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

Nadya V Pletneva; Eugene G Maksimov; Elena A Protasova; Anastasia V Mamontova; Tatiana R Simonyan; Rustam H Ziganshin; Konstantin A Lukyanov; Liya Muslinkina; Sergei Pletnev; Alexey M Bogdanov; Vladimir Z Pletnev

doi:10.1016/j.csbj.2021.05.017

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

Comput Struct Biotechnol J. 2021 May 11:19:2950-2959. doi: 10.1016/j.csbj.2021.05.017. eCollection 2021.

Affiliations

¹ Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia.
² Faculty of Biology, Lomonosov Moscow State University, 119992 Moscow, Russia.
³ Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow 121205, Russia.
⁴ Structural Biology Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
⁵ Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

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.

Keywords: Ala (A), alanine; Arg (R), arginine; Asn (R), asparagine; Chromophore maturation; DTT, dithiothreitol; EC, extinction coefficient; EET, excitation energy transfer; EGFP, enhanced green fluorescent protein; ESET, excited-state electron transfer; EYFP; EYFP, enhanced yellow fluorescent protein; Excitation energy transfer; FLIM, fluorescence lifetime imaging microscopy; FP, fluorescent protein; FQY, fluorescence quantum yield; FRET, Förster resonance energy transfer; FTIR, Fourier-transform infrared (spectroscopy; Femtosecond spectroscopy; Fluorescent proteins; GFP, green fluorescent protein; GYG, glycine-tyrosine-glycine; Gln (Q), glutamine; Glu (E), glutamic acid; Gly (G), glycine; His (H), histidine; IVA-cloning, in vivo assembly cloning; Leu (L), leucine; PBS, phosphate buffered saline; PCR, polymerase chain reaction; Phe (F), phenylalanine; REACh, resonance energy-accepting chromoprotein; Ser (S), serine; Structure-guided mutagenesis; Trp (W), tryptophan; Tryptophan fluorescence; Tyr (Y), tyrosine; Val (V), valine; X-ray structure; avGFP, Aequorea victoria green fluorescent protein; sfGFP, superfolder GFP.