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Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy
Published on: September 12, 2019
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Label-Free Analysis of Protein Aggregation and Phase Behavior
Zenon Toprakcioglu1, Pavankumar Challa1, Catherine Xu1
1Department of Chemistry , University of Cambridge , Lensfield Road , CB2 1EW , Cambridge , U.K.
ACS Nano
|November 19, 2019
Summary
Monitoring protein phase transitions, like amyloid fibril formation, can now be done using intrinsic tryptophan autofluorescence. This label-free method accurately tracks protein self-assembly and distinguishes various solid phases without external dyes.
Area of Science:
- Biophysics
- Biochemistry
- Materials Science
Background:
- Protein phase transitions, from soluble monomers to solid aggregates like amyloid fibrils, are crucial for biological functions and diseases.
- Current methods for monitoring protein aggregation often require extrinsic dyes that can interfere with the natural process.
- Proteins possess intrinsic fluorescence due to aromatic amino acids, offering a potential label-free detection mechanism.
Purpose of the Study:
- To demonstrate the utility of intrinsic tryptophan autofluorescence for monitoring protein self-assembly and phase transitions.
- To develop a label-free method for imaging protein nanofibrils using intrinsic fluorescence.
- To assess the correlation between intrinsic fluorescence microscopy and traditional dye-based methods.
Main Methods:
- Measurement of tryptophan autofluorescence in space and time for reconstituted silk fibroin, β-lactoglobulin, and lysozyme during fibrillar self-assembly.
- Utilizing a droplet-microfluidic approach to spatially confine protein self-assembly.
- Label-free imaging of protein nanofibrils via intrinsic fluorescence microscopy.
Main Results:
- Tryptophan autofluorescence increases as hydrophobic burial occurs during fibrillar self-assembly, indicating successful monitoring.
- Intrinsic fluorescence microscopy successfully images protein nanofibrils in a label-free manner.
- Microstructural analysis from intrinsic fluorescence microscopy correlates well with extrinsic dye-based methods.
- Protein autofluorescence can differentiate between various solid phases, including β-sheet-rich structures, spherulites, and crystals.
Conclusions:
- Intrinsic tryptophan autofluorescence provides an accurate, label-free method for monitoring protein self-assembly and phase transitions.
- The droplet-microfluidic approach combined with intrinsic fluorescence enables precise imaging and characterization of protein aggregates.
- This technique offers a versatile tool for studying diverse protein phase transition phenomena without perturbing the system with extrinsic labels.
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