Chemical synthesis of Shiga toxin subunit B using a next-generation traceless "helping hand" solubilizing tag.

The application of solid-phase peptide synthesis and native chemical ligation in chemical protein synthesis (CPS) has enabled access to synthetic proteins that cannot be produced recombinantly, such as site-specific post-translationally modified or mirror-image proteins (D-proteins). However, CPS is commonly hampered by aggregation and insolubility of peptide segments and assembly intermediates. Installation of a solubilizing tag consisting of basic Lys or Arg amino acids can overcome these issues. Through the introduction of a traceless cleavable linker, the solubilizing tag can be selectively removed to generate native peptide. Here we describe the synthesis of a next-generation amine-reactive linker N-Fmoc-2-(7-amino-1-hydroxyheptylidene)-5,5-dimethylcyclohexane-1,3-dione (Fmoc-Ddap-OH) that can be used to selectively introduce semi-permanent solubilizing tags ("helping hands") onto Lys side chains of difficult peptides. This linker has improved stability compared to its predecessor, a property that can increase yields for multi-step syntheses with longer handling times. We also introduce a new linker cleavage protocol using hydroxylamine that greatly accelerates removal of the linker. The utility of this linker in CPS was demonstrated by the preparation of the synthetically challenging Shiga toxin subunit B (StxB) protein. This robust and easy-to-use linker is a valuable addition to the CPS toolbox for the production of challenging synthetic proteins.

A monodisperse transmembrane α-helical peptide barrel.

The fabrication of monodisperse transmembrane barrels formed from short synthetic peptides has not been demonstrated previously. This is in part because of the complexity of the interactions between peptides and lipids within the hydrophobic environment of a membrane. Here we report the formation of a transmembrane pore through the self-assembly of 35 amino acid α-helical peptides. The design of the peptides is based on the C-terminal D4 domain of the Escherichia coli polysaccharide transporter Wza. By using single-channel current recording, we define discrete assembly intermediates and show that the pore is most probably a helix barrel that contains eight D4 peptides arranged in parallel. We also show that the peptide pore is functional and capable of conducting ions and binding blockers. Such α-helix barrels engineered from peptides could find applications in nanopore technologies such as single-molecule sensing and nucleic-acid sequencing.

Cyclic peptide analogs of 558-565 epitope of A2 subunit of Factor VIII prolong aPTT. Toward a novel synthesis of anticoagulants.

Novel anticoagulant therapies target specific clotting factors in blood coagulation cascade. Inhibition of the blood coagulation through Factor VIII-Factor IX interaction represents an attractive approach for the treatment and prevention of diseases caused by thrombosis. Our research efforts are continued by the synthesis and biological evaluation of cyclic, head to tail peptides, analogs of the 558-565 sequence of the A2 subunit of FVIII, aiming at the efficient inhibition of Factor VIIIa-Factor IXa interaction. The analogs were synthesized on solid phase using the acid labile 2-chlorotrityl chloride resin, while their anticoagulant activities were examined in vitro by monitoring activated partial thromboplastin time and the inhibition of Factor VIII activity. The results reveal that these peptides provide bases for the development of new anticoagulant agents.

Practical approaches to designing novel protein assemblies.

Molecular self-assembly offers a means by which sophisticated materials can be constructed with unparalleled precision. Designing self-assembling protein structures is of particular interest as a result of the unique functional capabilities of proteins. Custom-designed protein materials could lead to new possibilities in therapeutics, bioenergy, and materials science. Although the field was long hampered by the challenges involved in designing such complex molecules, novel approaches and computational tools have recently led to remarkable progress. Here we review recent design studies in the context of three fundamental aspects of self-assembling materials: subunit organization, subunit interactions, and regulation of assembly.

Construction of histidine-tagged yeast mitochondrial cytochrome c oxidase for facile purification of mutant forms.

