Native Protein Conformation Research Articles

Exploring the effect of nsSNPs in human YPEL3 gene in cellular senescence

YPEL3 that induces cellular senescence in both normal and tumour cells of humans may show altered expression under the influence of incidental mutations. In this study, we proposed the first structure of Native YPEL3 protein and its five possible deleterious mutants—V40M, C61Y, G98R, G108S, and A131T and predicted their deleterious effects to alter stability, flexibility and conformational changes in the protein. The MD simulation (RMSD, RMSF, Rg, h-bond and SASA) analysis revealed that the variants V40M, G98R and G108S increased the flexibility in protein, and variant V40M imparted more compactness to the protein.. In general, variants attributed changes in the native conformation and structure of the YPEL3 protein which might affect the native function of cellular senescence. The study provides opportunities for health professionals and practitioners in formulating précised medicines to effectively cure various cancers. We propose in-vitro or in-vivo studies should consider these reported nsSNPs while examining any malfunction in the YPEL3 protein.

High-Pressure Electrospray Ionization Yields Supercharged Protein Complexes from Native Solutions While Preserving Noncovalent Interactions.

Increasing charge state of protein complexes from native solutions while preserving noncovalent interactions in native mass spectrometry (MS) offers great opportunity to gain deeper insights into gas-phase protein structures. Several previous studies have disclosed the possibility of high pressure in supercharging small proteins, whereas its capability to supercharge large protein assemblies under native conditions and how it might affect protein structures remain open questions. Herein, we demonstrated that the high-pressure-induced supercharging strategy affords unique advantages of supercharging protein complexes with the highest charge state surpassing the Rayleigh limit (ZR) and concurrently preserving native-like topology. By examining 32 proteins and protein complexes with molecular weights (MWs) ranging from 8.58 to 801 kDa, we demonstrated that the increased average charge states of macromolecular ions have a strong dependence on the surface areas of native protein conformations and MWs. Factors that might contribute to the high-pressure-induced supercharging capability toward macromolecular ions were discussed. Furthermore, using collision cross section (CCS) variation as a function of charge state, we investigate the effects of gas pressure and charge states on gas-phase structures of proteins and protein complexes. Smaller proteins have the largest CCS variations once supercharged, while macromolecular protein complexes are less affected. The results revealed that both surface density of charge and charged surface basic residues contribute to the observed CCS-charge disciplines for all the macromolecules investigated. Taken together, the results presented here indicate that increasing gas pressure in the ion source affords a rapid, simple, and controlled supercharging method, offering the potency of facilitating further applications of native top-down MS analysis with improved transmission, fragmentation, and detection efficiency.

Use of supercritical carbon dioxide technology for fabricating a tissue engineering scaffold for anterior cruciate ligament repair

Tissue-engineered grafts may be useful in Anterior Cruciate Ligament (ACL) repair and provide a novel, alternative treatment to clinical complications of rupture, harvest site morbidity and biocompatibility associated with autografts, allografts and synthetic grafts. We successfully used supercritical carbon dioxide (Sc-CO2) technology for manufacturing a “smart” biomaterial scaffold, which retains the native protein conformation and tensile strength of the natural ACL but is decellularized for a decreased immunogenic response. We designed and fabricated a new scaffold exhibiting (1) high tensile strength and biomechanical properties comparable to those of the native tissue, (2) thermodynamically-stable extra-cellular matrix (ECM), (3) preserved collagen composition and crosslinking, (4) a decellularized material milieu with potential for future engineering applications and (5) proven feasibility and biocompatibility in an animal model of ligament reconstruction. Because of the “smart” material ECM, this scaffold may have the potential for providing a niche and for directing stem cell growth, differentiations and function pertinent to new tissue formation. Sc-CO2-related technology is advanced and has the capability to provide scaffolds of high strength and durability, which sustain a lifetime of wear and tear under mechanical loading in vivo.

