# Small Protein Domains as Potential Spin Labels for In Vitro, Cellular, and Light-Induced Dipolar EPR Spectroscopy

## TL;DR

This paper shows how small protein domains can act as spin labels for EPR spectroscopy, enabling distance measurements in cells using light.

## Contribution

The study introduces LOV domains as genetically encoded spin labels for dipolar EPR spectroscopy, enabling in-cell distance measurements.

## Key findings

- LOV domains can generate multiple FMN radicals upon blue light exposure for EPR spectroscopy.
- LOV1-LOV2 fusion proteins show distinct distances due to domain interactions and dimerization.
- Light-induced EPR measurements can infer structure and dynamics of LOV proteins in cells.

## Abstract

This study explores
the potential of small light-oxygen-voltage
(LOV) domains for utilization as protein spin labels in different
dipolar EPR spectroscopy methods. The distinctive photochemical properties
of selected LOV domain variants are exploited to generate a variety
of (meta)­stable flavin mononucleotide (FMN) radicals upon blue light
absorption. Three different radicals, FMN·–, FMNH·, and an FMN-methionine radical, and an excited
FMN triplet species, were generated. The FMN radicals were generated
in LOV single domains and two model LOV1-LOV2 fusion proteins, and
the latter proteins demonstrated that simple and effective orthogonal
spin labeling can be performed. Subsequently, dipolar EPR experiments
were conducted in aqueous solution and in cells with and without additional
light excitation, in order to measure the distances between
the FMN cofactor radicals, and to infer the structure and dynamics
of the LOV domain proteins. Interestingly, all LOV1-LOV2 fusion proteins
exhibit defined but largely distinct distances. This can be attributed
to two factors: the respective LOV domains have different interactions
with each other, and the presence of neutral FMN radicals leads to
dimerization of the LOV1 domains. Nevertheless, using LOV domains
as genetically encoded spin labels could offer numerous advantages.
As a true molecular biology concept, labeling and measurements can
be performed in any accessible cell type using light as the only stimulus.
Additionally, the various paramagnetic FMN states enable the measurement
of distances between two radicals, as well as between a radical and
a triplet state.