Interaction between colicin A pore-forming domain and its cognate immunity protein

213 p.La colicina A y en concreto el dominio formador de poros, pfColA es expresado por Escherichia coli y resulta tóxico para cepas relacionadas. Las bacterias productoras de la toxina expresan la proteína de inmunidad Cai que las protege de la misma toxina que producen. Cai inhibe la formación de poros y subsecuente muerte celular mediante una interacción directa hélice-hélice en la membrana interna de la bacteria. En el presente trabajo se analizó la interacción entre Cai y pfColA en diferentes entornos como micelas de detergente, vesículas lipídicas o en la membrana bacteriana. El estudio funcional en vesículas lipídicas demostró que pfColA es capaz de conducir protones y que esta actividad se ve inhibida por Cai. Esta actividad resultó ser dosis dependiente, saturable y de la que se deduce un complejo 1:1. El par Cai-pfColA fue co-expresado en la membrana interna de E. coli y se consiguió aislar el complejo integro en detergente de tipo alkilglucósido. Por último, la formación de complejo fue testada también en solución en micelas de detergente, donde se forma un complejo muy estable con una afinidad en el rango de micromolar.CSIC Instituto Biofisika Institutu

Residue-by-residue analysis of cotranslational membrane protein integration in vivo

We follow the cotranslational biosynthesis of three multispanning Escherichia coli inner membrane proteins in vivo using high-resolution force profile analysis. The force profiles show that the nascent chain is subjected to rapidly varying pulling forces during translation and reveal unexpected complexities in the membrane integration process. We find that an N-terminal cytoplasmic domain can fold in the ribosome exit tunnel before membrane integration starts, that charged residues and membrane-interacting segments such as re-entrant loops and surface helices flanking a transmembrane helix (TMH) can advance or delay membrane integration, and that point mutations in an upstream TMH can affect the pulling forces generated by downstream TMHs in a highly position-dependent manner, suggestive of residue-specific interactions between TMHs during the integration process. Our results support the ‘sliding’ model of translocon-mediated membrane protein integration, in which hydrophobic segments are continually exposed to the lipid bilayer during their passage through the SecYEG translocon

Proteinen tolestura tunel erribosomikoan

Proteins are synthesised as linear polymers and must fold into their native three-dimensional structure to perform various functions in the cell. Understanding protein folding is crucial because protein misfolding is at the origin of several neurodegenerative diseases. Protein folding can start cotranslationally, i.e. when the emerging peptide is still asso-ciated with the ribosome. Indeed, it has been shown that more than one third of the cell’s proteins fold in the limited space of the ribosome tunnel. Increasing evidence suggests that the ribosome plays a critical role in protein folding. The ribosome can facilitate protein compaction, cause the creation of non-visible media in solution or delay the onset of folding. However, the study of cotranslational folding presents serious difficulties, mainly due to the limitations of the different current techniques. Hence, most studies on protein folding are based on proteins in solution, which are carried out by unfolding and refolding the protein, without taking into account the role of the ribosome in this process. In this article, we summarised the techniques developed in recent years for the study of cotranslational protein folding.; Proteinak polimero lineal gisa sintetizatzen dira eta beren jatorrizko egitura tridimentsionalean tolestu behar dira zelulan hainbat funtzio betetzeko. Proteinen tolespena ulertzea funtsezkoa da, tolespen okerrak hainbat gaixotasun neuro-degeneratiboren jatorria direlako. Proteinen tolespena modu koitzultzailean has daiteke, hau da, sortzen ari den peptidoa erribosomari lotuta dagoenean oraindik. Izan ere, zelularen proteinen heren bat baino gehiago erribosomaren tunelaren espazio mugatuan tolesten direla frogatu da, hau da, erribosomaren gainazalarekiko interakzioek modulatuta eta erribosoma-tunelaren beraren mugen pean. Gero eta ebidentzia gehiagok iradokitzen dute erribosomak funtsezko zeregina duela proteinen tolespenean. Erribosomak proteina trinkotzea erraztu dezake, soluzioan ikusten ez diren bitartekoak sortzea eragin dezake edo tolestearen hasiera atzeratu dezake. Hala ere, proteinen koitzulpenezko tolesdura aztertzeak zailtasun handiak ditu, batik bat, egungo teknikek dituzten mugengatik. Hori dela eta, proteinen tolesteari buruzko ikerketa gehienak soluzioan dauden proteinetan oinarritzen dira, proteina tolestuz eta destolestuz egiten direnak, prozesu horretan erribosomak duen rola kontuan hartu gabe. Artikulu honetan, azken urteotan proteinen koitzulpenezko tolestura ikertzeko garatu diren tekniken laburpena egin da

