Biológiai makromolekulák statikus és dinamikus szerkezetvizsgálata multinukleáris, multidimenzionális NMR segítségével = Application of multinuclear, multidimensional NMR spectroscopy in static and dynamical structure determination of biological macromolecules

A multinukleáris, multidimenzionális NMR spektroszkópiát alkalmazva több impulzusszekvenciát adaptáltam. Jellemeztem néhány biológiailag fontos, gyógyszer hatóanyag jelölt nyílt láncú és ciklikus peptid oldatbeli viselkedését. Elvégeztem a 178 aminosavat tartalmazó homodimer dinein könnyű lánc jelazonosítását, vizsgáltam kötődését miozin fragmensekhez, meghatároztam a kötődési állandót. Megállapítottam, a szabad formában rendezetlen peptid fragmensek kötődés során adott szerkezetet vesznek fel. 31P NMR méréseket alkalmazva elvégeztem a dUTPáz által katalizált hidrolízis kinetikai vizsgálatát, azonosítottam a reakciótermékeket, kiszámoltam a pszeudoelsőrendű sebességi állandót, és megállapítottam, hogy a mechanizmus különbözik fémion jelenlétében és hiányában. | One goal of this project was the application of various multinuclear, and multidimensional NMR spectroscopy techniques. I adapted several pulse sequences during the investigation of the following topics: I characterised the solution structure and behaviour of several open-chained and cyclic peptides, that are drug candidates. I performed the spectral assignment of the homodimer dynein light chain (DLC), made up from 178 aminoacid residues. I studied the binding properties upon formation of a DLC-myosin complex, and determined the Kd binding constant. I investigated several myosin fragments, and realised that the instrinsecally unorganised peptide fragments adopt a definite structure upon binding. Making use of 31P NMR measurements I made the kinetic investigation of the hydrolisis reaction catalysed by the dUTPase enzyme. I characterized the reaction products, calculated the pseudo-first order kinetic constant, and proved that the reaction mechanism follows a different route in the presence and absence of metal ion

Pure shift amide detection in conventional and TROSY-type experiments of 13^{13}C,15^{15}N-labeled proteins

Large coupling networks in uniformly $^{13}$C,$^{15}$N-labeled biomolecules induce broad multiplets that even in flexible proteins are frequently not recognized as such. The reason is that given multiplets typically consist of a large number of individual resonances that result in a single broad line, in which individual components are no longer resolved. We here introduce a real-time pure shift acquisition scheme for the detection of amide protons which is based on 13C-BIRDr,X. As a result the full homo- and heteronuclear coupling network can be suppressed at low power leading to real singlets at substantially improved resolution and uncompromised sensitivity. The method is tested on a small globular and an intrinsically disordered protein (IDP) where the average spectral resolution is increased by a factor of ~ 2 and higher. Equally important, the approach works without saturation of water magnetization for solvent suppression and exchanging amide protons are not affected by saturation transfer

Investigation of the stomata size and frequency of grapevine (Vitis vinifera L.) cultivar ‘Kékfrankos’

Grapevine (Vitis vinifera L.) leaves show high morphological diversity alongside the shoot. This variability has been investigated in this study to explore the change in leaf size, leaf thickness, stomata density and stomata size among the 1st, 5th and 10th leaves on the main shoots and leaves on the laterals. Results showed that leaf size altered from the basal abaxial leaves to the middle of the shoot, while the laterals had the smallest leaves. Number of stomata also varied significantly regarding the different levels of the canopy. First leaves on the shoots had the least stomata per unit leaf area while this number increased above. In contrast with this the size, i.e. length and width of the stomata did not differ. Leaf thickness was the lowest on the leaves of the lateral shoots, while the values decreased from the 1st to the 10th nodes. These results raised the question about the ontogeny and heteroblasty of the grapevine foliage

