Amino acid residue at the 165th position tunes EYFP chromophore maturation. A structure-based design

• Published in: Computational and structural biotechnology journal Vol. 19; no. C; pp. 2950 - 2959
• Main Authors: Pletneva, Nadya V., Maksimov, Eugene G., Protasova, Elena A., Mamontova, Anastasia V., Simonyan, Tatiana R., Ziganshin, Rustam H., Lukyanov, Konstantin A., Muslinkina, Liya, Pletnev, Sergei, Bogdanov, Alexey M., Pletnev, Vladimir Z.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2021; Elsevier; Research Network of Computational and Structural Biotechnology
• Subjects: Chromophore maturation; Excitation energy transfer; EYFP; Femtosecond spectroscopy; Fluorescent proteins; Structure-guided mutagenesis; Tryptophan fluorescence; X-ray structure; Gly (G); Trp (W); Structure-guided mutagenesis; Val (V); DTT; FP; His (H); FLIM; Gln (Q); EGFP; GFP; REACh; Leu (L); GYG; FTIR; Fluorescent proteins; Tryptophan fluorescence; sfGFP; FRET; EYFP; EC; Ala (A); PBS; Excitation energy transfer; Ser (S); Arg (R); FQY; Phe (F); Femtosecond spectroscopy; avGFP; Asn (R); EET; Glu (E); Chromophore maturation; ESET; IVA-cloning; X-ray structure; Tyr (Y); PCR; Ser (S), serine; His (H), histidine; avGFP, Aequorea victoria green fluorescent protein; EET, excitation energy transfer; Leu (L), leucine; Asn (R), asparagine; IVA-cloning, in vivo assembly cloning; DTT, dithiothreitol; Gln (Q), glutamine; EC, extinction coefficient; Arg (R), arginine; FQY, fluorescence quantum yield; FTIR, Fourier-transform infrared (spectroscopy; Glu (E), glutamic acid; Val (V), valine; FLIM, fluorescence lifetime imaging microscopy; GYG, glycine-tyrosine-glycine; Ala (A), alanine; FRET, Förster resonance energy transfer; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; ESET, excited-state electron transfer; Gly (G), glycine; Phe (F), phenylalanine; PBS, phosphate buffered saline; Trp (W), tryptophan; Tyr (Y), tyrosine; FP, fluorescent protein; sfGFP, superfolder GFP; EYFP, enhanced yellow fluorescent protein; PCR, polymerase chain reaction; REACh, resonance energy-accepting chromoprotein
• ISSN: 2001-0370; 2001-0370
• DOI: 10.1016/j.csbj.2021.05.017

[Display omitted] For the whole GFP family, a few cases, when a single mutation in the chromophore environment strongly inhibits maturation, were described. Here we study EYFP-F165G – a variant of the enhanced yellow fluorescent protein – obtained by a single F165G replacement, and demonstrated multiple fluorescent states represented by the minor emission peaks in blue and yellow ranges (~470 and ~530 nm), and the major peak at ~330 nm. The latter has been assigned to tryptophan fluorescence, quenched due to excitation energy transfer to the mature chromophore in the parental EYFP protein. EYFP-F165G crystal structure revealed two general independent routes of post-translational chemistry, resulting in two main states of the polypeptide chain with the intact chromophore forming triad (~85%) and mature chromophore (~15%). Our experiments thus highlighted important stereochemical role of the 165th position strongly affecting spectral characteristics of the protein. On the basis of the determined EYFP-F165G three-dimensional structure, new variants with ~ 2-fold improved brightness were engineered.