TU Darmstadt / ULB / TUprints

Binding pocket stabilization by high-throughput screening of yeast display libraries

Lerma Romero, Jorge A. ; Meyners, Christian ; Christmann, Andreas ; Reinbold, Lisa M. ; Charalampidou, Anna ; Hausch, Felix ; Kolmar, Harald (2022)
Binding pocket stabilization by high-throughput screening of yeast display libraries.
In: Frontiers in Molecular Biosciences, 2022, 9
doi: 10.26083/tuprints-00022911
Article, Secondary publication, Publisher's Version

[img] Text
fmolb-09-1023131.pdf
Copyright Information: CC BY 4.0 International - Creative Commons, Attribution.

Download (3MB)
[img] Text (Supplement)
Table1.DOCX
Copyright Information: CC BY 4.0 International - Creative Commons, Attribution.

Download (2MB)
Item Type: Article
Type of entry: Secondary publication
Title: Binding pocket stabilization by high-throughput screening of yeast display libraries
Language: English
Date: 19 December 2022
Place of Publication: Darmstadt
Year of primary publication: 2022
Publisher: Frontiers Media S.A.
Journal or Publication Title: Frontiers in Molecular Biosciences
Volume of the journal: 9
Collation: 16 Seiten
DOI: 10.26083/tuprints-00022911
Corresponding Links:
Origin: Secondary publication DeepGreen
Abstract:

Protein dynamics have a great influence on the binding pockets of some therapeutic targets. Flexible protein binding sites can result in transient binding pocket formation which might have a negative impact on drug screening efforts. Here, we describe a protein engineering strategy with FK506-binding protein 51 (FKBP51) as a model protein, which is a promising target for stress-related disorders. High-throughput screening of yeast display libraries of FKBP51 resulted in the identification of variants exhibiting higher affinity binding of conformation-specific FKBP51 selective inhibitors. The gene libraries of a random mutagenesis and site saturation mutagenesis of the FK1 domain of FKBP51 encoding sequence were used to create a yeast surface display library. Fluorescence-activated cell sorting for FKBP51 variants that bind conformation-specific fluorescently labeled ligands with high affinity allowed for the identification of 15 different protein variants with improved binding to either, or both FKBP51-specific ligands used in the screening, with improved affinities up to 34-fold compared to the wild type. These variants will pave the way to a better understanding of the conformational flexibility of the FKBP51 binding pocket and may enable the isolation of new selective ligands that preferably and selectively bind the active site of the protein in its open conformation state.

Uncontrolled Keywords: protein engineering, transient binding pocket, yeast display, flow cytometry, FKBP, high-throughput screening
Status: Publisher's Version
URN: urn:nbn:de:tuda-tuprints-229113
Classification DDC: 500 Science and mathematics > 540 Chemistry
500 Science and mathematics > 570 Life sciences, biology
Divisions: Interdisziplinäre Forschungsprojekte > Centre for Synthetic Biology
07 Department of Chemistry > Clemens-Schöpf-Institut > Fachgebiet Biochemie
Date Deposited: 19 Dec 2022 12:47
Last Modified: 14 Nov 2023 19:05
SWORD Depositor: Deep Green
URI: https://tuprints.ulb.tu-darmstadt.de/id/eprint/22911
PPN: 503247103
Export:
Actions (login required)
View Item View Item