Impulsiveness in Alcohol Addiction and Pathological Gambling

Numerous conducted studies, as well as the daily clinical experience, proves the importance of the role that impulsiveness plays in the clinical course and the treatment response in both psychoactive substance addictions, such as alcohol use disorder and behavioral addictions, such as gambling addiction. In the daily practice, impulsiveness as a personality trait is observed either in the context of a determining, i.e. causing factor in the personality development or as a result of a developed addiction. Certain types of impulsiveness are more often present in certain types of addicts and their detection enables us to make a more precise diagnosis and sub-classification as well as a more adequate adaptation of the treatment protocol. According to the studies so far, the occurrence of impulsiveness significantly affects the occurrence of relapse in treated addicts. To a large extent it also determines the range of the treatment response to the applied treatment procedures. The objective of this review was to point out the specific features of the prevalence of certain impulsiveness elements in psychoactive substance addicts, such as alcohol addicts, and of behavioral addicts, such as gambling addicts, and to additionally emphasize their clinical, diagnostic, treatment and prognostic value

A fehérjefeltekeredés alapjai: a feltekeredés, a letekeredés és az aggregáció vizsgálata atomi szintű kísérleti és számítási módszerekkel = Unifying principles of protein folding: understanding folding, unfolding and aggregation at atomic-level by experimental and computational methods

Célkitűzésünk a fehérjefeltekeredés és konformációs állapotváltozások mélyebb megértése, alkalmasan megválasztott modellrendszerek - azaz különböző méretű és belső dinamikájú fehérjék - vizsgálatán keresztül. A kémiailag vagy bakteriális úton előállított fehérjék szerkezeti és dinamikai paramétereinek meghatározását követően olyan új variánsokat tervezünk, amelyek vagy még rendezettebbek, vagy a belsőleg rendezetlen fehérjék esetében, még rendezetlenebbé váltak. A vizsgált mini-, moduláris fehérjék, valamint a funkcionálisan rendezetlen rendszerek körét olyan fontos molekulák alkották, amelyek közvetlen kapcsolatban állnak a cukorbetegség, az agyi neurodegeneratív elváltozások, vagy az immunológia különböző tárgykörével. Kutatásaink során mind a téralkat, mind a fehérjék belső mozgékonyságának explicit jellemzésén keresztül értettük meg jobban az adott makromolekulák biológiai szerepét, fiziológiás jelentőségét. A peptidek és fehérjék racionális tervezése során nem csak mutánsokat és variánsokat, de nem-természetes aminosavakból felépülő „foldamereket” is terveztünk, majd állítottunk elő. Vizsgáltuk a béta-aminosavak és béta-peptidek beépíthetőségét és felhasználhatóságát a téralkat és mozgékonyság függvényében. Eredményeinket 33 angol nyelvű közleményben (IF=116,984), webszervereken, és több mint 40 hazai és nemzetközi tudományos fórumon és konferencián adtuk közre. | Our aim was to decipher and better understand the molecular details of protein folding and conformational switching achieved via selected polyamide nanosystems of different size and internal dynamics. Once the structural parameters of the chemically synthesized or bacterially expressed proteins were determined, new variants and mutants were designed. Thus, the polypeptide chain was made either more ordered for globular proteins, or less structured for the disordered proteins (IDP). The investigated mini-, modular, globular and unstructured proteins are involved in biologically important cellular processes associated with either Diabetes Mellitus, neurodegenerative or immunological diseases. Our goal was to better understand the biological role and function of these proteins by monitoring both their structure (NMR and X-ray crystallography) and internal dynamics (NMR). The rational design of selected peptides and proteins resulted in not only mutants and variants of the parent macromolecule, but also foldamers. The applicability of beta amino acids and beta peptides were in focus. Our results were published in international journals (33 articles, IF=116,984), webservers, and presented on over 40 scientific meetings

Selective 1Hα NMR methods to reveal functionally relevant proline cis/trans isomers in IDPs

It is important to identify proline cis/trans isomers that appear in several regulatory mechanisms of proteins, and to characterize minor species that are present due to the conformational heterogeneity in intrinsically disordered proteins (IDPs). To obtain residue level information on these mobile systems we introduce two H-1(alpha)-detected, proline selective, real-time homodecoupled NMR experiments and analyze the proline abundant transactivation domain of p53. The measurements are sensitive enough to identify minor conformers present in 4-15 % amounts; moreover, we show the consequences of CK2 phosphorylation on the cis/trans-proline equilibrium. Using our results and available literature data we perform a statistical analysis on how the amino acid type effects the cis/trans-proline distribution. The methods are applicable under physiological conditions, they can contribute to find key proline isomers in proteins, and statistical analysis results may help in amino acid sequence optimization for biotechnological purposes

