RESEARCH
RESEARCH
One particular area where solid state magic angle spinning (MAS) NMR is making substantial progress is the area of structure determination on proteins. Progress in both sample preparation and spectroscopic techniques is allowing for better and faster structural measurements. One productive approach is focussed on the preparation of nano- and microcrystals. These tend to be very well ordered, yielding excellent NMR data. At the same time the size requirements for SSNMR are much less demanding than for traditional X-ray crystallographic methods. Other immobilized or aggregated forms of proteins can also be studied, whether through association with membranes, or in amyloid fibrils.
The study of amyloid structure and formation is of particular interest, both for its medical significance (being implicated in diseases ranging from Alzheimer's to mad cow disease) and for its more basic contribution to our understanding of protein folding and misfolding. We willapply MAS SSNMR structural methods to examine the structural details of amyloid fibril formation.
As an illustration of the capabilities of SSNMR in this area:
here are some results from my postdoc
research at MIT. The GNNQQNY7-13
peptide
fragment of the yeast prion protein Sup35p forms both
(nano)crystals and
amyloid-like fibrils. Nelson et al. have determined the crystal
structure of its crystals, which have
been
proposed to reflect specific structural features common of amyloid
fibrils. Using MAS SSNMR we were able
to directly compare two different crystal forms and a number of
co-existing fibril forms via MAS SSNMR measurements[3].
(See also an earlier publication on DNP enhancement of the
crystalline material: [2])
The figures above show both transmission electron microscopy data on the crystals and fibrils formed by this peptide (a). Note that even the crystals are very small. The panels to the right (b) shows the (color coded) resonance positions in a two-dimensional solid state MAS NMR experiment on both types of samples. This particluar experiment (N-CO) shows the connectivities between directly bonded nitrogens and carbonyl carbons (which includes both the peptide bond and Gln/Asn side chains). These figures show the results for a segmentally labeled peptide GNNQQNY (underlined = 13C,15N-labeled). The relatively narrow peaks in the NMR data indicate that the crystals and fibrils have very homogeneous and well-defined structures. Interestingly each site reproducibly gives three signals indicating the presence of three distinct structural forms within the fibril samples. (for more details see [2])
Despite these structural variations between crystals and fibrils, and even within the fibril structural forms, the results do appear to support the idea of a common structural core, made up of an interdigitated 'steric zipper' involving the Gln and Asn side chains, but also highlighted a much increased structural and dynamical complexity in the fibrils. Clearly the direct structural characaterization of amyloid fibrils by MAS solid state NMR is essential to develop a thorough understanding of their characteristic features.