CD and NMR investigations on trifluoroethanol-induced step-wise folding of helical segment from scorpion neurotoxin.

A 14 amino acid residue peptide from the helical region of Scorpion neurotoxin has been structurally characterized using CD and NMR spectroscopy in different solvent conditions. 2,2,2-Trifluoroethanol (TFE) titration has been carried out in 11 steps from 0 to 90% TFE and the gradual stabilization of the conformation to form predominantly alpha-helix covering all of the 14 residues has been studied by 1H and 13C NMR spectroscopy. Detailed information such as coupling constants, chemical shift indices, NOESY peak intensities and amide proton temperature coefficients at each TFE concentration has been extracted and analysed to derive the step-wise preferential stabilization of the helical segments along the length of the peptide. It was found that there is a finite amount of the helical conformation in the middle residues 5-11 even at low TFE concentrations. It was also observed that > 75% TFE (v/v) is required for the propagation of the helix to the N and C termini and for correct packing of the side chains of all of the residues. These observations are significant to understanding the folding of this segment in the protein and may throw light on the inherent preferences and side chain interactions in the formation of the helix in the peptide.     

Mechanism by which the amyloid-like fibrils of a beta 2-microglobulin fragment are induced by fluorine-substituted alcohols

Although the formation of an alpha-helix or partial unfolding of proteins has been suggested to be important for amyloid fibrils to form in alcohols, the exact mechanism involved remains elusive. To obtain further insight into the development of amyloid fibrils, we used a 22-residue peptide, K3, corresponding to Ser20 to Lys41 of intact beta2-microglobulin. Although K3 formed an alpha-helix at high concentrations of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in 10 mM HCl (pH approximately 2), the helical content was not high, indicating a low preference to do so. The partly alpha-helical conformation was converted with time into a highly ordered beta-sheet with a fibrillar morphology as revealed by atomic force microscopy. Importantly, the TFE and HFIP-induced fibrillation exhibited a concentration dependence with a maximum at approximately 20 and approximately 10% (v/v), respectively, slightly below the concentrations at which these alcohols form dynamic clusters. Focusing on the similarity of the effects of alcohol on proteins with those of sodium dodecyl sulfate (SDS), we examined the effects of SDS on K3. SDS also induced fibrils to form with a maximum at approximately 4 mM, slightly below the critical micelle concentration. These results indicate that, with an increase in the concentration of hydrophobic cosolvent (TFE, HFIP, or SDS), a delicate balance of decreasing hydrophobic interactions and increasing polar interactions (i.e. H-bonds) in and between peptides leads to the formation of ordered fibrils with a bell-shaped concentration dependence.     

Involvement of electrostatic interactions in the mechanism of peptide folding induced by sodium dodecyl sulfate binding

Sodium dodecyl sulfate (SDS) has consistently been shown to induce secondary structure, particularly alpha-helices, in polypeptides, and is commonly used to model membrane and other hydrophobic environments. However, the precise mechanism by which SDS induces these conformational changes remains unclear. To examine the role of electrostatic interactions in this mechanism, we have designed two hydrophilic, charged amphipathic alpha-helical peptides, one basic (QAPAYKKAAKKLAES) and the other acidic (QAPAYEEAAEELAKS), and their structures were studied by CD and NMR. The design of the peptides is based on the sequence of the segment of residues 56-70 of human platelet factor 4 [PF4(56-70), QAPLYKKIIKKLLES]. Both peptides were unstructured in water, and in the presence of neutral, zwitterionic, or cationic detergents. However, in SDS at neutral pH, the basic peptide folded into an alpha-helix. By contrast, the pH needed to be lowered to 1.8 before alpha-helix formation was observed for the acidic peptide. Strong, attractive electrostatic interactions, between the anionic groups of SDS and the cationic groups of the lysines, appeared to be necessary to initiate the folding of the basic peptide. NMR analysis showed that the basic peptide was fully embedded in SDS-peptide micelles, and that its three-dimensional alpha-helical structure could be superimposed on that of the native structure of PF4(56-70). These results enabled us to propose a working model of the basic peptide-SDS complex, and a mechanism for SDS-induced alpha-helical folding. This study demonstrates that, while the folding of peptides is mostly driven by hydrophobic effects, electrostatic interactions play a significant role in the formation and the stabilization of SDS-induced structure.     

