Evidence for similar function of transmembrane segments in receptor and membrane-anchored proteins

Transmembrane (TM) regions of receptor proteins should have unique structural and/or chemical characteristics if these regions contain residues functional in TM signal transduction. However, in a survey of the membrane-occurring residues in 37 integral membrane proteins, we found that amino acid compositions of TM regions of receptor proteins (n = 11) could not be distinguished statistically from corresponding regions of membrane-anchored proteins (e.g., recognition molecules) with a functional external domain attached to a single hydrophobic membrane-spanning anchor segment (n = 16). TM regions in both categories of proteins differed from the compositions of TM regions in membrane-transport proteins (n = 10). The analysis implies that TM regions in receptor proteins may function mainly to anchor (and position) receptors in their cellular membranes, and therefore residues in receptors that participate in signal transduction need not be restricted to these regions. In addition to mechanisms involving receptor aggregation, ligand-activated conformational perturbation of a receptor external aqueous domain, resulting in membrane penetration of hydrophobic segment(s) of this domain to produce intramembranous contact with its cytoplasmic domain, is hypothesized as a further possible mode of signal transduction.     

Exploration of twin‐arginine translocation for expression and purification of correctly folded proteins in Escherichia coli

Historically, the general secretory (Sec) pathway of Gram‐negative bacteria has served as the primary route by which heterologous proteins are delivered to the periplasm in numerous expression and engineering applications. Here we have systematically examined the twin‐arginine translocation (Tat) pathway as an alternative, and possibly advantageous, secretion pathway for heterologous proteins. Overall, we found that: (i) export efficiency and periplasmic yield of a model substrate were affected by the composition of the Tat signal peptide, (ii) Tat substrates were correctly processed at their N‐termini upon reaching the periplasm and (iii) proteins fused to maltose‐binding protein (MBP) were reliably exported by the Tat system, but only when correctly folded; aberrantly folded MBP fusions were excluded by the Tat pathway's folding quality control feature. We also observed that Tat export yield was comparable to Sec for relatively small, well‐folded proteins, higher relative to Sec for proteins that required cytoplasmic folding, and lower relative to Sec for larger, soluble fusion proteins. Interestingly, the specific activity of material purified from the periplasm was higher for certain Tat substrates relative to their Sec counterparts, suggesting that Tat expression can give rise to relatively pure and highly active proteins in one step.     

Measuring the stability of partly folded proteins using TMAO

Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N-oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte-induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline-resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two-state equilibrium unfolding, we found that DeltaG degrees for unfolding depends linearly on TMAO concentration, and that the sensitivity of DeltaG degrees to urea (the m-value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline-resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of DeltaG degrees on each cosolvent are similar to those obtained by standard urea-induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full-length Notch ankyrin domain.     

Ultrasensitivity in multisite phosphorylation of membrane-anchored proteins

Cellular signaling is initially confined to the plasma membrane, where the cytoplasmic tails of surface receptors and other membrane-anchored proteins are phosphorylated in response to ligand binding. These proteins often contain multiple phosphorylation sites that are regulated by membrane-confined enzymes. Phosphorylation of these proteins is thought to be tightly regulated, because they initiate and regulate signaling cascades leading to cellular activation, yet how their phosphorylation is regulated is poorly understood. Ultrasensitive or switchlike responses in their phosphorylation state are not expected because the modifying enzymes are in excess. Here, we describe a novel mechanism of ultrasensitivity exhibited by multisite membrane-anchored proteins, but not cytosolic proteins, even when enzymes are in excess. The mechanism underlying this concentration-independent ultrasensitivity is the local saturation of a single enzyme by multiple sites on the substrate. Local saturation is a passive process arising from slow membrane diffusion, steric hindrances, and multiple sites, and therefore may be widely applicable. Critical to this ultrasensitivity is the brief enzymatic inactivation that follows substrate modification. Computations are presented using ordinary differential equations and stochastic spatial simulations. We propose a new role, to our knowledge, for multisite membrane-anchored proteins, discuss experiments that can be used to probe the model, and relate our findings to previous theoretical work.     

