Konformációs viszonyok és elektronszerkezet-változások vizsgálata fehérjékben = Investigation of conformational fluctuations and electronic structure variations in proteins

Feltérképeztük a foszfoglicerát kináz molekuláris felismerési folyamataiban szerepet játszó konformációs változásokat. Elvégeztük a foszfoinozitol kináz 3 alfa izoformájánnak 3D szerkezetpredikcióját, azonban a ligandumkötődés vizsgálatára a kooperáló partner elállása miatt nem került sor. Felderítettük a tetrahidribiopterin (BH4) kofaktor szerepe a nitrogén monoxid szintetáz (NOS) aktiválódásában. Meghatároztuk a NO kötőhelyét a nitroforin 4 (NP4) fehérje aktív helyén. Low-mode (LMOD) keresésen alapuló platfromfüggetlen konformerkereső és dokkoló eljárást dolgoztunk ki, amely az AMBER programcsomagban is hozzáférhető. Elvégeztük a kifejlesztett eljárás több konformerkereső módszerrel történő összehasonlító vizsgálatát. Eljárást dolgoztunk ki humán P450 2C9 ligandumok azonosítására és az izoforma specificitás vizsgálatára a 2C családban. Felderítettük a NADH kofaktor és NO kölcsönhatás szerepét a P450 NO-reduktáz fehérjében. A támogatott időszak alatt megkezdett hisztamin receptorok kutatását érintő vizsgálataink alapján új lipofil zsebet találtunk a H1 receptorban, ligandum információkkal segített homológiamodellezéssel előállítottuk a H4 receptor első atomi felbontású modelljét és eljárást dolgoztunk ki új H4 ligandunok azonosítására. Utóbbi eljárást az eddig ismert legkiterjedtebb szerkezet alapú szűrővizsgálatban alkalmazva új, kísérletileg is megerősített H4 ligandumokat azonosítottunk. | Conformational motions responsible for the substrate recognition in phosphoglycerate kinase have been explored. Homology model for phosphoinositol 3 kinase alpha isoform has been developed, however docking studies were suspended due to the changed interest of the partner (ComGenex Inc). The role of tetrahydrobiopterin cofactor in nitric oxide synthase has been clarified. The putative nitric oxide binding site in nitrophorin 4 has been identified. A platform-independent LMOD conformational search method has been developed and integrated to AMBER package at UCSF. The performance of this method has been evaluated and compared to other algorithms. A new virtual screening protocol has been developed for the identification of CYP 2C9 ligands. The protocol was useful for isofom specificity studies in the 2C family. The role of NADH-nitric oxide interaction in P450-No reductase has been investigated. A new lipophilic binding pocket in human histamine H1 receptor has been identified. The first atom-level model of human histamine H4 receptor has been constructed by ligand supported homology modelling. that was used to develop an effective virtual screening protocol. The protocol allowed us to perform the largest structure based virtual screening experiment using a screening database of more than 8 million compounds. Identified new chemical scaffolds showed submicromolar binding affinity towards the human histamine H4 receptor as revealed by experimental studies

Catalytically distinct states captured in a crystal lattice: the substrate-bound and scavenger states of acylaminoacyl peptidase and their implications for functionality

Acylaminoacyl peptidase (AAP) is an oligopeptidase that only cleaves short peptides or protein segments. In the case of AAP fromAeropyrum pernix(ApAAP), previous studies have led to a model in which the clamshell-like opening and closing of the enzyme provides the means of substrate-size selection. The closed form of the enzyme is catalytically active, while opening deactivates the catalytic triad. The crystallographic results presented here show that the open form of ApAAP is indeed functionally disabled. The obtained crystal structures also reveal that the closed form is penetrable to small ligands: inhibitor added to the pre-formed crystal was able to reach the active site of the rigidified protein, which is only possible through the narrow channel of the propeller domain. Molecular-dynamics simulations investigating the structure of the complexes formed with longer peptide substrates showed that their binding within the large crevice of the closed form of ApAAP leaves the enzyme structure unperturbed; however, their accessing the binding site seems more probable when assisted by opening of the enzyme. Thus, the open form of ApAAP corresponds to a scavenger of possible substrates, the actual cleavage of which only takes place if the enzyme is able to re-close.</jats:p

Achieving Functionality Through Modular Build-up: Structure and Size Selection of Serine Oligopeptidases

