Journal Article

FZJ-2021-05885

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Rational Design of a Split Flavin-Based Fluorescent Reporter

Yudenko, A. ; Smolentseva, A. ; Maslov, I. ; Semenov, O. ; Goncharov, I. M. ; Nazarenko, V. V. ; Maliar, N. L. ; Borshchevskiy, V. ; Gordeliy, V.FZJ* ; Remeeva, A. ; Gushchin, I. (Corresponding author)

2021
ACS Washington, DC

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Please use a persistent id in citations: doi:10.1021/acssynbio.0c00454

Abstract: Protein-fragment complementation assays are used ubiquitously for probing protein–protein interactions. Most commonly, the reporter protein is split in two parts, which are then fused to the proteins of interest and can reassemble and provide a readout if the proteins of interest interact with each other. The currently known split fluorescent proteins either can be used only in aerobic conditions and assemble irreversibly, or require addition of exogenous chromophores, which complicates the design of experiments. In recent years, light-oxygen-voltage (LOV) domains of several photoreceptor proteins have been developed into flavin-based fluorescent proteins (FbFPs) that, under some circumstances, can outperform commonly used fluorescent proteins such as GFP. Here, we show that CagFbFP, a small thermostable FbFP based on a LOV domain-containing protein from Chloroflexus aggregans, can serve as a split fluorescent reporter. We use the available genetic and structural information to identify three loops between the conserved secondary structure elements, Aβ-Bβ, Eα-Fα, and Hβ-Iβ, that tolerate insertion of flexible poly-Gly/Ser segments and eventually splitting. We demonstrate that the designed split pairs, when fused to interacting proteins, are fluorescent in vivo in E. coli and human cells and have low background fluorescence. Our results enable probing protein–protein interactions in anaerobic conditions without using exogenous fluorophores and provide a basis for further development of LOV and PAS (Per-Arnt-Sim) domain-based fluorescent reporters and optogenetic tools.

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Contributing Institute(s):

1. Strukturbiochemie (IBI-7)

Research Program(s):

1. 5241 - Molecular Information Processing in Cellular Systems (POF4-524) (POF4-524)

Appears in the scientific report 2021

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Database coverage:
; BIOSIS Previews ; Biological Abstracts ; Clarivate Analytics Master Journal List ; Essential Science Indicators ; IF < 5 ; JCR ; SCOPUS ; Science Citation Index Expanded ; Web of Science Core Collection

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