Enhancing the biophysical properties of mRFP1 through incorporation of fluoroproline

Title
Enhancing the biophysical properties of mRFP1 through incorporation of fluoroproline
Author(s)
윤형돈카나가벨디판쿠마나다라잔사라바난아야두라이[아야두라이]
Keywords
GREEN FLUORESCENT PROTEIN; SYNTHETIC BIOLOGY; SUBSTITUTION; CHROMOPHORE; STABILITY; DESIGN
Issue Date
201311
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Citation
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, v.440, no.4, pp.509 - 514
Abstract
Here we enhanced the stability and biophysical properties of mRFP1 through a combination of canonical and non-canonical amino acid mutagenesis. The global replacement of proline residue with (2S, 4R)--4fluoroproline [(4R)-FP] into mRFP1 led to soluble protein but lost its fluorescence, whereas (2S, 4S)-4-fluoroproline [(4S)-FP] incorporation resulted in insoluble protein. The bioinformatics analysis revealed that (4R)-FP incorporation at Pro63 caused fluorescence loss due to the steric hindrance of fluorine atom of (4R)-FP with the chromophore. Therefore, Pro63 residue was mutated with the smallest amino acid Ala to maintain non coplanar conformation of the chromophore and helps to retain its fluorescence with (4R)-FP incorporation. The incorporation of (4R)-FP into mRFP1-P63A showed about 2-3-fold enhancement in thermal and chemical stability. The rate of maturation is also greatly accelerated over the presence of (4R)-FP into mRFP1-P63A. Our study showed that a successful enhancement in the biophysical property of mRFP1-P63A[(4R)-FP] using non-canonical amino acid mutagenesis after mutating non-permissive site Pro63 into Ala. (C) 2013 Elsevier Inc. All rights reserved.
URI
http://hdl.handle.net/YU.REPOSITORY/28538http://dx.doi.org/10.1016/j.bbrc.2013.09.062
ISSN
0006-291X
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