Title:Addition of Mercury Causes Quenching of NIR Fluorescence Emission
Spectra of a Photoactivatable PAiRFP1 Protein
Volume: 23
Issue: 5
Author(s): Fakhrul Hassan*, Faez Iqbal Khan, Feng Juan, Abbas Khan and Dakun Lai
Affiliation:
- Faculty of Rehabilitation and Allied Health Sciences, Riphah International University, Islamabad 46000, Pakistan
Keywords:
PAiRFP1, photoactivation, NIR biosensor, molecular evolution, fluorescence emission, protein modeling.
Abstract:
Background: Biliverdin (BV) containing far-red light photoactivatable near-infrared fluorescent
protein (NIR-FP) named PAiRFP1 has been developed by directed molecular evolution from
one bathy bacteriophytochrome of Agrobacterium tumefaciens C58 called Agp2 or AtBphP2. Usually,
the fluorescence intensity of the NIR emission spectra of PAiRFP1 tends to increase upon repeated
excitation by far-red light.
Objective: This study aimed at exploring the role of PAiRFP1 and its mutants, such as V386A,
V480A, and Y498H, as NIR biosensors for the detection of Hg2+ ions in the buffer solutions.
Methods: In this study, we used PCR-based site-directed reverse mutagenesis, fluorescence spectroscopy,
and molecular modeling approaches on PAiRFP1 and its mutants.
Results: It was found that PAiRFP1 and its mutants experienced strong quenching of NIR fluorescence
emission spectra upon the addition of different concentrations (0-3μM) of mercuric chloride
(HgCl2).
Conclusion: We hypothesized that PAiRFP1 and its variants have some potential to be used as NIR
biosensors for the in vitro detection of Hg2+ ions in biological media. Moreover, we also hypothesized
that PAiRFP1 would be the best tool to use as a NIR biosensor to detect Hg2+ ions in living organisms
because of its higher signal-to-noise (SNR) ratio than other infra-red fluorescent proteins.