Chemistry Reference
In-Depth Information
of
15
N and
13
C even for small proteins. This will make full use of multidimensional
heteronuclear NMR experiments to increase the spectral resolution. Unlike other
proteins, expression of metalloproteins in a bacterial system usually requires
specific metal ions to be supplemented in the medium to induce overexpression
of the targeted proteins [
14
,
29
]. Alternatively, metal ions have to be incorporated
into the proteins after purification, particularly if the metal ions play structural
roles, as otherwise the proteins may not be stable for structural characterization. For
example, HypA from
Helicobacter pylori
precipitates easily in the absence of zinc
which serves a structural role [
15
]. However, caution has to be taken during metal
incorporation since excess metal ions may also cause protein aggregation.
A major bottleneck in solving protein structures by NMR is the highly peak-
picking and assignment of chemical shifts and NOEs. The strategy of the assignment
process and structure calculation can be found in an excellent review [
30
]. In general,
for a
15
N/
13
C-labeled protein, a series of double/triple resonance experiments are
recorded for resonance assignments. Backbone assignments are derived from HNCA,
HNCOCA, HNCACB, CBCACONH, HNCO, and HNCACO, whereas side-chain
protons and carbon atoms are assigned from HCCH-TOCSY, HCCH-COSY,
HBHACONH, C(CO)NH, and H(CCO)NH [
24
,
25
]. The chemical shifts of backbone
and side-chain are then used to assign NOEs (
15
N-/
13
C-HSQC-NOESY) to derive
inter-protein distance restraints. Usually the structure determination process goes
though several iterations of compiling a NOESY peak list, assignment of NOE
cross-peaks to sequence-specific interactions, structure generations and assessment,
refinement of NOESY peak lists, and reassignment of the cross-peaks, which can be
carried out automatically [
31
,
32
]. In addition to distance restraints, dihedral angle
restraints are usually obtained from several experiments, e.g., HNHA [
33
] or HNHB
[
34
], or predicated from TALOS, a program that empirically predicts backbone
angles (
) based on the chemical shifts of H
a
,C
a
,C
b
,C
0
,andN[
35
], as well as
the H-bond restraints derived from H-D exchange experiments. For elongated
macromolecules, residual dipolar couplings (RDC) as additional restraints are neces-
sary for structure determination. Information about RDC can be found in this topic. In
order to get relatively good quality for the structures, the numbers of NMR restraints
used for structural determination are usually of the order of 10-20 independent
interatomic distances per amino acid plus some dihedral restraints and as well as
atom-atom vector directions. The quality of calculated structures has to be evaluated
using programs PROCHECK, WHATIF, etc., and detailed description can be found
in a recent review [
13
].
In addition to the general strategies described above, metal-based NMR para-
meters are also of great help in the evaluation of structures of metalloproteins,
especially for those metalloproteins whose folding is highly metal-dependent. To
incorporate metal cluster constraints into structural calculation, residues that coordi-
nate to metal ions (e.g., Zn
2+
) must be identified first either by mutagenesis studies or
by physical characterization such as UV absorption spectroscopy, EXAFS, and NMR
spectroscopy [
36
-
38
]. Providing that metal coordination residues and geometries are
unveiled, metal cluster restraints can be obtained based on relevant crystal structures
of either macromolecules or small molecules. Usually, metal cluster restraints are not
f
,
C
