The Salmonella type III secretion system inner rod protein PrgJ is partially folded

The type III secretion system (T3SS) is essential in the pathogenesis of many bacteria. The inner rod is important in the assembly of the T3SS needle complex. However, the atomic structure of the inner rod protein is currently unknown. Based on computational methods, others have suggested that the Salmonella inner rod protein PrgJ is highly helical, forming a folded 3 helix structure. Here we show by CD and NMR spectroscopy that the monomeric form of PrgJ lacks a tertiary structure, and the only well-structured part of PrgJ is a short α-helix at the C-terminal region from residues 65-82. Disruption of this helix by glycine or proline mutation resulted in defective assembly of the needle complex, rendering bacteria incapable of secreting effector proteins. Likewise, CD and NMR data for the Shigella inner rod protein MxiI indicate this protein lacks a tertiary structure as well. Our results reveal that the monomeric forms of the T3SS inner rod proteins are partially folded.     

The most pathogenic transthyretin variant, L55P, forms amyloid fibrils under acidic conditions and protofilaments under physiological conditions.

The L55P transthyretin (TTR) familial amyloid polyneuropathy-associated variant is distinct from the other TTR variants studied to date and the wild-type protein in that the L55P tetramer can dissociate to the monomeric amyloidogenic intermediate and form fibril precursors under physiological conditions (pH 7.0, 37 degrees C). The activation barrier associated with L55P-TTR tetramer dissociation is lower than the barrier for wild-type transthyretin dissociation, which does not form fibrils under physiological conditions. The L55P-TTR tetramer is also very sensitive to acidic conditions, readily dissociating to form the monomeric amyloidogenic intermediate between pH 5.5-5.0 where the wild-type TTR adopts a nonamyloidogenic tetrameric structure. The formation of the L55P monomeric amyloidogenic intermediate involves subtle tertiary structural changes within the beta-sheet rich subunit as discerned from Trp fluorescence, circular dichroism analysis, and ANS binding studies. The assembly of the L55P-TTR amyloidogenic intermediate at physiological pH (pH 7.5) affords protofilaments that elongate with time. TEM studies suggest that the entropic barrier associated with filament assembly (amyloid fibril formation) is high in vitro, amyloid being defined by the laterally assembled four filament structure observed by Blake upon isolation of "fibrils" from the eye of a FAP patient. The L55P-TTR protofilaments formed in vitro bind Congo red and thioflavin T (albeit more weakly than the fibrils produced at acidic pH), suggesting that the structure observed probably represents an amyloid precursor. The structural continuum from misfolded monomer through protofilaments, filaments, and ultimately fibrils must be considered as a possible source of pathology associated with these diseases.     

NMR Structure of a Monomeric Intermediate on the Evolutionarily Optimized Assembly Pathway of a Small Trimerization Domain

Efficient formation of specific intermolecular interactions is essential for self-assembly of biological structures. The foldon domain is an evolutionarily optimized trimerization module required for assembly of the large, trimeric structural protein fibritin from phage T4. Monomers consisting of the 27 amino acids comprising a single foldon domain subunit spontaneously form a natively folded trimer. During assembly of the foldon domain, a monomeric intermediate is formed on the submillisecond time scale, which provides the basis for two consecutive very fast association reactions. Mutation of an intermolecular salt bridge leads to a monomeric protein that resembles the kinetic intermediate in its spectroscopic properties. NMR spectroscopy revealed essentially native topology of the monomeric intermediate with defined hydrogen bonds and side-chain interactions but largely reduced stability compared to the native trimer. This structural preorganization leads to an asymmetric charge distribution on the surface that can direct rapid subunit recognition. The low stability of the intermediate allows a large free-energy gain upon trimerization, which serves as driving force for rapid assembly. These results indicate different free-energy landscapes for folding of small oligomeric proteins compared to monomeric proteins, which typically avoid the transient population of intermediates.     

