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Biotin-labeled proteins are widely used as tools to study protein–protein interactions and proximity in living cells. Proteomic methods broadly employ proximity-labeling technologies based on protein biotinylation in order to investigate the transient encounters of biomolecules in subcellular compartments. Biotinylation is a post-translation modification in which the biotin molecule is attached to lysine or tyrosine residues. So far, biotin-based technologies proved to be effective instruments as affinity and proximity tags. However, the influence of biotinylation on aspects such as folding, binding, mobility, thermodynamic stability, and kinetics needs to be investigated. Here, we selected two proteins [biotin carboxyl carrier protein (BCCP) and FKBP3] to test the influence of biotinylation on thermodynamic and kinetic properties. Apo (without biotin) and holo (biotinylated) protein structures were used separately to generate all-atom structure-based model simulations in a wide range of temperatures. Holo BCCP contains one biotinylation site, and FKBP3 was modeled with up to 23 biotinylated lysines. The two proteins had their estimated thermodynamic stability changed by altering their energy landscape. In all cases, after comparison between the apo and holo simulations, differences were observed on the free-energy profiles and folding routes. Energetic barriers were altered with the density of states clearly showing changes in the transition state. This study suggests that analysis of large-scale datasets of biotinylation-based proximity experiments might consider possible alterations in thermostability and folding mechanisms imposed by the attached biotins.
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Biotinylation as a tool to enhance the uptake of small molecules in Gram-negative bacteria
Antibiotic resistance is a major public health concern. The shrinking selection of effective antibiotics and lack of new development is making the situation worse. Gram-negative bacteria more specifically pose serious threat because of their double layered cell envelope and effective efflux systems, which is a challenge for drugs to penetrate. One promising approach to breach this barrier is the “Trojan horse strategy”. In this technique, an antibiotic molecule is conjugated with a nutrient molecule that helps the antibiotic to enter the cell through dedicated transporters for the nutrient. Here, we explored the approach using biotin conjugation with a florescent molecule Atto565 to determine if biotinylation enhances accumulation. Biotin is an essential vitamin for bacteria and is obtained through either synthesis or uptake from the environment. We found that biotinylation enhanced accumulation of Atto565 in E . coli . However, the enhancement did not seem to be due to uptake through biotin transporters since the presence of free biotin had no observable impact on accumulation. Accumulated compound was mostly in the periplasm, as determined by cell fractionation studies. This was further confirmed through the observation that expression of streptavidin in the periplasm specifically enhanced the accumulation of biotinylated Atto565. This enhancement was not observed when streptavidin was expressed in the cytoplasm indicating no significant distribution of the compound inside the cytoplasm. Using gene knockout strains, plasmid complementation and mutagenesis studies we demonstrated that biotinylation made the compound a better passenger through OmpC, an outer membrane porin. Density functional theory (DFT)-based evaluation of the three-dimensional geometries showed that biotinylation did not directly stabilize the conformation of the compound to make it favorable for the entry through a pore. Further studies including molecular dynamics simulations are necessary to determine the possible mechanisms of enhanced accumulation of the biotinylated Atto565.
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- Award ID(s):
- 1709381
- PAR ID:
- 10309575
- Editor(s):
- van Veen, Hendrik W.
- Date Published:
- Journal Name:
- PLOS ONE
- Volume:
- 16
- Issue:
- 11
- ISSN:
- 1932-6203
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1371/journal.pone.0260023
