New Protein NMR Technique

• Residual Dipolar Couplings (RDCs)

• Pseudocontact Shifts (PCSs)

• Paramagnetic Relaxation Enhancement

(PRE)

生物大分子波谱学原理吴季辉

Residual Dipolar Couplings

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

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；

Alignment is critical to observe RDCs

Alignment of a DNA strand with respect to the static magnetic field, B0

Weak alignment can be induced by the intrinsic magnetic susceptibility anisotropy of the molecules

W B χ B

0χ

Alignment of paramagnetic proteins relative to B0

0χ

B0

diagmagnetic paragmagnetic

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Bicelle Bacteriophage Pf1

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

External alignment media

Purple Membrane

Orientation of the uniaxial alignment media is described by a director

Alignment mechanism

通常可以测定的 RDC

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.

JHNC΄ 的测定 : 脉冲序列 2D IPAP HNCO(Ottiger et al. 1998)

JCαC΄ 的测定 ph0=0ph1=1ph2=1 3ph3=3ph8=0 0 0 0 2 2 2 2 ph30=1 1 1 1 3 3 3 3 ph10=0 0 2 2ph11=0ph14=2ph15=0ph21=0ph9= 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 ph31=0 2 2 0 2 0 0 2 2 0 0 2 0 2 2 0

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΄

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， JCH 的复相依赖于 Δ1 的延迟。当 Δ1 取不同的时间，也就是分别在 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)

Determination of the molecular alignment requires the knowledge of the molecular structures a priori

2

3

3cos 1

2i i

N Hi i

iN H

kD

r

RDCs depends on the orientation of the internuclear vector relative to the principal axes system (PAS) of alignment

Alignment tensors

AZZ(1)

For axially symmetric alignment, permissible orientations will lie along the surface of a cone with semi-angle θ

Any single measured RDC (Dij) corresponds to a continuum of possible

bond orientations

2cossin1cos324

D 2212

21

320res

ij

zz

ij

ji Ar

h

The alignment tensor provides the basis for interpretation of RDCs

利用 Alignment Tensor 确定两个Domain 的相对取向

A single measured RDC restraintspossible vector orientations to a cone

Possible internuclear vector orientations correspond to the intersection of cones.

Ambiguity can be lifted by acquisition of RDCs using two or more alignment media

Multi-alignment RDCs are highly complementary

Underdetermined!

DIDC program: Tolman, J. R.; J. Am. Chem. Soc. 2002Briggman, K. B.; Tolman, J. R.; J. Am. Chem. Soc. 2003, 125, 10164-10165

Characterization of dynamics by RDCs

Generalized order parameters,Motional asymmetries

RDCs measured in five independent alignment media

Model free (Griesinger)

DIDC (Tolman)

Vector orientation is overdetermined

Peti, et. al.; J. Am. Chem. Soc. 2002, 124, 5822-5833

RDCs

15N spin relaxation

RDC based structure determination

Long range HN-HN

NOE or RDC

Pros:

Only require a minimum set of 2D experiments

Simplify spectrum interpretation

No side chain assignment

Structure and Dynamic studies simultaneously

Assessment of multi-aligning RDC datasets

Linear independence of two RDC datasets can be established by observing their degree of correlation.

Two datasets Three or more datasets

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

Singular value decomposition of the RDC data

SVD of the data 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

Principal axes of ubiquitin alignment in different media

1: Phage Pf1 2: Helfrich phase 3: PEG 4-9: Purple Membrane 10: Bicelles 11: CTAB-doped bicelles

PseudoContact Shifts (PCS)

Pseuocontact Shift—PCS

Pintacuda etal. Acc Chem Res 2007,Su etal, JACS, 2008/2009

Ln tags:

-CYVDTNNDGAYEGDEL

NO

OHO

OH

SH

NO

O-O

O-

N

O O-

O

O-

N

O

O-

O O-

Ln