Residue-by-residue analysis of cotranslational membrane protein integration in vivo

We follow the cotranslational biosynthesis of three multispanning Escherichia coli inner membrane proteins in vivo using high-resolution force profile analysis. The force profiles show that the nascent chain is subjected to rapidly varying pulling forces during translation and reveal unexpected complexities in the membrane integration process. We find that an N-terminal cytoplasmic domain can fold in the ribosome exit tunnel before membrane integration starts, that charged residues and membrane-interacting segments such as re-entrant loops and surface helices flanking a transmembrane helix (TMH) can advance or delay membrane integration, and that point mutations in an upstream TMH can affect the pulling forces generated by downstream TMHs in a highly position-dependent manner, suggestive of residue-specific interactions between TMHs during the integration process. Our results support the ‘sliding’ model of translocon-mediated membrane protein integration, in which hydrophobic segments are continually exposed to the lipid bilayer during their passage through the SecYEG translocon

Interaction between colicin A pore-forming domain and its cognate immunity protein

213 p.La colicina A y en concreto el dominio formador de poros, pfColA es expresado por Escherichia coli y resulta tóxico para cepas relacionadas. Las bacterias productoras de la toxina expresan la proteína de inmunidad Cai que las protege de la misma toxina que producen. Cai inhibe la formación de poros y subsecuente muerte celular mediante una interacción directa hélice-hélice en la membrana interna de la bacteria. En el presente trabajo se analizó la interacción entre Cai y pfColA en diferentes entornos como micelas de detergente, vesículas lipídicas o en la membrana bacteriana. El estudio funcional en vesículas lipídicas demostró que pfColA es capaz de conducir protones y que esta actividad se ve inhibida por Cai. Esta actividad resultó ser dosis dependiente, saturable y de la que se deduce un complejo 1:1. El par Cai-pfColA fue co-expresado en la membrana interna de E. coli y se consiguió aislar el complejo integro en detergente de tipo alkilglucósido. Por último, la formación de complejo fue testada también en solución en micelas de detergente, donde se forma un complejo muy estable con una afinidad en el rango de micromolar.CSIC Instituto Biofisika Institutu

Interaction between colicin A pore-forming domain and its cognate immunity protein

213 p.La colicina A y en concreto el dominio formador de poros, pfColA es expresado por Escherichia coli y resulta tóxico para cepas relacionadas. Las bacterias productoras de la toxina expresan la proteína de inmunidad Cai que las protege de la misma toxina que producen. Cai inhibe la formación de poros y subsecuente muerte celular mediante una interacción directa hélice-hélice en la membrana interna de la bacteria. En el presente trabajo se analizó la interacción entre Cai y pfColA en diferentes entornos como micelas de detergente, vesículas lipídicas o en la membrana bacteriana. El estudio funcional en vesículas lipídicas demostró que pfColA es capaz de conducir protones y que esta actividad se ve inhibida por Cai. Esta actividad resultó ser dosis dependiente, saturable y de la que se deduce un complejo 1:1. El par Cai-pfColA fue co-expresado en la membrana interna de E. coli y se consiguió aislar el complejo integro en detergente de tipo alkilglucósido. Por último, la formación de complejo fue testada también en solución en micelas de detergente, donde se forma un complejo muy estable con una afinidad en el rango de micromolar.CSIC Instituto Biofisika Institutu