Unveiling the thermodynamic signature underlying the interaction of human serum albumin with sub-micellar concentrations of a surface active ionic liquid

The interaction of the transport protein human serum albumin (HSA) has been studied with sub-micellar concentrations of the surface active ionic liquid 1-butyl-3-methylimidazolium octylsulfate ([BMIM][OS]). The prominent quenching of intrinsic Trp fluorescence of HSA together with a discernible hypsochromic shift of the maximum fluorescence wavelength from λem ~ 347 nm in native HSA to λem ~ 335 nm in the presence of ~15.6 mM of the [BMIM][OS] suggests disruption of the native protein conformation following interaction with the ionic liquid. This is further substantiated from far-UV circular dichroism spectroscopy which reveals the denaturing influence of [BMIM][OS] through lowering of α-helicity content of the protein. However, the major impetus of the present work lies in a detailed calorimetric investigation of the HSA-[BMIM][OS] interaction process. The isothermal titration calorimetry results delineates the predominant role of hydrophobic effect governing the interaction as characterized by negative change of heat capacity (∆Cp = − 160.65 ± 8.5 J mol−1 K−1). However, the temperature-dependent calorimetric results show an enthalpy-driven (ΔH < 0, exothermic) hydrophobic effect with gradually decreasing contribution from the entropy counterpart with increasing temperature. These results are counterintuitive to the notion of classical hydrophobic effect characterized by favorable entropy contribution (ΔS > 0) and unfavorable enthalpy contribution (ΔH > 0, endothermic), and are rationalized in terms of the ‘nonclassical hydrophobic effect’.

Experimental and theoretical investigations on the interaction of glucose molecules with myoglobin in the aqueous solution using theoretical and experimental methods

Osmolytes are generally well-known for the stabilization of proteins. The stabilizing impact of glucose on the dynamics and structure of myoglobin was probed through molecular simulation‚ docking and spectroscopic procedures. Using thermal stability examinations, the thermodynamic folding properties, point of melting temp. (Tm ), thermodynamic enthalpy change (ΔH°) and thermodynamic entropy change (ΔS°) were determined to find out the depiction of myoglobin folding. Glucose operated as an enhancer relative to myoglobin stabilization. The quenching static model was demonstrated by fluorescence spectroscopy. There was one binding site. According to the spectroscopy analysis, glucose was capable of protecting the native structural conformation of protein as well as preventing from protein unfolding. The fluorescence spectroscopy together with simulation through molecular docking method revealed that definitely hydrogen bonding plus van der Waals forces had major contributions to the stabilization of the myoglobin–glucose complex. Hence, the direct interactions contributed slightly to the stabilization impact whereas indirect interactions resulted from the hydration arise from a molecular mechanism primarily inducing the glucose stabilizing impacts. An elevation occurred in the Tm of the myoglobin–glucose complex because of the greater H-bond creation and limited surface hydrophobic activity. Our findings indicate that glucose was capable of protecting the native conformation of myoglobin, clearly describing that glucose stabilization is preferred to be omitted from myoglobin surface. This is because water is more inclined to provide desirable interacting with myoglobin functional groups as compared to glucose. Also, MD results confirmed that the structural changes of myoglobin is the effect of complex formation with glucose. Communicated by Ramaswamy H. Sarma

Superhydrophilic versus normal polydopamine coating: A superior and robust platform for synergistic antibacterial and antithrombotic properties

Blood-contacting devices including implantation devices, indwelling medical devices, and extracorporeal circuits have been at risk of thrombus and infections, which lead to treatment failure and serious adverse clinical events. In this study, we have reported a facile and robust mussel-inspired superhydrophilic coating for use on these blood-contacting devices to combat thrombosis and infections. The coating was fabricated mainly based on the rapid formation and accumulation of polydopamine (PDA) nanoparticles and in situ-synthesized using silver nanoparticles (Ag NPs) in the presence of sodium periodate. Therefore, the stability and long-term effects of superhydrophilicity were maintained because of the synergetic contribution of hydrophilic chemical components and surface topography of stacked nanoparticles. Different from mostly reported PDA coatings (with the water contact angle > 30°) that have good affinity to protein adsorption and cause blood coagulation, the superhydrophilic PDA coating in this work effectively reduced the nonspecific adsorption of proteins, maintained the native conformation of the adhered proteins, and further inhibited thrombus formation. Meanwhile, the superhydrophilic surface with Ag+ also demonstrated significant antibacterial properties against both Staphylococcus aureus (Gram-positive bacteria) and Pseudomonas aeruginosa (Gram-negative bacteria). Besides, the embedded Ag+ presented meaningful sustained release with less than 25% released amount even after 30 days incubation, suggesting effective and safe antibacterial ability in a low dose-dependent manner, due to the reduced protein adsorption platform supported killing and releasing of bacteria. Such a superhydrophilic surface-supported low-fouling platform will be proposed as a new strategy for combating thrombotic and infection, especially for blood-contacting devices.

