Expression, purification, and initial structural characterization of nonstructural protein 2B, an integral membrane protein of Dengue-2 virus, in detergent micelles

Dengue virus causes serious diseases affecting people in tropical and sub-tropical regions. The nonstructural (NS) protein 2B is an integral membrane protein and important for the regulation of viral protease NS3, which is significant for virus replication. The NS2B-NS3 complex is an important drug target for treating dengue fever. However, little is known about the structure of NS2B in its entirety. Herein, we describe the expression and purification of this integral membrane protein from cell membrane and inclusion bodies of Escherichia coli cells. The initial nuclear magnetic resonance (NMR) and circular dichroism (CD) results indicate that the purified protein adopts alpha-helical structures in LMPG and TDPC micelles.     

Design and biophysical characterization of a monomeric four-alpha-helix bundle protein Aα4 with affinity for the volatile anesthetic halothane

A monomeric four-α-helix bundle protein Aα₄ was designed as a step towards investigating the interaction of volatile general anesthetics with their putative membrane protein targets. The alpha helices, connected by glycine loops, have the sequence A, B, B′, A′. The DNA sequence was designed to make the helices with the same amino acid sequences (helix A and A′, B and B′, respectively) as different as possible, while using codons which are favorable for expression in E. coli. The protein was bacterially expressed and purified to homogeneity using reversed-phase HPLC. Protein identity was verified using MALDI–TOF mass spectrometry. Far-UV circular dichroism spectroscopy confirmed the predominantly alpha-helical nature of the protein Aα₄. Guanidinium chloride induced denaturation showed that the monomeric four-α-helix bundle protein Aα₄ is considerably more stable compared to the dimeric di-α-helical protein (Aα₂-L38M)₂. The sigmoidal character of the unfolding reaction is conserved while the sharpness of the transition is increased 1.8-fold. The monomeric four-α-helix bundle protein Aα₄ bound halothane with a dissociation constant (K_d) of 0.93 ± 0.02 mM, as shown by both tryptophan fluorescence quenching and isothermal titration calorimetry. This monomeric four-α-helix bundle protein can now be used as a scaffold to incorporate natural central nervous system membrane protein sequences in order to examine general anesthetic interactions with putative targets in detail.     

Molecular engineering of respiratory syncytial virus immunogen for rational redesign of prophylactic peptide vaccines against pediatric viral pneumonia

The immunogen FFL_001 of respiratory syncytial virus (RSV), a widely spread virus that infects the lungs and breathing passages, is a three-helix bundle protein of 115 amino acids long that consists of three helical arms H1, H2 and H3. Here, we attempted to perform molecular engineering of the immunogen, aiming to considerably reduce the protein scaffold of FFL_001 with only moderate activity loss. Structural analysis and molecular dynamics (MD) simulations revealed that two helices H2 and H3 of the FFL_001 three-helix bundle can directly interact with its cognate monoclonal antibody Motavizumab, while the remaining helix H1 plays a crucial role in stabilisation of the three-helix bundle conformation. Binding of the two split peptide segments separately representing FFL_001 two-helix bundle H2-H3 and Motavizumab-binding site to the antibody would incur considerable entropy penalty as compared to binding of the intact FFL_001, suggesting that peptide segments are highly flexible that exhibit a strong intrinsic disorder in solution. In this respect, a scheme was proposed to rationally redesign the immunogen protein scaffold by truncation and cyclisation of the three-helix bundle. The cyclisation was conducted on the spatially vicinal residue pairs in H2 and H3 helical arms by mutating the residue to cysteine and introducing a disulphide bond across them. Consequently, we obtained three cyclic peptides that were theoretically predicted to have strong binding potency towards Motavizumab. In order to substantiate the computational finding, binding affinity of the designed cyclic peptide and its linear counterpart was determined. Consistently, no binding can be found between the linear peptide and Motavizumab (K_d = n.d.), while a moderately high affinity was observed for cyclic peptide (K_d = 16.2 nM).     

