Effects of reverse genetic mutations on the spectral and photochemical behavior of a photoactivatable fluorescent protein PAiRFP1

Fakhrul Hassan; Faez Iqbal Khan; Honghong Song; Dakun Lai; Feng Juan

doi:10.1016/j.saa.2019.117807

Effects of reverse genetic mutations on the spectral and photochemical behavior of a photoactivatable fluorescent protein PAiRFP1

Spectrochim Acta A Mol Biomol Spectrosc. 2020 Mar 5:228:117807. doi: 10.1016/j.saa.2019.117807. Epub 2019 Nov 18.

Authors

Fakhrul Hassan¹, Faez Iqbal Khan², Honghong Song¹, Dakun Lai³, Feng Juan⁴

Affiliations

¹ School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China.
² School of Electronic Science and Engineering, University of Electronic Science and Technology of China, Chengdu, China. Electronic address: khanfaeziqbal@gmail.com.
³ School of Electronic Science and Engineering, University of Electronic Science and Technology of China, Chengdu, China. Electronic address: dklai@uestc.edu.cn.
⁴ School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China. Electronic address: fengjuan@uestc.edu.cn.

PMID: 31806482
DOI: 10.1016/j.saa.2019.117807

Abstract

Bacteriophytochrome photoreceptors (BphPs) containing biliverdin (BV) have great potential for the development of genetically engineered near-infrared fluorescent proteins (NIR FPs). We investigated a photoactivatable fluorescent protein PAiRFP1, was engineered through directed molecular evolution. The coexistence of both red light absorbing (Pr) and far-red light absorbing (Pfr) states in dark is essential for the photoactivation of PAiRFP1. The PCR based site-directed reverse mutagenesis, spectroscopic measurements and molecular dynamics (MD) simulations were performed on three targeted sites V386A, V480A and Y498H in PHY domain to explore their potential effects during molecular evolution of PAiRFP1. We found that these substitutions did not affect the coexistence of Pr and Pfr states but led to slight changes in the photophysical parameters. The covalent docking of biliverdin (cis and trans form) with PAiRFP1 was followed by several 100 ns MD simulations to provide some theoretical explanations for the coexistence of Pr and pfr states. The results suggested that experimentally observed coexistence of Pr and Pfr states in both PAiRFP1 and mutants were resulted from the improved stability of Pr state. The use of experimental and computational work provided useful understanding of Pr and Pfr states and the effects of these mutations on the photophysical properties of PAiRFP1.

Keywords: Bacteriophytochrome photoreceptors; Molecular dynamics simulation; PAiRFP1; Photoactivation.

MeSH terms

Agrobacterium
Bacterial Proteins / chemistry
Biliverdine / chemistry
Computational Biology / methods*
Directed Molecular Evolution
Escherichia coli / metabolism
Hydrogen Bonding
Luminescent Proteins / chemistry*
Molecular Conformation
Molecular Dynamics Simulation
Mutagenesis, Site-Directed
Mutation*
Photochemistry / methods*
Phytochrome / chemistry*
Principal Component Analysis
Protein Conformation
Protein Engineering
Protein Structure, Secondary
Red Fluorescent Protein
Reverse Genetics*
Signal-To-Noise Ratio
Software
Spectrophotometry, Ultraviolet
Surface Properties

Substances

Bacterial Proteins
Luminescent Proteins
Phytochrome
Biliverdine

Supplementary concepts

Agrobacterium fabrum