Enzymes of the prolyl oligopeptidase family (S9 family) recognize their substrates not only by the specificity motif to be cleaved but also by size - they hydrolyze oligopeptides smaller than 30 amino acids. They belong to the serine-protease family, but differ from classical serine-proteases in size (80 kDa), structure (two domains) and regulation system (size selection of substrates). This group of enzymes is an important target for drug design as they are linked to amnesia, schizophrenia, type 2 diabetes, trypanosomiasis, periodontitis and cell growth. By comparing the structure of various members of the family we show that the most important features contributing to selectivity and efficiency are: (i) whether the interactions weaving the two domains together play a role in stabilizing the catalytic triad and thus their absence may provide for its deactivation: these oligopeptidases can screen their substrates by opening up, and (ii) whether the interaction-prone β-edge of the hydrolase domain is accessible and thus can guide a multimerization process that creates shielded entrance or intricate inner channels for the size-based selection of substrates. These cornerstones can be used to estimate the multimeric state and selection strategy of yet undetermined structures

Assignment of vibrational circular dichroism cross-referenced electronic circular dichroism spectra of flexible foldamer building blocks: towards assigning foldamers’ pure chiroptical properties

The assignment of the most established ECD spectra of polypeptides and foldamers is either “evidence based” or rely on the 3D-structures of longer oligomers of limited internal dynamics, derived from NMR (or X-ray) data. Critics warn that using NMR and ECD side by side have severe limitations for flexible molecules as the explicit knowledge of the conformational ensembles is a challenge. Herein we present the old-new method of comparing ab initio computed and measured VCD data to validate both structures and, conf(i), and their relative weights, c(i), making up the conformational ensemble. Based on the array of {conf(i), c(i)} the pure ECD spectra, g(i)conf(i), can be ab initio calculated. The reconstructed spectrum Σc(i)*g(i)conf(i) can thus help to assign any experimental ECD counterpart. Here we present such a protocol successfully applied for flexible foldamer building blocks of sugar β-amino acid diamides. The epimeric pair of our model system was selected because these molecules are conformationally tunable by simple chemical modification (N-methylation) and thus, the robustness of our current approach could be probed. The initial H-bond (NH..O) eliminated by N-methylation reorients the amide plain influencing the chiroptical properties of the foldamer building block, a structural change successfully monitored by the VCD- and ECD-transition changes now assigned to pure conformers. The current method seems general and effective without requiring extensive CPU and spectroscopic resources

First principles calculation of the reaction rates for ligand binding to myoglobin: the cases of NO and CO

Ligand binding by proteins is among the most fundamental processes in nature. Among these processes the binding of small gas molecules, such as O2, CO and NO to heme proteins has traditionally received vivid interest, which was further boosted by their recently recognized significant role in gas sensing in the body. At the heart of the binding of these ligands to the heme group is the spin-forbidden reaction between high-spin iron(II) and the ligand yielding a low spin adduct. We use computational means to address the complete mechanism of CO and NO binding by myoglobin. As it involves several steps occurring on different time-scales, molecular dynamics simulations were performed to address the diffusion of the ligand through the enzyme, and DFT calculations in combination with statistical rate calculation to investigate the spin-forbidden reaction. The calculations yielded rate constants in qualitative agreement with experiment and revealed that the bottle-neck of NO and CO binding is different: for NO diffusion was found to be rate-limiting, while for CO the spin-forbidden step is the slowest

Predictable Conformational Diversity in Foldamers of Sugar Amino Acids

Systematic conformational search was carried out for monomers and homohexamers of furanoid β-amino acids: cis-(S,R) and trans-(S,S) stereoisomers of aminocyclopentane carboxylic acid (ACPC), two different aminofuranuronic-acids (AFU(alpha) and AFUβ), their isopropylidene derivatives (AFU(ip)) as well as the key intermediate β-aminotetrahydrofurancarboxylic acid (ATFC). Stereochemistry of the building blocks was chosen to match with that of natural sugar amino acid (xylose and ribose) precursors (XylAFU and RibAFU). Results show that hexamers of cis furanoid beta-amino acids show great variability: while hydrophobic cyclopentane (cis(ACPC)6), and hydrophilic (XylAFU(alpha)/(beta))6 foldamers favor two different zigzagged conformation as hexamers, the backbone fold turns into a helix in case of (cisATFC)6 (10-helix) and (XylAFU(ip))6 (14-helix). Trans stereochemistry resulted in hexamers exclusively of right-handed helix conformation, (H12P)6, regardless of their polarity. We found that the preferred oligomeric structure of XylAFU(alpha)/(beta) is conformationally compatible with beta-pleated sheets, while that of the trans/(S,S) units match with alpha-helices of proteins

