Interactions between mycoplasma lipid-associated membrane proteins and the host cells

Mycoplamas are a group of wall-less prokaryotes widely distributed in nature, some of which are pathogenic for humans and animals. There are many lipoproteins anchored on the outer face of the plasma membrane, called lipid-associated membrane proteins (LAMPs). LAMPs are highly antigenic and could undergo phase and size variation, and are recognized by the innate immune system through Toll-like receptors (TLR) 2 and 6. LAMPs can modulate the immune system, and could induce immune cells apoptosis or death. In addition, they may associate with malignant transformation of host cells and are also con-sidered to be cofactors in the progression of AIDS.

Lipid-associated membrane proteins of Mycoplasma penetrans induce production of proinflammatory cytokines in human monocytic cells

The aim of this study is to explore potential pathogenicity of Mycoplasma penetrans, and to investigate whether M.penetrans lipid-associated membrane proteins (LAMPs) could induce human monocytic cell line (THP-1) to produce some proinflammatory cytokines in vitro, including interleukin-1β(IL-1β) , tumor necrosis factor alpha (TNF-α) , and IL-8. THP-1 was stimulated with different concentrations of M .penetrans LAMPs and at different time to analyze the production of human IL-1β, TNF-αand IL-8. The protein levels of human IL-1β, TNF-αand IL-8 were measured by enzyme-linked immunoadsorbent assay (ELISA) and the mRNA levels of these proinflamrnatory cytokines were detected by reverse transcriptase -PCR (RT-PCR). It was demonstrated in the present study that the production of IL-1β, TNF-αand IL-8 increased in dose- and time-dependent manner after stimulation with M. penetrans LAMPs in THP-1 cells. M.penetrans LAMPs also induced the expression of IL-1β, TNF-αand IL-8 mRNA. The production of IL-1β, TNF-αand IL-8 and the expression of mRNA were down-regulated by pyrrolidine dithiocarbamate (PDTC). This study demonstrated that M. penetrans LAMPs can induce the production of proinflammatory cytokines in human monocytic cells in vitro, thus suggesting that it may be an important etiological factor.

Expression of inducible nitric oxide synthase induced by lipid-associated membrane proteins of Ureaplasma urealyticum is regulated by nuclear factor κB-mediated mechanism in murine macrophages

The aim was to investigate the molecular mechanisms responsible for the inducible nitric oxide synthase (iNOS) gene expression stimulated by lipid associated membrane proteins (LAMPs) of Ureaplasma urealyticum (U.urealyticum). Detection of NO, the expression of iNOS and the activation of nuclear factor κB (NF-κB) in direct response to U.urealyticum LAMPs in a murine macrophages, the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB and of cycloheximide (CHX), a protein synthase inhibitor were available. The results indicated that U.urealyticum LAMPs stimulated mouse macrophages to express iNOS and thus produce NO in dose- and time-dependent manner by activating NF-κB. The expression of iNOS, NO production and the activation of NF-κB were inhibited by U.urealyticum LAMPs combination with PDTC or CHX. In conclusion, our findings suggest that U.urealyticum may be an etiological factor to certain diseases due to its ability to stimulate the expression of iNOS, which is probably mediated through the activation of NF-κB.

Changes in milk fat globule membrane proteins along lactation stage of Laoshan dairy goat

The milk fat globule membrane(MFGM)is a complex structure with numerous functions,and its composition is affected by many factors.There have been few systematic investigations on goat MFGM proteome profiling during lactation.Individual milk samples from 15 healthy dairy goats were obtained at six lactation time points for investigation of the MFGM proteome using both data-independent acquisition(DIA)and data-dependent acquisition(DDA)proteomics techniques combined with multivariate statistical analysis.Using the DIA method,890 variably abundant MFGM proteins were discovered throughout the lactation cycle.From 1 to 240 d,butyrophilin subfamily 1 member A1,lipoprotein lipase,perilipin-2,and adipose triglyceride lipase were upregulated,while APOE,complement C3,clusterin,and IgG were downregulated.Furthermore,from 1 to 90 d,annexin A1,annexin A2,and antithrombin-ll were downregulated,then upregulated by d 240.Albumin had a high degree of connectedness,indicating that it was a key protein,according to protein-protein interaction research.Overall,our findings gave new insights into the biological features of MFGM protein in goat milk throughout lactation,which may aid in the creation of specialized MFGM products and infant formula.