Yeast CcO (cytochrome c oxidase) has been developed as a facile system for the production and analysis of mutants of a mitochondrial form of CcO for mechanistic studies. First, a 6H tag (His6 tag) was fused to the C-terminus of a nuclear-encoded subunit of CcO from yeast Saccharomyces cerevisiae. This allowed efficient purification of a WT (wild-type) mitochondrial CcO, 6H-WT (yeast CcO with a 6H tag on the nuclear-encoded Cox13 subunit), with a recovery yield of 45%. Its catalytic-centre activity [≈180 e·s(-1) (electrons per s)], UV-visible signatures of oxidized and reduced states and ability to form the P(M) ['peroxy' (but actually a ferryl/radical state)] and F (ferryl) intermediates confirm normal functioning of the histidine-tagged protein. Point mutations were introduced into subunit I of the 6H-WT strain. All mutants were screened for their ability to assemble CcO and grow on respiratory substrate. One such mutant [6H-E243DI (the 6H-WT strain with an additional mutation of E243D in mitochondrial DNA-encoded subunit I)] was purified and showed ~50% of the 6H-WT catalytic-centre activity, consistent with the effects of the equivalent mutation in bacterial oxidases. Mutations in both the D and the H channels affect respiratory growth and these effects are discussed in terms of their putative roles in CcO mechanism.

Development of a simple fed-batch process for the high-yield production of recombinant shiga toxin B-chain protein.

Shiga toxins are one of the very potent agents for causing dysentery, diarrhoea and haemolytic uremic syndrome with very low LD50. For better understanding of their biology, detection and neutralization, the components of toxins are needed to be expressed and purified in bulk amounts. However, following traditional expression procedures, this task is very tedious as the yield of the toxin is very low. In this manuscript, we have described the optimization of media for enhanced production of recombinant Shiga toxin B (rStx-B) chain protein in Escherichia coli. This protein is known to have neutralization ability against shiga toxins. Furthermore, fed-batch cultivation process in E. coli was also developed in the optimized medium. Expression was induced with 1 mM isopropyl-beta-thiogalactoside (IPTG). The purification of protein involved Ni-NTA affinity chromatography under native conditions followed by gel filtration chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell weight and purified protein of about 19.41 g/l (dry cell weight, 11.38 g/l) and 30 mg/l of culture, respectively. The purity of the recombinant StxB protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. Reactivity of this protein was determined by Western blotting as well as indirect ELISA using specific antibodies. These results establish the application of this protein for diagnosis of shiga toxin infection or for neutralizing the toxicity.

Role of the intra-A-chain disulfide bond of insulin-like peptide 3 in binding and activation of its receptor, RXFP2.

INSL3 is a member of the insulin-IGF-relaxin superfamily and plays a key role in male fetal development and in adult germ cell maturation. It is the cognate ligand for RXFP2, a leucine-rich repeat containing G-protein coupled receptor. To date, and in contrast to our current knowledge of the key structural features that are required for the binding of INSL3 to RXFP2, comparatively little is known about the key residues that are required to elicit receptor activation and downstream cell signaling. Early evidence suggests that these are contained principally within the A-chain. To further explore this hypothesis, we have undertaken an examination of the functional role of the intra-A-chain disulfide bond. Using solid-phase peptide synthesis together with regioselective disulfide bond formation, two analogs of human INSL3 were prepared in which the intra-chain disulfide bond was replaced, one in which the corresponding Cys residues were substituted with the isosteric Ser and the other in which the Cys were removed altogether. Both of these peptides retained nearly full RXFP2 receptor binding but were devoid of cAMP activity (receptor activation), indicating that the intra-A-chain disulfide bond makes a significant contribution to the ability of INSL3 to act as an RXFP2 agonist. Replacement of the disulfide bond with a metabolically stable dicarba bond yielded two isomers of INSL3 that each exhibited bioactivity similar to native INSL3. This study highlights the critical structural role played by the intra-A-chain disulfide bond of INSL3 in mediating agonist actions through the RXFP2 receptor.

Solution structure of a conformationally restricted fully active derivative of the human relaxin-like factor.

Analogous to insulin, the relaxin-like factor (RLF) must undergo a structural transition to the active form prior to receptor binding. Thus, the C-terminus of the B chain of RLF folds toward the surface of the central B chain helix, causing partial obliteration of the two essential RLF receptor-binding site residues, valine B19 and tryptophan B27. Via comparison of the solution structure of a fully active C-terminally cross-linked RLF analogue with the native synthetic human RLF (hRLF), it became clear that the cross-linked analogue largely retains the essential folding of the native protein. Both proteins exist in a major and minor conformation, as revealed by multiple resonances from tryptophan B27 and adjacent residues on the B chain helix. Notably, the minor conformation is significantly more highly populated in the chemically cross-linked RLF than it is in the hRLF. In addition, compared to the unmodified molecule, subtle differences are observed within the B chain helix whereby the cross-linked derivative shows a reduced level of hydrogen bonding and significant peak broadening at the binding site residue ValB19. On the basis of these observations, we suggest that the solution structure of the native hormone represents an inactive conformer and that a dynamic equilibrium exists between the C-terminally unfolded binding conformation and the inactive conformation of the RLF.