Deciphering the Hidden Informational Content of Protein Sequences: FOLDABILITY OF PROINSULIN HINGES ON A FLEXIBLE ARM THAT IS DISPENSABLE IN THE MATURE HORMONE*

Protein sequences encode both structure and foldability. Whereas the interrelationship of sequence and structure has been extensively investigated, the origins of folding efficiency are enigmatic. We demonstrate that the folding of proinsulin requires a flexible N-terminal hydrophobic residue that is dispensable for the structure, activity, and stability of the mature hormone. This residue (Phe^B1 in placental mammals) is variably positioned within crystal structures and exhibits ¹H NMR motional narrowing in solution. Despite such flexibility, its deletion impaired insulin chain combination and led in cell culture to formation of non-native disulfide isomers with impaired secretion of the variant proinsulin. Cellular folding and secretion were maintained by hydrophobic substitutions at B1 but markedly perturbed by polar or charged side chains. We propose that, during folding, a hydrophobic side chain at B1 anchors transient long-range interactions by a flexible N-terminal arm (residues B1–B8) to mediate kinetic or thermodynamic partitioning among disulfide intermediates. Evidence for the overall contribution of the arm to folding was obtained by alanine scanning mutagenesis. Together, our findings demonstrate that efficient folding of proinsulin requires N-terminal sequences that are dispensable in the native state. Such arm-dependent folding can be abrogated by mutations associated with β-cell dysfunction and neonatal diabetes mellitus.     

Folding propensities of synthetic peptide fragments covering the entire sequence of phage 434 Cro protein.

The phage 434 Cro protein, the N-terminal domain of its repressor (R1-69) and that of phage lambda (lambda6-85) constitute a group of small, monomeric, single-domain folding units consisting of five helices with striking structural similarity. The intrinsic helix stabilities in lambda6-85 have been correlated to its rapid folding behavior, and a residual hydrophobic cluster found in R1-69 in 7 M urea has been proposed as a folding initiation site. To understand the early events in the folding of 434 Cro, and for comparison with R1-69 and lambda6-85, we examined the conformational behavior of five peptides covering the entire 434 Cro sequence in water, 40% (by volume) TFE/water, and 7 M urea solutions using CD and NMR. Each peptide corresponds to a helix and adjacent residues as identified in the native 434 Cro NMR and crystal structures. All are soluble and monomeric in the solution conditions examined except for the peptide corresponding to the 434 Cro helix 4, which has low water solubility. Helix formation is observed for the 434 Cro helix 1 and helix 2 peptides in water, for all the peptides in 40% TFE and for none in 7 M urea. NMR data indicate that the helix limits in the peptides are similar to those in the native protein helices. The number of side-chain NOEs in water and TFE correlates with the helix content, and essentially none are observed in 7 M urea for any peptide, except that for helix 5, where a hydrophobic cluster may be present. The low intrinsic folding propensities of the five helices could account for the observed stability and folding behavior of 434 Cro and is, at least qualitatively, in accord with the results of the recently described diffusion-collision model incorporating intrinsic helix propensities.     

NMR structure of human thymosin alpha-1

800 MHz NMR structure of the 28-residue peptide thymosin alpha-1 in 40% TFE/60% water (v/v) has been determined. Restrained molecular dynamic simulations with an explicit solvent box containing 40% TFE/60% TIP3P water (v/v) were used, in order to get the 3D model of the NMR structure. We found that the peptide adopts a structured conformation having two stable regions: an alpha-helix region from residues 14 to 26 and two double β-turns in the N-terminal twelve residues which form a distorted helical structure.     