Dilute Bicelles for Glycosyltransferase Studies, Novel Bicelles with Phosphatidylinositol

Solution-state NMR can be used to study protein- lipid interactions, in particular, the effect that proteins have on lipids. One drawback is that only small assemblies can be studied, and therefore, fast-tumbling bicelles are commonly used. Bicelles contain a lipid bilayer that is solubilized by detergents. A complication is that they are only stable at high concentrations, exceeding the CMC of the detergent. This issue has previously been addressed by introducing a detergent (Cyclosfos-6) with a substantially lower CMC. Here, we developed a set of bicelles using this detergent for studies of membrane-associated mycobacterial proteins, for example, PimA, a key enzyme for bacterial growth. To mimic the lipid composition of mycobacterial membranes, PI, PG, and PC lipids were used. Diffusion NMR was used to characterize the bicelles, and spin relaxation was used to measure the dynamic properties of the lipids. The results suggest that bicelles are formed, although they are smaller than "conventional " bicelles. Moreover, we studied the effect of MTSL-labeled PimA on bicelles containing PI and PC. The paramagnetic label was shown to have a shallow location in the bicelle, affecting the glycerol backbone of the lipids. We foresee that these bicelles will be useful for detailed studies of protein-lipid interactions

Protein folding within the ribosomal tunnel

[EU] Proteinak polimero lineal gisa sintetizatzen dira eta beren jatorrizko egitura tridimentsionalean tolestu behar dira zelulan hainbat funtzio betetzeko. Proteinen tolespena ulertzea funtsezkoa da, tolespen okerrak hainbat gaixotasun neuro-degeneratiboren jatorria direlako. Proteinen tolespena modu koitzultzailean has daiteke, hau da, sortzen ari den peptidoa erribosomari lotuta dagoenean oraindik. Izan ere, zelularen proteinen heren bat baino gehiago erribosomaren tunelaren espazio mugatuan tolesten direla frogatu da, hau da, erribosomaren gainazalarekiko interakzioek modulatuta eta erribosoma-tunelaren beraren mugen pean. Gero eta ebidentzia gehiagok iradokitzen dute erribosomak funtsezko zeregina duela proteinen tolespenean. Erribosomak proteina trinkotzea erraztu dezake, soluzioan ikusten ez diren bitartekoak sortzea eragin dezake edo tolestearen hasiera atzeratu dezake. Hala ere, proteinen koitzulpenezko tolesdura aztertzeak zailtasun handiak ditu, batik bat, egungo teknikek dituzten mugengatik. Hori dela eta, proteinen tolesteari buruzko ikerketa gehienak soluzioan dauden proteinetan oinarritzen dira, proteina tolestuz eta destolestuz egiten direnak, prozesu horretan erribosomak duen rola kontuan hartu gabe. Artikulu honetan, azken urteotan proteinen koitzulpenezko tolestura ikertzeko garatu diren tekniken laburpena egin da.[EN] Proteins are synthesised as linear polymers and must fold into their native three-dimensional structure to perform various functions in the cell. Understanding protein folding is crucial because protein misfolding is at the origin of several neurodegenerative diseases. Protein folding can start co-translationally, i.e. when the emerging peptide is still associated with the ribosome. Indeed, it has been shown that more than one third of the cell’s proteins fold in the limited space of the ribosome tunnel. Increasing evidence suggests that the ribosome plays a critical role in protein folding. The ribosome can facilitate protein compaction, cause the creation of non-visible media in solution or delay the onset of folding. However, the study of co-translational folding presents serious difficulties, mainly due to the limitations of the different current techniques. Hence, most studies on protein folding are based on proteins in solution, which are carried out by unfolding and refolding the protein, without taking into account the role of the ribosome in this process. In this article, we summarised the techniques developed in recent years for the study of co-translational protein folding.Artikulu hau UPV/EHUk finantzatutako eta Eusko Jaurlaritzako Hezkuntza Sailak emandako dirulaguntzari esker egin da.Peer reviewe