Improved expression and purification of the correctly folded response regulator PlnC from lactobacilli

The response regulator PlnC is part of the signal transduction system that plays a key role in the regulation of bacteriocin production in Lactobacillus plantarum C11. In this study, we wanted to express high levels of the response regulator PlnC in a soluble and native form for purification and further studies. The protein was expressed as a fusion protein (fPlnC) containing an N-terminal Flag-tag to facilitate detection and purification. When the fusion gene, fplnC, was expressed in Escherichia coli BL21, nearly all (99%) of the recombinant protein ended up inside inclusion bodies as an incorrectly folded protein. By utilizing two different Gram-positive expression systems (SIP and NICE) in L. plantarum NC8 and Lactobacillus sakei Lb790, the expression of the soluble fPlnC was significantly increased, being 20-40 times more than that in E. coli BL21. Using the N-terminal tag, the expressed protein was purified by immunoprecipitation. By DNA-binding study (EMSA), we demonstrated that the fusion protein purified from the soluble pool was correctly folded as judged by its ability to bind specifically on regulated promoters. Using our approach, we estimate that about 1 mg of fPlnC can be purified from 11 of the bacterial culture.     

Membrane translocation of folded proteins

An ever-increasing number of proteins have been shown to translocate across various membranes of bacterial as well as eukaryotic cells in their folded states as a part of physiological and/or pathophysiological processes. Herein, we provide an overview of the systems/processes that are established or likely to involve the membrane translocation of folded proteins, such as protein export by the twin-arginine translocation system in bacteria and chloroplasts, unconventional protein secretion and protein import into the peroxisome in eukaryotes, and the cytosolic entry of proteins (e.g., bacterial toxins) and viruses into eukaryotes. We also discuss the various mechanistic models that have previously been proposed for the membrane translocation of folded proteins including pore/channel formation, local membrane disruption, membrane thinning, and transport by membrane vesicles. Finally, we introduce a newly discovered vesicular transport mechanism, vesicle budding and collapse, and present evidence that vesicle budding and collapse may represent a unifying mechanism that drives some (and potentially all) of folded protein translocation processes.     

Twin-arginine-dependent translocation of folded proteins

Twin-arginine translocation (Tat) denotes a protein transport pathway in bacteria, archaea and plant chloroplasts, which is specific for precursor proteins harbouring a characteristic twin-arginine pair in their signal sequences. Many Tat substrates receive cofactors and fold prior to translocation. For a subset of them, proofreading chaperones coordinate maturation and membrane-targeting. Tat translocases comprise two kinds of membrane proteins, a hexahelical TatC-type protein and one or two members of the single-spanning TatA protein family, called TatA and TatB. TatC- and TatA-type proteins form homo- and hetero-oligomeric complexes. The subunits of TatABC translocases are predominantly recovered from two separate complexes, a TatBC complex that might contain some TatA, and a homomeric TatA complex. TatB and TatC coordinately recognize twin-arginine signal peptides and accommodate them in membrane-embedded binding pockets. Advanced binding of the signal sequence to the Tat translocase requires the proton-motive force (PMF) across the membranes and might involve a first recruitment of TatA. When targeted in this manner, folded twin-arginine precursors induce homo-oligomerization of TatB and TatA. Ultimately, this leads to the formation of a transmembrane protein conduit that possibly consists of a pore-like TatA structure. The translocation step again is dependent on the PMF.     

Enhancing phage display of antibody fragments using gIII-amber suppression

The effect of utilizing Ex12 helper phage, a mutant M13K07 helper having two amber codons at the gIII (gIII-amber), in combination with Escherichia coli host strains belonging to the supE genotype on improving the phage display of antibody fragments was investigated. Because of an inefficient read-through of the UAG codons, Ex12 helper phage produced approximately 10% of the intracellular wt pIII in the supE host cells compared to M13K07. The phage progenies rescued from the supE XL-1 Blue MRF' strain carrying the recombinant phagemid, pCMTG-SP112, by Ex12 helper phage displayed both antibody-DeltapIII fusion and wt pIII at a ratio of 1:1.5, and achieved a 50-fold greater display of the antibody-DeltapIII compared to those obtained by a conventional phage rescue using M13K07. Additionally observed were a 100-fold increase in antigen-binding functionality and a drastic improvement on antigen-specific panning efficiency by the phage progenies. Our approach permits the display of at least one antibody fragment as well as more than one copy of wt pIII on the surface of recombinant phages, and this would make the phagemid-based phage display technology more practical and reliable.     

