Misfolded protein sensor method
A catalytic conformational sensor method for detecting abnormal proteins and proteinaceous particles. The method is based on the interaction of a peptide fragment or probe with an abnormal proteinaceous particle. The interaction catalyzes the transformation of the probe to a predominately beta sheet conformation and allows the probe to bind the abnormal proteinaceous particle. This in turn, catalyzes the propagation of a signal associated with the test sample-bound probe. As a result signals can be propagated even from samples containing very low concentrations of abnormal proteinaceous particles. The peptide probes can be designed to bind to a desired peptide sequence or can even be based on dendrimer structure to control further aggregate propagation.
1. An in vitro method for detecting a target protein having a predominantly β-sheet secondary structure, comprising:
forming a mixture by adding a propagation catalyst to a sample suspected of containing a target protein having a predominantly β-sheet secondary structure, wherein the propagation catalyst is a peptide that:
(i) has a predominantly alpha-helix and/or random coil secondary structure and interacts with protein having a predominantly β-sheet secondary structure;
(ii) undergoes a conformational shift that results in a decrease in alpha-helix and/or random coil secondary structure and an increase in β-sheet secondary structure upon contact with protein having a predominantly β-sheet secondary structure or upon contact with another such propagation catalyst that has undergone such a conformational shift; and
(iii) is labeled with an optically detectable moiety;
allowing the propagation catalyst and any target protein present in the sample to interact; and
detecting any increase in β-sheet secondary structure in the mixture by detecting the optically detectable moiety, the increase being due, at least in part, to an increase in β-sheet secondary structure of the propagation catalyst, wherein any such increase indicates the presence of target protein in the sample.
2. The method of claim 1 , wherein the optically detectable moiety is a fluorophore.
3. The method of claim 2 , wherein the fluorophore is selected from the group consisting of tryoptophan, 1-anilino-8-napthalene sulfonate (ANS) and Congo Red stain.
4. The method of claim 1 , wherein the peptide is labeled with an optically detectable moiety at each of its N-terminus and its C-terminus.
5. The method of claim 4 , wherein the optically detectable moieties at each of the N-terminus and C-terminus have the capacity to interact to form excimers.
6. The method of claim 5 , wherein, when the propagation catalyst undergoes said conformational shift, interaction between the optically detectable moieties at each of the N-terminus and C-terminus of the peptide probe results in excimer formation.
7. The method of claim 4 , wherein the optically detectable moieties at each of the N-terminus and C-terminus comprise a fluorescence energy transfer (FRET) pair.
8. The method of claim 7 , wherein, when the propagation catalyst undergoes said conformational shift, interaction between the optically detectable moieties at each of the N-terminus and C-terminus of the peptide probe results in fluorescence energy transfer (FRET).
9. The method of claim 4 , wherein the optically detectable moieties at each of the N-terminus and C-terminus comprise a fluorophore/quencher pair.
10. The method of claim 9 , wherein, when the propagation catalyst undergoes said conformational shift, interaction between the optically detectable moieties at each of the N-terminus and C-terminus of the peptide probe results in quenching of the fluorescence of the fluorophore.
11. The method of claim 4 , further comprising adjusting a reaction condition to increase or decrease interactions between the optically detectable moieties at each of the N-terminus and C-terminus.
12. The method of claim 11 , wherein the reaction condition is selected from the group consisting of: ionic strength of the sample, pH of the sample, concentration of the sample, temperature, and the presence or absence of nucleating agents.
13. The method of claim 4 , further comprising modifying the amino acid sequence of the propagation catalyst to increase or decrease interactions between the optically detectable moieties at each of the N-terminus and C-terminus.
14. The method of claim 1 , wherein the method further comprises, prior to the step of adding the propagation catalyst to the sample, the step of subjecting the sample to a disaggregation technique.
15. The method of claim 1 , wherein the detecting step comprises detecting aggregates comprising the propagation catalyst.
16. The method of claim 1 , wherein the target protein is associated with a disease.
17. The method of claim 16 , wherein the disease is selected from the group consisting of Alzheimer's Disease, Huntingon's Disease, and prion-associated diseases.
18. The method of claim 13 , wherein the target protein is selected from the group consisting of Aβ protein, huntingtin protein, transmissible spongiform, and prion proteins.
19. The method of claim 1 , wherein the sample comprises a biological sample from a subject.
20. The method of claim 1 , wherein the sample comprises a biological sample from a living subject.
21. The method of claim 1 , wherein the sample comprises a biological sample from a human subject.
22. The method of claim 1 , wherein the sample comprises blood, lymph, CSF, or tissue.
23. The method of claim 1 , wherein the sample comprises a biological sample and a solvent.