Three-dimensional structure in lipid micelles of the pediocin-like antimicrobial peptide sakacin P and a sakacin P variant that is structurally stabilized by an inserted C-terminal disulfide bridge

The three-dimensional structures in dodecylphosphocholine (DPC) micelles and in trifluoroethanol (TFE) of the pediocin-like antimicrobial peptide sakacin P and an engineered variant of sakacin P (termed sakP[N24C+44C]) have been determined by use of nuclear magnetic resonance spectroscopy. SakP[N24C+44C] has an inserted non-native activity- and structure-stabilizing C-terminal disulfide bridge that ties the C-terminus to the middle part of the peptide. In the presence of DPC, the cationic N-terminal region (residues 1-17) of both peptides has an S-shaped conformation that is reminiscent of a three-stranded antiparallel beta-sheet and that is more pronounced when the peptide was dissolved in TFE instead of DPC. The four positively charged residues located in the N-terminal part are found pointing to the same direction. For both peptides, the N-terminal region is followed by a well-defined central amphiphilic alpha-helix (residues 18-33), and this in turn is followed by the C-terminal tail (residues 34-43 for sakacin P and 34-44 for sakP[N24C+44C]) that lacks any apparent common secondary structural motif. In the presence of DPC, the C-terminal tails in both peptides fold back onto the central alpha-helix, thereby creating a hairpin-like structure in the C-terminal halves. The lack of long-range NOEs between the beta-sheet Nu-terminal region and the hairpin-like C-terminal half indicates that there is a flexible hinge between these regions. We discuss which implications such a structural arrangement has on the interaction with the target cell membrane.     

Conformational properties of five peptides corresponding to the entire sequence of glutathione transferase domain II

Five peptides matching the helices alpha4, alpha5, alpha6, alpha7, and alpha8, spanning the entire sequence of domain II of pG-STP1-1, have been synthesized and their conformations analyzed by far-UV CD spectroscopy. The results show that a5, a7, and a8 peptides are unstructured in water/2,2,2-trifluoroethanol (TFE) solutions. The a4-peptide also adopts random conformations in aqueous solvent. Moreover, the relative low helical content (20%), estimated for this peptide in the presence of 30% (v/v) TFE, suggests that the sequence of this protein fragment does not possess sufficient information for a strong helical propensity. On the contrary, the synthesized a6 peptide, in the presence of TFE, showed a relevant structural autonomy with a helical content (41%) which was significantly higher than that estimated, under the same conditions, for all other peptides. More in general in the presence of solvents less polar than water, the isolated a6 peptide shows the same helical conformation adopted by the corresponding alpha6-helix in the hydrophobic core of the protein. A n-capping box motif, strictly conserved at the N-terminal of the alpha6-helix of all GST and related protein including eucaryotic translation elongation factor (EF1gamma) and the yeast prion protein Ure2, plays an important role in the alpha-helix nucleation and stability of this protein fragment. The results suggest that the alpha6-helix might represent a nucleation site of GST folding and that the helical conformation of this region of the protein is an important requirement during earlier events of GST refolding.     

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin lactococcin G

The three-dimensional structures of the two peptides, lactococcin G-alpha (LcnG-alpha; contains 39 residues) and lactococcin G-beta (LcnG-beta, contains 35 residues), that constitute the two-peptide bacteriocin lactococcin G (LcnG) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles and TFE. In DPC, LcnG-alpha has an N-terminal alpha-helix (residues 3-21) that contains a GxxxG helix-helix interaction motif (residues 7-11) and a less well defined C-terminal alpha-helix (residues 24-34), and in between (residues 18-22) there is a second somewhat flexible GxxxG-motif. Its structure in TFE was similar. In DPC, LcnG-beta has an N-terminal alpha-helix (residues 6-19). The region from residues 20 to 35, which also contains a flexible GxxxG-motif (residues 18-22), appeared to be fairly unstructured in DPC. In the presence of TFE, however, the region between and including residues 23 and 32 formed a well defined alpha-helix. The N-terminal helix between and including residues 6 and 19 seen in the presence of DPC, was broken at residues 8 and 9 in the presence of TFE. The N-terminal helices, both in LcnG-alpha and -beta, are amphiphilic. We postulate that LcnG-alpha and -beta have a parallel orientation and interact through helix-helix interactions involving the first GxxxG (residues 7-11) motif in LcnG-alpha and the one (residues 18-22) in LcnG-beta, and that they thus lie in a staggered fashion relative to each other.     

Structural analysis of the heparin-binding site of the NC1 domain of collagen XIV by CD and NMR.

