Co-translational insertion and assembly of a multi-spanning membrane protein from Escherichia coli monitored in vivo

• Main Author: Krč, Ajda
• Other Authors: Novinec, Marko
• Format: Bachelor Thesis
• Language: English
• Published: 2020
• Subjects: polytopic membrane proteins; pulling forces; transmembrane α helix; co-translational insertion; politopični membranski proteini; vlečne sile; transmembranska α vijačnica; ko-translacijsko vstavljanje; Sila; sami
• Online Access: https://repozitorij.uni-lj.si/IzpisGradiva.php?id=118722; https://repozitorij.uni-lj.si/Dokument.php?id=133116&dn=; https://plus.si.cobiss.net/opac7/bib/27356163?lang=sl

Most membrane proteins are inserted into the membrane co-translationally. In bacteria, such as the gram-negative Escherichia coli (E. coli), the insertion usually follows the secretory (Sec) pathway with the Sec translocon as a key protein complex that enables partitioning of membrane proteins into the lipid bilayer. To monitor the process of co- translational insertion and folding of membrane proteins, an in vivo translation technique that adopts translational arrest peptides as “force sensors” has been developed recently. Using this technique, the “pulling forces” acting on the nascent polypeptide chain can be measured during integration of transmembrane helices into the membrane. In this study, the method was used to follow the insertion of the multi-spanning membrane protein BtuC, the transmembrane domain of the vitamin B12 translocase in E. coli. Previous work on BtuC suggests that the transmembrane helices (TMHs) insert more or less sequentially, one after the other, following the predicted insertion pattern based on ΔG calculations. However, there are discrepancies in the second part of the generated force profile which imply that apart from hydrophobicity, other forces might play a role in insertion of downstream TMHs of BtuC. Therefore, the aim was to see (1) whether upstream TMHs have an effect on insertion of downstream TMHs and (2) whether they can insert efficiently by themselves. Surprisingly, the results of the first part show no significant difference in pulling forces when the first four upstream TMHs are deleted from the BtuC constructs. This suggests that downstream TMHs of BtuC insert for the most part independently regardless of the presence or absence of the upstream TMHs. The only discrepancy is seen in TMH5, which seems to insert later. For that reason, we decided to test whether the N-terminally engineered hydrophobic Lep segment (TMH1) plays a role in insertion of BtuC constructs. Unfortunately, the results show very poor expression of BtuC constructs without the Lep segment. That indicates that upstream TMHs of BtuC cannot efficiently insert and/or are not stable enough by themselves, at least not in conditions used in the force assay in vivo. In conclusion, hydrophobicity represents the major force contributing to insertion of the multi-spanning membrane protein BtuC. Any other force that might act on transmembrane helices during the process cannot yet be detected using the force measurement assay in vivo. Večina bakterijskih membranskih proteinov se v membrano vstavi po kotranslacijskem mehanizmu. Pri bakterijah, kakršna je tudi gram negativna Escherichia coli (E.coli), vstavljanje navadno poteka po sekrecijskem (Sec) mehanizmu, kjer osrednje funkcije opravlja proteinski kompleks imenovan Sec translokon, ki omogoča prehod membranskih proteinov v membrano. Proces kotranslacijskega vstavljanja in zvijanja membranskih proteinov lahko spremljano z in vivo metodo, kjer s pomočjo peptidov, ki povzročijo začasno zastajanja translacije merimo sile, ki delujejo ob vgrajevanju transmembranskih vijačnic (TMV) v celično membrano. Diplomska naloga se ukvarja s spremljanjem kotranslacijskega vstavljanja in sestavljanja politopičnega membranskega proteina BtuC iz E.coli. BtuC je membranski del bakterijskega ABC transporterja s polnim imenom BtuC2D2(F), ki skrbi za prenos vitamina B12 (kobalamina) v celico. Pretekle raziskave na BtuC so pokazale, da se TMV tega proteina v membrano vstavljajo večinoma zaporedno, v skladu z napovedanimi izračunanimi hidrofobnimi vrednostmi (ΔG). Vendar pa neujemanja v drugem delu profila vlečne sile nakazujejo, da so poleg hidrofobnosti na vstavljanje TM regij vplivajo tudi drugi faktorji. Glavni raziskovalni vprašanji, ki sta vodili diplomsko delo sta torej: (1) ali se druga polovica membranskega proteina BtuC (TMV5-10) lahko vstavlja in sestavlja v membrano neodvisno od prve polovice proteina (TMV1-4) in (2) ali se druga polovica proteina lahko vstavi v membrano sama od sebe. Rezultati poskusov so nekoliko presenetljivi, saj profil vlečne sile drugega dela BtuC ne odstopa bistveno od profila vlečne sile celotnega proteina. To sicer potrjuje, da se vijačnice vstavljajo v membrano neodvisno druga od druge, oziroma da se drugi del proteina BtuC vstavi v membrano neodvisno od prvega dela, vseeno pa eksperimentalni podatki odstopajo od teoretično izračunanih vrednosti ΔG v drugem delu proteina. Glavna razlika med profiloma je vidna v primeru TMV5, ki se glede na eksperimentalne podatke v membrano vstavi pozneje kot sicer. Zato smo se odločili preveriti še, ali na vstavljanje konstruktov vpliva umetno dodan N-terminalni hidrofobni segment Lep. Na žalost so bili nivoji izražanja konstruktov brez hidrofobnih sekvenc Lep na N-koncu prenizki za nadaljnjo analizo podatkov. To nam pove, da se TMV membranskega proteina BtuC ne morejo vstaviti v membrano brez pomoči dodatne vodilne sekvence Lep, oziroma da konstrukti, ki so uporabljeni v metodi zaznavanja vlečne sile in vivo, sami od sebe niso dovolj stabilni. Iz zbranih rezultatov lahko zaključimo, da je hidrofobnost vseeno glavna sila, ki pripomore k vstavljanju in zvijanju membranskih proteinov v membrano. Katerih koli drugih interakcij, ki še delujejo na TMV zaenkrat še ne moremo zaznati z metodo zaznavanja vlečnih sil in vivo.