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 rate.

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).