NESG NMR Conference Call

Applications of RDCs andUse of Lanthanide Tags

Presenter: J. Prestegard, UGA

9/26/05

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)

• 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)

RDCs Come From the Dipolar Interaction Between Two Spins

15N

1HB0

r

HZNZ

2

3II

2

1θ3cos

r

C D

Brackets denote averaging – goes to zero without partial orientation

Inducing Order Using Liquid Crystalline MediaRestores Dipole Interaction in Solution

B0

Most versatile medium: C12E5:octanol

-1/4(J + D)

1/4(J + D)

-1/4(J + D)

1/4(J + D)E

J + D

RDCs are Easily Measured as Contributions to Multiplet Splittings

Some Other Experiments for 15N-1H RDC Measurement

• Tolman JR, Prestegard JH: Measurement of one-bond amide N-15-H-1 couplings. J. Magn. Reson., 1996, 112:245-252

• Ottiger M, Delaglio F, Bax A, Measurement of couplings using IPAP. JMR 1998,131: 373-378.   

• Kontaxis G, Clore GM, Bax A, TROSY-HSQC offsets. J. Magn. Reson., 2000 143:184-190.

Use of 15N-1H RDCs for Structure Validation and Refinement: TM112

rmsd = 1.693(NMR vs Crystal structure)

REsidual Dipolar Coupling Analysis Tool(REDCAT)

Valafar, H., & J.H. Prestegard (2004), J. Mag. Res. 167: 228-241

• Given a proposed structure and RDCs, calculates order tensor solutions.

• Finds best order tensor solution.

• Gives principal elements and Euler angles.

• Back-calculates RDCs.

• Estimates errors and helps identify problematic data.

Another program: Dosset, Hus, Marion & Blackledge (2001), JBNMR, 20: 223-231

Correlation of Experimental 1H-15N RDCs with Calculated RDCs from the NMR Structure of TM112

NMR model after correction (Qfactor=0.57773)

-10

-8

-6

-4

-2

0

2

4

6

-30 -25 -20 -15 -10 -5 0 5 10

expt RDC

ca

lc R

DC

Correlation of Experimental 1H-15N RDCs with Calculated RDCs from Crystal Structure of TM1112

x-Ray model after correction (Q-factor=0.373893)

-10

-8

-6

-4

-2

0

2

4

6

-30 -20 -10 0 10

expt RDC

ca

lc R

DC

Structure Refinement Using RDCsSchwieters CD et al. XPLOR-NIH, J. Magn. Res. 160 (1): 65-73 JAN 2003

Write RDCs in principal alignment frame:D = (Da/r3){(3cos2θ – 1)/r3 + (3/2)Rsin2θcos(2)}

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

plot of exp RDC vs calc RDC (Q-factor=0.519)

-10

-8

-6

-4

-2

0

2

4

6

8

10

-20 -15 -10 -5 0 5 10 15 20

exp RDC

ca

lc R

DC

expt RDC vs calc RDC after simulated annealing (2000K-20K) Q-factor = 0.157

-12

-10

-8

-6

-4

-2

0

2

4

6

8

10

-15 -10 -5 0 5 10

expt RDC

calc

RD

C

RMSD Before refinement : 0.711, After refinement: 0.672

Using RDCs directly in structure determination:PF1455 – A Protein with a Novel Fold?

• 10 kDa protein from Pyrococcus furiosus • No significant sequence identity to PDB

entries• No significant threading hits with

Genthreader or Prospect• RDC-Prospect finds a structural homolog• Can RDCs and backbone NOEs lead from

the homolog to a structure?

Data Collected:

• H-N RDCs – in phage - 63

• HaCa RDCs in phage - 61

• H-N RDCs – in C12E5 - 54

• Primary NOEs (45 sequential, 9 long range)

• Secondary NOEs (18 sequential, 19 long range)

• Ca shifts – 74

Refinement

• Started with 1cc8 as template• Constraints: NOE, RDC, torsion, radius of

gyration• Three rounds of simulated annealing (400K) in

vacuum • One round of simulated annealing in water• 20 independent runs – 1.4Å cluster• Structure moves 2.5Å rmsd from template• Ramachandran statistics: 56%, 30%, 13%, 1%

Plot of exp NH RDC vs calc RDC Q-factor = 0.14

-12

-10

-8

-6

-4

-2

0

2

4

6

8

-15 -10 -5 0 5 10

exp NH RDc

ca

lc N

H R

DC

T6_initial structure_ML51 (Q-factor = 0.75)

