# Development of bifunctional fluorescent probes and their application to α-helix labelling

## TL;DR

This paper introduces new fluorescent probes that improve the accuracy of studying protein dynamics by reducing labeling issues and enabling better spectroscopic measurements.

## Contribution

The development of bifunctional fluorescent probes that address orientational disorder and atropisomeric diastereoisomerism in protein labeling.

## Key findings

- The probes reduce orientational disorder, improving fluorescence polarization and FRET measurements.
- They solve the problem of atropisomeric diastereoisomerism in existing probes.
- Bio-orthogonal attachment groups are introduced to overcome limitations of traditional labeling methods.

## Abstract

The site selective modification of proteins and peptides is an important venture when it comes to the study of biological systems, such as in the determination of viable pharmacological targets and in the understanding of biomolecular mechanisms. In this paper we report on the development of novel bifunctional probes that allow for the unambiguous site-specific labelling of short peptides for spectroscopic measurements as demonstration of our future intentions to introduce these as functional labels for the study of protein dynamincs in situ. The symmetrical nature and bifunctional attachments of these probes to their targets significantly reduces their orientational disorder (i.e. ‘dye diffusion’), improving the accuracy and interpretation of established methods to study protein dynamics such as fluorescence polarization and Foerster Resonance Energy Transfer (FRET) measurements. In addition to solving a problem which has led to previous probes giving convoluted data owing to atropisomeric diastereoisomerism upon binding, we also introduce bio-orthogonal attachment groups that circumvent some of the drawbacks associated with the traditional labelling chemistries of thiol-reactive groups. These novel probes will be useful tools for future bulk and single-molecule spectroscopic experiments.

Novel bifunctional probes for α-helix labelling is described. These probes both solve an existing problem with current probes of this class, as well as introduce new orthogonality that will assist with the future study of dynamic proteins in situ.