Long range structural restraints

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Long range structural restraints
New Protein NMR Technique
Distance Orientation (e- ?H)
Mainly Orientation (bond vector)
Distance (e- ? H)
Clore, Chem. Rev. 2009
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Residual Dipolar Couplings
Residual Dipolar Couplings (RDC) are measured as
contributions to lines splittings in anisotropic
solution relative to isotropic solution.
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RDC????
?????????????????????????,?????????(?????)???????
?J,??????????(???????)??????????????JD????D?????
?????? ?????Ecosy???????????????????
?????????3??? 1 ?????????????,??????2????????????
2 ??????,??????????????,????????5??????,????????
?????????,???????????,????????????????,???????????
? 3 ??????????????,?????????????????????
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Alignment is critical to observe RDCs
Alignment of a DNA strand with respect to the
static magnetic field, B0
Alignment of paramagnetic proteins relative to B0
B0
diagmagnetic
paragmagnetic
Weak alignment can be induced by the intrinsic
magnetic susceptibility anisotropy of the
molecules
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External alignment media
Bicelle
Bacteriophage Pf1
Orientation of the uniaxial alignment media is
described by a director
Purple Membrane
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• Alignment of the target molecules is dictated by
  the interactions between the molecules and the
  alignment media
• Better control over the alignment level
• Multiple aligning conditions

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Alignment mechanism
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???????RDC
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Measurements of RDCs Frequency based methods
(IPAP)
Residual Dipolar Couplings (RDC) are measured as
contributions to lines splittings in anisotropic
solution relative to isotropic solution.
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JHNC????????2D IPAP HNCO(Ottiger et al. 1998)
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JCaC????
ph00 ph11 ph21 3 ph33 ph80 0 0 0 2 2 2 2
ph301 1 1 1 3 3 3 3 ph100 0 2
2 ph110 ph142 ph150 ph210 ph9 0 0 0 0 0 0 0
0 1 1 1 1 1 1 1 1 ph310 2 2 0 2 0 0 2 2 0 0 2
0 2 2 0
????????,??t1???Ca180?????
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JN C???? 3D TROSY-HNCO ??1JN C????
?????,?????????????IP-AP 15N,1H-HSQC?????????,?
?????????,????TROSY?????????????????????????1JN
C?
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JHaHN,JCaHa???
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JCH??? 3D CB(CA)CONH??JCH??
???????????????,????t1?????????a,b,c??180?1H??????
t1???,JCH???????1??????1??????,??????a,b,c,??,2?1
?eff?????,???????????????????????,?????????JCH???(
Chou and Bax 2001)
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Alignment tensors
RDCs depends on the orientation of the
internuclear vector relative to the principal
axes system (PAS) of alignment
Determination of the molecular alignment requires
the knowledge of the molecular structures a priori
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The alignment tensor provides the basis for
interpretation of RDCs
Any single measured RDC (Dij) corresponds to a
continuum of possible bond orientations
AZZ(1)
For axially symmetric alignment, permissible
orientations will lie along the surface of a cone
with semi-angle ?
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??Alignment Tensor????Domain?????
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Multi-alignment RDCs are highly complementary
Underdetermined!
Ambiguity can be lifted by acquisition of RDCs
using two or more alignment media
Possible internuclear vector orientations
correspond to the intersection of cones.
A single measured RDC restraints possible vector
orientations to a cone
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Characterization of dynamics by RDCs
RDCs measured in five independent alignment media
Generalized order parameters, Motional asymmetries
Model free (Griesinger)
DIDC (Tolman)
RDCs
15N spin relaxation
Vector orientation is overdetermined
Peti, et. al. J. Am. Chem. Soc. 2002, 124,
5822-5833
Briggman, K. B. Tolman, J. R. J. Am. Chem. Soc.
2003, 125, 10164-10165
DIDC program Tolman, J. R. J. Am. Chem. Soc.
2002
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Pseuocontact ShiftPCS
PseudoContact Shifts (PCS)
Ln tags
-CYVDTNNDGAYEGDEL
Pintacuda etal. Acc Chem Res 2007,Su etal, JACS,
2008/2009
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PRE Applications
Spin label
Clore Chem Rev 2009 Tang Nature 2007
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Protein conformational exchange
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Chemical exchange saturation transfer (CEST)