Different molten globule-like folding intermediates of hen egg white lysozyme induced by high pH and tertiary butanol

We have provided evidence that hen egg white lysozyme (HEWL) existed in alpha helical and beta structure dominated molten globule (MG) states at high pH and in the presence of tertiary butanol, respectively. Circular dichroism (CD), intrinsic fluorescence, ANS binding and acrylamide-induced fluorescence quenching techniques have been used to investigate alkali-induced unfolding of HEWL and the effect of tertiary butanol on the alkaline-induced state. At pH 12.75, HEWL existed as molten globule like intermediate. The observed MG-like intermediate was characterized by (i) retention of 77% of the native secondary structure, (ii) enhanced binding of ANS (approximately 5 times) compared to native and completely unfolded state, (iii) loss of the tertiary structure as indicated by the tertiary structural probes (near-UV, CD and Intrinsic fluorescence) and (iv) acrylamide quenching studies showed that MG state has compactness intermediate between native and completely unfolded states. Moreover, structural properties of the protein at isoelectric point (pI) and denatured states have also been described. We have also shown that in the presence of 45% tertiary butanol (t-butanol), HEWL at pH 7.0 and 11.0 (pI 11.0) existed in helical structure without much affecting tertiary structure. Interestingly, MG state of HEWL at pH 12.7 transformed into another MG state (MG2) at 20% t-butanol (v/v), in which secondary structure is mainly beta sheets. On further increasing the t-butanol concentration alpha helix was found to reform. We have proposed that formation of both alpha helical and beta sheet dominated intermediate may be possible in the folding pathway of alpha + beta protein.     

Effect of pH on the pore forming activity and conformational stability of ostreolysin, a lipid raft-binding protein from the edible mushroom Pleurotus ostreatus

Ostreolysin, a pore-forming protein from the edible oyster mushroom (Pleurotus ostreatus), is a member of the aegerolysin protein family, a novel group of small acidic proteins found in bacteria, molds, mushrooms, and plants. It binds to lipid rafts and interacts specifically with cholesterol-rich lipid domains. In this study, ostreolysin was classified as a single-domain all-beta-structured protein on the basis of cDNA sequencing. pH-induced and thermally induced unfolding of ostreolysin was studied by means of CD, UV absorption, and intrinsic tryptophan fluorescence to characterize conformational transitions associated with its functional properties, i.e., binding to lipid membranes, pore forming activity on lipid vesicles, and hemolysis. At 25 degrees C and between pH 6 and 9, ostreolysin adopted a monomeric and thermodynamically stable nativelike conformation, characterized by rigid tertiary structure and predominantly beta-sheet secondary structure. Between pH 2 and 3, the protein underwent an irreversible transition to a partially unfolded, molten globule-like state which bound ANS, and exhibited disrupted tertiary structure and enhanced non-native alpha-helical structure. Functional studies showed that, unlike colicins and some other bacterial pore-forming toxins, the acid-induced molten globule-like state of ostreolysin is not relevant for lipid binding and pore formation. Instead, the compact native state was necessary for binding to cholesterol/sphingomyelin multilamellar vesicles, optimally in the pH range from 6 to 7, and for pore formation and hemolysis, maximally between pH 7 and 8.     

Acid-induced partly folded conformation resembling a molten globule state of xylanase from an alkalothermophilic Bacillus sp.

Nonnative protein structures having a compact secondary, but not rigid tertiary structure, have been increasingly observed as intermediate states in protein folding. We have shown for the first time during acid-induced unfolding of xylanase (Xyl II) the presence of a partially structured intermediate form resembling a molten globule state. The conformation and stability of Xyl II at acidic pH was investigated by equilibrium unfolding methods. Using intrinsic fluorescence and CD spectroscopic studies, we have established that Xyl II at pH 1.8 (A-state) retains the helical secondary structure of the native protein at pH 7.0, while the tertiary interactions are much weaker. At variance, from the native species (N-state), Xyl II in the A-state binds 1-anilino-8-sulfonic acid (ANS) indicating a considerable exposure of aromatic side chains. Lower concentration of Gdn HCl are required to unfold the A-state. For denaturation by Gdn HCl, the midpoint of the cooperative unfolding transition measured by fluorescence for the N-state is 3.5 +/- 0.1 M, which is higher than the value (2.2 +/- 0.1 M) observed for the A-state at pH 1.8. This alternatively folded state exhibits certain characteristics of the molten globule but differs distinctly from it by its structural stability that is characteristic for native proteins.     

Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella

Shigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular "needle" that forms a rod-like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far-UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37 degrees C and MxiH having a value near 42 degrees C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (DeltaG degrees 0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m-values obtained for the urea-induced unfolding of the proteins suggest that they undergo only a small change in solvent-accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein-protein interactions.     