Disorder under stress: Role of polyol osmolytes in modulating fibrillation and aggregation of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) comprise ~30–40% of the proteome, have key roles in cellular processes, and have been reported to be involved in stress regulation working in synergy with osmolytes. Osmolytes are known to accumulate against various stresses in living systems and are known to stabilize the native conformation of globular proteins. However, little is known of their effect on IDPs and their mechanism of action is unclear. We have investigated the effect of a series of polyol osmolytes on the conformation, aggregation and fibrillation properties of the IDPs α and β-synuclein, involved in Parkinson's disease, using fluorescence, CD, light scattering and TEM. We observe inhibition of fibril and aggregate formation with increasing concentration as well as the number of hydroxyl groups in polyols as observed by light scattering measurements which correlates well with the increase in viscosity of solution with increasing number of OH groups in them. However, ThT assay, while indicating suppression of fibril formation at various concentrations of polyols, shows enhanced fibrillation at some other concentrations which could be due to the heterogeneity of the species formed that are ThT insensitive. Fibril formation was, thus, probed by using Nile red fluorescence which showed sensitivity towards the species formed. ANS binding fluorescence also indicates a decrease in the hydrophobicity of the fibrils with increasing number of OH groups in polyols. Polyols do not have any effect on the fibrillation of β-syn but lead to enhanced amorphous aggregate formation in presence of Ethylene Glycol and Glycerol and a reduction in the presence of Sorbitol. The net free energy of transfer of the proteins from water to Sorbitol is large and positive while it is relatively negligible in the case of Glycerol suggestive of greater preferential exclusion effect of Sorbitol in comparison with Glycerol in the case of IDPs as well. The results overall show differential and complex effect of osmolytes towards the fibrillation/aggregation properties of the two IDPs and suggest that an appropriate balance between the concentration and type of polyol or osmolyte would be required for the survival of organisms rich in IDPs under various stress conditions.

Characterization of Warfarin Inhibition Kinetics Requires Stabilization of Intramembrane Vitamin K Epoxide Reductases

Intramembrane enzymes are often difficult for biochemical characterization. Human vitamin K epoxide reductase (VKOR) is the target of warfarin. However, this intramembrane enzyme becomes insensitive to warfarin inhibition in vitro, preventing the characterization of inhibition kinetics for decades. Here we employ structural biology methods to identify stable VKOR and VKOR-like proteins and purify them to near homogeneity. We find that the key to maintain their warfarin sensitivity is to stabilize their native protein conformation in vitro. Reduced glutathione drastically increases the warfarin sensitivity of a VKOR-like protein from Takifugu rubripes, presumably through maintaining a disulfide-bonded conformation. Effective inhibition of human VKOR-like requires also the use of LMNG, a mild detergent developed for crystallography to increase membrane protein stability. Human VKOR needs to be preserved in ER-enriched microsomes to exhibit warfarin sensitivity, whereas human VKOR purified in LMNG is stable only with pre-bound warfarin. Under these optimal conditions, warfarin inhibits with tight-binding kinetics. Overall, our studies show that structural biology methods are ideal for stabilizing intramembrane enzymes. Optimizing toward their inhibitor-binding conformation enables the characterization of enzyme kinetics in difficult cases.