Total chemical synthesis and biophysical characterization of the minimal isoform of the KChIP2 potassium channel regulatory subunit.

The potassium channel accessory subunit KChIP2 associates with Kv4.2 channels in the cardiac myocyte and is involved in the regulation of the transient outward current (I(to)) during the early phase of repolarization of the action potential. As a first step to biophysically probe the mechanism of KChIP2, we have chemically synthesized its minimal isoform, KChIP2d, using Boc chemistry solid phase peptide synthesis in conjunction with native chemical ligation. The synthetic KChIP2d protein is primarily alpha-helical as predicted and becomes more structured upon binding calcium as assessed by (1)H-NMR and CD spectroscopy. Synthetic KChIP2d is in a monomer-dimer equilibrium in solution, and there is evidence for two monomer binding sites on an N-terminal peptide of Kv4.2. Planned future studies include the incorporation of fluorescent and spin labeled probes in KChIP2d to yield structural information in parallel with electrophysiologic studies to elucidate KChIP2d's mechanism of action.

Structure by design: from single proteins and their building blocks to nanostructures.

Nanotechnology realizes the advantages of naturally occurring biological macromolecules and their building-block nature for design. Frequently, assembly starts with the choice of a "good" molecule that is synthetically optimized towards the desired shape. By contrast, we propose starting with a pre-specified nanostructure shape, selecting candidate protein building blocks from a library and mapping them onto the shape and, finally, testing the stability of the construct. Such a shape-based, part-assembly strategy is conceptually similar to protein design through the combinatorial assembly of building blocks. If the conformational preferences of the building blocks are retained and their interactions are favorable, the nanostructure will be stable. The richness of the conformations, shapes and chemistries of the protein building blocks suggests a broad range of potential applications; at the same time, it also highlights their complexity. In this Opinion article, we focus on the first step: validating such a strategy against experimental data.

Inhibition of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infectivity by peptides analogous to the viral spike protein.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is the cause of an atypical pneumonia that affected Asia, North America and Europe in 2002-2003. The viral spike (S) glycoprotein is responsible for mediating receptor binding and membrane fusion. Recent studies have proposed that the carboxyl terminal portion (S2 subunit) of the S protein is a class I viral fusion protein. The Wimley and White interfacial hydrophobicity scale was used to identify regions within the CoV S2 subunit that may preferentially associate with lipid membranes with the premise that peptides analogous to these regions may function as inhibitors of viral infectivity. Five regions of high interfacial hydrophobicity spanning the length of the S2 subunit of SARS-CoV and murine hepatitis virus (MHV) were identified. Peptides analogous to regions of the N-terminus or the pre-transmembrane domain of the S2 subunit inhibited SARS-CoV plaque formation by 40-70% at concentrations of 15-30 microM. Interestingly, peptides analogous to the SARS-CoV or MHV loop region inhibited viral plaque formation by >80% at similar concentrations. The observed effects were dose-dependent (IC50 values of 2-4 microM) and not a result of peptide-mediated cell cytotoxicity. The antiviral activity of the CoV peptides tested provides an attractive basis for the development of new fusion peptide inhibitors corresponding to regions outside the fusion protein heptad repeat regions.

Initial synthesis and structure of an all-ferrous analogue of the fully reduced [Fe4S4]0 cluster of the nitrogenase iron protein.

The synthetic cubane-type iron-sulfur clusters [Fe(4)S(4)(SR)(4)](z) form a four-member electron transfer series (z = 3-, 2-, 1-, and 0), all members of which except that with z = 0 have been isolated and characterized. They serve as accurate analogues of protein-bound [Fe(4)S(4)(SCys)(4)](z) redox centers, which, in terms of core oxidation states, exhibit the redox couples [Fe(4)S(4)](3+/2+) and [Fe(4)S(4)](2+/1+). Clusters with the all-ferrous core [Fe(4)S(4)](0) have never been isolated because of their oxidative sensitivity. Recent work on the Fe protein of Azotobacter vinelandii nitrogenase has demonstrated the formation of the all-ferrous state upon reaction with a strong reductant. Treatment of the cyanide cluster [Fe(4)S(4)(CN)(4)](3-) with K[Ph(2)CO] in acetonitrile/tetrahydrofuran affords the all-ferrous cluster [Fe(4)S(4)(CN)(4)](4-), isolated as the Bu(4)N(+) salt. The x-ray structure demonstrates retention of a cubane-type structure with idealized D(2)(d) symmetry. The Mössbauer spectrum unambiguously demonstrates the [Fe(4)S(4)](0) oxidation state. Bond distances, core volumes, (57)Fe isomer shifts, and visible absorption spectra make evident the high degree of structural and electronic similarity with the fully reduced Fe protein. The attribute of cyanide ligation causes positive [Fe(4)S(4)](2+/1+) and [Fe(4)S(4)](1+/0) redox potential shifts, facilitating the initial isolation of an analogue of the [Fe(4)S(4)](0) protein site.

