Immunization with nanovaccines containing mutated K-Ras peptides and imiquimod aggravates heterotopic pancreatic cancer induced in mice

PurposeThe growing incidence and lethality of pancreatic cancer urges the development of new therapeutic approaches. Anti-tumoral vaccines can potentiate the immune response against the tumor, targeting specific antigens expressed only on tumor cells. In this work, we designed new vaccines for pancreatic cancer, composed by chitosan nanocapsules (CS NCs) containing imiquimod (IMQ) as adjuvant, and targeting the K-Ras mutation G12V.Experimental designWe tested the immunogenicity of our vaccines in mice, carrying different combinations of K-Ras mutated peptides. Then, we analyzed their prophylactic and therapeutic efficacy in mice bearing heterotopic pancreatic cancer.ResultsUnexpectedly, although good results were observed at short time points, the different combinations of our CS NCs vaccines seemed to potentiate tumor growth and reduce survival rate. We propose that this effect could be due to an inadequate immune response, partially because of the induction of a regulatory tolerogenic response.ConclusionOur results call for caution in the use of some NCs containing IMQ in the immunotherapy against pancreatic cancer

Polymeric Nanocapsules for Vaccine Delivery: Influence of the Polymeric Shell on the Interaction With the Immune System

The use of biomaterials and nanosystems in antigen delivery has played a major role in the development of novel vaccine formulations in the last few decades. In an effort to gain a deeper understanding of the interactions between these systems and immunocompetent cells, we describe here a systematic in vitro and in vivo study on three types of polymeric nanocapsules (NCs). These carriers, which contained protamine (PR), polyarginine (PARG), or chitosan (CS) in the external shell, and their corresponding nanoemulsion were prepared, and their main physicochemical properties were characterized. The particles had a mean particle size in the range 250–450 nm and a positive zeta potential (~30–40 mV). The interaction of the nanosystems with different components of the immune system were investigated by measuring cellular uptake, reactive oxygen species production, activation of the complement cascade, cytokine secretion profile, and MAP kinases/nuclear factor κB activation. The results of these in vitro cell experiments showed that the NC formulations that included the arginine-rich polymers (PR and PARG) showed a superior ability to trigger different immune processes. Considering this finding, protamine and polyarginine nanocapsules (PR and PARG NCs) were selected to assess the association of the recombinant hepatitis B surface antigen (rHBsAg) as a model antigen to evaluate their ability to produce a protective immune response in mice. In this case, the results showed that PR NCs elicited higher IgG levels than PARG NCs and that this IgG response was a combination of anti-rHBsAg IgG1/IgG2a. This work highlights the potential of PR NCs for antigen delivery as an alternative to other positively charged nanocarriersThis work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2011-30337-C02-02 and BIO2014-53091-C3-1-R). Financial support from the Xunta de Galicia (Centro singular de investigación de Galicia 2016–2019 and Grupo de referencia competitiva, ED431C 2016041) and the European Union (European Regional Development Fund—ERDF) is gratefully acknowledged. MP acknowledges fellowships from the Spanish Ministry of Education (FPU predoctoral grants program)S

Polymeric nanocapsules for vaccine delivery: influence of the polymeric shell on the interaction with the immune system

