<p>Two species of <i>Neisseria</i>, <i>N. meningitidis</i> and <i>N. gonorrhoeae</i>, are obligate human pathogens that cause meningitis and gonorrhea, respectively. Although generally asymptomatic, <i>N. meningitidis</i> can cause invasive meningococcal disease with high mortality rate. Due to emerging antibiotic resistance strains of <i>N. gonorrhoeae</i>, the Centers for Disease Control and Prevention (CDC) have designated it as an urgent threat to public health. Therefore, immediate interventions are required for fight against these Neisserial pathogens. Iron is an essential nutrient for all bacteria, including <i>Neisseria</i>. However, free iron is scarce in human, therefore, <i>Neisseria</i> have evolved to acquire iron from host proteins. These iron acquisition systems are immunogenic and important for infection and are promising therapeutic targets.</p> <p> In the host, lactoferrin sequesters free iron and limits iron availability to pathogens. However, <i>Neisseria</i> have evolved machinery to hijack iron directly from lactoferrin itself. Lactoferrin binding proteins, LbpA and LbpB, are outer membrane proteins that together orchestrate the acquisition of iron from lactoferrin. Additionally, LbpB serves an additional role in providing protection against host cationic antimicrobial peptides and innate immune response. Despite studies aimed at deciphering the roles of LbpA and LbpB, the molecular mechanisms underpinning iron acquisition and immune protection remain unknown. Here, we investigated the role of the lactoferrin binding proteins in iron acquisition and protection against cationic antimicrobial peptides. We obtained three-dimensional structures of <i>Neisseria</i> LbpA and LbpB in complex with lactoferrin using cryo-electron microscopy and X-ray crystallography. These structures show that both LbpA and LbpB bind to C-lobe of lactoferrin, albeit at distinct sites. Structural analyses show that while lactoferrin maintains its iron-bound closed conformation in the LbpB-lactoferrin complex, it undergoes a large conformational change from an iron-bound closed to an iron-free open conformation upon binding to LbpA. This observation suggest that LbpA alone can trigger the extraction of iron from lactoferrin. Our studies also provide an explanation for LbpB’s preference towards holo-lactoferrin over apo-lactoferrin and LbpA’s inability to distinguish between holo- and apo-lactoferrin. Furthermore, using mutagenesis and binding studies, we show that anionic loops in the C-lobe of LbpB contribute to binding the cationic antimicrobial peptide lactoferricin. Solution scattering studies of the LbpB-lactoferricin complex showed that LbpB undergoes a small conformational change upon peptide binding.</p> Together, our studies provide structural insights into the role of the lactoferrin binding proteins in iron acquisition and evasion of the host immune defenses. Moreover, this work lays the foundation for structure-based design of therapeutics against <i>Neisseria</i> targeting the lactoferrin binding proteins.

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance – PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol – using 2H solidstate nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.

Proteins are known to form functional clusters in plasma membranes. In order to identify individual proteins within clusters we developed a method to visualize by atomic force microscopy (AFM) the cytoplasmic surface of native plasma membrane, excised from Xenopus laevis oocyte and spread on poly-L-lysine coated glass. After removal of the vitelline membrane intact oocytes were brought in contact with coated glass and then rolled off. Inside-out oriented plasma membrane patches left at the glass surface were first identified with the lipid fluorescent marker FM1-43 and then scanned by AFM. Membrane patches exhibiting the typical phospholipid bilayer height of 5 nm showed multiple proteins, protruding from the inner surface of the membrane, with heights of 5 to 20 nm. Modelling plasma membrane proteins as spherical structures embedded in the lipid bilayer and protruding into the cytoplasm allowed an estimation of the respective molecular masses. Proteins ranged from 35 to 2,000 kDa with a peak value of 280 kDa. The most frequently found membrane protein structure (40/μm2) had a total height of 10 nm and an estimated molecular mass of 280 kDa. Membrane proteins were found firmly attached to the poly-L-lysine coated glass surface while the lipid bilayer was found highly mobile. We detected protein structures with distinguishable subunits of still unknown identity. Since X. laevis oocyte is a generally accepted expression system for foreign proteins, this method could turn out to be useful to structurally identify specific proteins in their native environment at the molecular level. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in inflammatory bowel disease. For the in situ delivery of IL-10 by Escherichia coli as carrier chassis, a modified transporter was designed with the ability to secrete biologically active IL-10. De novo DNA synthesis comprised a 561-bp fragment encoding the signal sequence of the E. coli outer membrane protein F fused in frame to an E. coli codon-optimized mature human IL-10 gene under control of a T7 promoter. The construct was overexpressed in E. coli laboratory strains, E. coli BL21 (DE3) and E. coli MDS42:T7. The mean concentrations of human IL-10 in the periplasm and culture supernatant of E. coli BL21 (DE3) were 355.8 ± 86.3 and 5.7 ± 1.7 ng/ml, respectively. The molecular mass of the recombinant E. coli-derived human IL-10 was 19 kDa, while under non-reducing conditions the native IL-10 dimer could be demonstrated. Reduction of tumor necrosis factor-α secretion in lipopolysaccharide-stimulated mouse macrophages and detection of the activated form of the transcription factor signal transducer and activator of transcription protein 3 proved the biological activity of the bacteria-produced human IL-10. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Understanding the function of membrane proteins is crucial to elucidate the molecular mechanisms by which transmembrane signaling based physiological processes,i. e., the interactions of extracellular ligands with membrane-bound receptors, are regulated. In this work, synthetic transmembrane segments derived from the visual photoreceptor rhodopsin, the full length system rhodopsin and mutants of opsin are used to study physical processes that underlie the function of this prototypical class-A G-protein coupled Receptor. The dependency of membrane protein hydration and protein-lipid interactions on side chain charge neutralization is addressed by fluorescence spectroscopy on synthetic transmembrane segments in detergent and lipidic environment constituting transmembrane segments of rhodopsin in the membrane. Results from spectroscopic studies allow us to construct a structural and thermodynamical model of coupled protonation of the conserved ERY motif in transmembrane helix 3 of rhodopsin and of helix restructuring in the micro-domain formed at the protein/lipid water phase boundary. Furthermore, synthesized peptides and full length systems were studied by time resolved FTIR-Fluorescence Cross Correlation Hydration Modulation, a technique specifically developed for the purpose of this study, to achieve a full prospect of time-resolved hydration effects on lipidic and proteinogenic groups, as well as their interactions. Multi-spectral experiments and time-dependent analyses based on 2D correlation where established to analyze large data sets obtained from time-resolved FTIR difference spectra and simultaneous static fluorescence recordings. The data reveal that lipids play a mediating role in transmitting hydration to the subsequent membrane protein response followed by water penetration into the receptor structure or into the sub-headgroup region in single membrane-spanning peptides carrying the conserved proton uptake site (monitored by the fluorescence emission of hydrophobic buried tryptophan). Our results support the assumption of the critical role of the lipid/water interface in membrane protein function and they prove in particular the important influence of electrostatics, i. e., side chain charges at the phase boundary, and hydration on that function. / Für die Aufklärung der molekularen Wirkungsweise von physiologischen, auf Signaltransduktion, d. h. dem Zusammenspiel von extrazellulären Reizen und membrangebundenen Rezeptoren, beruhenden Prozessen ist das Verständnis der Funktion von Membranproteinen unerlässlich. In dieser Arbeit werden von Rhodopsin abgleitete, synthetische transmembrane Segmentpeptide, Opsin-Mutanten und der vollständige Photorezeptor Rhodopsin untersucht, um die physikalischen Prozesse zu beleuchten, die der Funktionen dieses prototypischen Klasse-A G-Protein gekoppelten Rezeptors zugrunde liegen. Die Abhängigkeit der Membranprotein-Hydratation und der Lipid-Protein-Wechselwirkung von der Ladung einer Aminosäuren-Seitenkette wird erforscht. Hierzu werden synthetische, transmembrane Segmentpeptide in Lipid und Detergenz, als Modell transmembraner Segmente von Rhodopsin in der Membran mittels Fluoreszenzspektroskopie untersucht. Aus den erhaltenen Ergebnissen wird ein thermodynamisches und strukturelles Modell hergeleitet, welches die Kopplung der Protonierung des hochkonservierten ERY-Motivs in Transmembranhelix 3 von Rhodopsin an die Restrukturierung der Helix in der Mikroumgebung der Lipid-Wasser-Phasengrenze erklärt. Des Weiteren werden sowohl die Segementpeptide als auch die vollständigen Systeme Opsin und Rhodopsin mittels zeitaufgelöster FTIR-Fluoreszenz-Kreuzkorrelations-Hydratations-Modulation untersucht. Diese Technik wurde eigens zur Aufklärung von zeitabhängigen Hydratationseffekten auf Lipide und Proteine oder Peptide entwickelt. Dabei werden zeitaufgelöste FTIR Differenz-Spektren und gleichzeitig statische