Design and biophysical characterization of a monomeric four-alpha-helix bundle protein Aα(4) with affinity for the volatile anesthetic halothane

A monomeric four-α-helix bundle protein Aα(4) was designed as a step towards investigating the interaction of volatile general anesthetics with their putative membrane protein targets. The alpha helices, connected by glycine loops, have the sequence A, B, B', A'. The DNA sequence was designed to make the helices with the same amino acid sequences (helix A and A', B and B', respectively) as different as possible, while using codons which are favorable for expression in E. coli. The protein was bacterially expressed and purified to homogeneity using reversed-phase HPLC. Protein identity was verified using MALDI-TOF mass spectrometry. Far-UV circular dichroism spectroscopy confirmed the predominantly alpha-helical nature of the protein Aα(4). Guanidinium chloride induced denaturation showed that the monomeric four-α-helix bundle protein Aα(4) is considerably more stable compared to the dimeric di-α-helical protein (Aα(2)-L38M)(2). The sigmoidal character of the unfolding reaction is conserved while the sharpness of the transition is increased 1.8-fold. The monomeric four-α-helix bundle protein Aα(4) bound halothane with a dissociation constant (K(d)) of 0.93±0.02mM, as shown by both tryptophan fluorescence quenching and isothermal titration calorimetry. This monomeric four-α-helix bundle protein can now be used as a scaffold to incorporate natural central nervous system membrane protein sequences in order to examine general anesthetic interactions with putative targets in detail.     

Expression of foreign antigens on the surface ofEscherichia coli by fusion to the outer membrane protein TraT

ThetraT gene is one of the F factor transfer genes and encodes an outer membrane protein which is involved in interactions between anEscherichia coli and its surroundings. This protein was altered so as to permit the expression of foreign proteins on the outer membrane ofE. coli in this study. A 729-bp DNA fragment, including the leader and entire structural gene sequence oftraT, was amplified and obtained by PCR. This sequence was then subcloned downstream of thetac promoter of pDR540, resulting in a TraT expression vector, pT2. Here, we report that the expression of TraT protein, fused either with a partial pre-S antigen of hepatitis B virus (60 and 98 amino acids, respectively) or with the snake venom rhodostomin (72 amino acids), was successfully achieved on the outer membrane ofE. coli, using the pT2 plasmid. This result was demonstrated using dot blot and immunofluorescence analysis. This finding supports the notion that the pT2 plasmid can be used as anE. coli display system. This system can detect a foreign peptide of about 100 amino acid residues in length on the bacterial surface.     

A thioredoxin NbTRXh2 from Nicotiana benthamiana negatively regulates the movement of Bamboo mosaic virus

An up‐regulated gene derived from Bamboo mosaic virus (BaMV)‐infected Nicotiana benthamiana plants was cloned and characterized in this study. BaMV is a single‐stranded, positive‐sense RNA virus. This gene product, designated as NbTRXh2, was matched with sequences of thioredoxin h proteins, a group of small proteins with a conserved active‐site motif WCXPC conferring disulfide reductase activity. To examine how NbTRXh2 is involved in the infection cycle of BaMV, we used the virus‐induced gene silencing technique to knock down NbTRXh2 expression in N. benthamiana and inoculated the plants with BaMV. We observed that, compared with control plants, BaMV coat protein accumulation increased in knockdown plants at 5 days post‐inoculation (dpi). Furthermore, BaMV coat protein accumulation did not differ significantly between NbTRXh2‐knockdown and control protoplasts at 24 hpi. The BaMV infection foci in NbTRXh2‐knockdown plants were larger than those in control plants. In addition, BaMV coat protein accumulation decreased when NbTRXh2 was transiently expressed in plants. These results suggest that NbTRXh2 plays a role in restricting BaMV accumulation. Moreover, confocal microscopy results showed that NbTRXh2‐OFP (NbTRXh2 fused with orange fluorescent protein) localized at the plasma membrane, similar to AtTRXh9, a homologue in Arabidopsis. The expression of the mutant that did not target the substrates failed to reduce BaMV accumulation. Co‐immunoprecipitation experiments revealed that the viral movement protein TGBp2 could be the target of NbTRXh2. Overall, the functional role of NbTRXh2 in reducing the disulfide bonds of targeting factors, encoded either by the host or virus (TGBp2), is crucial in restricting BaMV movement.     