Impact of domain interchange on conformational stability and equilibrium folding of chimeric class micro glutathione transferases

Rat micro class glutathione transferases M1-1 and M2-2 are homodimers that share a 78% sequence identity but display differences in stability. M1-1 is more stable at the secondary and tertiary structural levels, whereas its quaternary structure is less stable. Each subunit in these proteins consists of two structurally distinct domains with intersubunit contacts occurring between domain 1 of one subunit and domain 2 of the other subunit. The chimeric subunit variants M(12), which has domain 1 of M1 and domain 2 of M2, and its complement M(21), were used to investigate the conformational stability of the chimeric homodimers M(12)-(12) and M(21)-(21) to determine the contribution of each domain toward stability. Exchanging entire domains between class micro GSTs is accommodated by the GST fold. Urea-induced equilibrium unfolding data indicate that whereas the class micro equilibrium unfolding mechanism (i.e., N(2) <--> 2I <--> 2U) is not altered, domain exchanges impact significantly on the conformational stability of the native dimers and monomeric folding intermediates. Data for the wild-type and chimeric proteins indicate that the order of stability for the native dimer (N(2)) is M2-2 > M(12)-(12) M1-1 approximately M(21)-(21), and that the order of stability of the monomeric intermediate (I) is M1 > M2 approximately M(12) > M(21). Interactions involving Arg 77, which is topologically conserved in GSTs, appear to play an important role in the stability of both the native dimeric and folding monomeric structures.     

Characterization of amyloidogenic intermediate states through a combined use of CD and NMR spectroscopy

Characterization of amyloidogenic intermediate states is of central importance in understanding the molecular mechanism of amyloid formation. In this study, we utilized CD and NMR spectroscopy to investigate secondary structure of the monomeric amyloidogenic intermediate of a β-structured SH3 domain, which was induced by trifluoroethanol (TFE). The combined biophysical studies showed that the native state SH3 domain is gradually converted to the amyloidogenic intermediate state at TFE concentrations of 20-26% (v/v) and the aggregation-prone state contains substantial amount of the β-sheet conformation (∼ 30%) with disordered (54%) and some helical characters (16%). Under weaker amyloidogenic conditions of higher TFE concentrations (> 40%), the β-sheet structures were gradually changed to helical conformations and the relative content of the helical and β-sheet conformations was highly correlated with the aggregation propensity of the SH3 domain. This indicates that the β-sheet characters of the amyloidogenic states may be critical to the effective amyloid formation.     

A two-step refolding of acid-denatured microbial transglutaminase escaping from the aggregation-prone intermediate

Microbial transglutaminase (MTG) is a monomeric globular enzyme made of 331 amino acid residues. The conformation of MTG was examined over the pH 2.0-6.0 region using circular dichroism (CD) and 1-anilino-8-naphthalenesulfonate (ANS). Under conditions of low ionic strength, a decrease of pH below 4 caused a stepwise unfolding with an intermediate exhibiting specific ANS-binding before full unfolding at pH 2.0. At high ionic strength, the decrease of pH led to only an intermediate without further unfolding. The intermediate corresponds to the molten globule state with a secondary structure similar to the native state but disordered tertiary structures. A pH- and NaCl concentration-dependent phase diagram showed that the fully unfolded state exists only under limited conditions of low pH and a low NaCl concentration. Although a refolding yield by the direct jump to pH 6.0 was low, a two-step refolding with incubation at pH 4.0, where MTG is marginally stable, and a subsequent jump to pH 6.0 improved the yield by suppressing the kinetic traps. We propose that the two-step refolding is useful for improving the yield of larger proteins with a high pI value.     

Quaternary structure of Dictyostelium discoideum nucleoside diphosphate kinase counteracts the tendency of monomers to form a molten globule

Multimeric enzymes that lose their quaternary structure often cease to be catalytically competent. In these cases, conformational stability depends on contacts between subunits, and minor mutations affecting the surface of the monomers may affect overall stability. This effect may be sensitive to pH, temperature, or solvent composition. We investigated the role of oligomeric structure in protein stability by heat and chemical denaturation of hexameric nucleoside diphosphate kinase from Dictyostelium discoideum and its P105G mutant over a wide range of pH. The wild-type enzyme has been reported to unfold without prior dissociation into monomers, whereas monomer unfolding follows dissociation for the P105G mutant (Giartosio et al. (1996) J. Biol. Chem. 271, 17845-51). We show here that these features are also preserved at alkaline pH, with the wild-type enzyme always hexameric at room temperature whereas the mutant dissociates into monomers at pH >or=10. In acidic conditions (pH 相似文献   

