The effect of hydrostatic pressure on membrane-bound proteins

Many cellular proteins are bound to the surfaces of membranes and participate in various cell signaling responses. Interactions between this group of proteins are in part controlled by the membrane surface to which the proteins are bound. This review focuses on the effects of pressure on membrane-associated proteins. Initially, the effect of pressure on membrane surfaces and how pressure may perturb the membrane binding of proteins is discussed. Next, the effect of pressure on the activity and lateral association of proteins is considered. We then discuss how pressure can be used to gain insight into these types of proteins.

X-ray diffraction of a gram-negative bacterial membrane mimetic

This thesis applies x-ray diffraction to measure he membrane structure of lipopolysaccharides and to develop a better model of a LPS bacterial melilbrane that can be used for biophysical research on antibiotics that attack cell membranes. \iVe ha'e Inodified the Physics department x-ray machine for use 3.'3 a thin film diffractometer, and have lesigned a new temperature and relative humidity controlled sample cell.\Ve tested the sample eel: by measuring the one-dimensional electron density profiles of bilayers of pope with 0%, 1%, 1G :VcJ, and 100% by weight lipo-polysaccharide from Pse'udo'lTwna aeTuginosa. Background VVe now know that traditional p,ntibiotics ,I,re losing their effectiveness against ever-evolving bacteria. This is because traditional antibiotic: work against specific targets within the bacterial cell, and with genetic mutations over time, themtibiotic no longer works. One possible solution are antimicrobial peptides. These are short proteins that are part of the immune systems of many animals, and some of them attack bacteria directly at the membrane of the cell, causing the bacterium to rupture and die. Since the membranes of most bacteria share common structural features, and these featuret, are unlikely to evolve very much, these peptides should effectively kill many types of bacteria wi Lhout much evolved resistance. But why do these peptides kill bacterial cel: '3 , but not the cells of the host animal? For gramnegative bacteria, the most likely reason is that t Ileir outer membrane is made of lipopolysaccharides (LPS), which is very different from an animal :;ell membrane. Up to now, what we knovv about how these peptides work was likely done with r !10spholipid models of animal cell membranes, and not with the more complex lipopolysa,echaricies, If we want to make better pepticies, ones that we can use to fight all types of infection, we need a more accurate molecular picture of how they \vork. This will hopefully be one step forward to the ( esign of better treatments for bacterial infections.

Surface display of proteins by Gram-negative bacterial autotransporters

Affiliation: Faculté de Médecine Vétérinaire, Université de Montréal

Analysis of proteins from membrane and soluble fractions of Giardia duodenalis trophozoites of two Brazilian axenic strains

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions

Background: Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results: We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (K-D) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a K-D of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions: We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro.

Vibrational spectroscopic and electrochemical investigations of multi-centered heme proteins in biomimetic membrane architectures

Membrane proteins play a major role in every living cell. They are the key factors in the cell’s metabolism and in other functions, for example in cell-cell interaction, signal transduction, and transport of ions and nutrients. Cytochrome c oxidase (CcO), as one of the membrane proteins of the respiratory chain, plays a significant role in the energy transformation of higher organisms. CcO is a multi centered heme protein, utilizing redox energy to actively transport protons across the mitochondrial membrane. One aim of this dissertation is to investigate single steps in the mechanism of the ion transfer process coupled to electron transfer, which are not fully understood. The protein-tethered bilayer lipid membrane is a general approach to immobilize membrane proteins in an oriented fashion on a planar electrode embedded in a biomimetic membrane. This system enables the combination of electrochemical techniques with surface enhanced resonance Raman (SERRS), surface enhanced reflection absorption infrared (SEIRAS), and surface plasmon spectroscopy to study protein mediated electron and ion transport processes. The orientation of the enzymes within the surface confined architecture can be controlled by specific site-mutations, i.e. the insertion of a poly-histidine tag to different subunits of the enzyme. CcO can, thus, be oriented uniformly with its natural electron pathway entry pointing either towards or away from the electrode surface. The first orientation allows an ultra-fast direct electron transfer(ET) into the protein, not provided by conventional systems, which can be leveraged to study intrinsic charge transfer processes. The second orientation permits to study the interaction with its natural electron donor cytochrome c. Electrochemical and SERR measurements show conclusively that the redox site structure and the activity of the surface confined enzyme are preserved. Therefore, this biomimetic system offers a unique platform to study the kinetics of the ET processes in order to clarify mechanistic properties of the enzyme. Highly sensitive and ultra fast electrochemical techniques allow the separation of ET steps between all four redox centres including the determination of ET rates. Furthermore, proton transfer coupled to ET could be directly measured and discriminated from other ion transfer processes, revealing novel mechanistic information of the proton transfer mechanism of cytochrome c oxidase. In order to study the kinetics of the ET inside the protein, including the catalytic center, time resolved SEIRAS and SERRS measurements were performed to gain more insight into the structural and coordination changes of the heme environment. The electrical behaviour of tethered membrane systems and membrane intrinsic proteins as well as related charge transfer processes were simulated by solving the respective sets of differential equations, utilizing a software package called SPICE. This helps to understand charge transfer processes across membranes and to develop models that can help to elucidate mechanisms of complex enzymatic processes.

