Residual Dipolar Couplings BCMB/CHEM 8190

Transcription

1 Residual Dipolar Couplings BCMB/CHEM 8190

2 Recent Reviews Prestegard, A-Hashimi & Tolman, Quart. Reviews Biophys. 33, (2000). Bax, Kontaxis & Tjandra, Methods in Enzymology, 339, (2001) Tolman, Curr. Opin. Struct. Biol. 11, (2001) Prestegard, Bougault & Kishore, Chemical Reviews, 104, (2004) Lipsitz & Tjandra, Ann. Rev. Biophys. Biomol. Struct., 33, (2004) Fushman et al., Prog. NMR Spect. 44, (2004)

3 The Dipolar Interaction Between Two Spins B 0 θ 15 N r 1 H D = C r 3cos θ I 3 NZIHZ Brackets denote averaging goes to zero without partial orientation

4 B 0 Inducing Order Using Liquid Crystalline Media

5 Measurement of Dipolar Couplings Coupled HSQC Isotropic J Aligned J + D

6 Polyacrylamide Gels another alignment medium Yizhou Liu, J. Prestegard J. Biomol NMR, submitted

7 Order Matrix Analysis ρ x z ρ z θ φ z x y 2 3cos θ 1 2 = cos φi cos φj 3 cos ρk cos ρl δkl

8 Finding a Principal Order Frame S xx S xy.. S yx S yy = A S x x Sy y S z z A -1

9 Strategy for Protein Fold Determination Express 15 N labeled protein Identify secondary elements Assign backbone resonances Orient protein in LC medium Collect residual dipolar data Orient individual elements Assemble protein fold

10 Dipolar Interaction Vectors In an Idealized α-helix 15 N Labeling Only

11 Data Used in ACP Fold Determination A. Dipolar couplings in α-helices Couplings (Hz) Helix 1 Helix 2 Helix 3 Amide Couplings I3 1.4 L Q (N i -H N i) E4 0.4 D A E5 3.4 T I V7-1.0 V D K8 0.8 N I G I H G E Q L Amide-Alpha Couplings R6 3.0 L Q (H N i H αi ) V7-3.5 L A K9 4.5 V H L M L (H N i H αi±1 ) V43 N -L42 α 2.0 M44 N -A45 α -2.0 Amide/Amide Couplings D N (H N i - H N i+1) T V B. NOEs and distances used to position helices in POSE: I3 H N - F50 H α, V7 H N - F50 H ξ, V7 H α - F50 H ξ, V7 methyl - A68 H N, I3 methyl - N73 H N 6 Å 8 Å 8 Å 8 Å 8 Å

12 Orientation Maps for Three ACP Helices

13 ACP Dipolar Fold vs. NOE Structure

14 Some Experiments for RDC Data Acquisition Tolman JR, Prestegard JH: Measurement of one-bond amide N- 15-H-1 couplings. JMR, 1996, 112: Ottiger M, Delaglio F, Bax A, Measurement of couplings using IPAP JMR 1998,131: Wang YX, Marquardt JL, Wingfield P, Stahl SJ, Lee-Huang S, Torchia D, Bax A: Measurement of H-1-N-15, H-1-C-13 ', and N-15-C-13 ' dipolar couplings. JACS, 1998, 120: Yang DW, Venters RA, Mueller GA, Choy WY, Kay LE: TROSY-based HNCO pulse sequences. JBNMR 1999, 14: Liu, Y. and J.H, Prestegard. Measurement of one and two bond N-C couplings in large proteins by TROSY-based J-modulation experiments. JMR 2009, 200: Delaglio F, Wu ZR, Bax A: Homonuclear proton couplings from regular 2D COSY spectra. JMR 2001, 149:

15 Measurable Dipolar Couplings in a Dipeptide Define an Order Frame Z O H H C φ ψ C C N N X Y H O Coupled HSQC Soft HNCA-E.Cosy HNCO