Expression and purification of N‐terminally acetylated Ssa1 chaperone from Saccharomyces cerevisiae

N‐terminal acetylation leads to proteomic diversity by constituting a cellular acetylome wherein proteins exhibit an alternate function and dynamic, allowing cells to react specifically to environmental stressors or cellular perturbation. N‐terminal acetylation is catalyzed by a collection of N‐terminal acetyltransferases (NATs), which transfer acetyl moieties from acetyl‐coenzyme A to α‐amino groups at specific N‐terminal residues. One theoretical target of N‐terminal acetylation is Ssa1, an Hsp70 class chaperone endogenous to Saccharomyces cerevisiae. Ssa1 works in tandem with its co‐chaperone Sis1 to restore the native conformations of aberrantly‐folded proteins through a transient ATP‐driven mechanism. The consequences of N‐terminal acetylation on Hsp70 function remain to be fully characterized. To further probe the functional and mechanistic changes associated with the N‐terminal acetylation of Hsp70, we developed an efficient, user‐friendly, one‐step purification of Ssa1 using previously constructed Protein‐A fusion yeast strains. Our results indicate that our method isolates high yields of pure acetylated and unacetylated Ssa1 without compromising enzymatic activity, allowing for future evaluation of the effects of N‐terminal acetylation on Hsp70 chaperone function and dynamic.Support or Funding InformationResearch supported by RI‐INBRE (#P20GM103430) from NIGMS, NIH.

Native ion mobility mass spectrometry reveals that small organic acid fragments impart gas-phase stability to carbonic anhydrase II.

A key element of studies that utilise ion mobility mass spectrometry (IM-MS) under native electrospray conditions for the analysis of protein-ligand binding is the maintenance of the native conformation of a protein during the removal of bulk solvent. Ruotolo and co-workers have demonstrated that the binding and subsequent dissociation of the anionic component of inorganic salts stabilise native protein conformations in the gas phase. In this study, we investigated the effect that organic acid fragments identified from a fragment-based drug discovery (FBDD) campaign might have on the gas-phase stability of carbonic anhydrase II (CA II). We utilised native IM-MS to monitor changes in the conformation of CA II in the absence and presence of four acidic fragments. By performing a series of collision-induced unfolding (CIU) experiments we determined the effect of fragment binding on the gas-phase stability of CA II. Binding and dissociation of acidic fragments result in increased gas-phase stability of CA II. CFU experiments revealed that the native-like compact gas-phase conformation of the protein is stable with higher degree of pre-activation when bound to a series of acidic fragments. Importantly, although acetate was present in high concentrations, the stabilising effect was not observed without the addition of the acidic fragments. Binding and subsequent dissociation of acidic fragments from CA II significantly delayed CIU in a manner which is probably analogous to the effect of inorganic anions. Furthermore, we saw a slightly altered stabilising effect between the different fragments investigated in this study. This suggests that the prevention of CIU by organic acids may be tuneable to specific properties of a bound ligand. These observations may open avenues to exploit IM-MS as a screening platform in FBDD.

Protein Binding on the Surface of Magnetic Nanoparticles

AbstractMagnetic nanoparticles (MNPs) are widely used in the areas of biology and biomedicine. The interaction between MNPs and proteins plays a crucial role in the bioapplication of MNPs, and the binding affinity of protein–MNPs is the manifestation of this interaction. The binding affinity of some proteins with MNPs modified in various ways is determined by fluorescence quenching. The results show that the binding affinity depends on the properties of both the MNPs and the proteins. The higher the surface curvature of MNPs, the larger the MNP, and the higher the binding affinity. No significant difference is found in binding affinity between MNPs with different modification methods. For proteins, the binding affinity depends on the properties of individual proteins, such as the amino acid sequence, the native protein conformation in solution, the isoelectric point, and surface potential. In general, the binding affinity is higher for proteins with cysteine residues on the surface. In addition, pH affects the binding affinity between proteins and MNPs; positively charged proteins and lower pH are more suitable for MNP binding due to electrostatic forces.