The use of biomaterials and nanosystems in antigen delivery has played a major role in the development of novel vaccine formulations in the last few decades. In an effort to gain a deeper understanding of the interactions between these systems and immunocompetent cells, we describe here a systematic in vitro and in vivo study on three types of polymeric nanocapsules (NCs). These carriers, which contained protamine (PR), polyarginine (PARG), or chitosan (CS) in the external shell, and their corresponding nanoemulsion were prepared, and their main physicochemical properties were characterized. The particles had a mean particle size in the range 250-450 nm and a positive zeta potential (~30-40 mV). The interaction of the nanosystems with different components of the immune system were investigated by measuring cellular uptake, reactive oxygen species production, activation of the complement cascade, cytokine secretion profile, and MAP kinases/nuclear factor κB activation. The results of these in vitro cell experiments showed that the NC formulations that included the arginine-rich polymers (PR and PARG) showed a superior ability to trigger different immune processes. Considering this finding, protamine and polyarginine nanocapsules (PR and PARG NCs) were selected to assess the association of the recombinant hepatitis B surface antigen (rHBsAg) as a model antigen to evaluate their ability to produce a protective immune response in mice. In this case, the results showed that PR NCs elicited higher IgG levels than PARG NCs and that this IgG response was a combination of anti-rHBsAg IgG1/IgG2a. This work highlights the potential of PR NCs for antigen delivery as an alternative to other positively charged nanocarriers.Ministerio de Ciencia e Innovación | Ref. SAF2011-30337-C02-02Ministerio de Economía y Competitividad | Ref. BIO2014-53091-C3-1-RXunta de Galicia | Ref. ED431C 201604

PVM/MA-shelled selol nanocapsules promote cell cycle arrest in A549 lung adenocarcinoma cells

Background: Selol is an oily mixture of selenitetriacylglycerides that was obtained as a semi-synthetic compound containing selenite. Selol is effective against cancerous cells and less toxic to normal cells compared with inorganic forms of selenite. However, Selol’s hydrophobicity hinders its administration in vivo. Therefore, the present study aimed to produce a formulation of Selol nanocapsules (SPN) and to test its effectiveness against pulmonary adenocarcinoma cells (A549). Results: Nanocapsules were produced through an interfacial nanoprecipitation method. The polymer shell was composed of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymer. The obtained nanocapsules were monodisperse and stable. Both free Selol (S) and SPN reduced the viability of A549 cells, whereas S induced a greater reduction in non-tumor cell viability than SPN. The suppressor effect of SPN was primarily associated to the G2/M arrest of the cell cycle, as was corroborated by the down-regulations of the CCNB1 and CDC25C genes. Apoptosis and necrosis were induced by Selol in a discrete percentage of A549 cells. SPN also increased the production of reactive oxygen species, leading to oxidative cellular damage and to the overexpression of the GPX1, CYP1A1, BAX and BCL2 genes. Conclusions: This study presents a stable formulation of PVM/MA-shelled Selol nanocapsules and provides the first demonstration that Selol promotes G2/M arrest in cancerous cells

Stealth monoolein-based nanocarriers for delivery of siRNA to cancer cells

While the delivery of small interfering RNAs (siRNAs) is an attractive strategy to treat several clinical con- ditions, siRNA-nanocarriers stability after intravenous administration is still a major obstacle for the development of RNA-interference based therapies. But, although the need for stability is well recognized, the notion that strong stabilization can decrease nanocarriers efficiency is sometimes neglected. In this work we evaluated two stealth functionalization strategies to stabilize the previously validated dioctade- cyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. The nanocarriers were pre- and post-pegylated, forming vectors with different stabilities in biological fluids. The stealth nanocarriers behavior was tested under biological mimetic conditions, as the production of stable siRNA-lipoplexes is determinant to achieve efficient intravenous siRNA delivery to cancer cells. Upon incubation in human serum for 2 h, by fluorescence Single Particle Tracking microscopy, PEG-coated lipo- plexes were found to have better colloidal stability as they could maintain a relatively stable size. In addi- tion, using fluorescence fluctuation spectroscopy, post-pegylation also proved to avoid siRNA dissociation from the nanocarriers in human serum. Concomitantly it was found that PEG-coated lipoplexes improved cellular uptake and transfection efficiency in H1299 cells, and had the ability to silence BCR-ABL, affecting the survival of K562 cells. Based on an efficient cellular internalization, good silencing effect, good siRNA retention and good col- loidal stability in human serum, DODAB:MO (2:1) siRNA-lipoplexes coated with PEG-Cer are considered promising nanocarriers for further in vivo validation. Statement of Significance This work describes two stealth functionalization strategies for the stabilization of the previously validated dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. These nanocarriers are capable of efficiently incorporating and delivering siRNA molecules to cells in order to silence genes whose expression is implicated in a pathological condition. The main objective was to functionalize these nanocarriers with a coating conferring protection to siRNA in blood without compromising its efficient delivery to cancer cells, validating the potential of DODAB:MO (2:1) siRNA-lipoplexes as therapeutic vec- tors. We show that the stealth strategy is determinant to achieve a stable and efficient nanocarrier, and that DODAB:MO mixtures have a very promising potential for systemic siRNA delivery to leukemic cells.FEDER through POFC-COMPETE and by national funds from FCT I.P. through the strategic funding UID/BIA/04050/2013 (CBMA) and PEst-C/FIS/UI0607/2013 (CFUM) and PTDC/QUI/69795/2006. We thank the support of the Frame Work Program 7 of the European Commission: BIOCAPS (316265, FP7/REGPOT) and Xunta de Galicia, Spain (Agrupamento INBIOMED, Grupo con potencial crecimiento) reference IF/00498/2012, scholarship SFRH/BD/68588/2010. NanoDelivery-I&D em Bionanotecnologia, Lda. for access to their equipment

