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Updated: Aug 13, 2025
Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy
Published on: June 27, 2014
Probing protein misfolding and dissociation with an infrared free-electron laser
Hisashi Okumura1, Takayasu Kawasaki2, Kazuhiro Nakamura3
1Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Okazaki, Aichi, Japan; Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki, Aichi, Japan; Department of Structural Molecular Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi, Japan.
Abstract:
Misfolding is observed in the mutant proteins that are causative for neurodegenerative disorders such as polyglutamine diseases. These proteins are prone to aggregate in the cytoplasm and nucleus of cells. To reproduce cells with the aggregated proteins, gene expression system is usually applied, in which the expression construct having the mutated DNA sequence of the interest is transfected into cells. The transfected DNA is finally converted into the mutant protein, which is gradually aggregated in the cells. In addition, a simple method to prepare the cells having aggregates inside has been recently applied. Peptides were first aggregated by incubating them in water. The aggregates are spontaneously taken up by cells because aggregated proteins generally transfer between cells. Peptides with different degrees of aggregation can be made by changing the incubation times and temperatures, which enables to examine contribution of aggregation to the toxicity to the recipient cells. Moreover, such cells can be used for therapeutic researches of diseases in which aggregates are involved. In this chapter, we show methods to induce aggregation of peptides. The functional analyses of the cells with aggregates are also described. Then, experimental dissociation of the aggregates produced using this method by mid infrared free electron laser irradiation and its theoretical support by molecular dynamics simulation are introduced as the therapeutic research for neurodegenerative disorders.
Insights
Researchers developed a new method to induce protein aggregation in cells for studying neurodegenerative diseases. This technique allows for controlled aggregation and potential therapeutic research into diseases like polyglutamine disorders.
Area of Science:
- Biochemistry
- Cell Biology
- Neuroscience
Background:
- Protein misfolding and aggregation are hallmarks of neurodegenerative disorders, including polyglutamine diseases.
- Aggregated proteins accumulate in cellular cytoplasm and nucleus, contributing to cellular dysfunction.
- Traditional methods to study aggregated proteins involve gene expression systems and DNA transfection.
Purpose of the Study:
- To present a simple and effective method for inducing peptide aggregation within cells.
- To explore the functional analysis of cells containing these induced aggregates.
- To investigate therapeutic strategies for neurodegenerative disorders involving protein aggregates.
Main Methods:
- Peptides are aggregated by incubation in water, with aggregation degree controlled by time and temperature.
- Aggregated peptides are spontaneously internalized by cells.
- Mid-infrared free electron laser irradiation is used for experimental dissociation of aggregates.
- Molecular dynamics simulations provide theoretical support for aggregate dissociation.
Main Results:
- The method allows for the creation of cells with intracellular protein aggregates.
- Varying aggregation conditions enables the study of aggregation's contribution to cellular toxicity.
- Successful dissociation of aggregates using free electron laser irradiation was demonstrated.
- Molecular dynamics simulations supported the experimental findings on aggregate dissociation.
Conclusions:
- A novel method for inducing and studying protein aggregation in cells has been established.
- This approach facilitates research into the mechanisms of neurodegenerative diseases.
- The findings support the potential of targeted physical methods, like laser irradiation, for therapeutic interventions.
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