions (relaxation) S2 is the order parameter for the slow motion, Sf2 is the order parameter for the fast motion, and te is the effective correlation time for the slow motion, t-1 = te-1 + tm -1. Michael Andrec, Gaetano T. Montelione, Ronald M. Levy Journal of Magnetic Resonance 139, 408–421 (1999) “Model-free” is the most popular method to calculate dynamic parameters 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Inversion Recovery: Measure T1 http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Carr-Purcell spin echo: Measure T2 http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Comparison of T1 and T2 relaxation http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Dependence of T1, T2 on Tumbling Time http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Chemical Exchange http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Chemical Exchange http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Example: GCN4-58 complexed with DNA -dynamics of complex formation dynamics of the basic leucine zipper domain of the dimeric yeast transcription factor GCN4 (GCN4-58) as it relates to DNA binding Ribbon diagram of GCN4-58 complexed with DNA. The section in blue is the binding domain. The section in red is the leucin zipper dimerization domain. The thicker and deeper purple regions indication the highest degree of disorder. low S2 indicate high flexibility. S2 can be used to estimate energetics. John Cavanagh and Mikael Akke nature structural biology • volume 7 number 1 • january 2000 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Dynamics in folded/unfolded lysozyme Arrows indicate oxidized (all disulfide bonds present) lysozyme Folded: 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Using NMR to identify residual structure Can in principle use all parameters: chemical shifts coupling constants HetNOE longitudinal relaxation rates (R1) transverse relaxation rates (R2) 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Chemical shift differences between unfolded lysozyme and random coil 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Relaxation Rates in Unfolded Lysozyme Unfolded lysozyme can be studied in 8 M urea. Unfolded lysozyme can also be studied without urea, if the disulfide bonds are reduced and the cysteines are derivatized to prevent them from forming disulfide bonds. 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Relaxation Rates in Unfolded Lysozyme What do you observe? 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Relaxation Rates in Unfolded Lysozyme Regions with higher relaxation rates are localized as clusters.  Presence of clusters of residual structure that are restricted in conformational space, thus relax faster. 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Analysis of the relaxation data Three means of analysis have been proposed: Model-free approach Cole-Cole distributions Gaussian clusters However: What gives rise to these clusters is not known. 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Relaxation Rates in Unfolded Lysozyme å = - 1 | int ) ( N j i rinsic e R l Random Coil Model of Segmental Motion å - + 2 | x b i Ae + Gaussian Distributions of Deviations 1. 2. 3. 4. 5. 6. There are six clusters of residual structure in HEWL-SME. 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Mapping of residual structure on the native structure How would you test what stabilizes these clusters? 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Hydrophobic clusters of residual structure 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

How would you test for the presence of long-range interactions How would you test for the presence of long-range interactions? Approach: Study effect of mutation 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Effect of mutation on chemical shifts 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Effect of mutation on relaxation rates A single point mutation, W62G in cluster 3, disrupts all clusters in reduced and methylated lysozyme. 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Effect of mutation on chemical shifts 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Effect of mutation on relaxation rates 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Model for unfolded ensemble 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Compactness by NMR 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Objectives of this Lecture and Practicum Resources Physical principle Sample requirements Parameters that are measured by NMR Dynamics by NMR Limitations Practical aspects Setup of NMR experiments (downstairs) 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

NMR spectroscopy Size Stability Sample homogeneity Need for labeling General limitations Size Stability Sample homogeneity Need for labeling Quantities and source of biomolecules 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Unfolded proteins have a small chemical shift dispersion. Resolution Wide versus small chemical shift dispersion folded unfolded Unfolded proteins have a small chemical shift dispersion. 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

1d 1H NMR spectra 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

HSQC spectra 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Example Solution NMR of DAGK 1H,15N-HSQC spectrum of a 120 aa long membrane protein in DPC micelles Diacylglycerol kinase: Charles R. Sanders, Frank Sonnichsen (2006) Solution NMR of membrane proteins: practice and challenges. Magn. Reson. Chem. 2006; 44: S24–S40 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Example Solution NMR of Rhodopsin It’s a headache. 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

