Impact of amino acid substitutions on the behavior of a photoactivatable near infrared fluorescent protein PAiRFP1.

ABSTRACT

A photoactivatable near-infrared fluorescent protein (NIR-FP) PAiRFP1 has been developed by 15 amino acid substitutions in its nonfluorescent template Agp2. In our previous communication, we investigated the role of three amino acids in PHY domain distal from BV molecule. The impact of the twelve amino acids in GAF domain, especially five residues near BV-binding pocket is unclear. In this paper, PCR based reverse mutagenesis, spectroscopic methods, molecular modelling and simulations have been employed to explore the roles of these substitutions during the molecular evolution of PAiRFP1. It was found that the residue L163 is important for protein folding in PAiRFP1. The residues F244 and C280 exerted remarkable effects on molar extinction coefficient, NIR fluorescence quantum yield, molecular brightness, fluorescence fold, and dark recovery rate. The residues F244 and V276 modulate the maximum absorption and emission peak position. The reverse mutant L168M exhibited a higher fluorescence fold than PAiRFP1. Additionally, the reverse mutants V203A, V294E, S218G and D127G possessed better spectral properties than PAiRFP1. This study is important for the rational design of a better BphP-based photoactivatable NIR-FPs.