Influencia de la movilidad de las hélices en la función de la bacteriorodopsina

La bacteriorodopsina (BR) es una proteína transportadora de protones que se encuentra en la membrana de la arqueobacteria H. salinarum. Consta de siete hélices α y un cromóforo, el retinal, unido covalentemente a la hélice G. Ha sido muy estudiada debido a su similitud con la rodopsina visual y otras proteínas de la familia de las GPCRs, además de formar parte de uno de los sistemas fotosintéticos más sencillos que se conocen. La BR se activa mediante la absorción de un fotón por parte del retinal, lo que proporciona la energía necesaria para realizar el fotociclo. El resultado final es el transporte neto de un protón desde el lado citoplasmático al lado extracelular. A pesar de que su estructura y función son bien conocidas, todavía hay aspectos funcionales que no han sido suficientemente caracterizados. En este sentido, la existencia de un posible movimiento de las hélices E, F y G en el lado citoplasmático durante el proceso de reprotonación ha sido ampliamente debatida. El hecho de que estos movimientos hayan sido detectados solamente en estructuras cristalográficas de mutantes ha llevado a pensar que podría tratarse de un artefacto. Asimismo, los trabajos de Resonancia Paramagnética Electrónica (EPR) en proteína marcada con una sonda de spin han dado valores muy diversos para la magnitud de estos movimientos, creando también dudas sobre su existencia y su implicación en la función. Este trabajo ha tenido como objetivo estudiar la influencia de la flexibilidad de las hélices E-F y F-G en la función de la BR. Para ello se ha expresado una quimera de la BR, denominada Loop5, donde se ha ampliado la longitud, y por tanto la flexibilidad, del bucle E-F mediante la sustitución del bucle nativo por su homólogo en la rodopsina. Por otro lado se ha restringido la flexibilidad de las hélices E-F y F-G mediante la expresión de dos dobles mutantes cisteína y la formación de un puente disulfuro interhélice: F153C(E)/R175C(F) y E166C(F)/A228C(G). Tanto el aumento de la flexibilidad del bucle E-F como la restricción de la movilidad de estas hélices y de las hélices F-G provocan una profunda alteración en la función de transporte. Asimismo el estudio de estos mutantes sitúa este cambio entre los intermediarios M y N y demuestra que es imprescindible para que pueda darse el cambio de accesibilidad desde el lado extracelular al lado citoplasmático, mecanismo que asegura que el transporte sea unidireccional. El aumento de flexibilidad del bucle E-F tiene como consecuencia el aceleramiento del intermediario M. El intermediario N aparece más temprano y tiene un mayor tiempo de vida media. Durante este intermediario sería cuando la proteína tiene una apertura hacia el lado citoplasmático. Al restringir la flexibilidad de las hélices E-F en el mutante F153C/R175C los cambios en el fotociclo no parecen tan relevantes, sin embargo el estudio del intermediario N mediante FTIR muestra cambios estructurales importantes respecto a la proteína nativa. La restricción de la flexibilidad de las hélices F-G en el mutante E166C/A228C provoca profundas alteraciones en el fotociclo favoreciendo la acumulación del intermediario M. Los intermediarios N y O (durante los cuales se reprotona la BR) no se forman y el fotociclo sigue otro camino alternativo para volver al estado basal. Además, el hecho de que el proceso de expulsión del protón (que tiene lugar en el lado extracelular) esté afectado indica la existencia de un mecanismo de cuerpo rígido durante el fotociclo. Por tanto, la flexibilidad de las hélices E, F y G tiene un papel muy relevante en la función de la BR, sugiriendo que este mecanismo es importante en proteínas que constan de 7 hélices transmembrana (7TM). La estrategia de formar un puente disulfuro interhélice en la BR, mediante la introducción