Protein aggregation as an antibiotic design strategy.

Taking advantage of the xenobiotic nature of bacterial infections, we tested whether the cytotoxicity of protein aggregation can be targeted to bacterial pathogens without affecting their mammalian hosts. In particular, we examined if peptides encoding aggregation-prone sequence segments of bacterial proteins can display antimicrobial activity by initiating toxic protein aggregation in bacteria, but not in mammalian cells. Unbiased in vitro screening of aggregating peptide sequences from bacterial genomes lead to the identification of several peptides that are strongly bactericidal against methicillin-resistant Staphylococcus aureus. Upon parenteral administration in vivo, the peptides cured mice from bacterial sepsis without apparent toxic side effects as judged from histological and hematological evaluation. We found that the peptides enter and accumulate in the bacterial cytosol where they cause aggregation of bacterial polypeptides. Although the precise chain of events that leads to cell death remains to be elucidated, the ability to tap into aggregation-prone sequences of bacterial proteomes to elicit antimicrobial activity represents a rich and unexplored chemical space to be mined in search of novel therapeutic strategies to fight infectious diseases.

Mechanical and Water Absorption Properties of Short Banana Fiber/Unsaturated Polyester/Molecular Sieves + ZnO Nanorod Hybrid Nanobiocomposites.

ZnO nanorods were prepared by the sol-gel method and characterized using UV-visible absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, powder X-ray diffraction (PXRD), thermogravimetric analysis/differential thermogravimetry (TGA/DTG), high-resolution transmission electron microscopy (HR-TEM), field emission scanning electron microscopy (FE-SEM), and energy-dispersive X-ray spectroscopy (EDAX). Banana fiber/polyester resin (BF/PE) biocomposites and BF/PE/MS/nano ZnO nanobiocomposites were made using the untreated and chemically treated (with NaOH, formic acid, acetic anhydride, hydrogen peroxide, and potassium permanganate) banana fiber (BF), unsaturated polyester resin (PE), molecular sieves (MS), and the prepared ZnO nanorods. The KMnO4, Ac2O, and NaOH treatments enhanced the thermal stability of the nanobiocomposites. Addition of 2% of ZnO nanorods increased the tensile strength of all of the chemically treated BF/PE/MS biocomposites. The chemical treatments alone decreased (NaOH-15.4 MPa; KMnO4-14.5 MPa; H2O2-9.9 MPa; Ac2O-7.9 MPa; HCOOH-6.9 MPa) the compressive strength of the untreated BF/PE/MS biocomposite (25.9 MPa). But the chemical treatment and addition of ZnO nanorods enhanced the compressive strength effectively (48.5, 41.6, 39.4, 37.0, and 34.6 MPa for NaOH, HCOOH, KMnO4, H2O2, and Ac2O treatments, respectively) compared to the untreated BF/PE/MS biocomposites (24.0 MPa). The H2O2 (69.0 MPa) and NaOH (62.9 MPa) treatments enhanced the flexural strength of the untreated BF/PE biocomposites (51.6 MPa). The addition of ZnO nanorods enhanced the flexural strength of all of the chemically treated (except NaOH) BF/PE/MS biocomposites (55.7, 59.4, 79.0, and 67.4 MPa for HCOOH, Ac2O, H2O2, and KMnO4 treatments, respectively). The impact strengths of the biocomposites were enhanced by both chemical treatments and addition of ZnO nanorods. The addition of ZnO nanorods decreased the water absorption of the biocomposites significantly from 24.3% for the untreated to a minimum of 14.5% for the H2O2-treated BF/PE/MS/ZnO nanobiocomposite.

Optical spectroscopic methods for probing the conformational stability of immobilised enzymes.

We report the development of biophysical techniques based on circular dichroism (CD), diffuse reflectance infrared Fourier transform (DRIFT) and tryptophan (Trp) fluorescence to investigate in situ the structure of enzymes immobilised on solid particles. Their applicability is demonstrated using subtilisin Carlsberg (SC) immobilised on silica gel and Candida antartica lipase B immobilised on Lewatit VP.OC 1600 (Novozyme 435). SC shows nearly identical secondary structure in solution and in the immobilised state as evident from far UV CD spectra and amide I vibration bands. Increased near UV CD intensity and reduced Trp fluorescence suggest a more rigid tertiary structure on the silica surface. After immobilised SC is inactivated, these techniques reveal: a) almost complete loss of near UV CD signal, suggesting loss of tertiary structure; b) a shift in the amide I vibrational band from 1658 cm(-1) to 1632 cm(-1), indicating a shift from alpha-helical structure to beta-sheet; c) a substantial blue shift and reduced dichroism in the far UV CD, supporting a shift to beta-sheet structure; d) strong increase in Trp fluorescence intensity, which reflects reduced intramolecular quenching with loss of tertiary structure; and e) major change in fluorescence lifetime distribution, confirming a substantial change in Trp environment. DRIFT measurements suggest that pressing KBr discs may perturb protein structure. With the enzyme on organic polymer it was possible to obtain near UV CD spectra free of interference by the carrier material. However, far UV CD, DRIFT and fluorescence measurements showed strong signals from the organic support. In conclusion, the spectroscopic methods described here provide structural information hitherto inaccessible, with their applicability limited by interference from, rather than the particulate nature of, the support material.

