SMS ENZYME · Exploring Structure-Function Relationship of Enzymes at Single Molecule Level: Fluorescence and Force Spectroscopy
7РП — „Хора“ (Действия „Мария Кюри“)
- Период
- 2010-05-14 → 2013-06-01
- Финансиране от ЕС
- 174 162 €
- Участници
- 1
- Схема
- MC-IIF
Линиите свързват координатора с партньорите.
Накратко на български
Дейността на единични ензими се анализира чрез използването на ДНК като шаблон. Това помага да се разбере как структурата на шаблона променя поведението на ензима и го отклонява от класическите биохимични модели.
Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.
Резултати накратко
Exploring Structure-Function Relationship of Enzymes at Single Molecule Level: Fluorescence and Force Spectroscopy
This project aimed to study the activity of a single enzyme. As an enzyme we used DNA in DNA-templated reactions. We could monitor the activity of this single enzyme and show that when the template was unstructured , it behaved as an ideal Michaelis-Menten enzyme (as taught in classical biochemistry text-books): the distribution of cycle times was exponential. However when the DNA template was structured, the enzyme displayed non-exponential behaviour as also observed in real enzymatic reactions. This demonstrates that the hoping of the DNA enzyme between various metastable conformation resulted in a non-classical Michaelis-Menten behaviour.
Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз
Цел на проекта
The relation between structure and functions is a central topic in enzymology. I propose to study that question at the single molecule level employing a novel optical and force spectroscopy. The enzyme will be linked to a glass surface employing PEG-Biotin-Streptavidin interaction. The location of a single enzyme will be determined by a dye attached to it. The substrate of the reaction, while being non-fluorescent, will be chosen so that its enzymatic reaction product is fluorescent. The time span between fluorescence bursts reflecting the distribution of enzymatic active states will indicate the turnover rate of the enzyme. Contrary to the expectation that an enzyme is characterised by a well defined catalytic rate, a stretched exponential function has been reported to be necessary to fit the the probability distribution of the time span between bursts. I will study the effect of temperature on this stretched exponential behaviour for different enzymes. I will explore the enzymes labelled at two specific position by dyes which can act as donor and acceptor for resonance energy transfer (FRET) and the distance dependent FRET efficiency will offer an insight into the conformation fluctuation of the enzyme. In parallel to the optical observation, I will apply a mechanical tension on the enzymes thereby altering the distribution of its active states. In collaboration with the group of Dr. Marc Baaden, starting from the known enzymatic structure we will study numerically how the tension affects the enzymatic conformations. I will study how this tension alters its functions, via the changes in the distribution of turnover times. These studies will offer an insight into the relation between the structural changes of an enzyme and its measured catalytic activity.
Оригинален текст от CORDIS (на английски).
Участници
- CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS · ParisКоординаторФранция
Връзки
Данни: CORDIS, © Европейски съюз