de dos cisteínas por mutagénesis dirigida, ha demostrado ser un buen método de estudio para determinar la relevancia de la flexibilidad de las hélices en la función de la proteína. Este método podría extrapolarse a otras proteínas 7TM.Bacteriorhodopsin (BR) is a proton pump protein found in the membrane of the archaebacterium H. salinarum. It comprises seven α helices and a retinal chromophore covalently bound to the helix G. It has been widely studied because of its similarity to visual rhodopsin and other GPCR proteins as well as being part of one of the simplest photosynthetic systems known. BR is activated by absorption of a photon by the retinal, which provides the energy needed to perform the photocycle. As a result there is a net transport of a proton from the cytoplasmic to the extracellular side. Although its structure and function are well known, there are still functional aspects that have not been sufficiently characterized. In this sense, the existence of some movements on the cytoplasmic side of helices E, F and G during the reprotonation pathway has been widely debated. The fact that these movements have been detected only in crystallographic structures of mutants has questioned their existence in the native protein. Likewise, Electronic Paramagnetic Resonance studies (EPR) in spin labeled protein have shown different values for the magnitude of these movements, creating doubts about their relevance in protein function. This work has been aimed at studying the influence of the flexibility of helices E-F and F-G in the function of BR. For this propose, a BR chimera has been expressed replacing the native E-F loop by its homologous in rhodopsin. This chimera, called Loop5, has an extended length and flexibility of the E-F loop. On the other hand the flexibility of the E-F and F-G helices has been restricted by the expression of two double-cysteine mutants and the formation of an interhelix disulfide bond: F153C (E)/ R175C (F) and E166C (F)/ A228C (G). Both the increased flexibility of the E-F loop as the restriction of mobility of E-F and F-G helices causes a deep alteration in the proton pumping activity. The study of these mutants has also shown that helix movements happen between M and N intermediates and prove to be essential to enable the change of accessibility from the extracellular to the cytoplasmic side. This mechanism ensures that the transport is unidirectional. The increased flexibility of the E-F loop in Loop5 mutant leads to M intermediate acceleration. The N intermediate appears earlier and has a longer half-life. During this intermediate the protein has already changed its accessibility form the extracellular to the cytoplasmic side. By restricting the flexibility of helices E-F in F153C/R175C mutant the photocycle has not been significantly altered. However, the study of the N intermediate by means of FTIR has showed significant structural changes compared to the native protein. Restricting the flexibility of the F-G helices in E166C/A228C mutant causes a deep alteration in the photocycle favouring M intermediate accumulation. The N and O intermediates (BR reprotonation) do not form and the photocycle follows an alternative pathway to return to the ground state. Moreover, the fact that the process of proton release (which takes place on the extracellular side) is concerned points to the presence of a rigid-body mechanism during BR photocycle. Therefore, the flexibility of the helix E, F and G has a major role in the function of BR, suggesting that this mechanism is important in 7 transmembrane helices proteins (7TM). The strategy of cross-linking two helices by means of an engineered disulphide bond in BR has proved to be a good method to determine the relevance of the flexibility of some helices in protein function. This method could be extrapolated to other 7TM protein family