新城疫病毒囊膜糖蛋白七肽重复区的融合作用

在新城疫病毒（Newcastle diseasevirus,NDV）膜融合的过程中融合糖蛋白（Fusion protein,F）的两段七肽重复区（Heptad repeat,HR）发挥着重要作用。这两段七肽重复区能够形成反相平行的六螺旋束结构,这被认为是融合蛋白融合后构象的核心结构。对融合作用的深入系统研究将有助于膜融合病毒的防控。     

Solid-state NMR characterization of conformational plasticity within the transmembrane domain of the influenza A M2 proton channel

Membrane protein function within the membrane interstices is achieved by mechanisms that are not typically available to water-soluble proteins. The whole balance of molecular interactions that stabilize three-dimensional structure in the membrane environment is different from that in an aqueous environment. As a result interhelical interactions are often dominated by non-specific van der Waals interactions permitting dynamics and conformational heterogeneity in these interfaces. Here, solid-state NMR data of the transmembrane domain of the M2 protein from influenza A virus are used to exemplify such conformational plasticity in a tetrameric helical bundle. Such data lead to very high resolution structural restraints that can identify both subtle and substantial structural differences associated with various states of the protein. Spectra from samples using two different preparation protocols, samples prepared in the presence and absence of amantadine, and spectra as a function of pH are used to illustrate conformational plasticity.     

Crystal structure of the Legionella effector Lem22

Legionella pneumophila is a pathogen causing severe pneumonia in humans called Legionnaires’ disease. Lem22 is a previously uncharacterized effector protein conserved in multiple Legionella strains. Here, we report the crystal structure of Lem22 from the Philadelphia strain, also known as lpg2328, at 1.40 Å resolution. The structure shows an up‐and‐down three‐helical bundle with a significant structural similarity to a number of protein‐binding domains involved in apoptosis and membrane trafficking. Sequence conservation identifies a putative functional site on the interface of helices 2 and 3. The structure is an important step toward a functional characterization of Lem22.     

NMR solution structure of ImB2, a protein conferring immunity to antimicrobial activity of the type IIa bacteriocin, carnobacteriocin B2

Bacteriocins produced by lactic acid bacteria are potent antimicrobial compounds which are active against closely related bacteria. Producer strains are protected against the effects of their cognate bacteriocins by immunity proteins that are located on the same genetic locus and are coexpressed with the gene encoding the bacteriocin. Several structures are available for class IIa bacteriocins; however, to date, no structures are available for the corresponding immunity proteins. We report here the NMR solution structure of the 111-amino acid immunity protein for carnobacteriocin B2 (ImB2). ImB2 folds into a globular domain in aqueous solution which contains an antiparallel four-helix bundle. Extensive packing by hydrophobic side chains in adjacent helices forms the core of the protein. The C-terminus, containing a fifth helix and an extended strand, is held against the four-helix bundle by hydrophobic interactions with helices 3 and 4. Most of the charged and polar residues in the protein face the solvent. Helix 3 is well-defined to residue 55, and a stretch of nascent helix followed by an unstructured loop joins it to helix 4. No interaction is observed between ImB2 and either carnobacteriocin B2 (CbnB2) or its precursor. Protection from the action of CbnB2 is only observed when ImB2 is expressed within the cell. The loop between helices 3 and 4, and a hydrophobic pocket which it partially masks, may be important for interaction with membrane receptors responsible for sensitivity to class IIa bacteriocins.     