Type XIV collagen, a fibril-associated collagen with interrupted triple helices (FACIT), interacts with the surrounding extracellular matrix and/or with cells via its binding to glycosaminoglycans (GAGs). To further characterize such interactions in the NC1 domain of chicken collagen XIV, we identified amino acids essential for heparin binding by affinity chromatography analysis after proteolytic digestion of the synthetic peptide NC1(84-116). The 3D structure of this peptide was then obtained using circular dichroism and NMR. The NC1(84-116) peptide appeared poorly structured in water, but the stabilization of its conformation by the interaction with hydrophobic surfaces or by using cosolvents (TFE, SDS) revealed a high propensity to adopt an alpha-helical folding. A 3D structure model of NC1(84-116), calculated from NMR data recorded in a TFE/water mixture, showed that the NC1-heparin binding site forms a amphipathic alpha-helix exhibiting a twisted basic groove. It is structurally similar to the consensus spatial alpha-helix model of heparin-binding [Margalit et al. (1993) J. Biol. Chem. 268, 19228-19231], except that the GAG binding domain of NC1 may be extended over 18 residues, that is, the NC1(94-111) segment. In addition, the formation of a hydrophobic groove upon helix formation suggests the contribution of additional sequences to ensure the stability of the GAG-binding domain. Overall the NC1(84-116) model exhibits a nativelike conformation which presents suitably oriented residues for the interaction with a specific GAG.     

Folding propensities of peptide fragments of myoglobin.

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.     

Stability and folding kinetics of a ubiquitin mutant with a strong propensity for nonnative beta-hairpin conformation in the unfolded state

A F45W mutant of yeast ubiquitin has been used as a model system to examine the effects of nonnative local interactions on protein folding and stability. Mutating the native TLTGK G-bulged type I turn in the N-terminal beta-hairpin to NPDG stabilizes a nonnative beta-strand alignment in the isolated peptide fragment. However, NMR structural analysis of the native and mutant proteins shows that the NPDG mutant is forced to adopt the native beta-strand alignment and an unfavorable type I NPDG turn. The mutant is significantly less stable (approximately 9 kJ mol(-1)) and folds 30 times slower than the native sequence, demonstrating that local interactions can modulate protein stability and that attainment of a nativelike beta-hairpin conformation in the transition state ensemble is frustrated by the turn mutations. Surprising, alcoholic cosolvents [5-10% (v/v) TFE] are shown to accelerate the folding rate of the NPDG mutant. We conclude, backed-up by NMR data on the peptide fragments, that even though nonnative states in the denatured ensemble are highly populated and their stability further enhanced in the presence of cosolvents, the simultaneous increase in the proportion of nativelike secondary structure (hairpin or helix), in rapid equilibrium with nonnative states, is sufficient to accelerate the folding process. It is evident that modulating local interactions and increasing nonnative secondary structure propensities can change protein stability and folding kinetics. However, nonlocal contacts formed in the global cooperative folding event appear to determine structural specificity.     

Structures of neuropeptide gamma from goldfish and mammalian neuropeptide gamma, as determined by 1H NMR spectroscopy.

Neuropeptide gamma belongs to tachykinin families which have a common C-terminal amino acid sequence (Phe-X-Leu-Met-NH2) and which induce various biological responses including salivation, hypotension, and contraction of gastrointestinal, respiratory, and urinary smooth muscle. In the present study, we present the solution structures of neuropeptide gamma (NPgamma) from gold fish (G-NPgamma) and mammalian NPgamma (M-NPgamma), as determined by nuclear magnetic resonance (NMR) spectroscopy in 50% trifluoroethanol (TFE)/water (1 : 1, v/v) solution and 200 mm sodium dodecyl sulfate (SDS) micelles. In aqueous TFE solution, G-NPgamma has a alpha-helical conformation in the region of His12-Met21 and a short helix in the N-terminal region, and has a beta-turn from Arg9 to Arg11 in between. In aqueous TFE solution, M-NPgamma also has alpha-helical conformations both in the C-terminal region and the N-terminal region and a beta-turn from His9 to Arg11 in between. In SDS micelle, the structure of G-NPgamma contains a stable alpha-helix from His12 to Met21 and a beta-turn from Arg9 to Arg11, while M-NPgamma has a short helix from Ser16 to Met21. The region from His12 to Met21 corresponds to the amino acid sequence of neurokinin A. Neuropeptide gamma may act as a precursor of neurokinin A and the post-translational processing of this peptide involves the enzymatic attack of the basic beta-turn region from residue 9 to residue 11 in the middle. From our relaxation study, it could be suggested that in fish system G-NPgamma induces the biological actions corresponding to those of substance P in mammalian system. The structures of G-NPgamma and M-NPgamma contain alpha-helical structures at the C-terminus and this helix seems to promote the affinity for NK1 and/or NK2 receptor.     

