residual dipolar couplings ;rdc
DESCRIPTION
Residual Dipolar Couplings ;RDC. Cheng-Kun Tsai 2005.05.14. Residual Dipolar Coupling. Introduction Theoretical Application. Introduction. NOE, Scalar J coupling --- local TROSY, Protein labeling strategies --- larger macromolecules RDC --- distance (short, long), angle. - PowerPoint PPT PresentationTRANSCRIPT
Residual Dipolar Couplings ;RDCResidual Dipolar Couplings ;RDC
Cheng-Kun Tsai2005.05.14
Cheng-Kun Tsai2005.05.14
Residual Dipolar CouplingResidual Dipolar Coupling
Introduction Theoretical Application
Introduction Theoretical Application
IntroductionIntroduction
NOE, Scalar J coupling --- local
TROSY, Protein labeling strategies --- larger macromolecules
RDC --- distance (short, long), angle
NOE, Scalar J coupling --- local
TROSY, Protein labeling strategies --- larger macromolecules
RDC --- distance (short, long), angle
ΞJ = JSI S ‧ I
TheoreticalTheoretical
Magnetic field:H(r) = ﹣μS/r3 + 3(r. μS) . r/r5
Dipolar coupling Hamiltonian:ΞD = - μI . H(r) = ( μI . μS/r3) – 3( μI . r)(μS . r)/r5
= γSγIβSβI {S . I/r3 – 3(S . r)(I . r)/r5}
S
Ir
If the spins I and S are heternuclear
Expand the equation and drop secondary terms
and
Then
In the “special” frame of reference defined
Define
P: “probability tensor”
Define
Note:
1. for example, in the static case
The principle z axis is parallel to the vector b
2. for a completely isotropically reorienting molecule
then then
A. Px = Py = 0.25 and Pz = 0.5B. Px = 0.2, Py = 0.3 and Pz = 0.5C. Px = Py = Pz = 1/3
Px2 + Py
2 + Pz2 = 1
P: “probability tensor”
Define “aligment tensor” A
Ax + Ay + Az = 0
A. Ax = Ay = -1/12, Az=1/6B. Ax = -2/15, Ay = -1/30, Az = 1/6C. Ax = Ay = Az =0
The calculation of the RDC constant D are expressed in various more or less complicated forms found in literature
The calculation of the RDC constant D are expressed in various more or less complicated forms found in literature
and
then
and
Define axial component Aa and rhombic component Ar
Saupe matrix (or order matrix) S
R: rhombicity of alignment tensor
η : asymmetry parameter
then
or
※Generalized order parameter S (0 S 1)≦ ≦
※ Maximum dipolar coupling
※ Magnitude of the residual dipolar coupling tensor
※Generalized degree of order (GDO)
and
motion ~ millisecond time scale
Dynamics:
= bx(t) . rx(t) + by(t) . ry(t)
+ bz(t) . rz (t)
, θ = θ (t)
then
anisotropies anisotropies
Residual dipolar couplings Complementary observables
1. chemical shift anisotropy (CSA)
2. pseudocontact shifts in paramagnetic systems
3. cross-correlated relaxation
Residual dipolar couplings Complementary observables
1. chemical shift anisotropy (CSA)
2. pseudocontact shifts in paramagnetic systems
3. cross-correlated relaxation
Dab = (J+D) - JDab = (J+D) - J
2H 1D spectrum of water deuterons in5% bicelle prepared in D2O at 35oC
(a) Isotropic spectrum 1JNH
(b) 4.5% (w/v) bicelle(c) 8% bicelle
Alignment mediaAlignment media
Liquid crystals --- 1963, Saupe Bicelles --- 1990s, Bacteriophage Polyacrylamide gels Other media
Liquid crystals --- 1963, Saupe Bicelles --- 1990s, Bacteriophage Polyacrylamide gels Other media
Bicelles Bacteriophage
Ref. RDC in structure determination of biomolecules, Chem. Rev. 2004, 104, 3519-3540
Alignment must be sufficient, but not so large Adjustment of media concentration Overall charge and charge distribution of a protein, in an
electrically charged medium The use of media-free, field-induced orientation of
biomolecules. Paramagnetic ions Diamagnetic anisotropy The option of using several alignment media Using multiple media, three reasons
Alignment must be sufficient, but not so large Adjustment of media concentration Overall charge and charge distribution of a protein, in an
electrically charged medium The use of media-free, field-induced orientation of
biomolecules. Paramagnetic ions Diamagnetic anisotropy The option of using several alignment media Using multiple media, three reasons
Data refinementData refinement
RMSD
--- improved Ramachandran plot
--- the most favored region improved
RMSD
--- improved Ramachandran plot
--- the most favored region improved
ApplicationsApplications
Structure refinement and domain orientations
DNA/RNA structure refinement
Conformation of small molecules and bound ligands
Structure refinement and domain orientations
DNA/RNA structure refinement
Conformation of small molecules and bound ligands
Structure refinement anddomain orientations
Structure refinement anddomain orientations
NMR structure and crystal structure
NMR structure refined with RDCs
(1) rat apo S100B(ββ), Ca2+-binding
(2) VEGF11-109
(3) Prp40
NMR structure and crystal structure
NMR structure refined with RDCs
(1) rat apo S100B(ββ), Ca2+-binding
(2) VEGF11-109
(3) Prp40
(1) rat apo S100B(ββ), Ca2+-binding(1) rat apo S100B(ββ), Ca2+-binding
A. Dimeric apo S100BB. Blue, rat, NMR with RDC yellow, rat green, bovine