Use of a β-lactamase fusion vector to investigate the organization of penicillin-binding protein 1B in the cytoplasmic membrane of Escherichia coli

The coding region for the mature form of TEM β–lactamase was fused to random positions within the coding region of the penicillin–binding protein 1B (PBP 1B) gene and the nucleotide sequences across the fusion junctions of 100 in–frame fusions were determined. All fusion proteins that contained at least the NH₂–terminal 94 residues of PBP 1B provided individual cells of E. coli with substantial levels of ampicillin resistance, suggesting that the β–lactamase moiety had been translocated to the periplasm. Fusion proteins that contained ≤ 63 residues of PBP 1B possessed β–lactamase activity, but could not protect single cells of E. coli from ampicillin, indicating that the 3–lactamase moiety of these fusion proteins remained in the cytoplasm. The β–lactamase fusion approach suggested a model for the organization of PBP 1B in which the protein is embedded in the cytoplasmic membrane by a single hydrophobic trans–membrane segment (residues 64–87), with a short NH₂–terminal domain (residues 1–63), and the remainder of the polypeptide (residues 68–844) exposed on the periplasmic side of the cytoplasmic membrane. The proposed model for the organization of PBP 1B was supported by experiments which showed that the protein was completely digested by proteinase K added from the periplasmic side of the cytoplasmic membrane but was only slightly reduced in size by protease attack from the cytoplasmic side of the membrane.     

The major heat-modifiable outer membrane protein CD is highly conserved among strains of Branhamella catarrhalis

The outer membrane of Branhamella catarrhalis contains a major, heat-modifiable outer membrane protein called CD which has epitopes on the surface of the intact bacterium. The gene encoding CD was cloned and expressed in Escherichia coli. The protein migrates in gels as a doublet, indicating that CD is encoded by single gene whose gene product has two stable conformations. The nucleotide sequence of the gene encoding CD was determined and shows homology with the OprF outer membrane protein of Pseudomonas species. The CD protein contains a proline-rich region, which appears to account for its aberrant migration in gels. Restriction fragment-length analysis of 30 isolates of B. catarrhalis with oligonucleotide probes corresponding to sequences in the CD gene produced identical patterns in Southern blot assays. The major heat-modifiable outer membrane protein CD shares homology with the OprF protein and is highly conserved among strains of B. catarrhalis.     

Sequence, expression and localization of the immunity protein for colicin B

Summary Cells of Escherichia coli containing the cbi locus on plasmids are immune to colicin B which kills cells by dissipating the membrane potential through pore formation in the cytoplasmic membrane. The nucleotide sequence of the cbi region was determined. It contains an open reading frame for a polypeptide consisting of 175 amino acids. The amino acid sequence is homologous to the primary structure of the colicin A immunity protein. This, and the strong homology between the pore-forming domains of colicins A and B suggests a common evolutionary origin for both colicins. The immunity protein could be identified following strong overexpression of cbi. The electrophoretically determined molecular weight of 20 000 was close to the calculated molecular weight of 20 185. The protein contains four large hydrophobic regions. The immunity protein was localized in the membrane fraction and was mainly contained in the cytoplasmic membrane. It is proposed that the immunity protein inactivates the colicin in the cytoplasmic membrane.     

Aggregation of model membrane proteins, modulated by hydrophobic mismatch, membrane curvature, and protein class

Aggregation of transmembrane proteins is important for many biological processes, such as protein sorting and cell signaling, and also for in vitro processes such as two-dimensional crystallization. We have used large-scale simulations to study the lateral organization and dynamics of lipid bilayers containing multiple inserted proteins. Using coarse-grained molecular dynamics simulations, we have studied model membranes comprising ∼7000 lipids and 16 identical copies of model cylindrical proteins of either α-helical or β-barrel types. Through variation of the lipid tail length and hence the degree of hydrophobic mismatch, our simulations display levels of protein aggregation ranging from negligible to extensive. The nature and extent of aggregation are shown to be influenced by membrane curvature and the shape or orientation of the protein. Interestingly, a model β-barrel protein aggregates to form one-dimensional strings within the bilayer plane, whereas a model α-helical bundle forms two-dimensional clusters. Overall, it is clear that the nature and extent of membrane protein aggregation is dependent on several aspects of the proteins and lipids, including hydrophobic mismatch, protein class and shape, and membrane curvature.     