Fluoreszenzsignale aufgenommen und diese zeitabhängigen multispektralen Datensätze mittels 2D Korrelation analysiert. Die Auswertung der Experimente enthüllt einen sequentiellen Hydratationsprozess. Dieser beginnt mit der Bildung von Wasserstoffbrückenbindungen an der Carbonylgruppe des Lipids, gefolgt von Strukturänderungen der Transmembranproteine und abgeschlossen durch das Eindringen von Wasser in das Proteininnere. Letzteres wird nachgewiesen durch die Fluoreszenz von Tryptophan im hydrophoben Peptid- oder Proteininneren. Die Ergebnisse dieser Arbeit unterstreichen die Annahme, dass Lipid-Protein-Wechselwirkungen eine entscheidende Rolle in der Funktion von Membranproteinen spielen und das insbesondere Elektrostatik, in Form von Ladungen an der Phasengrenze, und die Hydratisierung einen kritischen Einfluss auf diese Funktion haben.

The atomic force microscope (AFM) provides a powerful instrument for investigating and manipulating biological samples down to the subnanometer scale. In contrast to other microscopy methods, AFM does not require labeling, staining, nor fixation of samples and allows the specimen to be fully hydrated in buffer solution during the experiments. Moreover, AFM clearly compares in resolution to other techniques. In general, the AFM can be operated in an imaging or a force spectroscopy mode. In the present work, advantage was taken of this versatility to investigate single biomolecules and biomolecular assemblies. A novel approach to investigate the visco-elastic behavior of biomolecules under force was established, using dextran as an example. While a molecule tethered between a solid support and the cantilever tip was stretched at a constant velocity, the thermally driven oscillation of the cantilever was recorded. Analysis of the cantilever Brownian noise provided information about the visco-elastic properties of dextran that corresponded well to parameters obtained by alternative methods. However, the approach presented here was easier to implement and less time-consuming than previously used methods. A computer controlled force-clamp system was set up, circumventing the need for custom built analogue electronics. A commercial PicoForce AFM was extended by two computers which hosted data acquisition hardware. While the first computer recorded data, the second computer drove the AFM bypassing the manufacturer's microscope control software. To do so, a software-based proportional-integral-differential (PID) controller was implemented on the second computer. It allowed the force applied to a molecule to be held constant over time. After tuning of the PID controller, response times obtained using that force-clamp setup were comparable to those of the recently reported analogue systems. The performance of the setup was demonstrated by force-clamp unfolding of a pentameric Ig25 construct and the membrane protein NhaA. In the latter case, short-lived unfolding intermediates that were populated for less than 10 ms, could be revealed. Conventional single-molecule dynamic force spectroscopy was used to unfold the serine:threonine antiporter SteT from Bacillus subtilis, an integral membrane protein. Unfolding force patterns revealed the unfolding barriers stabilizing structural segments of SteT. Ligand binding did not induce new unfolding barriers suggesting that weak interactions with multiple structural segments were involved. In contrast, ligand binding caused changes in the energy landscape of all structural segments, thus turning the protein from a brittle, rigid into a more stable, structurally flexible conformation. Functionally, rigidity in the ligand-free state was thought to facilitate specific ligand binding, while flexibility and increased stability were required for conformational changes associated with substrate translocation. These results support the working model for transmembrane transport proteins that provide alternate access of the binding site to either face of the membrane. Finally, high-resolution imaging was exploited to visualize the extracellular surface of Cx26 gap junction hemichannels (connexons). AFM topographs reveal pH-dependent structural changes of the extracellular connexon surface in presence of HEPES, an aminosulfonate compound. At low pH (&amp;lt; 6.5), connexons showed a narrow and shallow channel entrance, which represented the closed pore. Increasing pH values resulted in a gradual opening of the pore, which was reflected by increasing channel entrance widths and depths. At pH &amp;gt; 7.6 the pore was fully opened and the pore diameter and depth did not increase further. Importantly, coinciding with pore gating a slight rotation of the subunits was observed. In the absence of aminosulfonate compounds, such as HEPES, acidification did not affect pore diameters and depths, retaining the open state. Thus, the intracellular concentration of taurine, a naturally abundant aminosulfonate compound, might be used to tune gap junction sensitivity at low pH.