Synergistic antitumoral effect of epigenetic inhibitors and gemcitabine in pancreatic cancer cells

Epigenetic modifications could drive some of the molecular events implicated in proliferation, drug resistance and metastasis of pancreatic ductal adenocarcinoma (PDAC). Thus, epigenetic enzyme inhibitors could be the key to revert those events and transform PDAC into a drug-sensitive tumor. We performed a systematic study with five different epigenetic enzyme inhibitors (1, UVI5008, MS275, psammaplin A, and BIX01294) targeting either Histone Deacetylase (HDAC) 1 or 1/4, DNA methyltransferase 3a (DNMT3a), Euchromatic histone lysine methyltransferase 2 (EHMT2), or Sirtuin 1 (SIRT1), as well as one drug that restores the p53 function (P53R3), in three different human PDAC cell lines (SKPC-1, MIA PaCa-2, and BxPC-3) using 2D and 3D cell cultures. The synergistic effect of these antitumoral drugs with gemcitabine was tested and the most efficient combinations were characterized by RNA-seq. The inhibition of HDAC1/4 (MS275), HDAC1/4/SIRT1/DNMT3a (UVI5008) or EHMT2 (BIX01294) induced a significant reduction on the cell viability, even in gemcitabine-resistance cells. The combination of UVI5008 or MS275 with gemcitabine induced a synergistic effect at low concentration and the RNA-Seq analysis revealed some synergy candidate genes as potential biomarkers. Reverting aberrant epigenetic modifications in combination with gemcitabine offers an alternative treatment for PDAC patients, with an important reduction of the therapeutic dose.Xunta de Galicia | Ref. ED431C 2020/02Xunta de Galicia | Ref. ED431G2019/06Ministerio de Economía, Industria y Competitividad | Ref. BIO2017-84974-RXunta de Galicia | Ref. ED481A-2021/364Xunta de Galicia | Ref. ED481A-2018/230Ministerio de Economía, Industria y Competitividad | Ref. PID2019-107855RB-I00Xunta de Galicia | Ref. ED431C 2017/6

Cracking the immune fingerprint of metal-organic frameworks

International audienceThe human body is in a never-ending chess game against pathogens. When the immune system, our natural defence tool, is weakened, these organisms are able to escape, overcoming the body's contingency plan, which results in the body going into a pathological state. To overcome this checkmate status, emerging nanomedicines have been successfully employed as one of the best tactics for boosting the immune response, manipulating the body's defence tools for the specific recognition/elimination of pathological cells via the active ingredient delivery. However, the vast majority of these drug-delivery systems (DDS) are considered to be exclusively passive vehicles, with nanoscale metal-organic frameworks (nanoMOFs) attracting a great deal of attention due to their versatility and ability to carry and deliver exceptional drug payloads and to modulate their biological bypass. Nonetheless, their intrinsic immunogenicity character has been never addressed. Considering the immense possibilities that nanoMOFs offer as a treatment platform, the present study aimed to unveil the immunological fingerprint of MOFs, including an in-deep evaluation of the cellular oxidation balance, the inflammation and recruitment of immune cells and the precise Th1/Th2 cytokine profile that is triggered. This study aims to gain insights that will make more feasible the design of customized immune-active MOF nanoplatforms according to targeted diseases, as the next ace up immune system sleeve. This journal i

Synergistic Effect of Metal Oxide Nanoparticles on Cell Viability and Activation of MAP Kinases and NFκB