Identification of initiation sites for T4 lysozyme folding using CD and NMR spectroscopy of peptide fragments

Using CD and 2D (1)H NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the (1)H NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Halpha chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and HD-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.     

Structure analysis of the fourth transmembrane domain of Nramp1 in model membranes

Nramp1 (natural resistance-associated macrophage protein 1) is an integral membrane protein with 12 putative transmembrane domains. As a proton-coupled divalent metal cation transporter, it is involved in defense against intracellular pathogens. Disease-causing mutation in Nramp1 occurring at glycine 169 located within the fourth transmembrane domain (TM4) suggests functional importance of this domain. In this paper, we study the three-dimensional structures of a peptide, corresponding to the TM4 of the wild-type Nramp1, in SDS micelles and 2, 2, 2-trifluoroethanol solvent using CD and NMR spectroscopies. We have found that an alpha-helix is predominantly induced in membrane-mimetic environments and the folding of the C-terminal residues is regulated by pH in SDS micelles. The peptide is embedded in SDS micelles and self-associated by coiled-coil interactions. The helix of the peptide in TFE is lengthened towards the N-terminus compared with those in SDS micelles at acidic pH and the self-association of the peptide is also observed in TFE. The fact that Mn(2+) ions are accessible to Asp-14 located in the interior of SDS micelles is found and the binding affinity is increased with increasing pH. The self-association of the peptide may provide a path by which Mn(2+) ions pass through the membrane.     

The helical structure propensity in the first helix of the histidine phosphocarrier protein of Streptomyces coelicolor

The bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), formed by a cascade of several proteins, mediates the uptake and phosphorylation of carbohydrates, and it is also involved in signal transduction. Its uniqueness in bacteria makes the PTS a target for new antibacterial drugs. These drugs can be obtained from peptides or proteins fragments able to interfere the first step of the protein cascade: the phosphorylation of the HPr protein by the enzyme EI. We designed a peptide comprising the active site and the first alpha-helix of HPr of S. coelicolor; we also obtained a fragment of HPr by protein engineering methods, comprising the first forty-eight residues and thus, containing the amino acids of the shorter peptide. Both fragments were disordered in aqueous solution, with a similar percentage of helical structure ( approximately 7 %), and an identical free energy of helix formation. In 40 % TFE, both fragments acquired native-like helical structure, stabilized by non-native hydrophobic interactions, as shown by the 2D-NMR assignments of the shorter peptide, and the presence of similar NOE contacts in both fragments. These findings, with the kinetic results in other members of the HPr family, highlight the importance of short- and long-range interactions during the folding reaction of HPr proteins. Based on the residual helical population, hypothesis about the inhibition capacity of the PTS by both fragments are discussed.     

Structural studies of a folding intermediate of bovine pancreatic ribonuclease A by continuous recycled flow

A new technique, continuous recycled flow (CRF) spectroscopy, has been developed for observing intermediates of any thermally induced, reversible reaction with a half-life of 10 s or longer. The structure can be probed by any spectroscopic method which does not perturb the system. Prolonged signal acquisitions of 8 h for ribonuclease A are possible. CRF was used to investigate the structure of the slow-folding intermediates of chemically intact ribonuclease A (RNase A) during thermal unfolding/folding under acidic conditions. The following conclusions were reached on the basis of the proton nuclear magnetic resonance and far-ultraviolet circular dichroism spectra of a folding intermediate(s): (A) The conformation of the detected folding intermediate(s) is similar to that of the heat-denatured protein. There is only limited formation of new structures. (B) The N-terminal alpha-helix is partially stable under these conditions and is in rapid (less than 10 ms) equilibrium with the denatured conformation. (C) There are long-range interactions between the hydrophobic residues of the N-terminal alpha-helix and the rest of the protein. These interactions persist well above the melting point. (D) An aliphatic methyl group reports on the formation of a new structure(s) that lie(s) outside of the N-terminal region. (E) The structures detected in chemically modified, nonfolding forms of the RNase A are also present in the folding intermediate(s). There are, however, additional interactions that are unique to chemically intact RNase A.     