Molecular dynamics simulation was used to study ion permeation through different membrane proteins embedded in a lipid bilayer (DMPC) with different saline solutions. The potential of mean force (PMF) for ion transport was obtained by umbrella sampling simulations. A revised MacKerell force field for tryptophan residues was studied using gramicidin A (gA) channel as a test model. The revised force field contribution to the Na+ PMF was consonant with the prediction from the experimental results, but in stark contrast to the prediction of the CHARMM force field, version 22, for the tryptophan side-chain. A new grid-based correction map algorithm by MacKerell group, called CMAP, was introduced into the CHARMM force field, version 31. The CMAP algorithm focused on optimizing phi, psi dihedral parameters for the peptide backbone. The CMAP corrections reduced the excessive translocation barrier. Decomposition demonstrated the reduction in the translocation barrier was due to effects on the K+ PMFH2O rather than on K+ PMFgA. The presence of negatively charged sulfonate group at the entrance and exit of the gA channel affected the depth and the location of the highly occupied sites. The negatively charged sulfonate group produced a strong attraction for the cations in the bulk towards the channel mouth. In the M2 transmembrane domain channel (M2-TMD), three M2-TMD structures were studied, differing only in whether the selectivity-filter (four His37 side-chains) was uncharged, +2 charged, or +3 charged. M2-TMD structural properties were compared with the structural properties of other models extracted from NMR and X-ray studies. The spontaneous cation and anion entry into the charged selectivity-filter was different from that into a neutral selectivity-filter. Cl- ions had a lower free-energy barrier in the selectivity-filter than either Na+ or NH4+ ions through the M2-TMD channel. NH4+ ions had a lower free-energy barrier in the selectivity-filter than Na+ ions. Based on accessible rotamer conformations, a revised conductance mechanism was proposed. In this conductance mechanism, the His37 side-chain functioned as an acceptor and donor group, whereas the Trp41 side-chain functioned as a carrying group.

<p>The pathological hallmarks of sickle cell disease originate from a single mutation of the beta hemoglobin gene resulting in a valine at position 6 instead of the canonical glutamic acid. This small change perpetuates many factors, manifesting into chronic embolic processes in the microvasculature, causing painful vaso-occlusive episodes and eventual organ failure. There have been numerous therapies developed to reduce the mortality of sickle cell ranging from agents to induce production of fetal hemoglobin to chronic blood transfusions. Although each of these options are effective at improving the quality of life for sickle cell patients, they only treat one aspect of the disease and, for some, become ineffective over time. In the hope of producing a better therapy, a better understanding of the pathogenesis of vaso-occlusive episodes is needed. While many models have been offered to account for these vaso-occlusive events, one recently proposed mechanism stems from the elevated tyrosine phosphorylation of the cytoplasmic domain of the major erythrocyte membrane protein, Band 3. Band 3 serves as a hub for many critical proteins in the red cell. It binds ankyrin, which associates the spectrin cortical cytoskeleton to the red cell membrane, deoxygenated hemoglobin, the kinases Wnk1 and OSR1, which regulate cation transport, and a glycolytic enzyme metabolon that regulates the production of ATP and glutathione. When Band 3 is tyrosine phosphorylated, each of these proteins dissociate, causing significant changes to red cell homeostasis. These changes include an accumulation of reactive oxygen species, vesiculation and release of prothrombotic microvesicles, leakage of cell free hemoglobin, and a decrease in cell volume. Normally, Band 3 exists in a predominantly unphosphorylated state, however, in sickle cell disease, Band 3 is abundantly tyrosine phosphorylated. Reduction in the tyrosine phosphorylation of Band 3 has been documented to prevent the release of microvesicles and hemoglobin from sickle cell red blood cells. Because these microvesicles and cell free hemoglobin contribute to the vaso-occlusive episodes in sickle cell patients, inhibiting the mechanism for their release offers a potential therapeutic option. But to accomplish this, the molecular cause for the elevated tyrosine phosphorylation in sickle cell disease must be identified. Since tyrosine phosphorylation is performed by a tyrosine kinase and removed by a tyrosine phosphatase, the elevation in phosphorylation must be due to changes in both of these processes. Unfortunately, the identity and nature of these kinases and phosphatases are poorly understood. In this dissertation, I identified the tyrosine kinases Syk, Lyn, and Src attributed to Band 3</p> <p>15</p> <p>phosphorylation that facilitates the release of microvesicles and hemoglobin in sickle cell red blood cells. Inhibition of Syk or one of the two Src family kinases is sufficient to prevent the destabilization of the red blood cell membrane. These kinases function in a hierarchy, where one of the three Src family kinase, Lyn phosphorylates Syk, activating it, and promoting the phosphorylation of Band 3 at tyrosines 8 and 21. Prevention of either phosphorylation event prevents the release of microvesicles and cell free hemoglobin. I also report the identification of PTP1B as the tyrosine phosphatase responsible for maintaining Band 3 in an unphosphorylated state. Interestingly, in sickle cell disease, this tyrosine phosphatase is proteolytically cleaved, resulting in a reduction in dephosphorylating potential. It has been reported previously that PTP1B is a substrate of the calcium dependent protease, calpain and that calpain inhibitors improve the cell morphology of sickle erythrocytes. Inhibition of this proteolytic process may offer an additional therapeutic option for the treatment of sickle cell disease.</p>

No description available.