Development of tularemic scFv antibody fragments using phage display

Polyclonal antibodies, as well as monoclonal antibodies are efficacious in providing protective immunity against Francisella tularensis. This study demonstrates the application of phage display libraries for the construction of monoclonal antibodies against F. tularensis. Novel single-chain fragment variable (scFv) antibodies were generated against a whole bacterial lysate of F. tularensis live vaccine strain using the human single fold scFv libraries I (Tomlinson I + J). A total of 20 clones reacted with the bacterial cell lysate. Further, the library contains two clones responsive to recombinant lipoprotein FTT1103Δsignal (F. tularensis subsp. tularensis Schu S4), which was constructed without a signal sequence. These positively-binding scFvs were evaluated by scFv-phage enzyme-linked immunosorbent assay (ELISA). Then, positive scFvs were expressed in a soluble form in Escherichia coli HB2151 and tested for positive scFvs by using scFv-ELISA.     

Secretion of correctly processed and folded pancreatic secretory trypsin inhibitor by Bacillus subtilis.

We constructed a plasmid, designated pNPP126, containing a DNA sequence encoding a fusion protein composed of Bacillus amyloliquefaciens neutral protease prepeptide (signal peptide) and human pancreatic secretory trypsin inhibitor (hPSTI), where the mature hPSTI is accurately fused to the 3'-terminal of the prepeptide coding region. It was observed that the strain Bacillus subtilis MT600 harboring pNPP126 could secrete a trypsin inhibitory activity into the culture medium. The N-terminal amino acid sequence, the amino acid composition and the stoichiometry of the purified hPSTI produced by B. subtilis were the same as those of natural hPSTI, indicating that the transformant B. subtilis MT600 (pNPP126) could efficiently secrete the correctly processed and folded hPSTI into the culture medium.     

A protein sequence fitness function for identifying natural and nonnatural proteins

The infinitesimally small sequence space naturally scouted in the millions of years of evolution suggests that the natural proteins are constrained by some functional prerequisites and should differ from randomly generated sequences. We have developed a protein sequence fitness scoring function that implements sequence and corresponding secondary structural information at tripeptide levels to differentiate natural and nonnatural proteins. The proposed fitness function is extensively validated on a dataset of about 210 000 natural and nonnatural protein sequences and benchmarked with existing methods for differentiating natural and nonnatural proteins. The high sensitivity, specificity, and percentage accuracy (0.81%, 0.95%, and 91% respectively) of the fitness function demonstrates its potential application for sampling the protein sequences with higher probability of mimicking natural proteins. Moreover, the four major classes of proteins (α proteins, β proteins, α/β proteins, and α + β proteins) are separately analyzed and β proteins are found to score slightly lower as compared to other classes. Further, an analysis of about 250 designed proteins (adopted from previously reported cases) helped to define the boundaries for sampling the ideal protein sequences. The protein sequence characterization aided by the proposed fitness function could facilitate the exploration of new perspectives in the design of novel functional proteins.     

Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones.

Disulfide bonds are normally formed after a polypeptide has been exported from the reducing environment of the cytoplasm into a more oxidizing compartment, such as the bacterial periplasm. Recently, we showed that in Escherichia coli trxB gor mutants, in which the reduction of thioredoxin and glutathione is impaired, the redox potential of the cytoplasm becomes comparable to that of the mammalian endoplasmic reticulum, thus allowing the formation of disulfide bonds in certain complex proteins (P. H. Bessette et al., 1999, Proc. Natl. Acad. Sci. USA 96, 13703-13708]. Here, we investigate the expression of a Fab antibody fragment in the bacterial cytoplasm. The effect of coexpressing cytoplasmic chaperones (GroEL/ES, trigger factor, DnaK/J), as well as signal sequenceless versions of periplasmic chaperones (DsbC and Skp), was examined. Skp coexpression was shown to have the most significant effect (five- to sixfold increase) on the yield of correctly folded Fab. A maximum yield of 0.8 mg Fab/L/OD(600) Fab was obtained, indicating that cytoplasmic expression may be a viable alternative for the preparative production of antibody fragments.     

Production of nonclassical inclusion bodies from which correctly folded protein can be extracted

