Structure and oligomerization of the periplasmic domain of GspL from the type II secretion system of Pseudomonas aeruginosa.

The ability of bacteria to infect a host relies in part on the secretion of molecular virulence factors across the cell envelope. Pseudomonas aeruginosa, a ubiquitous environmental bacterium causing opportunistic infections in humans, employs the type II secretion system (T2SS) to transport effector proteins across its cellular envelope as part of a diverse array of virulence strategies. General secretory pathway protein L (GspL) is an essential inner-membrane component of the T2SS apparatus, and is thought to facilitate transduction of the energy from ATP hydrolysis in the cytoplasm to the periplasmic components of the system. However, our incomplete understanding of the assembly principles of the T2SS machinery prevents the mechanistic deconvolution of T2SS-mediated protein secretion. Here we show via two crystal structures that the periplasmic ferredoxin-like domain of GspL (GspLfld) is a dimer stabilized by hydrophobic interactions, and that this interface may allow significant interdomain plasticity. The general dimerization mode of GspLfld is shared with GspL from Vibrio parahaemolyticus suggesting a conserved oligomerization mode across the GspL family. Furthermore, we identified a tetrameric form of the complete periplasmic segment of GspL (GspLperi) which indicates that GspL may be able to adopt multiple oligomeric states as part of its dynamic role in the T2SS apparatus.

Covalent defects restrict supramolecular self-assembly of homopolypeptides: case study of ß2-fibrils of poly-L-glutamic acid.

Poly-L-glutamic acid (PLGA) often serves as a model in studies on amyloid fibrils and conformational transitions in proteins, and as a precursor for synthetic biomaterials. Aggregation of PLGA chains and formation of amyloid-like fibrils was shown to continue on higher levels of superstructural self-assembly coinciding with the appearance of so-called ß2-sheet conformation manifesting in dramatic redshift of infrared amide I' band below 1600 cm(-1). This spectral hallmark has been attributed to network of bifurcated hydrogen bonds coupling C = O and N-D (N-H) groups of the main chains to glutamate side chains. However, other authors reported that, under essentially identical conditions, PLGA forms the conventional in terms of infrared characteristics ß1-sheet structure (exciton-split amide I' band with peaks at ca. 1616 and 1683 cm(-1)). Here we attempt to shed light on this discrepancy by studying the effect of increasing concentration of intentionally induced defects in PLGA on the tendency to form ß1/ß2-type aggregates using infrared spectroscopy. We have employed carbodiimide-mediated covalent modification of Glu side chains with n-butylamine (NBA), as well as electrostatics-driven inclusion of polylysine chains, as two different ways to trigger structural defects in PLGA. Our study depicts a clear correlation between concentration of defects in PLGA and increasing tendency to depart from the ß2-structure toward the one less demanding in terms of chemical uniformity of side chains: ß1-structure. The varying predisposition to form ß1- or ß2-type aggregates assessed by infrared absorption was compared with the degree of morphological order observed in electron microscopy images. Our results are discussed in the context of latent covalent defects in homopolypeptides (especially with side chains capable of hydrogen-bonding) that could obscure their actual propensities to adopt different conformations, and limit applications in the field of synthetic biomaterials.

An FT-IR study on packing defects in mixed ß-aggregates of poly(L-glutamic acid) and poly(D-glutamic acid): a high-pressure rescue from a kinetic trap.

Under favorable conditions of pH and temperature, poly(L-glutamic acid) (PLGA) adopts different types of secondary and quaternary structures, which include spiral assemblies of amyloid-like fibrils. Heating of acidified solutions of PLGA (or PDGA) triggers formation of ß(2)-type aggregates with morphological and tinctorial properties typical for amyloid fibrils. In contrast to regular antiparallel ß-sheet (ß(1)), the amide I' vibrational band of ß(2)-fibrils is unusually red-shifted below 1600 cm(-1), which has been attributed to bifurcated hydrogen bonds coupling C═O and N-D groups of the main chains to glutamic acid side chains. However, unlike for pure PLGA, the amide I' band of aggregates precipitating from racemic mixtures of PLGA and PDGA (ß(1)) is dominated by components at 1613 and 1685 cm(-1)-typically associated with intermolecular antiparallel ß-sheets. The coaggregation of PLGA and PDGA chains is slower and biphasic and leads to less-structured assemblies of fibrils, which is reflected in scanning electron microscopy images, sedimentation properties, and fluorescence intensity after staining with thioflavin T. The ß(1)-type aggregates are metastable, and they slowly convert to fibrils with the infrared characteristics of ß(2)-type fibrils. The process is dramatically accelerated under high pressure. This implies the presence of void volumes within structural defects in racemic aggregates, preventing the precise alignment of main and side chains necessary to zip up ladders of bifurcated hydrogen bonds. As thermodynamic costs associated with maintaining void volumes within the racemic aggregate increase under high pressure, a hyperbaric treatment of misaligned chains leads to rectifying the packing defects and formation of the more compact form of fibrils.