Stabilization of partially folded states of cytochrome c in aqueous surfactant: effects of ionic and hydrophobic interactions

The interaction of submicellar concentrations of sodium dodecyl sulfate (SDS) with horse heart cytochrome c has been found to stabilize two spectroscopically distinct partially folded intermediates at pH 7. The first intermediate is formed by the interaction of SDS with native cytochrome c, and this intermediate retains the majority of the secondary structure while the tertiary structure of the protein is lost. The unfolding of this intermediate with urea leads to the formation of a second intermediate, which is also formed on refolding of the unfolded protein (unfolded by urea) by SDS. The second intermediate retains about 50% of the native secondary structure with no tertiary structure of the protein. The second intermediate was found to be absent at low pH. While induction of helical structure of a protein by SDS in the native condition has been reported earlier, this is possibly the first report of the refolding of a protein in a strongly denaturing condition (in the presence of 10 M urea). The relative contributions of the hydrophobic and the electrostatic interactions of the surfactants with cytochrome c have been determined from the formation of the molten globule species from the acid-induced unfolded protein in the presence of SDS or lauryl maltoside.     

Trifluoroethanol-induced "molten globule" state in stem bromelain

2,2,2-Trifluoroethanol (TFE) denatures proteins but also stabilizes/induces alpha helical conformation in partially/completely unfolded proteins. As reported earlier from this laboratory, stem bromelain is known to exist as a partially folded intermediate (PFI) at pH 2.0. The effect of increasing concentration of TFE on the PFI of bromelain has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino 8-naphthalene sulfonic acid (ANS), and near-UV CD temperature transition. Far-UV CD spectra show considerable accumulation of secondary structure at 70% (v/v) concentration of TFE with spectral features resembling the pH 7.0 preparation. Interestingly the partially folded intermediate regained significant tertiary structure/interactions, with increasing concentration of TFE, and at 60% (v/v) TFE approached almost that of the pseudo native (pH 7.0) state. Further increase to 70% (v/v) TFE, however, resulted in complete loss of tertiary structure/interactions. Studies on tryptophan fluorescence also suggested the induction of some compact structure at 60% (v/v) concentration of TFE. The partially folded intermediate showed enhanced binding of the fluorescent probe (ANS) in the presence of 60% (v/v) TFE. Taken together these observations suggest a "molten globule" state between 60 and 70% (v/v) TFE. Thermal transition studies in the near-UV CD region indicated cooperative transition for PFI in the presence of 60% (v/v) TFE changing to noncooperative transition at 70% (v/v) TFE. This was accompanied by a shift in the midpoint of thermal denaturation (T(m)) from 58 to 51 degrees C. Gradual transition and loss of cooperative thermal unfolding in the 60-70% (v/v) range of TFE also support the existence of the molten globule state.     

Structure of bacteriophage P22 portal protein in relation to assembly: investigation by Raman spectroscopy.

Salmonella phage P22, which serves as an assembly paradigm for icosahedral double-stranded DNA viruses, packages its viral genome through a capsid channel (portal) comprising 12 copies of a 725-residue subunit. Secondary and tertiary structures of the portal subunit in monomeric and dodecameric states have been investigated by Raman spectroscopy using a His6-tagged recombinant protein that self-assembles in vitro [Moore, S. D., and Prevelige, P. E., Jr. (2001) J. Biol. Chem. 276, 6779-6788]. The portal protein exhibits Raman secondary structure markers typical of a highly alpha-helical subunit fold that is little perturbed by assembly. On the other hand, Raman markers of subunit side chains change dramatically with assembly, an indication of extensive changes in side chain environments. The cysteinyl Raman signature of the portal consists of a complex pattern of sulfhydryl stretching bands, revealing diverse hydrogen-bonding states for the four S-H groups per subunit (Cys 153, Cys 173, Cys 283, and Cys 516). Upon assembly, the population of strongly hydrogen-bonded S-H groups decreases, while the population of weakly hydrogen-bonded S-H groups increases, implying that specific intrasubunit S-H.X hydrogen bonds must be weakened to effect dodecamer assembly and that the molecular mechanism involves reorganization of subunit domains without appreciable changes in domain conformations. Comparison with other viral protein assemblies suggests an assembly process not requiring metastable intermediates. The recently published X-ray structure of the phi29 portal [Simpson, A. A., et al. (2000) Nature 408, 745-750] shows that residues 125-225 lining the channel surface form alpha-helical modules spaced by short beta-strands and turns; a surprisingly close secondary structure homology is predicted for residues 240-350 of the P22 portal, despite no apparent sequence homology. This motif is proposed as an evolutionarily conserved domain involved in DNA translocation.     