Human granulocyte-colony stimulating factor (hG-CSF), an important biopharmaceutical drug used in oncology, is currently produced mainly in Escherichia coli. Expression of human hG-CSF gene in E. coli is very low, and therefore a semisynthetic, codon-optimized hG-CSF gene was designed and subcloned into pET expression plasmids. This led to a yield of over 50% of the total cellular proteins. We designed a new approach to biosynthesis at low temperature, enabling the formation of "nonclassical" inclusion bodies from which correctly folded protein can be readily extracted by nondenaturing solvents, such as mild detergents or low concentrations of polar solvents such as DMSO and nondetergent sulfobetaines. FT-IR analysis confirmed different nature of inclusion bodies with respect to the growth temperature and indicated presence of high amounts of very likely correctly folded reduced hG-CSF in nonclassical inclusion bodies. The yield of correctly folded, functional hG-CSF obtained in this way exceeded 40% of the total hG-CSF produced in the cells and is almost completely extractable under nondenaturing conditions. The absence of the need to include a denaturation/renaturation step in the purification process allows the development of more efficient processes characterized by higher yields and lower costs and involving environment-friendly technologies. The technology presented works successfully at the 50-L scale, producing nonclassical inclusion bodies of the same quality. The approach developed for the production of hG-CSF could be extended to other proteins; thus, a broader potential for industrial exploitation is envisaged.     

Production of native, correctly folded bovine pancreatic trypsin inhibitor by Escherichia coli

A gene for bovine pancreatic trypsin inhibitor (BPTI) was fused to the coding sequence for the Escherichia coli alkaline phosphatase signal peptide and expressed in E. coli under the control of the alkaline phosphatase promoter. When induced in phosphate-depleted medium such cells produced a trypsin inhibitor that was indistinguishable from native, properly folded BPTI. In particular, the BPTI produced by E. coli had three disulfide bonds that appeared to be identical to those found in native BPTI, as assayed by sensitivity to iodoacetate, dithiothreitol, and urea. This expression/secretion system will make possible the production of variant BPTI molecules, thus allowing the perturbing effects of amino acid substitutions on BPTI folding, structure, and function to be assessed.     

In vitro selection of Jun-associated proteins using mRNA display

Although yeast two-hybrid assay and biochemical methods combined with mass spectrometry have been successfully employed for the analyses of protein–protein interactions in the field of proteomics, these methods encounter various difficulties arising from the usage of living cells, including inability to analyze toxic proteins and restriction of testable interaction conditions. Totally in vitro display technologies such as ribosome display and mRNA display are expected to circumvent these difficulties. In this study, we applied an mRNA display technique to screening for interactions of a basic leucine zipper domain of Jun protein in a mouse brain cDNA library. By performing iterative affinity selection and sequence analyses, we selected 16 novel Jun-associated protein candidates in addition to four known interactors. By means of real-time PCR and pull-down assay, 10 of the 16 newly discovered candidates were confirmed to be direct interactors with Jun in vitro. Furthermore, interaction of 6 of the 10 proteins with Jun was observed in cultured cells by means of co-immunoprecipitation and observation of subcellular localization. These results demonstrate that this in vitro display technology is effective for the discovery of novel protein–protein interactions and can contribute to the comprehensive mapping of protein–protein interactions.     

Charge effects on folded and unfolded proteins

We develop a theory for the effects of charge on the stabilization of globular proteins. The folding process is modeled as occurring through a fictitious intermediate state along a two-part thermodynamic pathway in which the molecule (i) increases its density and then (ii) rearranges its ionic groups to the protein surface. The equilibrium for the binding of protons in salt solutions is assumed to be driven by the electrical potential due to the charge distribution, in addition to the intrinsic binding affinity and bulk proton concentration. The potential is calculated for inside and outside a porous sphere model of the protein using the Poisson-Boltzmann relation, wherein the interior dielectric constant is taken to be a linear function of the chain density. The model predicts the slope of the titration curves for native myoglobin in agreement with experiments by Breslow and Gurd (1962). From the similar experiments on the unfolded state, and from the experiments of Privalov et al. (1986) on the intrinsic viscosity of the unfolded molecules, the theory shows that the unfolded state has a much higher density than a chain in a theta solvent and that the density increases with ionic strength. In addition, from the free energy of proton binding to the protein, we also calculate the electrostatic contributions to protein stability, a major contribution deriving from changes in ionization. We consider the example of the stability of myoglobin as a function of pH, ionic strength, and ionic groups buried in the native protein structure. We show that although maximum stability of most proteins should occur at their isoelectric point, the burial of nontitratable groups should lead to maximum stabilities at pH values other than the isoelectric point.     

Engineering model proteins for Photosystem II function

Our knowledge of Photosystem II and the molecular mechanism of oxygen production are rapidly advancing. The time is now ripe to exploit this knowledge and use it as a blueprint for the development of light-driven catalysts, ultimately for the splitting of water into O₂ and H₂. In this article, we outline the background and our approach to this technological application through the reverse engineering of Photosystem II into model proteins.