Abstract General
approaches for resonance assignments and structural analysis in uniformly
and differentially enriched proteins and protein assemblies by magic
angle spinning NMR spectroscopy are discussed using a 108-amino acid
residue E.coli thioredoxin as an example. It will be demonstrated
that a combination of multidimensional homo- and hetero-nuclear correlation
experiments yield nearly complete resonance assignments for the uniformly
enriched full-length protein as well as for two differentially enriched
non-covalent complexes formed by its complementary fragments. Secondary
and tertiary structure analysis is presented. The practical considerations
for high-resolution solid-state NMR studies of uniformly enriched
polypeptides are outlined, including sample preparation protocol,
instrumentation and pulse sequences. The potential of employing differential
labeling strategies for high-resolution structural studies of protein
interfaces and protein assemblies by solid-state NMR spectroscopy
is addressed. |