NMR Spectroscopy Question and Answer Session Judith Klein-Seetharaman Department of Structural Biology jks33@pitt.edu

What did we do? NMR instrumentation NMR sample preparation considerations Water signals Water suppression Detergents NMR setup – downstairs zg zgpr selabs hsqc Spectral processing: Topspin Software 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

What did we do then? Converting raw spectral formats to other formats using NMRpipe Example: HIV protease relaxation data Assignment of signals with one strategy HNCO HNCOCACBG HNCOCA HNCACB HNCA HNCACO Example: Ubiquitin assignment with NMRviewJ 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

What would be the next step? For determining a structure with NMR spectroscopy, after assignment, we would… Collect constraints: NOE Dipolar coupling Scalar coupling constants (gives dihedral angles) Solvent exchange Calculate a structure based on these constraints 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

What are other things one can do with NMR? For study of dynamics Measure and analyze T1, T2, HetNOE data We went through T1 analysis in computer lab (conversion of raw data with NMRpipe, analysis with NMRviewJ) T2 was your homework Other studies might include chemical shift perturbations Temperature titrations … 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Homework Assignments 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Homework Part 1 NMR instrumentation NMR sample preparation considerations Water signals Water suppression Detergents NMR setup – downstairs zg zgpr selabs hsqc Spectral processing: Topspin Software 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Experiments Run zg = records all 1H's in your sample zgpr = same as zg but with water suppression selabs = the way we set it up, it records only NH region hsqcfpgpf3gphwg = in short: hsqc = records all 1H's attached to 15N 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

click on Topspin icon and open the program 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

click on guest, expand MB3_Jan2007 directory This is the directory that contains all of the data that was acquired in class. 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Right click on MB3_Jan2007 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Select Expand & Show PULPROG/Title 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Samples Sample IDs Experiment IDs B 1-7 F 10-15 A 20-23 D 30-33 H 50-53 B 60, 61 F 70, 71 F 100-103 B 110-113 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Samples 40 = zg of sample E 41 = zgpr of sample E 1 = zgpr of Sample B 42 = selabs of Sample E 43 = hsqc of Sample E 50 = zg of Sample H 51 = zgpr of Sample H 52 = selabs of sample H 53 = hsqc of sample H 60 = zg of sample B 61 = zgpr of sample B 70 = zg of sample F 71 = zgpr of sample F -----------------experiments done with comp bio students 100 = zg of sample F 101 = zgpr of sample F 102 = selabs of sample F 103 = hsqc of sample F 110 = zg of sample B 111 = zg of sample B 112 = selabs of sample B 113 = hsqc of sample B Samples 1 = zgpr of Sample B 2 = zgpr of Sample B 3 = hsqc of Sample B 4 = zg of Sample B 5 = zg of Sample B 6 = selabs of Sample B 7 = selabs of Sample B 10 = zg of Sample F 11 = selabs of Sample F 12 = zgpr of Sample F 13 = hsqc of sample F 14 = hsqc of sample F 15 = hsqc of sample F ----- experiments done with MB3 students: 20 = zgpr of Sample A 21 = zg of sample A 22 = selabs of sample A 23 = hsqc of sample A 30 = zg of Sample D 31 = zgpr of Sample D 32 = selabs of Sample D 33 = hsqc of Sample D 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Solving the Mystery of the Samples Drag the experiment that you want to analyze into the main purple window area. If you do not see a spectrum, you need to type efp This will process the data so that you can see the spectrum. In the case of the hsqc it will also ask you for an experiment number where to store the processed data, you can use the default or enter a number, e.g. 2. Another alternative is to type “rser 1” that will retrieve the first slice of the 2d hsqc experiment also. 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

To see what acquisition parameters were chosen, click on the AcquPars tab, you will see a window: 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Notice that you can scroll down to see many fields under this tab. If you type ased only those parameters relevant to the specific experiment are shown: 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Some tips If you want to move more quickly from experiment to experiment, type re and then the experiment number, e.g. re 21 gives the experiment stored in directory 21 To save a picture of a spectrum select File, Export and then give it a file name extension indicating which format you would like (e.g. .jpg) Note that you may have to phase your spectrum. 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Phasing 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Sample A. 20-23 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Overlay Sample A From these spectra, we can conclude that we do not have 15N label, it i a water sample. It does not have detergent in it, or the detergent is deuterated. 22 = selabs 21 = zg 20 = zgpr 23 = hsqc 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Sample B. 1-7, 60-61, 110-113 3 = hsqc 8 = selabs 4 = zg 2 = zgpr 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Sample D. 30-33 33 = hsqc 32 = selabs 31 = zgpr 30 = zg 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Sample E. 40-43 42 = selabs 41 = zgpr 40 = zg 43 = hsqc 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Sample F. 10-15, 70-71, 100-103 103 = hsqc 102 = selabs 101 = zgpr 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Sample H. 50-53 52 = selabs – disregard this spectrum 53 = hsqc 51 = zgpr 50 = zg 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Parameters to watch out for ns rg will affect actual intensity measured. 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Several different assignment strategies exist Most easily automated: HNCO HNCOCACB HNCOCA HNCACB HNCA HNCACO 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Homework Part 2 Converting raw spectral formats to other formats using NMRpipe Example: HIV protease relaxation data Assignment of signals with one strategy HNCO HNCOCACBG HNCOCA HNCACB HNCA HNCACO Example: Ubiquitin assignment with NMRviewJ 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

This is a cluster: 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

HNCO 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

HNCA 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

HNCACO 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

HNCOCACB, similar: CBCA(CO)NH Not in HNCOCACB experiment 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

HNCOCA 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

HNCACB 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

What are other things one can do with NMR? For study of dynamics Measure and analyze T1, T2, HetNOE data We went through T1 analysis in computer lab (conversion of raw data with NMRpipe, analysis with NMRviewJ) T2 was your homework Other studies might include chemical shift perturbations Temperature titrations … 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Dynamics in folded/unfolded lysozyme Smaller rates – more flexible 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

T1 for HIV protease 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

T2 for HIV protease 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

T2 for HIV protease 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

From last class in case there are questions 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

NMR parameters Chemical Shift H2O methyl aromatic Trp-side-chain NH OH Backbone NH aliphatic Side-chain HN Ha Spectrum see handout 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

NMR of membrane proteins In lipid bilayer: In detergent micelle: http://www.elmhurst.edu/~chm/vchembook/558micelle.html 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Problems! Detergent peaks 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 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

Selective excitation 1H Chemical Shift [ppm] 1H Chemical Shift [ppm] 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] 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

2d HSQC 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

1d projection of HSQC 12/9/2018 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session