Spiral superstructures of amyloid-like fibrils of polyglutamic acid: an infrared absorption and vibrational circular dichroism study.

Amyloid fibrils, which are often associated with certain degenerative disorders, reveal a number of intriguing spectral properties. However, the relationship between the structure of fibrils and their optical traits remains poorly understood. Poly(L-glutamic) acid is a model polypeptide shown recently to form amyloid-like fibrils with an atypical infrared amide I' band at 1595 cm(-1), which has been attributed to the presence of bifurcated hydrogen bonds coupling C═O and N-D groups of the main chains to glutamate side chains. Here we show that this unusual amide I' band is observed only for fibrils grown from pure enantiomers of the polypeptide, whereas fibrils precipitating from equimolar mixtures of poly(L-glutamic) and poly(D-glutamic) acids have amide I' bands at 1684 and 1612 cm(-1), which are indicative of a typical intermolecular antiparallel ß-sheet. Pure enantiomers of polyglutamic acid form spirally twisted superstructures whose handedness is correlated to the amino acid chirality, while fibrils prepared from the racemate do not form scanning electron microscopy (SEM)-detectable mesoscopically ordered structures. Vibrational circular dichroism (VCD) spectra of ß-aggregates prepared from mixtures of all L- or D-polyglutamic acid in varying ratios indicate that the enhancement of VCD intensity correlates with the presence of the twisted superstructures. Our results demonstrate that both IR absorption and enhanced VCD are sensitive to subtle packing defects taking place within the compact structure of amyloid fibrils.

Bifurcated hydrogen bonds stabilize fibrils of poly(L-glutamic) acid.

Model fibrillating homopolypeptides have been providing many insightful analogies to the clinically important phenomena of protein misfolding and amyloidogenesis. Here we show that the beta(2) structural variant of poly(l-glutamic) acid forms fibrils with an amyloid-like morphology, ability to enhance fluorescence of thioflavin T, and seeding properties. The beta(2) fibrils are formed upon heating of aqueous solutions of alpha-helical poly(l-glutamic) acid, which leads to a significant increase of pD (pH) of unbuffered samples and a concomitant precipitation of fibrils with unusual infrared traits: amide I' band being dramatically red-shifted to 1596 cm(-1), and the -COOD stretching band split into two peaks around 1730 and 1719 cm(-1). We are proposing that formation of three-center hydrogen bonds involving bifurcated peptide carbonyl acceptors (>C=O) and main chains' NH, as well as side chains' -COOH proton donors is likely to underlie the observed infrared characteristics of beta(2) fibrils. Such bonds provide additional conformational constraints in a tightly packed environment around glutamate side chains resulting in the decreased overall acidity of the polypeptide. The presence of bifurcated hydrogen bonds in amyloid fibrils may be an overlooked factor in fibrils' robustness, thermodynamic stability and the ability to propagate their own growth.

De novo refolding and aggregation of insulin in a nonaqueous environment: an inside out protein remake.

While thermodynamic penalties associated with protein-water interactions are the key driving force of folding, perturbed hydration of destabilized protein molecules may trigger aggregation, which in vivo often causes cellular and histological damage. Here we show, that the denatured state of an alpha-helical protein, insulin, converts to a non-native beta-sheet-rich structure upon de novo "refolding" in an anhydrous environment. The beta-pleated conformer precipitates from solutions of DMSO-denatured insulin upon dilution with chloroform. DMSO destroys hydrogen bond network of the native protein acting as a strong acceptor of main chain hydrogen bonds. Upon the addition of chloroform, which is a weak hydrogen bond donor per se, competitive hydrogen bonds between DMSO and chloroform are formed. This leads to the release of unfolded insulin molecules. In the absence of water, the imminent saturation of polypeptide's dandling hydrogen bonds does not produce the native and predominantly alpha-helical state but a beta-sheet-rich structure, which is morphologically and spectrally distinct from insulin amyloid fibrils. Unlike insulin fibrils, the beta-sheet conformer is metastable and refolds spontaneously to the native form in an aqueous environment. This implies that "folding" in the absence of water results in inefficient burial of hydrophobic side-chains, and thermodynamic frustration at the water-protein interface.