An outer membrane protein (OmpA) of Escherichia coli can be translocated across the cytoplasmic membrane of Bacillus subtllis

The translocation of secretory proteins derived from a Gram-positive (Staphylococcus hyicus prolipase) or a Gram-negative (Escherichia coli pre-OmpA protein) bacterium across the cytoplasmic membrane was studied in E. coli and Bacillus subtilis. in both microorganisms, the prolipase was found to be secreted across the plasma membrane when either the pre-prolipase signal peptide (38 amino acids in length) or the pre-OmpA signal peptide (21 amino acids in length) was used. Expression of the gene encoding the authentic pre-OmpA protein in B. subtilis resulted in the translocation of mature OmpA protein across the plasma membrane. Processing of the OmpA precursor in B. subtilis required the electrochemical potential and was sensitive to sodium azide, suggesting that the B. subtilis SecA homologue was involved in the translocation process. The mature OmpA protein, which was most likely present in an aggregated state, was fully accessible to proteases in protoplasted cells. Therefore, our results clearly demonstrate that an outer membrane protein can be secreted by B. subtilis, supporting the notion that the basic mechanism of protein translocation is highly conserved in Gram-positive and Gram-negative bacteria.     

Actin bundling and polymerisation properties of eukaryotic elongation factor 1 alpha (eEF1A), histone H2A-H2B and lysozyme in vitro

Elongation factor 1 alpha (eEF1A) is a positively charged protein which has been shown to interact with the actin cytoskeleton. However, to date, a specific actin binding site within the eEF1A sequence has not been identified and the mechanism by which eEF1A interacts with actin remains unresolved. Many protein–protein interactions occur as a consequence of their physicochemical properties and actin bundle formation has been shown to result from non-specific electrostatic interaction with basic proteins. This study investigated interactions between actin, eEF1A and two other positively charged proteins which are not regarded as classic actin binding proteins (namely lysozyme and H2A–H2B) in order to compare their actin organising effects in vitro. For the first time using atomic force microscopy (AFM) we have been able to image the interaction of eEF1A with actin and the subsequent bundling of actin in vitro. Interestingly, we found that eEF1A dramatically increases the rate of polymerisation (45-fold above control levels). We also show for the first time that H2A–H2B has remarkably similar effects upon actin bundling (relative bundle size/number) and polymerisation (35-fold increase above control levels) as eEF1a. The presence of lysozyme resulted in bundles which were distinct from those formed due to eEF1A and H2A–H2B. Lysozyme also increased the rate of actin polymerisation above the control level (by 10-fold). Given the striking similarities between the actin bundling and polymerisation properties of eEF1A and H2A–H2B, our results hint that dimerisation and electrostatic binding may provide clues to the mechanism through which eEF1A-actin bundling occurs.     

Structure and function study of paramyxovirus fusion protein heptad repeat peptides

Paramyxovirus might adopt a molecular mechanism of membrane fusion similar to that of other class I viruses in which the heptad repeat (HR) regions of fusion protein (F) HR1 and HR2 form a six-helix bundle structure inducing membrane fusion. In this study, we examined the structure and function of HR1 and HR2 from the avian paramyxovirus-2 (APMV-2) F protein. The study showed that APMV-2 HR1 and HR2 formed a stable six-helix bundle. Only a soluble APMV-2 HR2 peptide showed potent and specific virus-cell fusion inhibition activity. Cross-inhibiting activity with APMV-1 (Newcastle disease virus, NDV) was not found. A possible mechanism of membrane fusion inhibition by the paramyxovirus HR2 peptide is discussed.     

