Chemistry Reference
In-Depth Information
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
2 Basic Experimental Techniques Used in Solid-State NMR ............................... 189
2.1 Magic-Angle Spinning ................................................................ 189
2.2 High Power Heteronuclear Decoupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
2.3 Cross-Polarization ..................................................................... 190
2.4 Recoupling in MAS Solid-State NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
3 Protein Structure Determination by MAS Solid-State NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
3.1 Labeling Strategy and Sample Environments ........................................ 191
3.2 Three-Dimensional Structure Determination ........................................ 194
3.3 Ligand Conformation and Binding . . . ................................................ 196
3.4 Structural Changes upon Activation ................................................. 199
3.5 Protein Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
3.6 Sensitivity Enhancement .............................................................. 203
3.7 Structure Determination Based on Chemical Shifts ................................. 204
4 Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
1
Introduction
Membrane proteins are a large, diverse group of proteins, representing about
20-30% of the proteomes of most organisms, serving a multitude of cellular
functions, and more than 40% of drug targets [
1
]. For example, membrane proteins
often serve as receptors or provide channels for polar or charged molecules to pass
through the cell membrane and maintain a variety of biological processes [
2
].
Knowledge of a membrane protein structure enables us insight into its function
and dynamics, and can be used for further rational drug design. Therefore it is
always desirable to have an accurate picture of protein structure in the highest
resolution possible. However, owing to their intrinsic hydrophobicity, flexibility,
and instability, many fewer structures have been solved by X-ray crystallography
for membrane proteins compared to soluble proteins [
3
,
4
]. Moreover, the confor-
mational change of the transmembrane helices upon activation increase the diffi-
culty of capturing the activation state of a membrane protein to a higher resolution
by X-ray crystallography [
5
,
6
]. In contrast, solid-state NMR (SSNMR) is a suitable
technique to study molecular structure and interactions at atomic level in a variety
of sample forms; it can be used to determine a membrane protein structure and
probe its conformational dynamics in the native membrane environments.
Over the past several years, SSNMR has made tremendous progress, showing its
capability of determining membrane protein structure, ligand binding, and protein
dynamic conformation on a variety of time scales at atomic resolution. Many
membrane proteins have been investigated by magic-angle spinning (MAS)
SSNMR, factors investigated including the following: activation, inhibition, and
dynamics of the potassium channel KcsA-Kv1.3 [
7
-
11
]; structure, ligand confor-
mation, activation, and dynamics of the G protein coupled receptor - rhodopsin
[
12
-
25
]; protonation switch mechanism of the human H1 receptor[
26
];
the