Molecular dissection of ø29 scaffolding protein function in an in vitro assembly system

An in vitro assembly system was developed to study prolate capsid assembly of phage ?29 biochemically, and to identify regions of scaffolding protein required for its functions. The crowding agent polyethylene glycol can induce bacteriophage ?29 monomeric capsid protein and dimeric scaffolding protein to co-assemble to form particles which have the same geometry as either prolate T=3 Q=5 procapsids formed in vivo or previously observed isometric particles. The formation of particles is a scaffolding-dependent reaction. The balance between the fidelity and efficiency of assembly is controlled by the concentration of crowding agent and temperature. The assembly process is salt sensitive, suggesting that the interactions between the scaffolding and coat proteins are electrostatic. Three N-terminal ?29 scaffolding protein deletion mutants, Delta 1-9, Delta 1-15 and Delta 1-22, abolish the assembly activity. Circular dichroism spectra indicate that these N-terminal deletions are accompanied by a loss of helicity. The inability of these proteins to dimerize suggests that the N-terminal region of the scaffolding protein contributes to the dimer interface and maintains the structural integrity of the dimeric protein. Two C-terminal scaffolding protein deletion mutants, Delta 79-97 and Delta 62-97, also fail to promote assembly. However, the secondary structure and the dimerization ability of these mutants are unchanged relative to wild-type, which suggests that the C terminus is the likely site of interaction with the capsid protein.     

Structural characterization of a highly stable cysteine protease ervatamin C.

Ervatamin C, a novel cysteine protease, belongs to alpha + beta class of proteins, probably with two domains, and retains both secondary and tertiary structures along with biological activity over a wide range of pH (2-12). Under neutral conditions, GuHCl and temperature-induced unfolding was cooperative with high transition midpoints and shows no structural changes in the presence of urea reflecting a remarkable stability. The fluorescence emission maximum at 350 nm suffers a blue shift of 4-5 nm upon lowering the pH and a red shift of 5 nm under denatured conditions. Unfolding transition curves at pH 2.0 are non-coincidental indicating the presence of intermediates in the unfolding pathway. At extremely low pH, the enzyme loses all the tertiary structure and proteolytic activity but retains a predominant secondary structure and a strong binding to ANS. GuHCl-induced unfolding of the enzyme in this intermediate state is noncooperative and indicates sequential unfolding of the domains.     

Distinct structural elements in the first membrane-spanning segment of the epithelial sodium channel

Epithelial Na+ channels (ENaCs) comprise three subunits that have been proposed to be arranged in either an alpha2betagamma or a higher ordered configuration. Each subunit has two putative membrane-spanning segments (M1 and M2), intracellular amino and carboxyl termini, and a large extracellular loop. We have used the TOXCAT assay (a reporter assay for transmembrane segment homodimerization) to identify residues within the transmembrane segments of ENaC that may participate in important structural interactions within ENaC, with which we identified a candidate site within alphaM1. We performed site-directed mutagenesis at this site and found that, although the mutants reduced channel activity, ENaC protein expression at the plasma membrane was unaffected. To deduce the role of alphaM1 in the pore structure of ENaC, we performed tryptophan-scanning mutagenesis throughout alphaM1 (residues 110-130). We found that mutations within the amino-terminal part of alphaM1 had effects on activity and selectivity with a periodicity consistent with a helical structure but no effect on channel surface expression. We also observed that mutations within the carboxyl-terminal part of alphaM1 had effects on activity and selectivity but with no apparent periodicity. Additionally, these mutants reduced channel surface expression. Our data support a model in which the amino-terminal half of alphaM1 is alpha-helical and packs against structural element(s) that contribute to the ENaC pore. Furthermore, these data suggest that the carboxyl-terminal half of alphaM1 may be helical or assume a different conformation and may be involved in tertiary interactions essential to proper channel folding or assembly. Together, our data suggest that alphaM1 is divided into two distinct regions.