Whole cells of Vibrio cholerare serotype Inaba and serotype Ogawa (strains I389 and O395) were injected into rabbits to obtain antiserum. The antiserums were used for immune reaction with antigenic components of 25 strains of V.cholerae isolated from five provinces of Vietnam and the two standard strains I389 and O395 by Western-blot technique. Analysis of immune hybrid results showed that there were 11 antigenic components with molecular weights approximately 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa and 20kDa. In which the antigens of 45kDa, 42kDa, 31kDa and 20kDa were similar to OmpT, OmpS, Omp-31kDa and TcpA that have been considered as vaccine-candidate antigens. Among 25 V.cholerae strains, there were 6 antigenic components in common including 79kDa, 62kDa, 45kDa, 35kDa, 31kDa and 20kDa. 23/25 strains contained 42kDa antigen; 5/25 strains contained 38kDa and 23kDa antigens; 11/25 had 26kDa antigen. In addition, 7/25 strains contained antigens identical to V.cholerae I389 serotype Inaba; 6/25 strains contained antigens of I389 and O395; 12/25 strains had changes of antigenic components. These changes were actually the lack of antigens, not appearing new antigens. These results are considered as basis for researches about immune response and prevention of cholera disease. / Toàn bộ tế bào của các chủng Vibrio cholerare typ huyết thanh Inaba và typ huyết thanh Ogawa (chủng I389 và O395) được sử dụng để gây miễn dịch trên thỏ để thu kháng huyết thanh. Các kháng huyết thanh được dùng để thực hiện phản ứng miễn dịch với các thành phần kháng nguyên của 25 chủng V.cholerae phân lập từ 5 tỉnh thành của Việt Nam và hai chủng chuẩn I389 và O395 bằng kỹ thuật Western-blot. Phân tích kết quả lai miễn dịch cho thấy, có tổng số 11 thành phần kháng nguyên có kích thước khoảng 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa và 20kDa. Các kháng nguyên này chủ yếu là các protein màng ngoài (Omp) và kháng nguyên lông (TcpA). Trong đó các kháng nguyên 45kDa, 42kDa, 31kDa và 20kDa trùng với các kháng nguyên OmpS, OmpT, Omp-31kDa và TcpA được xem là những kháng nguyên dự tuyển vacxin tả. Có 6 kháng nguyên chung giữa 25 chủng với kích thước 79kDa, 62kDa, 45kDa, 35kDa, 31kDa và 20kDa. 7/25 chủng có các kháng nguyên giống với kháng nguyên của chủng V. cholerae I389 typ huyết thanh Inaba; 6/25 chủng có các kháng nguyên giống với kháng nguyên của cả hai chủng V.cholerae I389 và O395; 12/25 chủng có sự biến đổi thành phần kháng nguyên. Tuy nhiên, sự biến đổi này thực chất là sự thiếu hụt chứ không phải là sự xuất hiện các thành phần kháng nguyên mới. Các kết quả nghiên cứu này có thể được xem là nền tảng ban đầu cho các nghiên cứu về miễn dịch và dự phòng bệnh tả.