Recent advances and application of epitope imprinted materials

As artificial antibodies, protein-imprinted materials offer many advantages such as long-term storage stability, potential reusability, resistance to harsh environments and low cost. However, they suffer from obvious disadvantages, when whole proteins are used as templates. First, it is difficult to retain the native conformation of template proteins during the polymerization process. Second, it is unavailable to obtain pure proteins, especially low abundance proteins as the templates. Third, the relatively large imprinted sites are able to bind a range of smaller non-targets, resulting in reduced selectivity. Epitopes are the regions within the structures of globular proteins that can be classified as antigenic determinants. The epitope’s structural specificity usually represents the entire protein. And, they can be easily obtained through artificial synthesis. Currently, as an alternative to the target protein, epitopes have been successfully employed as templates to fabricate the recognition sites for several proteins. Compared with protein templates, epitope templates are much more robust and stable during the imprinting process. Moreover, epitope templates could be obtained in large scale with low cost, even for the rare proteins. In this review, we critically and comprehensively survey recent advances and applications in epitope imprinting. The epitope imprinting techniques have been developed into several formats, epitope bulk imprinting, grafted epitope surface imprinting, constrained epitope surface imprinting and synergic epitope surface imprinting according to how the template is presented in the imprinted materials. For the epitope bulk imprinting, binding sites were formed throughout a bulk material. The grafted epitope surface imprinting is achieved by immobilizing epitope on the film or particle surface and then polymerizing with monomers and crosslinkers to form a state-of-the-art recognition sites on the polymer or silica surface. In the constrained epitope surface imprinting, the epitope is confined between the matrix support and the final polymer matrix. Then the matrix support is removed to expose the recognition sites, usually by simply peeling imprinted polymer from the matrix or dissolving the matrix. To improve the affinities and selectivity, additional affinities were integrated into the epitope imprinted surface, which results in the technique of synergic epitope surface imprinting. More recently, application of epitope-imprinted materials has also increased substantially with the rapid development of the life sciences, although they are not as widely used as whole protein molecule imprinting. These are demonstrated in the aspect of the peptide recognition, protein recognition and cell recognition. Finally, we summarized the remaining challenges arising from the intrinsic properties of epitope imprinting, and proposed its future development direction.

Translational diffusion of unfolded and intrinsically disordered proteins.

Translational (or self-diffusion) coefficient in dilute solution is inversely proportional to the size of a diffusing molecule, and hence self-diffusion coefficient measurements have been applied to determine the effective hydrodynamic radii for a range of native and nonnative protein conformations. In particular, translational diffusion coefficient measurements are useful to estimate the hydrodynamic radius of natively (or intrinsically) disordered proteins in solution, and, thereby, probe the compactness of a protein as well as its change when environmental parameters such as temperature, solution pH, or protein concentration are varied. The situation becomes more complicated in concentrated solutions. In this review, we discuss the translational diffusion of disordered proteins in dilute and crowded solutions, focusing primarily on the information provided by pulsed-field gradient NMR technique, and draw analogies to well-structured globular proteins and synthetic polymers.

Ligand binding to a remote site thermodynamically corrects the F508del mutation in the human cystic fibrosis transmembrane conductance regulator

Many disease-causing mutations impair protein stability. Here, we explore a thermodynamic strategy to correct the disease-causing F508del mutation in the human cystic fibrosis transmembrane conductance regulator (hCFTR). F508del destabilizes nucleotide-binding domain 1 (hNBD1) in hCFTR relative to an aggregation-prone intermediate. We developed a fluorescence self-quenching assay for compounds that prevent aggregation of hNBD1 by stabilizing its native conformation. Unexpectedly, we found that dTTP and nucleotide analogs with exocyclic methyl groups bind to hNBD1 more strongly than ATP and preserve electrophysiological function of full-length F508del-hCFTR channels at temperatures up to 37 °C. Furthermore, nucleotides that increase open-channel probability, which reflects stabilization of an interdomain interface to hNBD1, thermally protect full-length F508del-hCFTR even when they do not stabilize isolated hNBD1. Therefore, stabilization of hNBD1 itself or of one of its interdomain interfaces by a small molecule indirectly offsets the destabilizing effect of the F508del mutation on full-length hCFTR. These results indicate that high-affinity binding of a small molecule to a remote site can correct a disease-causing mutation. We propose that the strategies described here should be applicable to identifying small molecules to help manage other human diseases caused by mutations that destabilize native protein conformation.

The Role of HSF1 Protein in Malignant Transformation.