The third Helix
RMSD: 1.04A to 0.29ARamachandran Plot: 76 to 86%(the most favored region)
(2) Vascular endothelial growth factor, VEGF11-109(2) Vascular endothelial growth factor, VEGF11-109
VEGF11-109 + v107, peptide antagonists, v107(GGNECDAIRMWEWECFERL)N terminus of VEGF11-109RMSD: 0.60 to 0.37A
(a) grey, solution structure red, NMR with RDC(b) cyan, crystal structure red, NMR with RDC
(3) The yeast splicing factor pre-mRNA processing protein 40, Prp40
(3) The yeast splicing factor pre-mRNA processing protein 40, Prp40
(a) WW1 domain, , Solution structure (b) WW2 domain(e) Structure with RDC
RMSD: 1.14 to 0.55A
No solution structure a homologous structure
, a closely related molecule
, a crystal structure
fitting of RDCs
(1) Ca2+-ligated CaM
(2) hemoglobin
No solution structure a homologous structure
, a closely related molecule
, a crystal structure
fitting of RDCs
(1) Ca2+-ligated CaM
(2) hemoglobin
(1) Calmodulin / CaM, a ubiquitous Ca2+ binding protein(1) Calmodulin / CaM, a ubiquitous Ca2+ binding protein
Blue, 1 Å crystal structure (1EXR)Red, Ca2+–CaM solution structure with RDC
(2) hemoglobin(2) hemoglobin
Crystal structure:T, tense state; R, relaxed state ; R2, second conformation
dark, R crystalmedium, solution with RDClight, R2 crystal
Relative domain orientations
(1) B and C domains of BL
(2) three fingers in TFIIIA
(3) MalBP
(4) T4 lysozyme
Relative domain orientations
(1) B and C domains of BL
(2) three fingers in TFIIIA
(3) MalBP
(4) T4 lysozyme
(1) B and C domains of barley lection (BL)(1) B and C domains of barley lection (BL)
A. X-ray structureB. NMR with RDC
(2) three fingers in TFIIIA, transcription factor IIIA(2) three fingers in TFIIIA, transcription factor IIIA
Cyan: without dipolar restraints
Yellow: with dipolar restraints
Red: crystal structure refined with NOE and dipolar restraints.
(3) MalBP, maltodextrin-binding protein(3) MalBP, maltodextrin-binding protein
(a) apo-state (crystal)(b) bound to β-cyclodextrin (inactive ligand)(c) bound to maltotriose (natural ligand)
(4) T4 lysozyme(4) T4 lysozyme
(a) WT lysozyme X-ray (b) M6I mutant X-ray Red , with RDC
DNA/RNA structure refinementDNA/RNA structure refinement
NMR – lack the elaborate tertiary structure
, less proton dense X-ray – misinterpretations of the global feature RDCs
NMR – lack the elaborate tertiary structure
, less proton dense X-ray – misinterpretations of the global feature RDCs
RDCs from RNA molecules
(1) A-tract DNA – curvature
(2) A-tract DNA -- both local and global structure
RDCs from RNA molecules
(1) A-tract DNA – curvature
(2) A-tract DNA -- both local and global structure
(1) A-tract DNA – curvature (1) A-tract DNA – curvature
DNA sequence:d(CGCGAATCGCGAATTCGCG)2
Blue, NMR with RDCRed, X-ray
Note:b) is rotated by 90° around the helix axis relative to a)
(2) A-tract DNA – both local and global structure (2) A-tract DNA – both local and global structure
10mer DNA strcture(GCGAAAAAAC)
(a) only NOE and sugar pucker constraints(b) NOE, sugar pucker, and RDC constraints(c) NOE, sugar pucker, backbone torsion angle , and RDC constraints
RDCs from RNA molecules
(1) RNA and tRNA
(2) hammerhead ribozyme, Mg2+
(3) IRE
RDCs from RNA molecules
(1) RNA and tRNA
(2) hammerhead ribozyme, Mg2+
(3) IRE
(2) hammerhead ribozyme, Mg2+(2) hammerhead ribozyme, Mg2+
(A) Solution conformation derived from dipolar coupling data in the absence of Mg2+.(B) X-ray structure in the presence of Mg2+
Conformation of small molecules and bound ligands
Conformation of small molecules and bound ligands
(1) AMM bound to ManBPA (2) LacNAc binds to lectin protein Galectin-3 (3) trimannoside at the glycosidic linkages
(1) AMM bound to ManBPA (2) LacNAc binds to lectin protein Galectin-3 (3) trimannoside at the glycosidic linkages
(1) AMM (a-methyl mannoside)bound to ManBPA (mannose-binding protein-A)
(1) AMM (a-methyl mannoside)bound to ManBPA (mannose-binding protein-A)
Yellow spheres correspond to Ca2.Black and red shperes to carbon and oxygen, respectively, of AMM, and MBP is represented by ribbon diagram.
(2) LacNAc binds to lectin protein Galectin-3(2) LacNAc binds to lectin protein Galectin-3
green ribbon, Solution structure of galectin-3C in the absence of ligand
magenta ribbon, compared to the X-ray crystal structure with LacNAc bound
ConclusionsConclusions
1. to obtain dipolar couplings on macromolecules in solution, the potential for refining protein structures was immediatelyobvious.
2. focused on the structural applications, researchers are also beginning to exploit RDCs in solution NMR for their dynamics information content.
3. have established a framework to determine interfragment motion, to calculate amplitudes of interdomain motion, and to separate the dynamic contribution to the measured RDC to determine the effective values of θ and ψ