Activation of nuclear factor κB and induction of inducible nitric oxide synthase by lipid-associated membrane proteins isolated from Mycoplasma penetrans

Background This study was designed to investigate the potential pathogenicity of Mycoplasma penetrans (M. penetrans) and its molecular mechanisms responsible for the induction of iNOS gene expression in mouse macrophages stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. penetrans.Methods Mouse macrophages were stimulated with M. penetrans LAMPs to assay the production of nitric oxide (NO). The expression of inducible nitric oxide synthase (iNOS) was detected by RT-PCR and Western blotting. The activity of nuclear factor κB (NF-κB) and the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, on the production of nitric oxide and the expression of iNOS were also assessed in mouse macrophages treated with M. penetrans LAMPs by indirect immunofluorescence and Western blotting.Results M. penetrans LAMPs stimulated mouse macrophages to produce nitric oxide in a dose- and time-dependent manner. The mRNA and protein levels of iNOS were also upregulated in response to LAMP stimulation and inhibited by PDTC treatment. M. penetrans LAMPs were found to trigger NF-κB activation, a possible mechanism for the induction of iNOS expression.Conclusion This study demonstrated that M. penetrans may be an important etiological factor of certain diseases due to the ability of M. penetrans LAMPs to stimulate the expression of iNOS, which is probably mediated through the activation of NF-κB.

Demethylation of miR-34a upregulates expression of membrane palmitoylated proteins and promotes the apoptosis of liver cancer cells

BACKGROUND Liver cancer is a common cancer and the main cause of cancer-related deaths worldwide.Liver cancer is the sixth most common cancer in the world.Although miR-34a and palmitoyl membrane palmitoylated protein(MPP2)are reportedly involved in various cell processes,their precise roles in liver cancer are still unclear.AIM To investigate the expression of micro RNA 34a(miR-34a),methylation of the miR-34a promoter and the expression of MPP2 in liver cancer cells and their related mechanisms.METHODS Together,78 cases of liver cancer tissues and 78 cases of adjacent tissues were collected.The methylation degree of miR-34a promoter in liver cancer/paracancerous tissue and liver cancer cells/normal liver cells,and the expression levels of miR-34a and MPP2 in the above samples were detected.Demethylation of liver cancer cells or transfection of liver cancer cells with miR-34a mimetic was performed.The MPP2 overexpression vector was used to transfect liver cancer cells,and the changes in proliferation,invasion,apoptosis,migration,and other biological functions of liver cancer cells after the above interventions were observed.Double luciferase reporter genes were used to detect the targeting relationship between miR-34a and MPP2.RESULTS Clinical samples showed that the expression levels of miR-34a and MPP2 in liver cancer tissues were lower than those in the normal tissues.The methylation degree of miR-34a promoter region in liver cancer cells was higher than that in normal liver cells.After miR-34a demethylation/mimetic transfection/MPP2 overexpression,the apoptosis of liver cancer cells was increased;the proliferation,invasion and migration capabilities were decreased;the expression levels of caspase 3,caspase 9,E-cadherin,and B-cell lymphoma 2(Bcl-2)-associated X protein were increased;and the expression levels of Bcl-2,N-cadherin,andβ-catenin were decreased.Double luciferase reporter genes confirmed that MPP2 is targeted by miR-34a.Rescue experiments showed that small interfering MPP2 could counteract the promoting effect of miR-34a demethylation on apoptosis and the inhibitory effect on cell proliferation,invasion,and migration.CONCLUSION miR-34a demethylation upregulates the expression level of MPP2 in liver cancer cells and promotes the apoptosis of liver cancer cells.miR-34a demethylation is a potential method for liver cancer treatment.