Trypanosomal TAC40 constitutes a novel subclass of mitochondrial -barrel proteins specialized in mitochondrial genome inheritance

Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a β-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum–mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a β-barrel protein of the mitochondrial porin family that mediates a DNA–cytoskeleton linkage that is essential for mitochondrial DNA inheritance.

High-resolution atomic force microscopy imaging of rhodopsin in rod outer segment disk membranes.

Atomic force microscopy (AFM) is a powerful imaging technique that allows recording topographical information of membrane proteins under near-physiological conditions. Remarkable results have been obtained on membrane proteins that were reconstituted into lipid bilayers. High-resolution AFM imaging of native disk membranes from vertebrate rod outer segments has unveiled the higher-order oligomeric state of the G protein-coupled receptor rhodopsin, which is highly expressed in disk membranes. Based on AFM imaging, it has been demonstrated that rhodopsin assembles in rows of dimers and paracrystals and that the rhodopsin dimer is the fundamental building block of higher-order structures.

Membrane trafficking from lysosomes to the plasma membrane regulates phosphatidylserine externalization during apoptosis

Programmed cell death is characterized by tightly controlled temporal and spatial intracellular Ca2+ responses that regulate the release of key proapoptotic proteins from mitochondria to the cytosol. Since apoptotic cells retain their ability to exclude membrane impermeable dyes, it is possible that the cells evoke repair mechanisms that, similar to those in normal cells, patch any damaged areas of the plasma membrane that preclude dye permeation. One critical distinction between plasma membrane repair in normal and apoptotic cells is the preservation of membrane lipid asymmetry. In normal cells, phosphatidylserine (PS) retains its normal asymmetric distribution in the inner membrane leaflet. In apoptotic cells, PS redistributes to the outer membrane leaflet by a Ca2+ dependent mechanism where it serves as a recognition ligand for phagocytes(1). In this study Ca 2+-specific fluorescent probes were employed to investigate the source of Ca2+ required for PS externalization. Experiments employing Rhod2-AM, calcium green 1, fura2-AM and the aqueous space marker FITC-dextran, demonstrated that exogenous Ca2+ imported with endocytotic vesicles into the cell was released into the cytosol in an apoptosis dependent manner. Labeling of the luminal side of the endocytotic vesicles with FITC-annexin 5, revealed that membrane lipid asymmetry was disrupted upon endosome formation. Specific labeling of the lysosomal luminal surface with the non-exchangeable membrane lipid probe, N-rhodamine-labeled-phosphatidylethanolamine (N-Rho-PE) and the lysosomal specific probe, lysotracker green, facilitated real-time monitoring of plasma membrane-to-endosome-to-lysosome transitions. Enforced elevation of cytosolic [Ca2+] with ionophore resulted in the redistribution of N-Rho-PE and PS from the inner membrane leaflet to the PM outer membrane leaflet. Identical results were obtained during apoptosis, however, the redistribution of both N-RhoPE and PS was dependent on the release of intra-lysosomal Ca2+ to the cytosol. Additional experiments suggested that lipid redistribution was dependent on the activity of lysosomal phospholipase A2 activity since lipid trafficking was abolished in the presence of chloroquine and lipase inhibitors. These data indicate that endosomal/lysosomal Ca2+ and the fusion of hybrid organelles to the plasma membrane regulates the externalization of PS during apoptosis. ^

Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope

Vaccinia virus (VV) produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Here we have investigated the resistance of EEV and IMV to neutralization by complement in the absence of immune antibodies. When EEV is challenged with complement from the same species as the cells used to grow the virus, EEV is resistant to neutralization by complement, whereas IMV is not. EEV resistance was not a result of EEV protein B5R, despite its similarity to proteins of the regulators of complement activation (RCA) family, or to any of the other EEV proteins tested (A34R, A36R, and A56R gene products). EEV was sensitive to complement when the virus was grown in one species and challenged with complement from a different species, suggesting that complement resistance might be mediated by host RCA incorporated into the EEV outer envelope. This hypothesis was confirmed by several observations: (i) immunoblot analysis revealed that cellular membrane proteins CD46, CD55, CD59, CD71, CD81, and major histocompatibility complex class I antigen were detected in purified EEV but not IMV; (ii) immunoelectron microscopy revealed cellular RCA on the surface of EEV retained on the cell surface; and (iii) EEV derived from rat cells expressing the human RCA CD55 or CD55 and CD59 were more resistant to human complement than EEV derived from control rat cells that expressed neither CD55 nor CD59. These data justify further analysis of the roles of these (and possible other) cellular proteins in EEV biology.

The dimensions of the protein import channels in the outer and inner mitochondrial membranes

Most mitochondrial proteins are imported into mitochondria through transmembrane channels composed largely, and perhaps exclusively, of proteins. We have determined the effective internal diameter of the protein import channel in the mitochondrial outer membrane to be between 20 Å and 26 Å during translocation. The diameter of the import channel in the inner membrane is smaller than the diameter of the outer membrane import channel. These results were obtained by measuring the effect of rigid steric bulk introduced into precursor proteins on import.

CooB plays a chaperone-like role for the proteins involved in formation of CS1 pili of enterotoxigenic Escherichia coli

CS1 pili serve as the prototype of a class of filamentous appendages found on the surface of strains of enterotoxigenic Escherichia coli. The four genes needed to synthesize functional CS1 pili in E. coli K12 are: cooA, which encodes the major pilin protein; cooD, which encodes a minor pilin protein found at the tip of the structure; cooC, which encodes a protein found in the outer membrane of piliated bacteria; and cooB. We show here that CooB, which is required for pilus assembly but is not part of the final structure, stabilizes CooA, CooC, and CooD. We previously reported that CooB is complexed with CooA in the periplasm and show here that CooB also is found complexed with CooD in the periplasm. CooB is associated with the membrane fraction only in the presence of CooC, suggesting that these two proteins also interact. This suggests that although it has no homology to known chaperone proteins, CooB serves a chaperone-like role for assembly of CS1.

An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding.

We have isolated a major integral membrane protein from Golgi-derived coatomer-coated vesicles. This 24-kDa protein, p24, defines a family of integral membrane proteins with homologs present in yeast and humans. In addition to sequence similarity, all p24 family members contain a motif with the characteristic heptad repeats found in coiled coils. When the yeast p24 isoform, yp24A, is knocked out in a strain defective for vesicle fusion, a dramatic reduction in the accumulation of transport vesicles is observed. Together, these results indicate a role for this protein family in the budding of coatamer-coated and other species of coated vesicles.

Disulfide oxidoreductase activity of Shigella flexneri is required for release of Ipa proteins and invasion of epithelial cells.

Secretion of IpaB, IpaC, and IpaD proteins of Shigella flexneri, essential for the invasion of epithelial cells, requires a number of proteins encoded by the spa and mxi loci on the large plasmid. Introduction of dsbA::Tn5 into S.flexneri from Escherichia coli K-12 reduced invasiveness, which resulted from a decrease in the capacity to release IpaB, IpaC, and IpaD proteins into the external medium. Examination of the surface-presented Ipa proteins of the dsbA mutant, however, revealed Ipa proteins at levels similar to those on wild-type cells. Since the defective phenotype was similar to that of the spa32 mutant of S. flexneri and the Spa32 sequence possessed two Cys residues, the effect of dsbA mutation of the folding structure of Spa32 under reducing conditions and on the surface expression of Spa32 was investigated. The results indicated that Spa32 was a disulfide-containing protein whose correctly folded structure was required for its presentation on the outer membrane. Indeed, replacing either one of the two Cys residues in Spa32 with Ser by site-directed mutagenesis reduced its capacity to release Ipa proteins into the external medium and led to the accumulation of Spa32 protein in the periplasm. These results indicated that the DsbA protein performs an essential function during the invasion of mammalian cells, by facilitating transport of the Spa32 protein across the outer membrane.

Afffinity tag excision strategies for Car9-tagged proteins

Thesis (Master's)--University of Washington, 2016-06