Anisotropy resolved multidimensional emission spectroscopy (ARMES): A new tool for protein analysis

Area of Science:

• Biophysics
• Analytical Chemistry
• Protein Science

Background:

• Protein structural analysis via intrinsic fluorescence is hindered by overlapping emission spectra.
• Distinguishing signals from multiple fluorophores within proteins presents a significant challenge.

Purpose of the Study:

• To demonstrate the utility of Anisotropy Resolved Multidimensional Emission Spectroscopy (ARMES) for resolving complex protein fluorescence.
• To analyze the chemical and thermal denaturation of human serum albumin (HSA) using ARMES.

Main Methods:

• Anisotropy Resolved Multidimensional Emission Spectroscopy (ARMES) combined with chemometric analysis.
• Multivariate Curve Resolution (MCR) applied to polarized emission data.
• Total Synchronous Fluorescence Scan (TSFS) for comprehensive fluorophore information.

Main Results:

• ARMES successfully resolved contributions from four distinct emitters in HSA: tryptophan (Trp) fluorescence, solvent-exposed tyrosine (Tyr) fluorescence, Tyr in a hydrophobic environment, and Trp room temperature phosphorescence (RTP).
• MCR analysis, anisotropy data, and literature validation confirmed the assignments of these emitters.
• The study observed expected transitions during HSA unfolding, validating the ARMES methodology.

Conclusions:

• ARMES provides a powerful new methodology for comprehensive intrinsic fluorescence analysis of proteins.
• This technique is applicable to any protein containing multiple intrinsic fluorophores, overcoming spectral overlap issues.
• ARMES offers detailed insights into protein structure, environment, and conformational changes during denaturation.