Biomedical Engineering Reference
In-Depth Information
inhibitor could be detected. Selective labelling of amino acid residues can be a
powerful alternative to uniform labelling that can enable sequential assignment
of even very large proteins. In one particular example, the 723-residue Malate
Synthase was labelled uniformly along the backbone while the side-chains of
isoleucine, leucine and valine residues were selectively labelled with a linear
pattern of
13
C,
2
H where the terminal methyl groups were additionally
protonated.
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Such a scheme can enable through-bond assignment of the
backbone and nearly complete assignment of the methyl resonances. Since
methyl-containing residues are typically well distributed at ligand-binding
sites,
4
the method of Tugarinov and co-workers could be used to determine
structures of even very large ligand-protein complexes in an efficient manner.
Nonetheless, through-bond assignment techniques are insensitive and time-
demanding for large proteins. Direct assignment of selectively labelled methyl
resonances could make the approach highly efficient. Very recently, the group
of Clore
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has used paramagnetic relaxation enhancement (PRE) to achieve
direct, stereospecific assignment of methyl resonances of the 27 kDa N-
terminal domain of the E. coli protein Enzyme 1. Five different site-specific
cysteine mutants of the protein were used to attach nitroxide-radical-
containing tags. The distance-dependent PRE rate was measured for each
methyl using a simple HMQC experiment and the information was used in a
Metropolis Monte Carlo calculation to determine the assignments. The PRE
assignments were then used to compare experimental vs. predicted methyl-
methyl NOEs for validation purposes. While the technique clearly requires the
generation of multiple cysteine mutants (the authors suggest one mutant per 6
kDa of protein), where feasible it should lead to rapid resonance assignment.
In many cases the structure of a protein may be known but obtaining
crystals with a ligand bound, particularly the weak binding ligands typical of
FBDD, may not be possible. Paramagnetic tags provide one attractive
approach to solving this issue. The group of Otting
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first showed that it is
possible to calculate the structure of a ligand in rapid exchange with a protein
to which a lanthanide ion is bound at an intrinsic metal-binding site. If the
orientation of the paramagnetic tensor is known with respect to the protein
coordinates, then both distance and angular information can be derived from
the magnitude of the PCS. A more recent example of the approach applied to a
protein in which a lanthanide-binding tag has been fused to the protein was
provided in the section on paramagnetism for ligand discovery.
18
The
additional example of a non-covalently bound peptide tag was also provided.
17
These methods are particularly exciting as once the tagged protein is available
and the tensor orientation determined, structure constraints are rapidly
determined by titrating the ligand or tag and acquiring simple 1D
1
H spectra of
the ligand. In principle then, the methods should be quite efficient.
While X-ray crystallography remains the structural method of choice when
available, it may have shortcomings. A recent example is provided by the
structure of the growth factor receptor-bound protein 2 (Grb2) SH2 domain.
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The crystal structure consisted of a domain-swapped dimer to which inhibitors