Circular dichroism studies of subtilisin Carlsberg immobilised on micron sized silica particles.

Immobilised enzymes are widely used in industry, but the reasons for loss of activity of such biocatalysts are usually not known. We have used circular dichroism (CD) to investigate the structure of one such system, i.e., subtilisin Carlsberg (SC) immobilised on silica gel particles (60 microm). A number of technical problems have to be overcome in order to obtain appropriate data from which conclusions can be drawn. A rotating cell holder has been developed to avoid sedimentation of the silica particles during the collection of spectra. By moving the cell holder as close as possible to the detector window, the effects of differential scattering can be minimised. However, the effects of absorption flattening limit the extent to which reliable quantitative information on secondary structure content can be obtained from far UV CD studies. We have used an empirical approach based on absorbance units derived from the high-tension voltage to correct for absorption flattening effects. After applying the correction there was satisfactory agreement with the solution spectra. Comparison of the fresh and used (inactive) SC-silica gel spectra in organic media reveals substantial change in the secondary structure. Additional evidence for loss of native conformation is provided by the significant decrease in the near UV CD spectrum. These results for the first time clearly demonstrate the origin of enzyme instability in the immobilised state.

On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg.

BACKGROUND: Enzymes have been extensively used in organic solvents to catalyze a variety of transformations of biological and industrial significance. It has been generally accepted that in dry aprotic organic solvents, enzymes are kinetically trapped in their conformation due to the high-energy barrier needed for them to unfold, suggesting that in such media they should remain catalytically active for long periods. However, recent studies on a variety of enzymes demonstrate that their initial high activity is severely reduced after exposure to organic solvents for several hours. It was speculated that this could be due to structural perturbations, changes of the enzyme's pH memory, enzyme aggregation, or dehydration due to water removal by the solvents. Herein, we systematically study the possible causes for this undesirable activity loss in 1,4-dioxane. RESULTS: As model enzyme, we employed the protease subtilisin Carlsberg, prepared by lyophilization and colyophilization with the additive methyl-beta-cyclodextrin (MbetaCD). Our results exclude a mechanism involving a change in ionization state of the enzyme, since the enzyme activity shows a similar pH dependence before and after incubation for 5 days in 1,4-dioxane. No apparent secondary or tertiary structural perturbations resulting from prolonged exposure in this solvent were detected. Furthermore, active site titration revealed that the number of active sites remained constant during incubation. Additionally, the hydration level of the enzyme does not seem to affect its stability. Electron paramagnetic resonance spectroscopy studies revealed no substantial increase in the rotational freedom of a paramagnetic nitroxide inhibitor bound to the active site (a spin-label) during incubation in neat 1,4-dioxane, when the water activity was kept constant using BaBr2 hydrated salts. Incubation was also accompanied by a substantial decrease in Vmax/KM. CONCLUSION: These results exclude some of the most obvious causes for the observed low enzyme storage stability in 1,4-dioxane, mainly structural, dynamics and ionization state changes. The most likely explanation is possible rearrangement of water molecules within the enzyme that could affect its dielectric environment. However, other mechanisms, such as small distortions around the active site or rearrangement of counter ions, cannot be excluded at this time.

Solubis: a webserver to reduce protein aggregation through mutation.

Protein aggregation is a major factor limiting the biotechnological and therapeutic application of many proteins, including enzymes and monoclonal antibodies. The molecular principles underlying aggregation are by now sufficiently understood to allow rational redesign of natural polypeptide sequences for decreased aggregation tendency, and hence potentially increased expression and solubility. Given that aggregation-prone regions (APRs) tend to contribute to the stability of the hydrophobic core or to functional sites of the protein, mutations in these regions have to be carefully selected in order not to disrupt protein structure or function. Therefore, we here provide access to an automated pipeline to identify mutations that reduce protein aggregation by reducing the intrinsic aggregation propensity of the sequence (using the TANGO algorithm), while taking care not to disrupt the thermodynamic stability of the native structure (using the empirical force-field FoldX). Moreover, by providing a plot of the intrinsic aggregation propensity score of APRs corrected by the local stability of that region in the folded structure, we allow users to prioritize those regions in the protein that are most in need of improvement through protein engineering. The method can be accessed at http://solubis.switchlab.org/.

Structural hot spots for the solubility of globular proteins.

Natural selection shapes protein solubility to physiological requirements and recombinant applications that require higher protein concentrations are often problematic. This raises the question whether the solubility of natural protein sequences can be improved. We here show an anti-correlation between the number of aggregation prone regions (APRs) in a protein sequence and its solubility, suggesting that mutational suppression of APRs provides a simple strategy to increase protein solubility. We show that mutations at specific positions within a protein structure can act as APR suppressors without affecting protein stability. These hot spots for protein solubility are both structure and sequence dependent but can be computationally predicted. We demonstrate this by reducing the aggregation of human α-galactosidase and protective antigen of Bacillus anthracis through mutation. Our results indicate that many proteins possess hot spots allowing to adapt protein solubility independently of structure and function.