The intracellular localization of the microvillus 110K protein,a component considered to be involved in side-on membrane attachment of F-actin

The microfilament bundle of intestinal epithelial cell microvilli is known to contain four major associated proteins in addition to actin. Of particular interest is a polypeptide of molecular weight 110000 (110K protein), since it is assumed to provide the lateral attachment of the bundle to the inner side of the plasma membrane. 110K protein was purified by SDS gel electrophoresis and used to elicit antibodies. Antigen-affinity-purified IgGs were used to study the intracellular organization of 110K protein by immunocytochemical procedures. The results are consistent with the proposed membrane attachment function for the 110K protein. It is absent from the terminal web level and restricted to that part of the core filament bundle which underlies the plasma membrane of the microvilli.     

Unfolding pathway of the colicin E1 channel protein on a membrane surface

The channel-forming domain of colicin E1 is composed of a soluble helical bundle which, upon membrane binding, unfolds to form an extended, two-dimensional helical net in the membrane interfacial layer. To characterize the pathway of unfolding of the protein and the structure of the surface-bound intermediate, the time-course of intra-protein distance changes and unfolding on a millisecond time-scale were determined from the kinetics of changes in the efficiency of fluorescence resonance energy transfer, and of the donor-acceptor overlap integral, between each of six individual tryptophan residues and a Cys-conjugated energy transfer acceptor (C509-AEDANS). Comparison of the rate constants revealed the following order of events associated with unfolding of the protein at the membrane surface: (A) movement of the hydrophobic core helices VIII-IX, coincident with a small change in Trp-Cys509 distances of the outer helices; (B) unfolding of surface helices in the helical bundle in the order: helix I, helices III, IV, VI, VII, and helix V; (C) a slow (time-scale, seconds) condensation of the surface-bound helices. The rate of protein unfolding events increased with increasing anionic lipid content. Unfolding did not occur below the lipid thermal phase transition, indicating that unfolding requires mobility in the interfacial layer. The structure of the two-dimensional membrane-bound intermediate in the steady-state was inferred to consist of a quasi-circular arrangement of eight helices embedded in the membrane interfacial layer and anchored by the hydrophobic helical hairpin. The pathway of unfolding of the colicin channel at the membrane surface, catalyzed by electrostatic and hydrophobic forces, is the first described for a membrane-active protein. It is proposed that the pathway and principles described for the colicin protein are relevant to membrane protein import.     

Self-assembly of a simple membrane protein: coarse-grained molecular dynamics simulations of the influenza M2 channel

The transmembrane (TM) domain of the M2 channel protein from influenza A is a homotetrameric bundle of α-helices and provides a model system for computational approaches to self-assembly of membrane proteins. Coarse-grained molecular dynamics (CG-MD) simulations have been used to explore partitioning into a membrane of M2 TM helices during bilayer self-assembly from lipids. CG-MD is also used to explore tetramerization of preinserted M2 TM helices. The M2 helix monomer adopts a membrane spanning orientation in a lipid (DPPC) bilayer. Multiple extended CG-MD simulations (5 × 5 μs) were used to study the tetramerization of inserted M2 helices. The resultant tetramers were evaluated in terms of the most populated conformations and the dynamics of their interconversion. This analysis reveals that the M2 tetramer has 2× rotationally symmetrical packing of the helices. The helices form a left-handed bundle, with a helix tilt angle of ∼16°. The M2 helix bundle generated by CG-MD was converted to an atomistic model. Simulations of this model reveal that the bundle's stability depends on the assumed protonation state of the H37 side chains. These simulations alongside comparison with recent x-ray (3BKD) and NMR (2RLF) structures of the M2 bundle suggest that the model yielded by CG-MD may correspond to a closed state of the channel.