Az aktin kölcsönhatása aktin-kötő fehérjékkel és peptidekkel: fluoreszcencia spektroszkópiai vizsgálatok = The Interaction of Actin with Actin-Binding Proteins and Peptides as Revealed by Spectroscopic Methods

Az OTKA K60968 pályázat keretei között az eredeti terveinknek megfelelően tanulmányoztuk az aktin monomereknek és filamentumoknak más fehérjékkel és peptidekkel való kölcsönhatásait. A kutatások során elsősorban fluoreszcencia spektroszkópiai módszereket alkalmaztunk, de az adott kérdéskörtől függően ezen módszerek eredményeit kiegészítettük elektron paramágneses rezonancia spektroszkópiai és kalorimetriai vizsgálatokkal is. Részletes vizsgálatokban jellemeztük az aktin filamentumoknak a forminokkal való kölcsönhatását, és megállapítottuk, hogy a forminok kötődésével a filamentumok szerkezete lazábbá válik. Azt is megfigyeltük, hogy a forminok által kiváltott konformációs módosulásokat a tropomiozin vagy a miozin kötődése megszünteti. Tanulmányoztunk és leírtunk továbbá egy eddig nem jellemzett formin családot, a DAAM forminokat. Ezen vizsgálataink mellett jellemeztük és értelmeztük az aktinnak a kölcsönhatását egyes mérgező toxinokkal, valamint új megfigyeléseket tettünk az aktin-miozin kölcsönhatás szerkezeti és kinetikai sajátságait illetően is. | According to the project plans we have studied the interactions between actin monomers / filaments and actin binding proteins and peptides. In these investigations we applied fluorescence spectroscopic methods, in conjunction with electron paramagnetic resonance and calorimetric assays. We described in details the interaction between actin filaments and formin, and found that the binding of formins made the actin filaments ore flexible. We also observed that the formin induced conformational changes were reversed by the binding of tropomyosin or myosin. We studied and described a novel formin family, the DAAM formins. Furthermore, we described the interactions between actin and toxic actin-binding peptides, and also characterised the interactions of myosin with actin in terms of the conformational and kinetic properties

Photocycle alteration and increased enzymatic activity in genetically modified photoactivated adenylate cyclase OaPAC

Photoactivated adenylate cyclases (PACs) are light activated enzymes that combine blue light sensing capacity with the ability to convert ATP to cAMP and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a blue light sensing domain using flavin which undergoes a structural reorganization after blue-light absorption. This minor structural change then is translated toward the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP. As cAMP is a key second messenger in numerous signal transduction pathways regulating various cellular functions, PACs are of great interest in optogenetic studies. The optimal optogenetic device must be “silent” in the dark and highly responsive upon light illumination. PAC from Oscillatoria acuminata is a very good candidate as its basal activity is very small in the dark and the conversion rates increase 20-fold upon light illumination. We studied the effect of replacing D67 to N, in the blue light using flavin domain. This mutation was found to accelerate the primary electron transfer process in the photosensing domain of the protein, as has been predicted. Furthermore, it resulted in a longer lived signaling state, which was formed with a lower quantum yield. Our studies show that the overall effects of the D67N mutation lead to a slightly higher conversion of ATP to cAMP, which points in the direction that by fine tuning the kinetic properties more responsive PACs and optogenetic devices can be generated