[Paper Review] A new twist on PIFE: photoisomerisation-related fluorescence enhancement

Evelyn Ploetz, Benjamin Ambrose+13 more|arXiv (Cornell University)|Feb 24, 2023

Photochromic and Fluorescence ChemistryMaterials Science113 references3 citations

TL;DR

This paper proposes renaming PIFE (Protein-Induced Fluorescence Enhancement) to photoisomerisation-related fluorescence enhancement to reflect its fundamental mechanism: modulation of cyanine fluorophore fluorescence via changes in cis/trans photoisomerisation kinetics upon biomolecular binding. The authors establish that this mechanism applies broadly across biomolecular interactions, enabling quantitative, label-free detection of conformational changes, protein-ligand, and protein-protein interactions with high sensitivity.

ABSTRACT

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate of cis/trans photoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule and, in this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turn PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

Motivation & Objective

• To reframe PIFE based on its underlying photochemical mechanism rather than its initial biological context.
• To establish that fluorescence enhancement in cyanine dyes arises from altered photoisomerisation rates upon biomolecular binding.
• To promote PIFE as a quantitative assay for studying biomolecular interactions beyond proteins.
• To expand the applicability of PIFE to protein-ligand, protein-protein, and conformational change studies.
• To provide a unified mechanistic framework for fluorescence enhancement across diverse biomolecular systems.

Proposed method

• Analyzing the photochemistry of cyanine fluorophores, particularly their cis/trans isomerisation dynamics.
• Linking changes in isomerisation rates to measurable fluorescence enhancement upon binding to biomolecules.
• Using single-molecule fluorescence techniques to quantify fluorescence enhancement in real time.
• Applying the PIFE mechanism to diverse biomolecular systems, including nucleic acids and protein complexes.
• Developing a theoretical framework to model fluorescence enhancement as a function of isomerisation kinetics.
• Validating the mechanism across multiple experimental systems to confirm generality.

Experimental results

Research questions

• RQ1 How does biomolecular binding modulate the photoisomerisation dynamics of cyanine fluorophores?
• RQ2 To what extent is fluorescence enhancement in PIFE universally applicable beyond protein interactions?
• RQ3 Can PIFE be quantitatively modeled based on isomerisation rate changes rather than protein-specific effects?
• RQ4 What are the limits and sensitivities of PIFE in detecting conformational changes in biomolecules?
• RQ5 How can PIFE be adapted for studying protein-ligand and protein-protein interactions?

Key findings

• Fluorescence enhancement in PIFE arises from reduced cis/trans photoisomerisation rates upon binding, not from static quenching or environmental changes.
• The mechanism is general and applies to interactions with proteins, nucleic acids, and other biomolecules, not just proteins.
• PIFE can be quantitatively modeled using kinetic parameters of isomerisation, enabling predictive applications.
• The method enables real-time detection of conformational changes in biomolecules with single-molecule sensitivity.
• PIFE is effective for studying protein-ligand and protein-protein interactions due to its sensitivity to binding-induced dynamics.
• The reclassification of PIFE as photoisomerisation-related fluorescence enhancement provides a more accurate and broadly applicable mechanistic foundation.