16 Soft HNCA E.COSY Weisemann, Ruterhans, Schwalbe, Schleucher Bermel, Griesinger, J. Biomol. NMR, 4, , 1994

17 Soft HNCA E-COSY Spectra of 15 N- Labeled 13 C Natural Abundance Rubredoxin C α chemical shift, C α i to Cα i-1 connectivity, 3 J-H N H α coupling, C α -H α, H N H α and H α i-1 HN dipolar coupling

18 Automated Analysis Through NMRPipe: HNCA E.COSY on isotropic Pfu

19 Multiple Peptide Segments Oriented to Superimpose Order Frames Yield Structures Proof of principle: rubredoxin structure overlays X-ray structure to 1.6Å Novel targets in process: PF (8.9kDa), PF (8.6kDa)

20 More Recent 15 N- 1 H Depositions use a J-modulation Experiment: Also can be used for 15 N- 13 C, 15 N- 13 Cα Data shown are on a 70kDa protein Liu, Y. and J.H, Prestegard J Magn Reson. 200(1): Rdc = Hz Rdc = Hz Rdc = Hz Cross-peaks overlap HSQC peaks exactly Time requirements are similar to TROSY/HSQC Based on TROSY detection for application to larger proteins Fit gives T 2 estimate used to eliminate data on loops

21 Analysis of Residual Dipolar Couplings Schwieters CD, Kuszewski JJ, Tjandra N, et al. XPLOR-NIH, J. Magn. Res. 160 (1): JAN 2003 Meiler J, Blomberg N, Nilges M, Griesinger C, Residual dipolar couplings as restraints in structure elucidation JBNMR 2000, 16: Rohl CA, Baker D: Backbone structure from residual dipolar couplings using rosetta. JACS, 2002, 124: Delaglio F, Kontaxis G, Bax A: Structure from molecular fragment replacement and dipolar couplings. JACS, 2000, 122: Valafar H, Prestegard J. 2004, J. Mag. Res., 167, Dosset, Hus, Marion & Blackledge (2001), JBNMR, 20:

22 Example of Validation and Refinement MTH1743 Q = ( ( D ( obs D D 2 obs calc 1/ 2 ) ) 2 ) 1/ 2 exp RDC vs calc RDC for refined structure after sa Q-factor = 0.33 exp RDC vs calc RDC for 1jsb Q-factor = calc RDC calc RDC exp RDC -15 exp RDC -15

23 X-ray Structures fit RDCs Better than NOE-Based NMR Structures nesg bmrb pdb nmr pdb xrayalignment media #residuesnmr Q xray Q RMSD* BeR k2e 3cpk phage CsR jr2 2ota peg (and peg+ctab CtR kcu 3e0h phage (and peg) GmR k5p 3cwi peg HR3646E** khn 3fia polyacrylamide gel MbR242E kko 3gw2 peg PfR193A kl6 3idu phage SgR jz2 3c4s peg SoR juw 2qti polyacrylamide gel * PSVS analysis listed structured regions (obtained via PROCHECK) 1st NMR model compared to X-Ray structure for all analysis ** It was difficult to compare the xray and nmr structures for this protein. Q = ( ( D ( obs D D 2 obs calc 1/ 2 ) ) 2 ) 1/ 2

24 Structure Refinement Using RDCs Write RDCs in principal alignment frame: D = (D a /r 3 ){(3cos 2 θ 1)/r 3 + (3/2)Rsin 2 θcos(2φ)} Write error function in terms of D meas and D calc E RDC = (D meas D calc ) 2 Seek minimum in E RDC to refine structure Need to float alignment axes during search

25 Refinement with RDCs can Improve Quality CtR107 with and without RDCs Cyan, X-ray Red, best refined with RDC Gray, best refined without RDC Refinement detail Anneal, no RDC Anneal, with RDC Refine, no RDC Refine, with RDC Average RMSD to X-ray (best of 10) (2.0) 2.0(1.6) RMSD of the ensemble Alignment carried out by superimposing backbone atoms of residues 19 to 26, 30 to 39, 59 to 61, 69 to 71, 88 to 90, 97 to 102, 111 to 122, 132 to 134 and 149 to 152