H2020Индивидуална стипендия2021–2024

DisCharge · Exploring molecular determinants of charged disordered protein interactions, phase separation and function from the test-tube to the cell

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

Период: 2021-09-01 → 2024-04-29
Финансиране от ЕС: 191 149 €
Участници: 1
Схема: MSCA-IF

Линиите свързват координатора с партньорите.

Накратко на български

Взаимодействията между силно заредени протеини, като ProTa и H1, се анализират от епруветката до клетката. Това помага да се разбере как последователността на протеините определя тяхното поведение и регулирането на генетичната информация.

Този кратък обзор е генериран от изкуствен интелект

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

Резултати накратко

Exploring molecular determinants of charged disordered protein interactions, phase separation and function from the test-tube to the cell

The overall objective of the project is to uncover specificity determinants of interaction and phase separation of highly charged IDPs using ProTa/H1 interaction as a model. Only a multidisciplinary study spanning several complexity regimes, which is currently lacking, can uncover such specificity determinants. It is hypothesized that physiologically, ProTa/H1 interaction plays out in LLPS-driven heterochromatin assemblies leading to transcriptional regulation. ProTa/H1 interactions and phase behaviour will be probed with a set of systematically designed variants in incrementally complex milieu, from the test-tube to the cell, to reveal how the sequence dictates interactions and phase behaviour in-vitro and attribute a given function in-cell. This will shed light on specificity determinants of interaction and function for such highly charged IDPs. In order to achieve this goal first we first uncovered the thermodynamic basis of interaction ProTa and H1 using a combination of single molecule FRET spectroscopy, calorimetery, integrative data analysis and analytical polymer theory. We discovered that a key driving force for this interaction is the entropy of released counter ions akin to synthetic poly-electrolytes. We further found out the complex to be experimentally dynamic at all conditions even when the affinity is expected to be very high, that is there is an affinity dynamics decoupling in such types of complexes, and we rationalized this behavior using simple physical principles. Furthermore, we characterized the full phase diagram of ProTa/H1 and optimized conditions for performing single molecule fluorescence experiments in such condensates. Some technical challenges were identified in experiments on ternary phases of nucleic acids/ProTa/H1 as well as for in-cellulo experiments, and strategies to develop these were addressed. Also, for the all the above mentioned accomplishments robust biochemical strategies for purification and labeling of ProTa and H1 variants, the latter being exceedingly degradation prone and consequently difficult to purify, were established.

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

Цел на проекта

Biological molecular recognition is centred around the structure-function paradigm, involving interfacial complementary shapes and non-covalent forces. Intrinsically disordered proteins (IDPs), constituting up to 40% of the proteome, evade the structure-function paradigm, owing to their lack of persistent structure. Despite some IDPs forming folded complexes, numerous IDPs form disordered complexes stabilized by multivalent interactions. An extreme example is the recently discovered disordered high-affinity complex formed by two highly charged IDPs, the nucleosome binding linker histone (H1) and its nuclear chaperone prothymosin-alpha (ProTa), both key regulators of chromatin-structure and -function. IDPs are also often implicated in liquid-liquid phase separation (LLPS) driven assemblies that facilitate numerous biochemical interactions. The specificity question in IDPs becomes particularly pertinent for such charge driven disordered interactions and associated LLPS processes considering the great abundance of such highly charged disordered stretches in our proteome. Specific physiological function of most IDP interactions and associated LLPS processes imply an important role of specificity, albeit encoded differently from folded proteins. Only a multidisciplinary study spanning several complexity regimes, which is currently lacking, can uncover such altered specificity. I hypothesize that physiologically, ProTa/H1 interaction plays out in LLPS-driven heterochromatin assemblies leading to transcriptional regulation. I will probe ProTa/H1 interactions and phase behaviour with a set of systematically designed variants in incrementally complex milieu, from the test-tube to the cell, to reveal how the sequence dictates interactions and phase behaviour in-vitro and attribute a given function in-cell. This will shed light on specificity determinants of interaction and function for such highly charged IDPs.

Оригинален текст от CORDIS (на английски).

Участници

UNIVERSITAT ZURICH · ZurichКоординаторШвейцария