A phosphoserine-lysine salt bridge within an alpha-helical peptide, the strongest alpha-helix side-chain interaction measured to date.

Phosphorylation is ubiquitous in control of protein activity, yet its effects on protein structure are poorly understood. Here we investigate the effect of serine phosphorylation in the interior of an alpha-helix when a salt bridge is present between the phosphate group and a positively charged side chain (in this case lysine) at i,i + 4 spacing. The stabilization of the helix is considerable and can overcome the intrinsically low preference of phosphoserine for the interior of the helix. The effect is pH dependent, as both the lysine and phosphate groups are titratable, and so calculations are given for several charge combinations. These results, with our previous work, highlight the different, context-dependent effects of phosphorylation in the alpha-helix. The interaction between the phosphate(2)(-) group and the lysine side chain is the strongest yet recorded in helix-coil studies. The results are of interest both in de novo design of peptides and in understanding the structural modes of control by phosphorylation.

Myasthenia gravis patients, but not healthy subjects, recognize epitopes that are unique to the epsilon-subunit of the acetylcholine receptor.

Myasthenia gravis (MG) is an autoimmune disease characterized by deficits in neuromuscular transmission due to antibody-mediated damage of the acetylcholine receptor (AChR). We examined the in vitro immune response of peripheral blood mononuclear cells isolated from MG patients (n=38) and healthy nonmyasthenic subjects (n=31) to epitopes on the alpha-, epsilon-, and gamma-chains of the AChR. The epsilon- and gamma-epitopes tested represent regions with little sequence homology to the alpha-chain, and little sequence homology between the epsilon- and gamma-chains. No differences were observed in the immune response of MG patients and healthy subjects to any of the alpha-chain epitopes tested. Serial studies of the immune response to the alpha-peptides suggest that epitope spread does occur over time. Cells from MG patients were stimulated by the epsilon- and gamma-chain peptides, although the response was weaker than that to the alpha-peptides. Cells from healthy subjects showed reactivity to gamma-chain peptides only; none of the healthy subjects responded to the epsilon-chain peptides tested. Differences between the epsilon- and gamma-chains may be important in the development of MG, because only MG patients respond to epitopes that are unique to the epsilon-subunit.

The intermediate chain of cytoplasmic dynein is partially disordered and gains structure upon binding to light-chain LC8.

The N-terminal domain of dynein intermediate chain, IC(1-289), is highly disordered, but upon binding to dynein light-chain LC8, it undergoes a significant conformational change to a more ordered structure. Using circular dichroism and fluorescence spectroscopy, we demonstrate that the change in conformation is due to an increase in the helical structure and to enhanced compactness in the environment of tryptophan 161. An increase in helical structure and compactness is also observed with trimethylamine-N-oxide (TMAO), a naturally occurring osmolyte used here as a probe to identify regions with a propensity for induced folding. Global protection of IC(1-289) from protease digestion upon LC8 binding was localized to a segment that includes residues downstream of the LC8-binding site. Several smaller constructs of IC(1-289) containing the LC8-binding site and one of the predicted helix or coiled-coil segments were made. IC(1-143) shows no increase in helical structure upon binding, while IC(114-260) shows an increase in helical structure similar to what is observed with IC(1-289). Binding of IC(114-260) to LC8 was monitored by fluorescence and native gel electrophoresis and shows saturation of binding, a stoichiometry of 1:1, and moderate binding affinity. The induced folding of IC(1-289) upon LC8 binding suggests that LC8 could act through the intermediate chain to facilitate dynein assembly or regulate cargo-binding interactions.

