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

Computational and Structural Biotechnology Journal

卷:19

Vladimir Z. Pletnev¹  Konstantin A. Lukyanov²  Tatiana R. Simonyan²  Anastasia V. Mamontova²  Elena A. Protasova³  Eugene G. Maksimov³  Nadya V. Pletneva⁴  Alexey M. Bogdanov⁴  Rustam H. Ziganshin⁴  Liya Muslinkina⁵  Sergei Pletnev⁶ 

[1] Corresponding authors at: Depatment of biophotonics (both), Laboratory of genetically encoded molecular tools ( A.M.B.), Laboratory of of X-ray study ( V.Z.P.).;

[2] Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow 121205, Russia;

[3] Faculty of Biology, Lomonosov Moscow State University, 119992 Moscow, Russia;

[4] Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia;

[5] Structural Biology Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA;

[6] Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA;

关键词: Fluorescent proteins;    EYFP;    Chromophore maturation;    X-ray structure;    Femtosecond spectroscopy;    Excitation energy transfer;   

DOI  :  

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown