Enhancing the biophysical properties of mRFP1 through incorporation of fluoroproline.
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)-4-fluoroproline [(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.
School of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk, South Korea.
Kanagavel
K
Deepankumar
Saravanan Prabhu
SP
Nadarajan
Niraikulam
N
Ayyadurai
Hyungdon
H
Yun
D055592:Biophysical Phenomena; D005453:Fluorescence; D006358:Hot Temperature; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D016296:Mutagenesis; D011392:Proline / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics; D055550:Protein Stability; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D011392:Proline; D000097573:Red Fluorescent Protein; C431803:fluoro-proline
(2S, 4R)-4-fluoroproline; (2S, 4S)-4-fluoroproline; (4R)-FP; (4S)-FP; Biophysical properties; FP; GdmCl; MM; NCAA; Non-canonical amino acid incorporation; Thermal stability; fluoroproline; guanidine hydrochloride; mRFP1; mRFP1-P63A; minimal media; non-canonical amino acid incorporation