1. Amino acid residue at the 165th position tunes EYFP chromophore maturation. A structure-based design
- Author
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Vladimir Z. Pletnev, Eugene G. Maksimov, Anastasia V. Mamontova, Elena A. Protasova, Rustam H. Ziganshin, Konstantin A. Lukyanov, Nadya V. Pletneva, Sergei Pletnev, Tatiana R. Simonyan, Liya Muslinkina, and Alexey M. Bogdanov
- Subjects
Yellow fluorescent protein ,ESET, excited-state electron transfer ,Crystal structure ,FRET, Förster resonance energy transfer ,Biochemistry ,Green fluorescent protein ,His (H), histidine ,0302 clinical medicine ,PCR, polymerase chain reaction ,Structural Biology ,Phe (F), phenylalanine ,Structure-guided mutagenesis ,GFP, green fluorescent protein ,0303 health sciences ,Gln (Q), glutamine ,biology ,EYFP, enhanced yellow fluorescent protein ,Chemistry ,Glu (E), glutamic acid ,Fluorescence ,EGFP, enhanced green fluorescent protein ,Arg (R), arginine ,Computer Science Applications ,FQY, fluorescence quantum yield ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Tyr (Y), tyrosine ,Femtosecond spectroscopy ,Gly (G), glycine ,FTIR, Fourier-transform infrared (spectroscopy ,FP, fluorescent protein ,Ala (A), alanine ,Biotechnology ,Research Article ,sfGFP, superfolder GFP ,Asn (R), asparagine ,Stereochemistry ,Biophysics ,GYG, glycine-tyrosine-glycine ,03 medical and health sciences ,Excitation energy transfer ,Leu (L), leucine ,PBS, phosphate buffered saline ,Tryptophan fluorescence ,Genetics ,medicine ,030304 developmental biology ,ComputingMethodologies_COMPUTERGRAPHICS ,IVA-cloning, in vivo assembly cloning ,REACh, resonance energy-accepting chromoprotein ,EET, excitation energy transfer ,Chromophore maturation ,Triad (anatomy) ,Ser (S), serine ,Chromophore ,Fluorescent proteins ,Val (V), valine ,EC, extinction coefficient ,DTT, dithiothreitol ,EYFP ,Trp (W), tryptophan ,FLIM, fluorescence lifetime imaging microscopy ,biology.protein ,avGFP, Aequorea victoria green fluorescent protein ,X-ray structure ,Femtochemistry ,TP248.13-248.65 - Abstract
Graphical abstract, 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.
- Published
- 2021