-10

-8

-6

-4

-2

0

2

4

6

-14 -12 -10 -8 -6 -4 -2 0 2 4 6 8

exp NH RDC

calc

NH

RD

C

Comparison of RDC (phage HN) before and after refinementpink symbols excluded – validation set

Structure of PF1455

RDCs can be Collected Without Alignment Media: Lanthanide Tagged Proteins:

RDC = -(hB2)/(1203r3kT) [½Δ(3cos2θ-1) + ¾sin2θcos]

Note: B2 dependence

Ln3+

1 2

3

Ikegami, T., et al. (2004) J. Biomol. NMR 29:339-349.Wohnert, J., et al. (2003) J. Am. Chem. Soc. 125:13338-13339.

Construct for Lanthanide Tagged EB1

MGHHHHHHG*ENLYFQG**YIDTNNDGWYEGDELLA*SAVVYSTSVTSDNLSRHDMLAWINESLQLNLTKIEQLCSGAAYCQFMDMLFPGSIALKKVKFQAKLEHEYIQNFKILQAGFKRMGVDKIIPVDKLVKGKFQDNFEFVQWFKKFFDANYDGKDYDPVAARQGQETAVAPSLVAPALNKPKKPLTSSSAAPQRPISTQRTAAAPKAGPGVVRKNPGVGNGDDEAAELMQQVNVLKLTVEDLEKERDFYFGKLRNIELICQENEGENDPVLQRIVDILYATDEGFVIPDEGGPQEEQEEY

Wohnert, J., et al. (2003) J. Am. Chem. Soc. 125:13338-13339.

TROSY-HSQC correlations give RDC data.900 MHz Field-Induced Alignment

Paramagnetic Systems Give Other Complementary Information

Bertini, I., et al. (2002). Concepts in Magnetic Resonance 14: 259-286.

PCS 1

12r 3[ ax(3cos 2 1) rh sin2 cos2]

PRE 15

(o

4)2 1

r 6

Bo2H

2 (gJB )4 J 2(J 1)2

(2kBT )2(4 r 3 r

1 H2 r

2)

CCR 1

30(

o

4)2 Bo

2H2 (gJB )2 J(J 1)

rNH3 kBT

(3cos2 1)

r3(4 r 3 r

1 H2 r

2)

RDC 1

120 2

Bo2HS2

rNH3 kBT

[ax(3cos2 1) rh sin2 cos2]

Comparison of Lu3+ and Dy3+ Complexes of Tagged Q15691 gives Pseudo-Contact Shifts

CharacteristicDiagonal shifts

15-20 Å

20-25 Å

Ln3+

Paramagnetic Enhancement of Spin Relaxation:

Distance MappingOver 30Å

Provides Validation of Assignments

Lanthanide -Tagged Hum-Q-15691

Test Case:Assignment of Glycine Subset of

Amino Acids in Q15691

RDC(Hz) PCS(Hz) Assign r(exp) r(model) -0.9 -103 Gly 58 19 28 1.2 -152 Gly 98 16 16 2.3 1 Gly 103 14 16 -3.0 -44 Gly 116 36 37 7.1 -86 Gly 138 21 25

Acknowledgements

T. WeldeghiorghisSilvia MariJohn GlushkaHomay ValafarGreg Benison

Fang TianNitin JainKristen MayerSonal BansalPeter Leblond

NIGMS

http://secnmr.org

Example of Multiple Coupling Experiment: Soft HNCA – E.COSY

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

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

C chemical shift, Ci to C

i-1 connectivity, 3J-HNH coupling, C-H, HNH and H

i-1HN dipolar coupling

Inadvertent Mixing of α and β States of Hα can give Systematic Errors in J+D

+ f =

J+D too small J+D correct

Inclusion of RDCs Improves Accuracy of Structures

No Dipolar Data With Dipolar Data

Structure 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

SAΔ41 0·71 NA 0·65 NA

3 1

23

2cos cos cos cos cos

i j k l kl

Order Matrix Analysis

x y

z

z

x

z

Finding a Principal Order Frame

Sxx Sxy ..Syx Syy .... .. ..

= ASx’x’

Sy’y’

Sz’z’

A-1

Simple Modification of Soft HNCA – E.COSYAllows scaled addition of sum and difference E.COSY

to compensate for relaxation effects

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

+/- 90