Antimicrobial Susceptibility and Characterization of Outer Membrane Proteins of Aeromonas hydrophila Isolated in China

Aeromonas hydrophila isolates from clinical cases （n=43） were tested against 8 antimicrobial agents and typed by outer membrane protein （OMP） pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin （MICs, ≥16 μg mL-1） and sulfamonomethoxine （MICs≥64 μg mLl）, but susceptible to norfloxacin （MICs,≤0.5 μg mL-1）. There was a high incidence of resistance to erythromycin （90.70%） and tylosin （93.02%）, while a low incidences of resistance to ciprofloxacin （2.33%）, enrofloxacin （2.33%） and florfenicol （4.65%）. Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective profiles. The strains with the similar OMP profiles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to florfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to florfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to fluoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.

Study on screening potential allergenic proteins from infant milk powders based on human mast cell membrane chromatography and histamine release assays

Cow's milk allergy is mainly observed in infants and young children. Most allergic reactions affect the skin, followed by the gastrointestinal and respiratory systems. Conventional diagnosis is based on positive allergy studies and evaluation of parameters including IgE and IgG1 levels, acute allergic skin response and anaphylactic shock reactions. We developed a cell membrane chromatographic(CMC)method based on human mast cells(HMC-1) for screening potential allergens in infant formula milk powders(IFMP). HMC-1 cell membranes were extracted and mixed with silica to prepare cell membrane chromatography columns(10 mm ? 2 mm i.d., 5 mm). Under the conditions of 0.2 mL/min flow rate and214 nm detection wavelength, human breast milk showed no retention. However, IFMP showed clear retention. The retained fractions were collected and analyzed through matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS). Four major milk proteins, i.e., α-casein, β-casein, α-lactalbumin, and β-lactoglobulin A, were identified. Furthermore, these proteins and β-lactoglobulin B showed clear retention on HMC-1/CMC columns. To test the degranulation effects of the five proteins, histamine and β-hexosaminidase release assays were carried out. All five proteins induced HMC-1 cells to release histamine and β-hexosaminidase. Also, we established a reversed phase liquid chromatographic(RPLC) method for the determination of the five proteins in IFMP and the results showed that 90% proteins in IFMP were α-casein and β-casein. We concluded that cow's milk proteins may be potential allergens and caseins cause more β-casein allergic risk than other proteins. This conclusion was consistent with other studies.

Generation and Identification of Monoclonal Antibody Against Porcine Adipocyte Plasma Membrane Proteins

Production of monoclonal antibody against porcine adipocyte plasma membrane proteins to explore a new way of controlling body fat deposition and improving carcass quality is discussed in this article. Membrane proteins of pig adipocyte plasma membrane proteins were extracted with the help of sucrose density gradient centrifugation, and two kinds of proteins were obtained. The monoclonal antibody （designated 3B2 and 3F3） of IgG1 and IgG2b subclass against adipocyte membrane proteins were produced by immunization, with adipocyte membrane proteins as an antigen, and its titer was 1：105 detected by enzyme-linked immunoadsorbent assay （ELISA）. The cell strains were identified by analyzing the number of chromosomes, the heat stability, the acid and alkali, the types and subtypes of immnoglobulin, and its peculiarities and affinities. Through identification, the chromosome number of hybridoma cell strains was from 80 to 100 and the strains formed good hybridomas colonies. The strains＇ affinity constants were 4.63 × 10^9 and 3.75 × 10^9 （mol L^-1）-1, respectively. At the same time, the McAb secreted was stable to environmental factors, such as, temperature, acid, alkali and so on. The monoclonal antibodies had been obtained and their specificity to porcine adipocyte plasma membrane proteins had been identified.

