Detecting misfolded proteins
Improperly folded proteins
cause debilitating illnesses such as Parkinson’s, Alzheimer’s and Huntington’s
diseases as well as prion diseases. Biological imaging and detection methods can
enable scientists to fully understand the structures of misfolded proteins and the
mechanisms underlying protein misfolding.
Vladimir N. Uversky at Indiana University
School of Medicine in Indianapolis and his colleagues at the University of Nebraska
Medical Center in Omaha have reviewed methods for studying protein misfolding disorders.
Since misfolded protein aggregates
were discovered by light microscopy, various techniques have advanced collective
knowledge of them. Polarization light microscopy showed that these aggregates have
an ordered submicroscopic structure; cryoelectron microscopy suggested that they
consist of several fibrils; and atomic force microscopy (AFM) showed that they consist
of at least three fibrils. On the basis of x-ray diffraction data, it was proposed
that the fibrils form a water-filled nanotube. Total internal reflection fluorescence
microscopy showed that fibril growth occurs in a cooperative process and at a constant
Sol-gel encapsulation can separate
individual proteins for single-molecule studies using various spectroscopic techniques,
but imaging with Förster resonance energy transfer (FRET) provides the highest
resolution. The method, for example, has demonstrated that the aggregation and fibrillation
of α-syn-uclein, a protein involved in Parkinson’s disease, requires
several oligomers and involves competing pathways. Three-color FRET, a more recent
development, can provide additional structural information.
AFM enables continuous monitoring of
the self-assembly of misfolded protein fibrils and reveals single-molecule interactions.
Performing the technique in tapping mode showed how fibrils self-assemble. Use of
the method also led to the discovery that the interprotein interactions are stronger
among misfolded proteins than among native proteins, and the strength of the interactions
promotes the formation of aggregates.
Because AFM can detect an increase
in protein interaction before macroscopic protein aggregation occurs, the reviewers
believe that this technique can be used as a novel diagnostic tool.
Adding lipophilic coatings to biological
labels can allow the molecules to cross the blood-brain barrier, and conjugating
them to antibodies can enable binding to target molecules. The authors remarked
that these modifications could provide diagnostic information for Parkinson’s
disease, whereas current assessments of the affliction only exclude other conditions.
(Journal of Proteome Research, October 2006, pp. 2505-2522).
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