Biomedical Engineering Reference
In-Depth Information
sequences frequently rely on scalar couplings to transfer magnetisation
between nuclei of interest. Such transfer steps require periods in which nuclear
magnetisation is the transverse plane and therefore subject to transverse
relaxation. Thus, complicated pulse sequences that correlate nuclei via weak
scalar couplings or that require multiple transfers mediated by scalar couplings
become less effective and less sensitive for larger proteins.
Resonance assignment of proton, carbon and nitrogen nuclei in the
polypeptide backbone is a critical first step in many NMR studies of protein
structure, dynamics or interactions. A common starting point is a two-
dimensional (2D) ( 1 H, 15 N) heteronuclear correlation spectrum acquired, for
example, using the Heteronuclear Single Quantum Coherence (HSQC)
experiment. An in-depth assessment of NMR data requires being able to
locate each NH cross-peak to a unique site in the target protein. This is
achieved by determining sequence-specific resonance assignments. There are
numerous experimental strategies that facilitate backbone resonance assign-
ment, many of which make use of uniform isotope-labelling strategies and
multi-dimensional heteronuclear NMR experiments. While these approaches
work well for smaller proteins (,25 kDa; Figure 1.1), they cease to be
applicable when the molecular weight increases as the transverse magnetisation
relaxes more rapidly.
Figure 1.1
Large proteins and protein complexes studied by NMR spectroscopy with
the aid of methyl-specific labelling. Surface representations of the three-
dimensional structures of proteins discussed in the text plotted as a
function of their complex molecular weight. PDB codes used: ubiquitin,
1ubq; maltose binding protein, (MBP), 1dmb; malate synthase G (MSG),
1p7t; SecA, 2vda; TET2, 2wzn; proteasome, 3okj; proteasome-activator
complex, 1z7q. Molecular weight ranges to which different
isotope-
labelling strategies are suited are indicated at the bottom.
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