The heat shock transcription factor, HSF1, is the main regulator of the proteotoxic stress response that orchestrates the adaptation of cells to stress conditions such as elevated temperature, oxidative stress, and proteotoxic stress. As such, HSF1 regulates a large number of stress response-related genes, primarily those encoding heat shock proteins (HSPs). HSPs are molecular chaperones involved in the acquisition of native protein conformations and the prevention of protein degradation, and they also contribute to the removal of denatured proteins via the proteasome. Representative members of the HSP family are HSP70 and HSP90. The stress response is a highly conserved mechanism across all eukaryotes, and HSF1 has been linked to a number of physiological processes (ribosomal biogenesis, translation, transcription, cell cycle, and metabolism) and pathological disorders (neurodegenerative disorders such as Parkinson´s and Alzheimer´s diseases). HSF1 activation is also prominent in different types of cancer (prostate, breast, colorectal carcinoma etc.) where it correlates with tumor aggressiveness and poor prognosis. HSF1 is therefore considered a diagnostic and prognostic marker and is currently being targeted to develop new cancer therapies. Several inhibitors of HSF1 have already been synthesized, but their molecular mechanism (s) of action, specificity those of HSF1, nontoxicity in healthy tissues, and their efficacy in targeting tumor cells remain to be elucidated. This review summarizes known mechanisms of HSF1 regulation and activation, the role of HSF1 during malignant transformation, and the potential of designing small molecule HSF1 inhibitors for cancer therapy. Key words: HSF1 transcription factor - molecular chaperones - cellular stress - tumor transformation - cancer This work was supported by the project MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 10. 8. 2018.

Quantitative Evaluation of Native Protein Folds and Assemblies by Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS)

Hydrogen deuterium exchange mass spectrometry (HDX-MS) has significant potential for protein structure initiatives but its relationship with protein conformations is unclear. We report on the efficacy of HDX-MS to distinguish between native and non-native proteins using a popular approach to calculate HDX protection factors (PFs) from protein structures. The ability of HDX-MS to identify native protein conformations is quantified by binary structural classification such that merits of the approach for protein modelling can be quantified and better understood. We show that highly accurate PF calculations are not a prerequisite for HDX-MS simulations that are capable of effectively discriminating between native and non-native protein folds. The simulations can also be performed directly on unique structures facilitating high-throughput evaluation of many alternate conformations. The ability of HDX-MS to classify the conformations of homo-protein assemblies is also investigated. In contrast to protein monomers, we show a significant lack of correspondence between the simulated and experimental HDX-MS data for these systems with a subsequent decrease in the ability of HDX-MS to identify native states. However, we demonstrate surprisingly high diagnostic ability of the simulated data for assemblies in which a significant proportion of the individual chains occupy protein-protein interfaces. We relate this to the number of peptides that can sample alternate subunit orientations and discuss these observations within the larger context of applying HDX-MS to evaluate protein structures.Graphical

BMP-2 and type I collagen preservation in human deciduous teeth after demineralization.

Great interest has recently been focused on tooth and tooth derivatives as suitable substrates for the treatment of alveolar bone defects. Here, we propose the use of demineralized baby teeth (BT) as potential grafting materials for bone augmentation procedures. Particles of human BT (Ø < 1 mm) were demineralized by means of a chemical/thermal treatment. Demineralized BT particles were thoroughly characterized by scanning electron microscopy/energy dispersive X-ray analyses to evaluate the effects of the demineralization on BT topography and mineral phase composition, and by enzyme-linked immunosorbent assays (ELISA) to quantify collagen and bone morphogenetic protein-2 (BMP-2) protein contents. The response of SAOS-2 cells to exogenous BMP-2 stimulation was evaluated to identify the minimum BMP-2 concentration able to induce osteodifferentiation in vitro (alkaline phosphatase (ALP) activity). The demineralization treatment led to a dramatic decrease in relative Ca and P content (%) of ≈75% with respect to the native BT particles, while preserving native protein conformation and activity. Interestingly, the demineralization process led to a rise in the bioavailability of BMP-2 in BT particles, as compared to the untreated counterparts. The BMP-2 content found in demineralized BT was also proved to be very effective in enhancing ALP activity, thus in the osteodifferentiation of SAOS-2 cells in vitro, as confirmed by cell experiments performed upon exogenously added BMP-2. In this study we demonstrate that the BMP-2 content found in demineralized BT is very effective in inducing cell osteodifferentiation, and strengthens the idea that BTs are very attractive bioactive materials for bone-grafting procedures.