How can you test your hypothesis? What is signal 1? How can you test your hypothesis? 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Assignment of Signal 1 NMR Spectroscopy Black: original spectrum, red: C-terminus, green: N-terminus (after AspN cleavage) An enzyme was used to cleave off the C-terminus at the site indicated below: 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Traditional Solution NMR Approaches Problems with full-length membrane proteins in detergents Size – is not the only problem (Trosy does not work for helical membrane proteins) Conformational exchange – fluctuations in the detergent micelle environment lead to fast relaxation thus signal decay Spin diffusion – cannot deuterate samples from mammalian cells Problem: Traditional assignment strategies using triple resonance experiments (13C,15N,1H) don’t work Klein-Seetharaman et al. (2004) PNAS 101, 3409-13. 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Traditional Solution NMR Approaches Problems with full-length membrane proteins in detergents Detergent signals cause dynamic range problems (Detergent signals cause spectral overlap) Detergent deuteration is often not feasible Problem: 1H,1H NOESY spectra do not show protein signals 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

1H Chemical Shift [ppm] 1H Chemical Shift [ppm] Evidence I: Selective excitation A. Selective excitation of the NH region using 90 degree pulse followed by direct observation. B. Selective excitation of the same region as in A. Using excitation sculpting. Backbone NH Tryptophan side chain NH 20 15 10 5 10 5 -5 1H Chemical Shift [ppm] 1H Chemical Shift [ppm] 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

You might also want to develop your own biophysical approaches… 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Large-scale expression system for rhodopsin: HEK293 cells 19F NMR Spectroscopy Approach Advantages of 19F NMR no natural background in proteins high sensitivity sensitive to differences in environment General Method for Attachment of 19F Label Rho SH +Dithiodipyridine Rho S S N Disulfide CF CH SH Exchange When people hear “solution NMR spectroscopy of a membrane protein” – the first thing to say is “That’s not possible.” At the time there was no precedent for a study of a GPCR protein. Limitation was believed to be that of size (48kD + micelles), fast relaxation of the signals. So the first thing I did, was to demonstrate that NMR was at all applicable to the study of rhodopsin. I did this using a 19F NMR approach. Advantages… I had to develop a method for attachment using cysteine mutants… These studies were only made possible by the development of a large-scale expression system by Phil Reeves in my advisor’s laboratory. We could prepare on the order of 10mg per sample of mutant. 3 2 (TET) Rho S S CH CF 2 3 Large-scale expression system for rhodopsin: HEK293 cells 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

19F NMR Spectroscopy Qualitative Changes dark light Dark Light (3') 23' 43' 63' 10.5 10.0 9.5 9.0 8.5 Chemical shift (ppm relative to TFA) Here are the results. Distinct chemical shifts. Distinct changes. This demonstrates that 19F solution NMR is indeed applicable to study of rhodopsin. 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

19F-19F Nuclear Overhauser Effect Proximity 19F-19F Nuclear Overhauser Effect S S CH CF 2 3 S S CH CF 2 3 Rho How can we use NMR to obtain more quantitative information? One approach that contains distance information: NOE. NOE between two 19F labels, indeed observed. 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Magic angle spinning http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Enhancing resolution in solid state NMR http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Aligned: Magic angle spinning: 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Current Status of solid state NMR http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

REDOR http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Objectives of this Lecture and Practicum Resources Physical principle Sample requirements Parameters that are measured by NMR Dynamics by NMR Limitations Practical aspects Setup of NMR experiments (downstairs) 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

The components of an NMR spectrometer A magnet Probehead(s) Radiofrequency sources Amplifiers Analog to digital converters The lock system The shim system A computer 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

The Shim System http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Monitoring Shimming http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Monitoring shimming http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Objectives of this Lecture and Practicum Resources Physical principle Sample requirements Parameters that are measured by NMR Dynamics by NMR Limitations Practical aspects Setup of NMR experiments (downstairs) Bring your coats!! 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

Outlook for next week Lecture: The structure determination pipeline Practical analysis of NMR data in computer lab: Topspin NMRpipe NMRviewJ 9/22/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture