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Dinamično induciran transport vode preko membranskih koprenašalcev
ID Sever, Marko (Author), ID Merzel, Franci (Mentor) More about this mentor... This link opens in a new window, ID Mravljak, Janez (Comentor)

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Abstract
V zadnjem desetletju se je razumevanje molekulskih mehanizmov membranskih prenašalcev dramatično povečalo, predvsem na račun eksperimentalno določenih struktur konformer, ki ustrezajo različnim funkcionalnim stanjem membranskih prenašalcev. Še vedno pa le-teh ni dovolj za natančen opis mehanističnih lastnosti tovrstnih proteinov. Membranski koprenašalni proteini so podvrženi velikim strukturnim preureditvam, ki so na splošno povezane z dogodki vezave in so bistvene za njihovo biološko funkcijo. Z združevanjem strukturnega znanja s kemičnimi, biofizikalnimi in računalniškimi pristopi se je začelo pojavljati jasnejše razumevanje različnih načinov transporta majhnih molekul in sorodnih zvrsti. Računalniški pristop predstavlja v tem primeru pomembno orodje, ki omogoča proučevanje delovanja teh sistemov na molekulskem nivoju in proučevanje sklopitev biofizikalnih lastnosti sistemov z eksperimentalno merljivimi količinami. Membranski prenašalci se lahko, poleg njihove glavne vloge specifičnih prenašalcev za majhne molekule in ione, obnašajo tudi kot vodni kanali. Vendar pa pogosto v različnih sistemih ni natančno znan niti položaj glavne poti prehoda vode v proteinu niti njihov funkcionalni mehanizem delovanja. Na splošno je celoten proces transporta substratov zagotovljen s prehodi »izmeničnega dostopa« med dvema glavnima konformacijskima stanjema: navzven obrnjena (OF) in navznoter obrnjena (IF) stanja, pri katerih je dostopnost substrata premaknjena z ene strani membrane na drugo. V okviru naše študije smo se osredotočili na transportne mehanizme vode v človeškem natrij glukoznem koprenašalcu 1 (SGLT1), pri študiju katerega pa je bilo do sedaj prikazanih veliko nasprotujočih hipotez o delovanju, ki po našem mnenju niso bile dovolj podprte z natančno biofizikalno študijo mehanizma. Namen študije je pojasniti vodni transport v omenjenem prenašalcu kot splošen mehanizem, ki bi se ga dalo prenesti tudi na druge membranske prenašalne sisteme, kot tudi na ostale proteinske in neproteinske sisteme. S študijo interne proteinske dinamike z uporabo metod atomistične molekulske dinamike, razvoja metod za zasledovanje difuzijskega in osmotsko pogojenega prehoda vode in specifičnih implementacij analize glavnih komponent (PCA) smo pojasnili mehanizem vodnega transporta v preiskovanem prenašalnem proteinu. Raziskovali smo protein SGLT1, vgrajen v membrano v različnih konformacijskih stanjih, ob prisotnosti ali odsotnosti substratov ter v obliki kompleksov zaviralec-protein. Natančno smo razklopili načine domenskega gibanja transporterja na posamezne komponente, ki dobro opisujejo v literaturi opisan karakterističen način gibanja t.i. "zibajočega snopa" (ang. rocking-bundle), in analizirali vpliv prisotnosti ali odsotnosti ligandov ali zaviralcev na fleksibilnostne lastnosti posameznega načina gibanja, kot tudi celokupnega domenskega gibanja. Ugotovili smo, da ima na prehod vode vpliv domensko gibanje prenašalnega proteina. Ovrednotili smo učinek uporabe zaviralcev na biofizikalne lastnosti proteina in ugotovili, da v primeru vodnega transporta svoj učinek najmočneje izražajo alosterično na mestu, ki ni v neposrednem stiku z vezavnim mestom za sladkorni del vezavnega mesta zaviralca. Predlagamo, da je pri načrtovanju novih zaviralcev smiselno upoštevati tudi kriterij za utrditev strukture oziroma gibanja prenašalca ob vezavi z zaviralcem. Med simulacijo molekulske dinamike smo zaznali prehod naravnega sladkornega liganda v potencialen nov vezavni žep v SGLT1. Ovrednotili smo biofizikalne lastnosti sistema, v kolikor je sladkorni ligand vezan v osnovnem ali pa v alternativnem vezavnem mestu. Glede na podano možnost vezave liganda v alternativno vezavno mesto smo izvedli virtualno rešetanje knjižnice spojin in z uporabo v tem delu razvitih metod napovedali potencialni učinek izbranega zadetka.

Language:Slovenian
Keywords:membranski proteini, molekulska dinamika, kvaziharmonska analiza, interna dinamika, vodni transport, SGLT1, vSGLT
Work type:Doctoral dissertation
Organization:FFA - Faculty of Pharmacy
Year:2023
PID:20.500.12556/RUL-153453 This link opens in a new window
Publication date in RUL:07.01.2024
Views:413
Downloads:38
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Secondary language

Language:English
Title:Dynamically induced water transport through membrane cotransporters
Abstract:
In the last decade, the understanding of the molecular mechanisms of membrane transporters has increased dramatically, mainly through experimentally determined conformer structures corresponding to different functional states of membrane transporters. However, these are still insufficient to accurately describe the mechanistic properties of these proteins. Membrane co-transporter proteins undergo major structural rearrangements, generally related to binding events, which are essential for their biological function. By combining structural knowledge with chemical, biophysical and computational approaches, a clearer understanding of the different modes of transport of small molecules and related species is beginning to emerge. In this case, the computational approach represents an important tool that allows the study of the functioning of these systems at the molecular level and the coupling of the biophysical properties of the systems to experimentally measurable quantities. In addition to their main role as specific carriers for small molecules and ions, membrane carriers can also behave as water channels. However, often in different systems, neither the position of the main water passage pathway in the protein nor their functional mechanism of action is precisely known. In general, the whole process of substrate transport is ensured by "alternating access" transitions between two main conformational states: the outward-facing (OF) and the inward-facing (IF) states, in which the substrate accessibility is shifted from one side of the membrane to the other. In the context of our study, we focused on the water transport mechanisms in human sodium-glucose co-transporter 1 (SGLT1), the study of which has so far shown numerous conflicting hypotheses of action, which in our opinion have not been sufficiently supported by a detailed biophysical study of the mechanism. The aim of this study is to elucidate the water transport in this transporter as a general mechanism that could be transferred to other membrane transporters as well as to other protein and non-protein systems. By studying the internal protein dynamics using atomistic molecular dynamics methods, the development of methods to trace diffusion- and osmotically-dependent water transport and specific implementations of the principle component analysis (PCA) method, we have elucidated the mechanism of water transport in the carrier protein under investigation. The membrane-embedded SGLT1 protein was investigated in different conformational states, including the states in the presence of ions and substrates, and in the form of inhibitor-protein complexes. We have precisely decomposed the transporter's domain motion modes into individual components that describe the characteristic rocking-bundle motion mode described in the literature and analysed the influence of the presence or absence of ligands or inhibitors on the flexibility properties of each mode as well as of the overall domain motion. We evaluated the effect of the use of inhibitors on the biophysical properties of the protein and found that, in the case of aqueous transport, they express their effect allosterically at a site that is not in direct contact with the binding site for the sugar moiety of the inhibitor binding site. We suggest that the design of new inhibitors should take into account a requirement for the stiffening of protein structure and dynamics upon binding with inhibitor. Molecular dynamics simulations have detected the transition of the natural sugar ligand to a potential new binding pocket in SGLT1. We have evaluated the biophysical properties of the system insofar as the sugar ligand is bound in either the primary or the alternative binding site. Given an indication of the possibility of ligand binding in the alternative binding site, we performed a virtual search of the compound library and used the developed methods to predict the potential effect of the selected hit.

Keywords:membrane proteins, molecular dynamics, quasiharmonic analysis, internal dynamics, water transport, SGLT1, vSGLT

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