药物化学chap7h_RDC

Determination of the molecular alignment requires the knowledge of the molecular structures a priori
The alignment tensor provides the basis for interpretation of RDCs
1 2
2
1 1 sin 2 cos 2 2
For axially symmetric alignment, permissible orientations will lie along the surface of a cone with semi-angle θ
利用Alignment Tensor确定两个 Domain的相对取向
Bicelle Bacteriophage Pf1
Purple Membrane
Orientation of the uniaxial alignment media is described by a director
- - - - - - - - -
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
B0
χ0
diagmagnetic
χ0
paragmagnetic
W B χ B
Weak alignment can be induced by the intrinsic magnetic susceptibility anisotropy of the molecules
External alignment media
Long range HN-HN
NOE or RDC
Assessment of multi-aligning RDC datasets
Two datasets Three or more datasets
Linear independence of two RDC datasets can be established by observing their degree of correlation.
Any single measured RDC (Dij) corresponds to a continuum of possible bond orientations
AZZ(1)
D
res ij
0 i j h 2 3 Azz 4 2 rij
3 cos
Characterization of dynamics by RDCs
RDCs measured in five independent alignment media
Model free (Griesinger) DIDC (Tolman)
Generalized order parameters, Motional asymmetries
Pseuocontact Shift—PCS
PseudoContact Shifts (PCS)
Ln tags:
HO N O
SH
O
-
O N O O O Ln
-
O
O
-
O
-
O
N
N O OO
-
OH
O
-CYVDTNNDGAYEGDEL
Pintacuda etal. Acc Chem Res 2007,Su etal, JACS, 2008/2009
Alignment tensors
DNi H i k rN3 H
i i
3cos 2 i 1 2
RDCs depends on the orientation of the internuclear vector relative to the principal axes system (PAS) of alignment
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
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
Residual Dipolar Couplings (RDC) are measured as contributions to lines splittings in anisotropic solution relative to isotropic solution.
RDC核磁实验
残留的偶极耦合的测量是应用以下原理：在不去偶的前提下，先在各向同 性条件下（多为水溶液）测定标量耦合常数J，然后在各向异性条件下（即 弱定向介质中）测定标量耦合和偶极耦合的总和J+D；偶极耦合D由两种情 况的差值得到。 有两类实验：Ecosy类型测定谱峰的裂分；测定一对峰的强度比值
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
同前一个序列一样，只是t1期间 的Ca180度脉冲取消
JN C΄的测定: 3D TROSY-HNCO 定量1JN C΄相关谱
对于小蛋白，可以用前边所述的修饰版本的IP-AP [15N,1H]-HSQC实验；而 对于大蛋白质，由于谱峰堆积比较严重，最好使用TROSY版本的实验。从两 个隔行扫描的谱中的相对谱峰强度得到1JN C΄
Dot products are not sufficient to assess the linear dependency of three or more datasets. Singular value decomposition is an effective way to assess independence
生物大分子波谱学原理 吴季辉
New Protein NMR Technique
• Residual Dipolar Couplings (RDCs) • Pseudocontact Shifts (PCSs) • Paramagnetic Relaxation Enhancement
(PRE)
Residual Dipolar Couplings
JHaHN,JCaHa的测定
JCH的测定: 3D CB(CA)CONH定量JCH实验
实验应用隔行扫描的方式执行三次，分别是在t1演化期中的不同时期a,b,c使用180度 1H脉冲。在恒时t1演化期，J 的复相依赖于Δ1的延迟。当Δ 取不同的时间，也就是 CH 1 分别在a,b,c,点时，2Δ1+Δeff也就不一样，也就得到三个不同强度的谱。根据谱峰信 噪比的比较，就可以通过公式得到JCH的大小(Chou and Bax 2001)
使用不同定向介质的3个理由： 1 同一个耦合常数对应多个角度，所以 至少需要2个不同的介质的数据来区分； 2 在某些情况下，尽管有多个核间矢量 可以观测到，但是无法得到至少5个独立 的成份，这时采用不同的介质可以提供 新的约束，虽然新介质导入新的张量， 但是这个对于整个刚性分子是公共的， 所以还是可能补充新的约束； 3 介质可能会诱导某些构象的取向，但 是不太可能若干不同的介质会发生同样 的诱导；
Principal axes of ubiquitin alignment in different media
1: Phage Pf1 2: Helfrich phase 4-9: Purple Membrane
3: PEG 10: Bicelles
11: CTAB-doped bicelles
JHNC΄的测定:脉冲序列2D IPAP HNCO(Ottiger et al. 1998)
JCαC΄的测定
ph0=0 ph1=1 ph2=1 3 ph3=3 ph8=0 0 0 0 2 2 2 2 ph30=1 1 1 1 3 3 3 3 ph10=0 0 2 2 ph11=0 ph14=2 ph15=0 ph21=0 ph9= 0 0 0 0 0 0 0 0 1 1 1 11111 ph31=0 2 2 0 2 0 0 2 2 0 020220
Singular value decomposition of the RDC matrix D allows one to judge independence of the RDC data and to signal average across datasets. It is also the basis by which independent OLC-RDC datasets can be constructed