Selectivity of aggregation-determining interactions.

Protein aggregation is sequence specific, favoring self-assembly over cross-seeding with non-homologous sequences. Still, as the majority of proteins in a proteome are aggregation prone, the high level of homogeneity of protein inclusions in vivo both during recombinant overexpression and in disease remains surprising. To investigate the selectivity of protein aggregation in a proteomic context, we here compared the selectivity of aggregation-determined interactions with antibody binding. To that purpose, we synthesized biotin-labeled peptides, corresponding to aggregation-determining sequences of the bacterial protein ß-galactosidase and two human disease biomarkers: C-reactive protein and prostate-specific antigen. We analyzed the selectivity of their interactions in Escherichia coli lysate, human serum and human seminal plasma, respectively, using a Western blot-like approach in which the aggregating peptides replace the conventional antibody. We observed specific peptide accumulation in the same bands detected by antibody staining. Combined spectroscopic and mutagenic studies confirmed accumulation resulted from binding of the peptide on the identical sequence of the immobilized target protein. Further, we analyzed the sequence redundancy of aggregating sequences and found that about 90% of them are unique within their proteome. As a result, the combined specificity and low sequence redundancy of aggregating sequences therefore contribute to the observed homogeneity of protein aggregation in vivo. This suggests that these intrinsic proteomic properties naturally compartmentalize aggregation events in sequence space. In the event of physiological stress, this might benefit the ability of cells to respond to proteostatic stress by allowing chaperones to focus on specific aggregation events rather than having to face systemic proteostatic failure.

Increasing the potency of an alhydrogel-formulated anthrax vaccine by minimizing antigen-adjuvant interactions.

Aluminum salts are the most widely used vaccine adjuvants, and phosphate is known to modulate antigen-adjuvant interactions. Here we report an unexpected role for phosphate buffer in an anthrax vaccine (SparVax) containing recombinant protective antigen (rPA) and aluminum oxyhydroxide (AlOH) adjuvant (Alhydrogel). Phosphate ions bind to AlOH to produce an aluminum phosphate surface with a reduced rPA adsorption coefficient and binding capacity. However, these effects continued to increase as the free phosphate concentration increased, and the binding of rPA changed from endothermic to exothermic. Crucially, phosphate restored the thermostability of bound rPA so that it resembled the soluble form, even though it remained tightly bound to the surface. Batches of vaccine with either 0.25 mM (subsaturated) or 4 mM (saturated) phosphate were tested in a disease model at batch release, which showed that the latter was significantly more potent. Both formulations retained their potency for 3 years. The strongest aluminum adjuvant effects are thus likely to be via weakly attached or easily released native-state antigen proteins.

Biophysical characterisation of thermal-induced precipitates of recombinant anthrax protective antigen: evidence for kinetically trapped unfolding domains in solid-state.

Insoluble aggregation or precipitation is one of the most common degradation pathways observed for biotherapeutics; despite this, the structural mechanisms by which this occurs remain poorly understood due to difficulties associated with biophysical characterisation of protein particulates. To address this knowledge gap, we developed a solid-state circular dichroism (CD) technique, which allows in situ measurements of the secondary and tertiary structural changes associated with the formation of visible therapeutic protein aggregates. We demonstrate how solid-state CD, in conjunction with other biophysical and computational methods can aid in gaining valuable insights into the mechanisms and pathways of thermal-induced precipitation of Bacillus anthracis recombinant protective antigen (rPA), the primary immunogen of anthrax subunit vaccine. Using these methods, we show the domains d3 and d4 are the most labile of the four structurally distinct domains of rPA and play the critical role in nucleating the cascade of unfolding and aggregation. During the assembly process, the domains d1 and d2 become kinetically trapped within the insoluble aggregate and reveal previously intractable distinct tertiary structural elements of the rPA native structure. These findings reveal a uniquely detailed insight into the role of rPA domains on protein stability and provide a mechanistic framework for thermal-induced unfolding and precipitation. It also shows that solid-state CD provides a novel approach in characterising protein precipitation that may facilitate rational improvements to the stability of biopharmaceuticals.

Proteomic methods applied to the analysis of immobilized biocatalysts.

Methods adapted from proteomics can directly characterize proteins present in immobilized biocatalysts. Complete hydrolysis followed by HPLC analysis of Tyr and Phe estimates total protein bound, and is preferable to conventional difference methods, as tested with subtilisin Carlsberg on silica. This new method shows that various treatments give quantitative desorption of proteins immobilized by adsorption. Intact desorbed proteins may be analyzed by electrospray mass spectrometry. The Candida antarctica lipase B from Novozyme 435 was shown to be heavily glycosylated, while the lipase from Lipozyme RM IM was a mixture of four N-terminally truncated forms. Peptides from selective cleavage were analyzed by tandem mass spectrometry, leading to automatic identification of proteins present. A second major protein present in Lipozyme RM IM was thus found to be alpha-amylase from Aspergillus oryzae. These methods should be valuable complements to activity measurements in understanding immobilized enzyme activity and stability.