Immunoproteomics of membrane proteins of Shigella flexneri 2a 2457T

AIM: To screen the immunogenic membrane proteins ofShigella flexneri 2a 2457T.METHODS: The routine two-dimensional polyacrylamidegel electrophoresis (2-DE) and Western blotting werecombined to screen immunogenic proteins of S. flexneri2a 2457T. Serum was gained from rabbits immunizedwith the same bacteria. Immunogenic spots werecut out from the polyacrylamide gel and digested bytrypsin in-gel. Matrix-assisted laser desorption/ionizationtime of flight-mass spectrometry (MALDI-TOF-MS)was performed to determine the molecular weight ofpeptides. Electrospray ionization (ESI-MS/MS) wasperformed to determine the sequences of the interestingpeptides.RESULTS: A total of 20 spots were successfullyidentified from Coomassie brilliant blue stained gelsrepresenting 13 protein entries, 5 known antigens and8 novel antigens. A hypothetical protein (YaeT) wasdetected, which might be a candidate target of vaccine.CONCLUSION: Membrane proteins of S. flexneri 2a2457T were successfully observed by 2-DE. Severalknown and novel antigens were identified by massspectrum.

Antibody Therapies Targeting Complex Membrane Proteins

In analyses of protein families that may serve as drug targets,membrane-associated G-protein-coupled receptors(GPCRs)dominate,followed by ion channels,transporters,and—to a lesser extent—membrane-bound enzymes.However,various challenges put such membrane proteins among key groups of underutilized opportunities for the application of therapeutic antibodies.Antibodies hold the promise of exquisite specificity,as they are able to target even specific conformations of a particular membrane protein,as well as adaptability through engineering into various antibody formats.However,the ease of raising and isolating specific,effective antibodies targeting membrane proteins depends on many factors.In particular,the generation of specific antibodies is easier when targeting larger,simpler,extracellular domains with greater uniqueness of amino acid sequence.The rareness of such ideal conditions is illustrated by the limited number of approved biologics for targeting GPCRs and other complex membrane proteins.Challenges in developing antibodies to complex membrane proteins such as GPCRs,ion channels,transporters,and membrane-bound enzymes can be addressed by the design of the antigen,antibody-generation strategies,lead optimization technologies,and antibody modalities.A better understanding of the membrane proteins being targeted would facilitate mechanism-based drug discovery.This review describes the advantages and challenges of targeting complex membrane proteins with antibodies and discusses the preparation of membrane protein antigens and antibody generation,illustrated by select examples of success.

Water relations and an expression analysis of plasma membrane intrinsic proteins in sensitive and tolerant rice during chilling and recovery

寒心的损害的症状是在射击的水赤字的开发，源于水运输和蒸发的不平衡。在这个工作，二个米饭变化(Oryza sativa L。var。Wasetoitsu 和 Somewake ) 幼苗是在 7 度 C 的 chilled，在 28 度 C 由恢复列在后面。基于生长显型和电解质漏测试， Somewake 被显示是一个寒心容忍的变化，和 Wasetoitsu 寒心敏感的。寒心的压力显著地减少了叶子，积聚的蒸发和渗透的根的相对的水内容(RWC ) 在两个变化的水力的电导率(Lp ) 。但是当回到了 28 度 C 时， Somewake 的水关系平衡更好恢复了。所有 11 血浆膜的 mRNA 表示侧面内在的蛋白质(果仁) ， aquaporins 的亚群，被有 TaqMan 次要的林子文件夹(MGB ) 的 -PCR 探查的即时反向的抄写(RT ) 随后决定源于米饭 var。Nipponbare 在寒心的处理和恢复期间。大多数 PIP 基因在低温度是下面调整的，并且在温暖的温度恢复了。在 Somewake 和 Wasetoitsu 的一些果仁的相对表示在平行减少了在期间寒心。然而在恢复期间， OsPIP1 的相对表示； 1， OsPIP2； 1， OsPIP2； 7 在射击和 OsPIP1； 1， OsPIP2； 1 比 Wasetoitsu 在 Somewake 在根是显著地更高的。这在在寒心的条件以后重建水平衡支持果仁的角色。我们讨论 aquaporin 果仁亚科的成员在植物起的多样化的作用寒心的忍耐取决于 aquaporin isoforms，植物织物和寒心的持续时间的阶段。

Determination of the genus-specific antigens in outer membrane proteins from the strains of Leptospira interrogans and Leptospira biflexa with different virulence

Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups.The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence,and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972).OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results:All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa.A positive protein fragment with approximately 32 KDa confirmed by Western blot,was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira.Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.

Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 k Da, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purifi ed recombinant protein was used to vaccinate the grass carp, C tenopharyngodon idella. Following vaccination of the fi sh their Ig M antibody levels were examined, as was the expression of I g M, Ig D and Ig Z immunoglobulin genes and other genes such as MHC Iα and MHC I I β, which are also involved in adaptive immunity. Interleukin genes( IL), including I L- 1β, IL- 8 and I L- 10, and type I and type II interferon(I FN) genes were also examined. At 3 and 4 weeks post-vaccination(wpv), signifi cant increases in Ig M antibody levels were observed in the fi sh vaccinated with the recombinant fusion protein, and an increase in the expression levels of I g M, Ig D and Ig Z genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fi sh. At four wpv, the fi sh were challenged with F. column a re, and the vaccinated fi sh showed a good level of protection against this pathogen, with 39% relative percent survival(RPS) compared with the control group. It can be concluded, therefore, that the fi ve OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fi sh and protection against F. columnare.

Multiscale molecular dynamics simulations of membrane remodeling by Bin/Amphiphysin/Rvs family proteins

Membrane curvature is no longer thought of as a passive property of the membrane; rather, it is considered as an ac- tive, regulated state that serves various purposes in the cell such as between cells and organelle definition. While transport is usually mediated by tiny membrane bubbles known as vesicles or membrane tubules, such communication requires complex interplay between the lipid bilayers and cytosolic proteins such as members of the Bin/Amphiphysin/Rvs （BAR） superfam- ily of proteins. With rapid developments in novel experimental techniques, membrane remodeling has become a rapidly emerging new field in recent years. Molecular dynamics （MD） simulations are important tools for obtaining atomistic information regarding the structural and dynamic aspects of biological systems and for understanding the physics-related aspects. The availability of more sophisticated experimental data poses challenges to the theoretical community for devel- oping novel theoretical and computational techniques that can be used to better interpret the experimental results to obtain further functional insights. In this review, we summarize the general mechanisms underlying membrane remodeling con- trolled or mediated by proteins. While studies combining experiments and molecular dynamics simulations recall existing mechanistic models, concurrently, they extend the role of different BAR domain proteins during membrane remodeling pro- cesses. We review these recent findings, focusing on how multiscale molecular dynamics simulations aid in understanding the physical basis of BAR domain proteins, as a representative of membrane-remodeling proteins.

Extraction and identification of membrane proteins from black widow spider eggs

The eggs of oviparous animals are storehouses of maternal proteins required for embryonic development. Identification and molecular characterization of such proteins will provide much insight into the regulation of embryonic development. We previously analyzed soluble proteins in the eggs of the black widow spider （Latrodectus tredecimguttatus）, and report here on the extraction and mass spectrometric identification of the egg membrane proteins. Comparison of different lysis solutions indicated that the highest extraction of the membrane proteins was achieved with 3%-4% sodium laurate in 40 mmol/L Tris-HCI buffer containing 4% CHAPS and 2% DTT （pH 7.4）. SDS-PAGE combined with nLC- MS/MS identified 39 proteins with membranelocalization annotation, including those with structural, catalytic, and regulatory activities. Nearly half of the identified membrane proteins were metabolic enzymes involved in various cellular processes, particularly energy metabolism and biosynthesis, suggesting that relevant metabolic processes were active during the embryonic development of the eggs. Several identified cell membrane proteins were involved in the special structure formation and function of the egg cell membranes. The present proteomic analysis of the egg membrane proteins provides new insight into the molecular mechanisms of spider embryonic development.

Immunoproteomic Analysis of Bordetella bronchiseptica Outer Membrane Proteins and Identification of New Immunogenic Proteins

