Issue 6, 2017

Designing brighter near-infrared fluorescent proteins: insights from structural and biochemical studies

Abstract

Brighter near-infrared (NIR) fluorescent proteins (FPs) are required for multicolor microscopy and deep-tissue imaging. Here, we present structural and biochemical analyses of three monomeric, spectrally distinct phytochrome-based NIR FPs, termed miRFPs. The miRFPs are closely related and differ by only a few amino acids, which define their molecular brightness, brightness in mammalian cells, and spectral properties. We have identified the residues responsible for the spectral red-shift, revealed a new chromophore bound simultaneously to two cysteine residues in the PAS and GAF domains in blue-shifted NIR FPs, and uncovered the importance of amino acid residues in the N-terminus of NIR FPs for their molecular and cellular brightness. The novel chromophore covalently links the N-terminus of NIR FPs with their C-terminal GAF domain, forming a topologically closed knot in the structure, and also contributes to the increased brightness. Based on our studies, we suggest a strategy to develop spectrally distinct NIR FPs with enhanced brightness.

Graphical abstract: Designing brighter near-infrared fluorescent proteins: insights from structural and biochemical studies

Supplementary files

Article information

Article type
Edge Article
Submitted
23 Feb 2017
Accepted
11 Apr 2017
First published
04 May 2017
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2017,8, 4546-4557

Designing brighter near-infrared fluorescent proteins: insights from structural and biochemical studies

M. Baloban, D. M. Shcherbakova, S. Pletnev, V. Z. Pletnev, J. C. Lagarias and V. V. Verkhusha, Chem. Sci., 2017, 8, 4546 DOI: 10.1039/C7SC00855D

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