Understanding the folding and stability of a zinc finger-based full sequence design protein with replica exchange molecular dynamics simulations

Full sequence design protein FSD-1 is a designed protein based on the motif of zinc finger protein. In this work, its folding mechanism and thermal stability are investigated using the replica exchange molecular dynamics model with the water molecules being treated explicitly. The results show that the folding of the FSD-1 is initiated by the hydrophobic collapse, which is accompanied with the formation of the C-terminal alpha-helix. Then the folding proceeds with the formation of the beta-hairpin and the further package of the hydrophobic core. Compared with the beta-hairpin, the alpha-helix has much higher stability. It is also found that the N-capping motif adopted by the FSD-1 contributes to the stability of the alpha-helix dramatically. The hydrophobic contacts made by the side chain of Tyr3 in the native state are essential for the stabilization of the beta-hairpin. It is also found that the folding of the N-terminal beta-hairpin and the C-terminal alpha-helix exhibits weak cooperativity, which is consistent with the experimental data. Meanwhile, the folding pathway is compared between the FSD-1 and the target zinc finger peptide, and the possible role of the zinc ion on the folding pathway of zinc finger is proposed. Proteins 2007. (c) 2007 Wiley-Liss, Inc.     

Structure and assembly of the pseudopilin PulG

The pseudopilin PulG is one of several essential components of the type II pullulanase secretion machinery (the Pul secreton) of the Gram-negative bacterium Klebsiella oxytoca. The sequence of the N-terminal 25 amino acids of the PulG precursor is hydrophobic and very similar to the corresponding region of type IV pilins. The structure of a truncated PulG (lacking the homologous region), as determined by X-ray crystallography, was found to include part of the long N-terminal alpha-helix and the four internal anti-parallel beta-strands that characterize type IV pilins, but PulG lacks the highly variable loop region with a disulphide bond that is found in the latter. When overproduced, PulG forms flexible pili whose structural features, as visualized by electron microscopy, are similar to those of bacterial type IV pili. The average helical repeat comprises 17 PulG subunits and four helical turns. Electron microscopy and molecular modelling show that PulG probably assembles into left-handed helical pili with the long N-terminal alpha-helix tightly packed in the centre of the pilus. As in the type IV pilins, the hydrophobic N-terminal part of the PulG alpha-helix is necessary for its assembly. Subtle sequence variations within this highly conserved segment seem to determine whether or not a type IV pilin can be assembled into pili by the Pul secreton.     

Helical structure of dermaseptin B2 in a membrane-mimetic environment

Dermaseptins are antimicrobial peptides from frog skin that have high membrane-lytic activity against a broad spectrum of microorganisms. The structure of dermaseptin B2 in aqueous solution, in TFE/water mixtures, and in micellar and nonmicellar SDS was analyzed by CD, FTIR, fluorescence, and NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin B2 is unstructured in water, but helical conformations, mostly in segment 3-18, are stabilized by addition of TFE. SDS titration showed that dermaseptin B2 assumes nonhelical structures at SDS concentrations far below the critical micellar concentration and helical structures at micellar concentrations. Dermaseptin B2 bound to SDS micelles (0.4 mM peptide, 80 mM SDS) adopts a well-defined amphipathic helix between residues 11-31 connected to a more flexible helical segment spanning residues 1-8 by a flexible hinge region around Val9 and Gly10. Experiments using paramagnetic probes showed that dermaseptin B2 lies near the surface of SDS micelles and that residue Trp3 is buried in the SDS micelle, but close to the surface. A slow exchange equilibrium occurs at higher peptide/SDS ratios (2 mM peptide, 80 mM SDS) between forms having distinct sets of resonances in the N-terminal 1-11 segment. This equilibrium could reflect different oligomeric states of dermaseptin B2 interacting with SDS micelles. Structure-activity studies on dermaseptin B2 analogues showed that the N-terminal 1-11 segment is an absolute requirement for antibacterial activity, while the C-terminal 10-33 region is also important for full antibiotic activity.