Apr 04

Residual Dipolar Couplings in Structure Determination – Recent Reviews • Prestegard, A-Hashimi & Tolman, Quart. Reviews Biophys. 33, 371-424 (2000). • Bax, Kontaxis & Tjandra, Methods in Enzymology, 339, 127-174 (2001) • Tolman, Curr. Opin. Struct. Biol. 11, 532-539 (2001) • Prestegard, Bougault & Kishore, Chemical Reviews, 104, 3519-3540 (2004) • Lipsitz & Tjandra, Ann. Rev. Biophys. Biomol. Struct., 33, 387-413 (2004) • Fushman et al., Prog. NMR Spect. 44, 189-214 (2004)

The Dipolar Interaction Between Two Spins B0

θ

1H

r 15N

C 3cos2θ − 1 INZIHZ D= 3 r 2

Brackets denote averaging – goes to zero without partial orientation

Inducing Order Using Liquid Crystalline Media B0

Measurement of Dipolar Couplings – Coupled HSQC Isotropic

J

Aligned J+D

Order Matrix Analysis z ρx

ρz

φz

θ x

3 cos2 θ − 1 2 ⋅ = ⋅ ⋅

⎡cos φi cos φj ⎢ ⋅ ⎢ ⎢ ⋅ ⎢ ⋅ ⎣

y

⋅ ⋅ ⋅⎤ ⎡3 cos ρk ⋅ ⋅ ⋅⎥ ⎢ ⎥×⎢ ⋅ ⋅ ⋅⎥ ⎢ ⎥ ⎢ ⋅ ⋅ ⋅⎦ ⎣

cos ρl − δkl ⎤ ⎥ ⋅ ⎥ ⎥ ⋅ ⎥ ⋅ ⎦

Finding a Principal Order Frame Sxx Sxy .. Syx Syy .. .. .. ..

Sx’x’ =

A

A-1

Sy’y’ Sz’z’

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

Dipolar Interaction Vectors In an Idealized α-Helix 15N

Labeling Only

Data Used in ACP Fold Determination A. Dipolar couplings in α-helices

Couplings (Hz) Helix 2

Helix 1

Helix 3

Amide Couplings (Ni-HNi)

I3 E4 E5 V7 K8 I10 I11 G12 E13 Q14 L15

1.4 0.4 3.4 -1.0 0.8 2.0 -0.3 2.1 2.1 0.0 -1.9

L37 D38 T39 V40

-2.6 1.6 -0.3 -2.6

Q66 A67 I69 D70 N73 G74 H75

8.2 7.7 6.8 6.1 7.4 5.5 7.7

Amide-Alpha Couplings (HNi – Hαi)

R6 V7 K9 L15

3.0 -3.5 4.5 0.0

L37 L42 V43 M44 L46 V43N-L42α M44N-A45α

0.4 0.0 3.5 2.0 -2.0 2.0 -2.0

Q66 A68 H75

0.0 -8.5 -9.0

D38 T39 V40

2.0 2.0 2.0

N73

2.0

(HNi – Hαi±1) Amide/Amide Couplings (HNi - HNi+1) B. NOEs and distances used to position helices in POSE:

I3 HN - F50 Hα, V7 HN - F50 Hξ, V7 Hα - F50 Hξ, V7 methyl - A68 HN, I3 methyl - N73 HN 6Å 8Å 8Å 8Å 8Å

Orientation Maps for Three ACP Helices

ACP Dipolar Fold vs. NOE Structure

Measurable Dipolar Couplings in a Dipeptide Define an Order Frame Z

O C

φ

N H

H C

ϕ

C O

H N X

Coupled HSQC Soft HNCA-E.Cosy HNCO

Y

Some Experiments for RDC Data Acquisition • Tolman JR, Prestegard JH: Measurement of one-bond amide N15-H-1 couplings. JMR, 1996, 112:245-252 • Ottiger M, Delaglio F, Bax A, Measurement of couplings using IPAP JMR 1998,131: 373-378. • 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, 120:7385-7386. • Yang DW, Venters RA, Mueller GA, Choy WY, Kay LE: TROSY-based HNCO pulse sequences. JBNMR 1999, 14:333343. • Tian F, Bolon PJ, Prestegard JH: Homonuclear residual dipolar couplings from CT-COSY. JACS, 1999, 121:7712-7713. • Delaglio F, Wu ZR, Bax A: Homonuclear proton couplings from regular 2D COSY spectra. JMR 2001, 149:276-281.

Soft HNCA – E.COSY

Weisemann, Ruterhans, Schwalbe, Schleucher Bermel, Griesinger, J. Biomol. NMR, 4, 231-240, 1994

Soft HNCA E-COSY Spectra of 15NLabeled 13C Natural Abundance Rubredoxin

Cα chemical shift, Cαi to Cαi-1 connectivity, 3J-HNHα coupling, Cα-Hα, HNHα and Hαi-1HN dipolar coupling

Automated Analysis Through NMRPipe: HNCA E.COSY on isotropic Pfu-1016054

http://spin.niddk.nih.gov/bax/software/NMRPipe/

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: PF1496972 (8.9kDa), PF1016054(8.6kDa)

Analysis of Residual Dipolar Couplings • Schwieters CD, Kuszewski JJ, Tjandra N, et al. XPLOR-NIH, J. Magn. Res. 160 (1): 65-73 JAN 2003 • Meiler J, Blomberg N, Nilges M, Griesinger C, Residual dipolar couplings as restraints in structure elucidation JBNMR 2000, 16: 245-252. • Rohl CA, Baker D: Backbone structure from residual dipolar couplings using rosetta. JACS, 2002, 124:2723-2729. • Delaglio F, Kontaxis G, Bax A: Structure from molecular fragment replacement and dipolar couplings. JACS, 2000, 122:2142-2143. • Valafar H, Prestegard J. 2004, J. Mag. Res., 167, 228-241 http://www.ccrc.uga.edu/web/CarbResource/Software/ • Dosset, Hus, Marion & Blackledge (2001), JBNMR, 20: 223231

Structure Refinement Using RDCs Write RDCs in principal alignment frame: D = (Da/r3){(3cos2θ – 1)/r3 + (3/2)Rsin2θcos(2φ)} Write error function in terms of Dmeas and Dcalc ERDC = (Dmeas – Dcalc)2 Seek minimum in ERDC to refine structure – Need to float alignment axes during search

Example of Validation and Refinement – MTH1743

10

10

5

5

0 -15

-10

exp RDC vs calc RDC for 1jsb Q-factor = 0.23

-5

0 -5

5

calc RDC

calc RDC

exp RDC vs calc RDC for refined structure after sa Q-factor = 0.33

10

0 -15

-10

-5

0 -5

-10

-10

-15

-15

ex p RDC

ex p RDC

5

10

Inclusion of RDCs Improves Accuracy of Structures No Structure

Dipolar Data

With Dipolar Data

Precision (Å) Accuracy (Å) Precision (Å) Accuracy (Å)

GB1

2·88

4·33

1·37

1·12

BAF

1·31

1·37

1·23

0·91

CVN

1·67

1·53

1·23

1·10

FK506

0·67

1·12

0·29

0·74

GATA-1

0·76

NA

0·68

NA

KH

0·39

NA

0·16

NA

SA∆41

0·71

NA

0·65

NA