Shortened insulin analogues: marked changes in biological activity resulting from replacement of TyrB26 and N-methylation of peptide bonds in the C-terminus of the B-chain.

The role of three highly conserved insulin residues PheB24, PheB25, and TyrB26 was studied to better understand the subtleties of the structure-function relationship between insulin and its receptor. Ten shortened insulin analogues with modifications in the beta-strand of the B-chain were synthesized by trypsin-catalyzed coupling of des-octapeptide (B23-B30)-insulin with synthetic peptides. Insulin analogues with a single amino acid substitution in the position B26 and/or single N-methylation of the peptide bond at various positions were all shortened in the C-terminus of the B-chain by four amino acids. The effect of modifications was followed by two types of in vitro assays, i.e., by the binding to the receptor of rat adipose plasma membranes and by the stimulation of the glucose transport into the isolated rat adipocytes. From our results, we can deduce several conclusions: (i) the replacement of tyrosine in the position B26 by phenylalanine has no significant effect on the binding affinity and the stimulation of the glucose transport of shortened analogues, whereas the replacement of TyrB26 by histidine affects the potency highly positively; [HisB26]-des-tetrapeptide (B27-B30)-insulin-B26-amide and [NMeHisB26]-des-tetrapeptide (B27-B30)-insulin-B26-amide show binding affinity 529 and 5250%, respectively, of that of human insulin; (ii) N-methylation of the B24-B25 peptide bond exhibits a disruptive effect on the potency of analogues in both in vitro studies regardless the presence of amino acid in the position B26; (iii) N-methylation of the B23-B24 peptide bond markedly reduces the binding affinity and the glucose transport of respective analogue [NMePheB24]-des-tetrapeptide (B27-B30)-insulin-B26-amide.

New insight into the structure and regulation of the plant vacuolar H+-ATPase.

Plant cells are characterized by a highly active secretory system that includes the large central vacuole found in most differentiated tissues. The plant vacuolar H+-ATPase plays an essential role in maintaining the ionic and metabolic gradients across endomembranes, in activating transport processes and vesicle dynamics, and, hence, is indispensable for plant growth, development, and adaptation to changing environmental conditions. The review summarizes recent advances in elucidating the structure, subunit composition, localization, and regulation of plant V-ATPase. Emerging knowledge on subunit isogenes from Arabidopsis and rice genomic sequences as well as from Mesembryanthemum illustrates another level of complexity, the regulation of isogene expression and function of subunit isoforms. To this end, the review attempts to define directions of future research on plant V-ATPase.

A novel method for the rational construction of well-defined immunogens: the use of oximation to conjugate cholera toxin B subunit to a peptide-polyoxime complex.

Cholera toxin B subunit (CTB), capable of binding to all mucous membranes in its pentameric form, is a potential carrier of mucosal vaccines. In our previous work we reported that the N-terminus of CTB, a threonine, could in principle undergo oxidation and oximation to form conjugates with a cascade of immunogenic peptides. In this study, we set up a model by chemically coupling CTB to a polyoxime that possessed five copies of influenza virus-derived peptides displayed in comblike form. The construct was reconstituted into pentameric form when eluted from a Superdex column after conjugation, and the pentameric nature of this CTB-viral peptide complex was confirmed by SDS-PAGE. GM(1)-ELISA assay showed that the binding properties of CTB-viral peptide complex were increased 4-5-fold over native CTB.

Substructure synthesis method for simulating large molecular complexes.

This paper reports a computational method for describing the conformational flexibility of very large biomolecular complexes using a reduced number of degrees of freedom. It is called the substructure synthesis method, and the basic concept is to treat the motions of a given structure as a collection of those of an assemblage of substructures. The choice of substructures is arbitrary and sometimes quite natural, such as domains, subunits, or even large segments of biomolecular complexes. To start, a group of low-frequency substructure modes is determined, for instance by normal mode analysis, to represent the motions of the substructure. Next, a desired number of substructures are joined together by a set of constraints to enforce geometric compatibility at the interface of adjacent substructures, and the modes for the assembled structure can then be synthesized from the substructure modes by applying the Rayleigh-Ritz principle. Such a procedure is computationally much more desirable than solving the full eigenvalue problem for the whole assembled structure. Furthermore, to show the applicability to biomolecular complexes, the method is used to study F-actin, a large filamentous molecular complex involved in many cellular functions. The results demonstrate that the method is capable of studying the motions of very large molecular complexes that are otherwise completely beyond the reach of any conventional methods.