Graduate theses
Current Record: 30 of 179
|
Title |
Εργαλεία φυσικής και στατιστικής ανάλυσης για την αποκάλυψη αλλοστερικών μονοπατιών παρακολουθώντας τη δυναμική των πρωτεϊνών |
Alternative Title |
Physical and statistical analysis tools to unravel allosteric pathways by monitoring protein dynamics |
Author
|
Τριάντου, Ειρήνη
|
Thesis advisor
|
Χαραλαμπίδης, Δημήτρης
|
Abstract |
The tools from the physical and statistical sciences rendered biological research quantitative.
In this thesis, a long-standing fundamental biological question has been addressed by adopting
such tools. Allostery represents the means by which biological information is propagatted over
distances. This signal propagation is essential to all life processes, and for that reason allostery
has been defined over the years as “the second secret of life”. Here, I tried to decipher the
means by which the signal from a binding event to one protein domain is transmitter to an
adjacent domain that is detached from the first one. After binding, the two domains approach
each other. I such a way a two-state model system is generated: an open free state and a closed
liganded one. The open state is the inactive one, whereas the closed one activates biological
processes, alike transport and chemoreception.
To identify the allosteric network connecting the ligand (or effector) to the docking and the distant domain,
I employed Statistical Coupling Analysis (SCA). Such a tool allows to identify groups of amino
acids that change following common patterns, or in others words co -evolve. Co-evolving amino
acids are likely part of a unique network, an allosteric network propagating th e allosteric event.
To monitor the movement of one domain relative to the other, I used single -molecule Förster
Resonance Energy Transfer (smFRET). This technique is a "spectroscopic ruler" based on
measuring the energy transfer between an energy donating fluorophore and an energy accepting
one. Its dynamic range is comparable to the size and changes in protein structures (Angstrom),
determining the distance between fluorophores. The structural changes (Structural dynamics)
between the two domains generating the two-state model system have been determined by
labeling site-specifically the two protein domains. FRET has been determined by a custommade confocal arrangement that I contributed in setting it up. I additionally adapted available
analysis tools to convert photon counts rates to FRET efficiencies and ultimately to interdomain distances.
The allosteric network connecting the two domains with the ligand has been retrieved and structural
dynamics of the wild-type protein were determined. Moreover, I observed that when the network was
disrupted, allosteric signal propagation was abolished. In such a case the protein was unable to adopt the
closed-liganded structure, thus remaining in the open inactive state, despite ligand binding.
|
Language |
Greek |
Issue date |
2023-07-28 |
Collection
|
School/Department--School of Sciences and Engineering--Department of Physics--Graduate theses
|
|
Type of Work--Graduate theses
|
Permanent Link |
https://elocus.lib.uoc.gr//dlib/c/4/6/metadata-dlib-1689314164-40977-6693.tkl
|
Views |
177 |