Protein Dynamics Relaxation techniques

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1 Protein Dynamics Relaxation techniques Daniel Mathieu Bruker Users Meeting 2016, Karlsruhe Innovation with Integrity

2 Proteins aren t exactly rock solid Users meeting

3 Characterizing Dynamic Processes using NMR Real time NMR Lineshape analysis Exchange Spectroscopy, EXSY, zz-exchange CPMG or R1rho Relaxation Dispersion Residual Dipolar Couplings / PREs Relaxation Analysis CEST (Chemical Exchange Saturation Transfer) Users meeting

4 Exchanging Proteins E p A = k BB k AA +k BA p B = k AB k AA +k BA state B state A A B Users meeting

5 How CEST works A B Users meeting

6 What does this look like? Basically CW absorption spectra Users meeting

7 Pulse program (pseudo)3d Experiment with a varying 15 N B 1 field offset Vallurupalli et al. J. Am. Chem. Soc. 2012, 134, Users meeting

8 Experimental setup Users meeting

9 Experimental parameters Users meeting

10 Processed result Users meeting

11 CEST Analysis Users meeting

12 Sample Information Users meeting

13 Data Selection Users meeting

14 Data Analysis Users meeting

15 Result Users meeting

16 2 H T 1 and T 1 ρ measurements in CH 2 D Methyl groups Kay et al 1995 J. Am. Chem. Soc., 117, Randomly deuterated sample Andrew L. Lee University of North Carolina at Chapel Hill Users meeting

17 Side Chain relaxation Roughly ubiquitine sized U- 13 C, randomly 50% 2 H labeled 2 mm in 90% H 2 O 10% D 2 O 310 K 500 MHz CP-TCI T 1ρ using a 1 khz spinlock field Users meeting

18 Deuterium relaxation measurements 2H z C z 4H z C z D z CH 2 D groups are selected by an x-filter type element The relaxation rate of H z C z D z/y is measured, not just D z/y A reference for the T 1 relaxation rate of H z C z is required Kay et al J. Am. Chem. Soc. 2002, 124, Users meeting

19 Measurements T 1 experiment shown for 3 different delays T relax (2 ms, 25 ms, 120 ms) pseudo3d experiment Measurement time for 8 delays and 4 replicates ~ 24 hours (T 1 + T 1 ρ) Users meeting

20 Determination of S 2 and τ e Measurement of only two parameters per methyl group Not sufficient to fit the order parameter and the internal correlation time Measurement is repeated at a different B 0 field strengths Analysis is fully integrated in the ProteinDynamicsCenter (2.0+) Users meeting

21 2 H Relaxation Analysis DynamicsCenter P. Neidig Users meeting

22 T 1 and T 1 ρ Analysis 500 and 700 MHz Users meeting

23 Side chain modeling setup Users meeting

24 Results Calculation of order parameters S 2 and the internal correlation times τ e Users meeting

25 CPMG relaxation dispersion Kay et al 2008 J. Biomol. NMR, 41, The HEROINE experiment Ban, Lee, Griesinger et al 2013 J. Biomol. NMR, 57, Users meeting

26 Relaxation dispersion What and Why? The dependency of effective transverse relaxation on an applied spinlock field which averages exchange contributions Relaxation dispersion is a method to characterize exchange processes by NMR Can be used to characterize invisible states Can yield dynamic and thermodynamic parameters Users meeting

27 Principles E p A = k BB k AA +k BA p B = k AB k AA +k BA state B state A A B Users meeting

28 Measurement of CT CPMG relaxation dispersion One reference experiment is recorded without a CPMG train Multiple CPMG fields for a constant relaxation period T are recorded Relaxation rates are determined from the ratio of the intensities of the reference and every individual CPMG experiment. This type of measurement saves a lot of time compared to individual CPMG or R 1 ρ relaxation measurements at every field. CPMG pulse trains deposit a lot of power into the sample which leads to heating Scan wise interleaved measurement Dummy pulses in every scan to achieve temperature compensation Users meeting

29 Outcome in the presence of exchange Exchange rates R ex contribute to the effective rate R 2,eff Exchange contributions can be changed by variation of the CPMG field In case of a two-site exchange, this can be described analytically The lowest possible field strength is limited by the length of T (longer means lower fields) The highest possible field strength is limited by the hardware capabilities for the same time T Kay et al J. Biomol. NMR 2008, 41, Users meeting

30 Why even higher power? Really High power! R 20? R 20? R 20! Higher power for the individual pulses reduces off-resonance effects One of the fitting parameters is R 20 (exchange-free R 2 ) This parameter is extracted from the plateau towards higher field strength In a lot of cases the plateau is not yet reached So why not just measure the end point? Users meeting

31 Experimental conditions All measurements were performed on u- 13 C, 15 N Ubiquitin (1.5 mm in 90% H 2 O 10% D 2 O) 500 MHz CP-TCI 293 K 80 µs π-pulses during CPMG trains Users meeting

32 The HEROINE experiment Heteronuclear Rotating Overhauser Invaded Exchange High power T 1 ρ measurement Recorded for different spinlock offsets to get as close to the on resonance condition as possible The corresponding CPMG experiment with lower power is temperature compensated with respect to the HEROINE experiment Users meeting

33 Setup Both experiments in one sequence Flag decides whether the HEROINE or CT CPMG experiment is executed Two lists (one for CPMG frequencies, one for T 1 ρ spin lock periods) All calculations e.g. temperature compensation are done in the pulse program -DLABEL_R Users meeting

34 Results: 5 khz spin-lock up to 125 ms T 1 type analysis (e.g. when using PDC) Clear dependency on the offset Users meeting

35 Temperature compensated CT-CPMG measurements Spin-lock fields up to 1 khz for 80 ms (using 6.25 khz π-pulses) Temperature compensated to match the HEROINE experiment Users meeting

36 Thanks Peter Neidig Wolfgang Bermel Clemens Anklin Martine Monette Helena Kovacs Andrew L. Lee Lewis Kay s Lab Christian Griesinger s Lab You for your attention Users meeting

37 Innovation with Integrity Copyright 2011 Bruker Bruker Corporation. Corporation. All rights reserved. All rights reserved.