Magasabbrendű talamikus magvak serkentő és gátló kontrollja = Excitatory and inhibitory control of higher order thalamic nuclei

Az OTKA pályázat során leírtunk és karakterizáltunk egy új gátlás-típust a talamuszban. E gátlórendszer axonvégződései és az általuk közvetített gátlás különbözött a talamuszban jól ismert gátlórendszerek tulajdonságaitól. Az axonterminálisok ultrastruktúrája és az általuk beidegzett célelemek hatékony gátlásra utaltak. Élettani kísérletek igazolták az anatómiai predikciókat és kimutatták, hogy ez a gátlás típus hatékony információ átvitelre képes magas frekvenciás impulzusok esetén is, mikor az ismert gátlópálya hatékonysága jelentősen csökken. Az új gátlás-típus képes megakadályozni a beidegzett talamikus sejtek működését, illetve képes kiváltani a karaterisztikus visszacsapó választ. Az új gátlás-típust sikerült azonosítani több pálya esetén, köztük főemlősökben a Parkinson-kórban érintett talamikus bemenetek esetében is. A pályázat során részletesen vizsgáltuk az új gátló pálya eredő sejtjeinek szerkezetét és működését. Egy új gátlópálya leírása, melyet egyedi működési mechanizmusok jellemeznek felveti a lehetőségét e pálya szelektív modulációjának. Olyan drogok, melyek ezen a speciális gátlóterminálison hatnak segíthetnek azon tünetek enyhítésén, melyeket e pályák aberráns aktivitása okoz (pl. Parkinson-kór, krónikus fájdalom). | During the OTKA project we discovered and characterized a novel inhibitory element in the thalamus. The structure of the nerve endings, their mode of action and the activity of the nerve cells were all different from the previously described inhibitory pathways in this brain centre. The anatomy of the nerve terminals and the nerve elements they contacted indicated powerful inhibitory action. Physiological measurements verified the anatomical predictions and demonstrated that these connections faithfully transfer inhibitory signals even at very high frequency, when the effectiveness of other inhibitory pathways is much reduced. We demonstrated that these inhibitory inputs are indeed able to silence thalamic neurons or induce strong, so-called, ?postinhibitory rebound? activity. The morhology and the actvity of the parent cells of these pathways has also been extensively characterized. We described several of these pathways in different thalamic nuclei most importantly in those known to be involved in Parkinson's disease. Discovering a separate class of inhibitory pathways in the thalamus with distinct mode of action raises the hope for their selective modulation. Drugs which acts on these terminals can help to alleviate the symptoms linked to the aberrant activity of these specialized inhibitory pathways

The mechanism of the reverse recovery-step, phosphate release, and actin activation of Dictyostelium myosin II.

The rate-limiting step of the myosin basal ATPase (i.e. in absence of actin) is assumed to be a post-hydrolysis swinging of the lever arm (reverse recovery step), that limits the subsequent rapid product release steps. However, direct experimental evidence for this assignment is lacking. To investigate the binding and the release of ADP and phosphate independently from the lever arm motion, two single tryptophan-containing motor domains of Dictyostelium myosin II were used. The single tryptophans of the W129+ and W501+ constructs are located at the entrance of the nucleotide binding pocket and near the lever arm, respectively. Kinetic experiments show that the rate-limiting step in the basal ATPase cycle is indeed the reverse recovery step, which is a slow equilibrium step (k(forward) = 0.05 s(-1), k(reverse) = 0.15 s(-1)) that precedes the phosphate release step. Actin directly activates the reverse recovery step, which becomes practically irreversible in the actin-bound form, triggering the power stroke. Even at low actin concentrations the power stroke occurs in the actin-attached states despite the low actin affinity of myosin in the pre-power stroke conformation