Bordetella bronchiseptica is a Gram-negative pathogen that causes acute and chronic respiratory infection in a variety of animals. To identify useful antigen candidates for diagnosis and subunit vaccine of B. bronchiseptica, immunoproteomic analysis was adopted to analyse outer membrane proteins of it. The outer membrane proteins extracted from B. bronchiseptica were separated by two-dimensional gel electrophoresis and analyzed by Western blotting for their reactivity with the convalescent serum against two strains. Immunogenic proteins were identified by matrix-assisted laser desorption/ionization time of flight-mass spectrometry（MALDI-TOF-MS）, a total of 14 proteins are common immunoreactive proteins, of which 1 was known antigen and 13 were novel immunogenic proteins for B. bronchiseptica. Putative lipoprotein gene was cloned and recombinantly expressed. The recombinant protein induced high titer antibody, but showed low protective indices against challenges with HB（B. bronchiseptica strain isolated from a infected rabbit）. The mortality of mice was 80% compared to 100% of positive controls. The identification of these novel antigenic proteins is an important resource for further development of a new diagnostic test and vaccine for B. bronchiseptica.

Secondary Structure Changes and Thermal Stability of Plasma Membrane Proteins of Wheat Roots in Heat Stress

The wheat roots membrane separates the cell from the environment around it and encloses the cell contents. The pro-tein secondary structure and thermal stability of the plasma membrane of wheat root have been characterized in D2O buffer from 20°C to 90°C by Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Quantitative analysis of the amide I band (1700 - 1600 cm–1) showed that the plasma membrane proteins contains 41% α-helix, 16% β-sheet, 18% turn, and 25% disorder structures at 20°C. At elevated temperatures from 25°C up to 90°C, the α-helix and the β-sheet structure unfold into turns and the disorder structure, with a major conformational transition occurring at 50°C. There is a rapid decline in H+-ATPase activity of plasma membrane from 35°C to 55°C and it remain very low level H+-ATPase activity of PM from 55°C to 90°C. Therefore the protein conformational transition was one of reasons of loses H+-ATPase activity of plasma membrane.

Solid-State NMR Spectroscopic Approaches to Investigate Dynamics, Secondary Structure and Topology of Membrane Proteins

Solid-state NMR spectroscopy is routinely used to determine the structural and dynamic properties of both membrane proteins and peptides in phospholipid bilayers [1-26]. From the perspective of the perpetuated lipids, 2H solid-state NMR spectroscopy can be used to probe the effect of embedded proteins on the order and dynamics of the acyl chains of phospholipid bilayers [8-13]. Moreover, 31P solid-state NMR spectroscopy can be used to investigate the interaction of peptides, proteins and drugs with phospholipid head groups [11-14]. The secondary structure of 13C = O site-specific isotopically labeled peptides or proteins inserted into lipid bilayers can be probed utilizing 13C CPMAS solid-state NMR spectroscopy [15-18]. Also, solid-state NMR spectroscopic studies can be utilized to ascertain pertinent informa- tion on the backbone and side-chain dynamics of 2H- and 15N-labeled proteins, respectively, in phospholipid bilayers [19-26]. Finally, specific 15N-labeled amide sites on a protein embedded inside oriented bilayers can be used to probe the alignment of the helices with respect to the bilayer normal [2]. A brief summary of all these solid-state NMR ap- proaches are provided in this minireview.

Do membrane proteins cluster without binding between molecules?

Clustering is a basic event for the initiation of immune cell responses, and simulation analyses of clustering of membrane proteins have been performed. It was claimed that a cluster is formed by the self-assembly induced by protein dimerization with a high binding speed (Woolf and Linderman, Biophys. Chem. 104, 217-227, 2003). We examined the cluster formation with Monte Carlo simulation using two algorithms. The first was that simulation processes were divided into two substeps. All proteins were subjected to movement in the first substep, followed by reaction in the second substep. The second algorithm was that proteins were first selected to react and proteins which did not react were subjected to movement. The self-assembly induced by dimerization was simulated only with the second algorithm. In this algorithm, monomers dissociated from dimers do not move because these monomers are not selected for movement, and a large proportion of such monomers are selected to form dimers in the next step. The self-assembly was again simulated with the first algorithm containing the conditions that monomers dissociated from dimers did not move in the next movement substep. This algorithm seems to be far removed from natural conditions. Thus, it is inferred that the self-assembly induced by dimerization is unlikely in situ, and that some interaction between proteins is required for cluster formation. In contrast to algorithms in previous simulations, our results suggest that it is more appropriate that proteins move to the same direction for a while and reflect when the collision occurs.