The route from the folded to the amyloid state: exploring the potential energy surface of a drug-like miniprotein

The amyloid formation of the folded segment of a variant of Exenatide (a marketed drug for Type-2 Diabetes Mellitus ) was studied by ECD and NMR. We found that the optimum temperature for E5 protein amyloidosis coincides with body temperature and requires well below physiological salt concentration. Decomposition of the ECD spectra and its barycentric representation on the folded-unfolded-amyloid potential energy surface allowed us to monitor the full range of molecular transformation of amyloidogenesis. We identified points of no return ( e.g. T =37°C, pH =4.1, c E5 =250µM, c NaCl =50mM, t >4-6 h) which will inevitably gravitate into the amyloid-state. The strong B-type FUV-ECD spectra and an unexpectedly strong NUV-ECD signal (Θ ~275-285nm ) indicate that the amyloid phase of E5 is built from monomers of quasi -elongated backbone structure ( φ ~-145°, ψ ~+145°) with strong interstrand Tyr↔Trp interaction. Misfolded intermediers and the buildup of "toxic" early-stage oligomers leading to self-association were identified and monitored as function of time. Results indicate that the amyloid transition is triggered by subtle misfolding of the α-helix exposing aromatic and hydrophobic side chains that may provide the first centers for an intermolecular reorganization. These initial clusters provide the spatial closeness and sufficient time for a transition to the β-structured amyloid nucleus thus the process follows a nucleated growth mechanism

Fémek szerepe a fehérjeszerkezetben és - működésben = The role of metals in protein structure and function

Fehérjekrisztallográfia, mágneses magrezonancia-spektroszkópia és molekulamodellezés segítségével vizsgáltuk az összefüggéseket néhány metalloprotein, valamint egy új típusú, rendezetlen fehérje szerkezete és működése között. Hatékony módszert fejlesztettünk ki a reakcióút kvantummechanikai számítására enzimekben. Tisztáztuk a DNS javításában fontos szerepet játszó dUTPáz által katalizált reakció legtöbb részletét. Meggyőző bizonyítékokat szolgáltattunk arra, hogy az enzimatikus foszfáthidrolízis során a dUTPázban nagy energiájú, trigonális bipiramisos elrendeződésű intermedier keletkezik. Kimutattuk, hogy a KAR-2 nevű molekula más, biszindol típusú ligandumoktól eltérő módon kötődik a kalmodulinhoz, ez magyarázza különleges fiziológiai hatását. Elvégeztük a hemoglobin hem-csoportjainak normál koordináták szerinti analízisét, amiből következtetéseket vontunk le a szerkezetre vonatkozóan. A deformációk azt mutatják, hogy a hem csoport szerkezete érzékeny a molekula távoli részében kötődő effektor jelenlétére, ami az allosztérikus szabályozás hatásmechanizmusának a tercier szerkezettel való kapcsolatát támasztja alá. A közelmúltban egy új agy-specifikus fehérjét izoláltunk, melynek átlagos rendezetlensége 46-47%, tehát szerkezet nélkülinek tekinthető. Részletes vizsgálatokat végeztünk e fehérje, illetve különböző fehérjékkel képezett komplexe szerkezetére vonatkozóan. | We investigated the relationship between the structure and activity of some metalloproteins and a new unfolded protein. We developed an efficient method for the quantum mechanical calculation of the reaction path in enzymes. Most details of the reaction catalysed by dUTPase, playing an important role in DNA repair, have been clarified. We provided convincing evidence that during enzymatic phosphate hydrolysis a high-energy, trigonal bipyramidal intermediate is formed. We have shown that the molecule KAR-2, in contrast to other bisindole-type ligands, has a different binding mode to calmodulin, which explains its special physiological effect. We performed the normal co-ordinate analysis of the hem groups of haemoglobin and derived conclusions on their structure. The deformations indicate that the structure of the hem group is sensitive to the presence of an effector bound in a distant region of the molecule. This finding supports the relation between the allosteric mechanism of action and the tertiary structure. Recently we isolated a new brain-specific protein, which is 46 to 47 per cent disordered, i.e. it can be considered as unfolded. We made detailed studies on the structure of this protein and its complex with others