Protein aggregation probed by two-photon fluorescence correlation spectroscopy of native tryptophan

Area of Science:

• Biophysics
• Biochemistry
• Spectroscopy

Background:

• Fluorescence correlation spectroscopy (FCS) is valuable for studying protein aggregation.
• A key limitation of FCS is the need for fluorescent labeling of proteins.
• Intrinsic protein fluorescence offers a potential alternative to exogenous labeling.

Purpose of the Study:

• To develop and validate a label-free FCS method using intrinsic tryptophan fluorescence for protein aggregation studies.
• To investigate the aggregation of tryptophan-containing proteins using two-photon excitation.
• To assess the utility of fluorescence lifetime measurements as a complementary parameter in aggregation studies.

Main Methods:

• Utilizing intrinsic tryptophan fluorescence excited by two-photon spectroscopy.
• Applying fluorescence correlation spectroscopy (FCS) to monitor protein dynamics.
• Measuring tryptophan fluorescence lifetime in parallel with FCS.

Main Results:

• Demonstrated effective use of intrinsic tryptophan fluorescence for label-free FCS analysis of protein aggregation.
• Observed clear aggregation signatures in barstar protein at pH 3.5 using two-photon tryptophan FCS.
• Obtained complementary information on the aggregation process through parallel tryptophan fluorescence lifetime analysis.

Conclusions:

• Intrinsic tryptophan fluorescence with two-photon excitation provides a powerful label-free approach for FCS studies of protein aggregation.
• This method is capable of probing soluble aggregates of unmodified proteins.
• The technique offers significant potential for understanding the role of soluble aggregates in diseases like amyloid toxicity.