H2020Индивидуална стипендия2015–2017

MECHANOPROTEASES · Single Molecule Study of Protease Mechano-Specificity

„Хоризонт 2020“ — Действия „Мария Склодовска-Кюри“

Период: 2015-04-01 → 2017-03-31
Финансиране от ЕС: 195 455 €
Участници: 1
Схема: MSCA-IF-EF-ST

Линиите свързват координатора с партньорите.

Накратко на български

Механизмите на ензима TEV се изследват чрез атомно-силова спектроскопия, за да се види как той разпознава и разрязва конкретни протеини. Това помага да се разбере как протеазите избягват грешни цели и разграждат само правилните молекули в клетката.

Този кратък обзор е генериран от изкуствен интелект

Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.

Резултати накратко

Single Molecule Study of Protease Mechano-Specificity

Fundamental mechanisms in biology rely on the association and recognition among proteins. Especially, enzymes associate specifically to their substrate in order to catalyze precise biochemical reactions in vivo. Proteases form a large family of enzymes found in all life kingdoms and are responsible for the cleavage of peptide bonds. Because a vast number of different proteases are prompt to catalyze various maturation or degradation reactions in the cellular environment, it is therefore important for proteases to have a limited range of potential substrates and avoid promiscuous off-target cleavages. We proposed to uncover at a single molecule level the kinetic and thermodynamic details of the specific recognition and digestion of the endoprotease TEV. The objective of this project is to design a new type of experiments in order to monitor the proteolytic reaction at the single molecule level. In our experiments, the mechanical unfolding of the substrate is accelerated by an Atomic Force Spectrometer in order to activate the reaction and measure the rates of the reaction.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз

Цел на проекта

Single-molecule enzymology offers new possibilities to dissect catalytic reactions that were previously unapproachable using biochemistry techniques conducted in the bulk. In particular, recent discoveries conducted at the single molecule level, such as the unanticipated force-mediated protein degradation pathway in the proteasome, highlight the close relation between mechanical forces and proteolysis in vivo. While much has been discovered about protein enzymology in the recent decades, the question of how mechanical force affects enzymatic catalysis remains vastly elusive. The main goal of this proposal is to understand the mechanobiology of proteolysis at the single molecule level. We will use the newly developed force-clamp spectroscopy technique, together with molecular biology engineering techniques and bioinformatics structural analysis to elucidate the molecular mechanisms that underlie protease catalysis under mechanical force. Successful enzymatic activity relies on the enzyme:substrate (E:S) assembly. Upon mechanical unfolding, proteins unveil their buried substrate sites, also called cryptic sites, thus favoring the formation of the E:S complex and ultimately permitting the subsequent chemical reaction. A key feature of recent mechano-chemistry experiments at the single bond level is that the rate at which the reduction of a protein disulfide bond occurs in the presence of a nucleophile is exponentially dependent on the stretching force. Hence, it is tempting to speculate that, in the case of an enzymatic reaction, the catalytic rate will be also force-dependent. We anticipate that the curved geometry of the bound substrate inhibits the E:S assembly at high-forces, implying a novel mechano-specificity character of proteases. Within a multidisciplinary approach, here we propose a series of innovative experiments to directly probe the effect of force on the kinetics of protease hydrolysis.

Оригинален текст от CORDIS (на английски).

Участници

KING'S COLLEGE LONDON · LondonКоординаторОбединеното кралство