In recent years, there has been an increase in the production of several types of nanoparticles (Nps) for different purposes. Several studies have been performed to analyse the toxicity induced by some of these individual Nps, but data are scarce on the potential hazards or beneficial effects induced by a range of nanomaterials in the same environment. The purpose of the study described here was to evaluate the toxicological effects induced by in vitro exposure of human cells to ZnO Nps in combination with different concentrations of other metal oxide Nps (Al2O3, CeO2, TiO2 and Y2O3). The results indicate that the presence of these Nps has synergistic or antagonistic effects on the cell death induced by ZnO Nps, with a quite marked beneficial effect observed when high concentrations of Nps were tested. Moreover, analysis by Western blot of the main components of the intracellular activation routes (MAPKs and NFκB) again showed that the presence of other Nps can affect cell activation. In conclusion, the presence of several Nps in the same environment modifies the functional activity of one individual Np. Further studies are required in order to elucidate the effects induced by combinations of nanomaterials

Influencia de la movilidad de las hélices en la función de la bacteriorodopsina

Descripció del recurs: el 23 de novembre de 2010La bacteriorodopsina (BR) es una proteína transportadora de protones que se encuentra en la membrana de la arqueobacteria H. salinarum. Consta de siete hélices α y un cromóforo, el retinal, unido covalentemente a la hélice G. Ha sido muy estudiada debido a su similitud con la rodopsina visual y otras proteínas de la familia de las GPCRs, además de formar parte de uno de los sistemas fotosintéticos más sencillos que se conocen. La BR se activa mediante la absorción de un fotón por parte del retinal, lo que proporciona la energía necesaria para realizar el fotociclo. El resultado final es el transporte neto de un protón desde el lado citoplasmático al lado extracelular. A pesar de que su estructura y función son bien conocidas, todavía hay aspectos funcionales que no han sido suficientemente caracterizados. En este sentido, la existencia de un posible movimiento de las hélices E, F y G en el lado citoplasmático durante el proceso de reprotonación ha sido ampliamente debatida. El hecho de que estos movimientos hayan sido detectados solamente en estructuras cristalográficas de mutantes ha llevado a pensar que podría tratarse de un artefacto. Asimismo, los trabajos de Resonancia Paramagnética Electrónica (EPR) en proteína marcada con una sonda de spin han dado valores muy diversos para la magnitud de estos movimientos, creando también dudas sobre su existencia y su implicación en la función. Este trabajo ha tenido como objetivo estudiar la influencia de la flexibilidad de las hélices E-F y F-G en la función de la BR. Para ello se ha expresado una quimera de la BR, denominada Loop5, donde se ha ampliado la longitud, y por tanto la flexibilidad, del bucle E-F mediante la sustitución del bucle nativo por su homólogo en la rodopsina. Por otro lado se ha restringido la flexibilidad de las hélices E-F y F-G mediante la expresión de dos dobles mutantes cisteína y la formación de un puente disulfuro interhélice: F153C(E)/R175C(F) y E166C(F)/A228C(G). Tanto el aumento de la flexibilidad del bucle E-F como la restricción de la movilidad de estas hélices y de las hélices F-G provocan una profunda alteración en la función de transporte. Asimismo el estudio de estos mutantes sitúa este cambio entre los intermediarios M y N y demuestra que es imprescindible para que pueda darse el cambio de accesibilidad desde el lado extracelular al lado citoplasmático, mecanismo que asegura que el transporte sea unidireccional. El aumento de flexibilidad del bucle E-F tiene como consecuencia el aceleramiento del intermediario M. El intermediario N aparece más temprano y tiene un mayor tiempo de vida media. Durante este intermediario sería cuando la proteína tiene una apertura hacia el lado citoplasmático. Al restringir la flexibilidad de las hélices E-F en el mutante F153C/R175C los cambios en el fotociclo no parecen tan relevantes, sin embargo el estudio del intermediario N mediante FTIR muestra cambios estructurales importantes respecto a la proteína nativa. La restricción de la flexibilidad de las hélices F-G en el mutante E166C/A228C provoca profundas alteraciones en el fotociclo favoreciendo la acumulación del intermediario M. Los