NMR Studies of Metalloproteins - NMR of Proteins and Small Biomolecules - page 69

of 81.4 kDa were achieved [ 16 ]. Recently, a few breakthroughs have been made in
this field. This includes the first NMR structure of a seven-helix transmembrane
protein determined in membrane-mimetic environments [ 17 ] and the first structure
determined in living cells by in-cell NMR [ 18 ]. Apart from its application in structural
studies, NMR spectroscopy is also able to monitor the internal motion of biomolecules
ranging over times from subnanoseconds to beyond seconds. Characterization of
dynamics of biomolecules, such as folding transition, will be a great help for our
understanding of the biological function of biomolecules.
Application of NMR spectroscopy in studies of metalloproteins is in principle
the same as other proteins if the proteins contain diamagnetic metal ions. In the
case of paramagnetic metalloproteins, things become more challenging since para-
magnetic metals affect longitudinal and transverse relaxation rates of observed
nuclei. However, recent advances in the hardware and methodology have enable
structures of such metalloproteins to be determined, these advances including
13 C-detected experiments, solid-state NMR, and the discovery of paramagnetic
relaxation enhancement (PRE) [ 19 - 21 ]. In this review, we will address mainly
the application of NMR spectroscopy in studies of metalloproteins including the
contribution of NMR to structural characterization of metalloproteins, and special
attention will be devoted to the utilization of NMR in characterization/identification
of the metal binding sites and their coordination environments as well as to probing
conformational changes of metalloproteins upon metal binding and release. The
techniques used for paramagnetic metalloproteins will also be included briefly since
a number of reviews in this field can be found [ 19 - 22 ]. A systematic review of the
application of metal NMR spectroscopy will not be made and interested readers are
directed elsewhere [ 23 ].
2 The Contribution of NMR to Structural Metalloproteins
2.1 Conventional Method for Structure Determination
of Metalloproteins
NMR spectroscopy can be applied to characterize structurally diamagnetic metal
containing metalloproteins similar to other proteins. Amongst the structures deposited
in the Brookhaven Protein Data Bank (PDB), ca. 15% of the structures were resolved
by NMR. Here, we will give a brief summary since the detailed methodology can be
found in numerous references [ 11 , 24 - 26 ].
In structural studies of proteins/metalloproteins, concentrations of about 1 mM
are typically required and proteins must be soluble and stable over a period of time
(weeks). For small proteins with several tens of amino acids, e.g. metallothionine
[ 27 , 28 ], it is sufficient to use 15 N-labeled samples to determine structures of the
proteins. However, if proteins can be overexpressed in a bacterial system (e.g.,
Escherichia coli ), it is desirable to overexpress the protein with uniform enrichment