A highly efficient, one-step purification of the Hsp70 chaperone Ssa1

Chaperone proteins are required to maintain the overall fold and function of proteins in the cell. As part of the Hsp70 family, Ssa1 acts to maintain cellular proteostasis through a variety of diverse pathways aimed to preserve the native conformation of target proteins, thereby preventing aggregation and future states of cellular toxicity. Studying the structural dynamics of Ssa1 in vitro is essential to determining their precise mechanisms and requires the development of purification methods that result in highly pure chaperones. Current methods of expressing and purifying Ssa1 utilize affinity tagged constructs expressed in Escherichia coli, however, expression in an exogenous source produces proteins that lack post-translational modifications leading to undesired structural and functional effects. Current protocols to purify Ssa1 from Saccharomyces cerevisiae require large amounts of starting material, multiple steps of chromatography, and result in low yield. Our objective was to establish a small-scale purification of Ssa1 expressed from its endogenous source, Saccharomyces cerevisiae, with significant yield and purity. We utilized a protein A affinity tag that was previously used to purify large protein complexes from yeast, combined with magnetic Dynabeads that are conjugated with rabbit immunoglobulin G (IgG). Our results show that we can produce native, highly pure, active Ssa1 via this one-step purification with minimal amounts of starting material, and this Ssa1-protein A fusion does not alter cellular phenotypes. This methodology is a significant improvement in Ssa1 purification and will facilitate future experiments that will elucidate the biochemical and biophysical properties of Hsp70 chaperones.

PH and Heat Resistance of the Major Celery Allergen Apig1.

The major celery allergen Apig1 is a member of the pathogenesis-related 10 class protein family. This study aims to investigate the impact of heat and pH on the native protein conformation required for Immunoglobulin E (IgE) recognition. Spectroscopic methods, MS and IgE-binding analyses are used to study the effects of pH and thermal treatment on Apig1.0101. Heat processing results in a loss of the native protein fold via denaturation, oligomerization, and precipitation along with a subsequent reduction of IgE recognition. The induced effects and timescales are strongly pH dependent. While Apig1 refolds partially into an IgE-binding conformation at physiological pH, acidic pH treatment leads to the formation of structurally heat-resistant, IgE-reactive oligomers. Thermal processing in the presence of a celery matrix or at pH conditions close to the isoelectric point (pI = 4.63) of Apig1.0101 results in almost instant precipitation. This study demonstrates that Apig1.0101 is not intrinsically susceptible to heat treatment in vitro. However, the pH and the celery matrix strongly influence the stability of Apig1.0101 and might be the main reasons for the observed temperature lability of this important food allergen.

Global Protein Stabilization Does Not Suffice to Prevent Amyloid Fibril Formation.

Mutations or cellular conditions that destabilize the native protein conformation promote the population of partially unfolded conformations, which in many cases assemble into insoluble amyloid fibrils, a process associated with multiple human pathologies. Therefore, stabilization of protein structures is seen as an efficient way to prevent misfolding and subsequent aggregation. This has been suggested to be the underlying reason why proteins living in harsh environments, such as the extracellular space, have evolved disulfide bonds. The effect of protein disulfides on the thermodynamics and kinetics of folding has been extensively studied, but much less is known on its effect on aggregation reactions. Here, we designed a single point mutation that introduces a disulfide bond in the all-α FF domain, a protein that, despite being devoid of preformed β-sheets, forms β-sheet-rich amyloid fibrils. The novel and unique covalent bond in the FF domain dramatically increases its thermodynamic stability and folding speed. Nevertheless, these optimized properties cannot counteract the inherent aggregation propensity of the protein, thus indicating that a high global protein stabilization does not suffice to prevent amyloid formation unless it contributes to hide from exposure the specific regions that nucleate the aggregation reaction.