intermediarios N y O (durante los cuales se reprotona la BR) no se forman y el fotociclo sigue otro camino alternativo para volver al estado basal. Además, el hecho de que el proceso de expulsión del protón (que tiene lugar en el lado extracelular) esté afectado indica la existencia de un mecanismo de cuerpo rígido durante el fotociclo. Por tanto, la flexibilidad de las hélices E, F y G tiene un papel muy relevante en la función de la BR, sugiriendo que este mecanismo es importante en proteínas que constan de 7 hélices transmembrana (7TM). La estrategia de formar un puente disulfuro interhélice en la BR, mediante la introducción de dos cisteínas por mutagénesis dirigida, ha demostrado ser un buen método de estudio para determinar la relevancia de la flexibilidad de las hélices en la función de la proteína. Este método podría extrapolarse a otras proteínas 7TM.Bacteriorhodopsin (BR) is a proton pump protein found in the membrane of the archaebacterium H. salinarum. It comprises seven α helices and a retinal chromophore covalently bound to the helix G. It has been widely studied because of its similarity to visual rhodopsin and other GPCR proteins as well as being part of one of the simplest photosynthetic systems known. BR is activated by absorption of a photon by the retinal, which provides the energy needed to perform the photocycle. As a result there is a net transport of a proton from the cytoplasmic to the extracellular side. Although its structure and function are well known, there are still functional aspects that have not been sufficiently characterized. In this sense, the existence of some movements on the cytoplasmic side of helices E, F and G during the reprotonation pathway has been widely debated. The fact that these movements have been detected only in crystallographic structures of mutants has questioned their existence in the native protein. Likewise, Electronic Paramagnetic Resonance studies (EPR) in spin labeled protein have shown different values for the magnitude of these movements, creating doubts about their relevance in protein function. This work has been aimed at studying the influence of the flexibility of helices E-F and F-G in the function of BR. For this propose, a BR chimera has been expressed replacing the native E-F loop by its homologous in rhodopsin. This chimera, called Loop5, has an extended length and flexibility of the E-F loop. On the other hand the flexibility of the E-F and F-G helices has been restricted by the expression of two double-cysteine mutants and the formation of an interhelix disulfide bond: F153C (E)/ R175C (F) and E166C (F)/ A228C (G). Both the increased flexibility of the E-F loop as the restriction of mobility of E-F and F-G helices causes a deep alteration in the proton pumping activity. The study of these mutants has also shown that helix movements happen between M and N intermediates and prove to be essential to enable the change of accessibility from the extracellular to the cytoplasmic side. This mechanism ensures that the transport is unidirectional. The increased flexibility of the E-F loop in Loop5 mutant leads to M intermediate acceleration. The N intermediate appears earlier and has a longer half-life. During this intermediate the protein has already changed its accessibility form the extracellular to the cytoplasmic side. By restricting the flexibility of helices E-F in F153C/R175C mutant the photocycle has not been significantly altered. However, the study of the N intermediate by means of FTIR has showed significant structural changes compared to the native protein. Restricting the flexibility of the F-G helices in E166C/A228C mutant causes a deep alteration in the photocycle favouring M intermediate accumulation. The N and O intermediates (BR reprotonation) do not form and the photocycle follows an alternative pathway to return to the ground state. Moreover, the fact that the process of proton release (which takes place on the extracellular side) is concerned points to the presence of a rigid-body mechanism during BR photocycle. Therefore, the flexibility of the helix E, F and G has a major role in the function of BR, suggesting that this mechanism is important in 7 transmembrane helices proteins (7TM). The strategy of cross-linking two helices by means of an engineered disulphide bond in BR has proved to be a good method to determine the relevance of the flexibility of some helices in protein function. This method could be extrapolated to other 7TM protein family