Biomedical Engineering Reference
In-Depth Information
methyl groups in highly perdeuterated proteins. Clearly, even using these
approaches, it is not (yet) possible to elucidate global 3D structures of proteins
of this size. However, as shown in Table 1.2, solution NMR spectroscopy has
been successfully used to characterise function, local structure and dynamics in
a wide range of complicated supramolecular protein systems.
The methyl-labelling schemes described in this chapter are versatile and
robust, and their application is becoming more and more widespread.
Furthermore, the prices of isotope-labelled metabolic precursors have
decreased considerably in the last 10 years. One of the great advantages of
these protocols is the possibility of performing combinatorial labellingin
which more than one amino acid type is labelled at one time. For example, as
outlined in Table 1.2, there are many reports of simultaneous isotope-labelling
of leucine, valine and isoleucine d
1
-methyl groups in the same protein.
Despite many important advances in methyl labelling of large proteins,
much work remains to be done. One current limitation concerns leucine and
valine and the inability to selectively label one of these amino acids but not the
other. Leucine and valine are commonly found in proteins and both have two
methyl groups. Therefore, even when stereospecific labelling is employed, the
high number of observable signals can cause considerable spectral overlap.
Isotope-labelling of both leucine and valine, e.g.,witha-ketoisovalerate or
acetolactate, also limits the maximum overall level of deuteration (and
therefore the experimental sensitivity) that can be achieved. Thus, labelling
strategies that target one of these two amino acids not the other would be very
welcome.
Threonine remains the only methyl-group containing amino acid for which a
suitable labelling strategy does not currently exist. Threonine containstwo
chiral centres and consequently schemes for the synthesis of [a,b-
2
H,
c
2
-
13
C
1
H
3
]-labelled samples are complicated and expensive. Using [
13
C,
15
N]-
labelled threonine is a possibility, but as the size of the target system increases,
deuteration of the C
a
and C
b
sites becomes more and more necessary.
Furthermore, threonine is a component of several amino acid biosynthesis
pathways (e.g., isoleucine, Figure 1.4), and thus direct addition to minimal
media would result in detrimental levels of isotope scrambling. A viable
precursor-based strategy for specific labelling of threonine methyl groups has
yet to be described.
Methyl-labelling strategies and the labelling patterns achievable using
metabolic precursors are also offering tremendous opportunities for solid-
state NMR spectroscopy of proteins. Recent examples include analysis of the
relaxation properties of methyl groups,
50,51
and the measurement of proton-
proton distance restraints for structure calculations.
52
The use of methyl-
specific isotope-labelling has not been limited to solution NMR studies of large
proteins. There have been several reports detailing the use of metabolic
precursors for the characterisation of smaller proteins. For example, methyl-
specific labelling has been successfully used in the analysis of biomolecular
interaction surfaces via chemical shift mapping
53,54
or the production of
