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

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)2950-2959
Number of pages10
JournalComputational and Structural Biotechnology Journal
Volume19
DOIs
Publication statusPublished - Jan 2021

Keywords

  • Chromophore maturation
  • Excitation energy transfer
  • EYFP
  • Femtosecond spectroscopy
  • Fluorescent proteins
  • Structure-guided mutagenesis
  • Tryptophan fluorescence
  • X-ray structure

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