Biomedical Engineering Reference
In-Depth Information
The problem of low
1
H signal overlap has now been largely overcome
through the preparation of protein samples enriched with low natural
abundance, spin-K isotopes of carbon and/or nitrogen.
1
Many NMR
experiments have since been written that utilise the large signal dispersion of
13
Cor
15
N nuclei to separate the signals of scalar-coupled nuclei over multiple
dimensions.
2,3
Furthermore, in addition to resolving spectral congestion,
isotope enrichment introduces more NMR-visible probes into the moleculesof
interest and allows a multitude of structural and dynamic information to be
accessed from their NMR signals.
Isotopic enrichment of proteins can take two forms: uniform or selective. In
the most commonly used approach the recombinant target protein is over-
expressed from E. coli grown in an isotopically enriched minimal-expression
medium containing uniformly labelled [
13
C]glucose and/or [
15
N]ammonium
chloride or sulphate, as the only carbon and nitrogen sources. The resulting
protein product is isotopically enriched at the same level as the expression
medium. Uniform labelling approaches were developed towards the end of the
1980s (ref. 4) and since have become routine and robust. In the last 20 years,
the price of isotopically enriched reagents has decreased considerably making
uniform labelling a common practice in structural biology laboratories.
Isotope-labelling of individual amino acids or groups of amino acids can
also be performed. Residue-specific isotope labelling is achieved by supple-
menting the expression medium with isotopically enriched amino acids.
5
This
approach is somewhat limited in vivo as a result of the scrambling of the
isotope-labelled sites by bacterial metabolic pathways. As an alternative,
isotope-labelled amino acids can be used in combination with cell-free in vitro
expression systems, which essentially alleviate isotopic dilution.
6,7
1.1.2 General Considerations for NMR Studies of Larger
Proteins
Over the past 20 years an enormous array of multi-dimensional heteronuclear
NMR experiments have been designed that can extract structural or dynamic
information about isotopically enriched proteins.
2
The strategy of combining
isotope-labelling with tailored NMR experiments has been so successful that it
has encouraged NMR spectroscopists to study larger and more complicated
biomolecular systems. However, as the size of protein targets increases new
problems arise.
The lifetime of the excited state in NMR spectroscopy is predominantly
affected by the overall molecular tumbling rate. As molecular size increases the
tumbling rate slows and this leads to an increase in the rate at which transverse
magnetisation relaxes. As the linewidth of an NMR signal is proportional to
the transverse relaxation rate, NMR spectra of larger molecules which tumble
more slowly are characterised by broad NMR signals.
The short lifetime of transverse relaxation in large proteins severely affects
the sensitivity, effectiveness and scope of NMR experiments. NMR pulse
