Oral Collagen Drink for Antiaging: Antioxidation, Facilitation of the Increase of Collagen Synthesis, and Improvement of Protein Folding and DNA Repair in Human Skin Fibroblasts.

This work unveils a fish collagen drink for improvement of skin aging. Previous studies frequently discussed the skin aging from the angle of the representative characteristics of collagen loss and the oxidative-induced expression of proteolytic enzymes matrix metalloproteinases (MMPs), but few groups comprehensively investigated the efficacy of oral hydrolyzed collagen for enhancing protein folding and DNA repair as well as improving notable cell behaviors. To delineate the broad perspective on delaying skin aging, we inspected the collagen drink-treated fibroblast cells from the molecular and cellular aspects. The results show that the collagen drink could perform the compact antiaging effects on ROS inhibition, the facilitation of the synthesis of extracellular matrix (ECM) proteins, the increase of mitochondrial activity, and improvement of the gene expression regarding correct protein folding, DNA mismatch repair (MMR) and base excision repair (BER). Although the experimental results are built on the cellular models, we believe that the positive outcomes can provide more details on the influence of oral hydrolyzed collagen supplement for antiaging. In short, we have successfully proved that the synergistic effect of the collagen drink could not only reduce the oxidative damage but also ameliorate the cell functionality to compensate the harmful effects induced by UVA.

Visible-light-driven photocontrol of the Trp-cage protein fold by a diazocine cross-linker.

Diazocines are characterized by extraordinary photochemical properties rendering them of particular interest for switching the conformation of biomolecules with visible light. Current developments afford synthetic access to unprecedented diazocine derivatives promising particular opportunities in photocontrol of proteins and biological systems. In this work, the well-established approach of photocontrolling the secondary structure of α-helices was exploited using a diazocine to reversibly fold and unfold the tertiary structure of a small protein. The protein of choice was the globulary folded Trp-cage, a widely used model system for the elucidation of protein folding pathways. A specifically designed, short and rigid dicarboxy-functionalized diazocine-based cross-linker was attached to two solvent-exposed side chains at the α-helix of the miniprotein through the use of a primary amine-selective active ester. This cross-linking strategy is orthogonal to the common cysteine-based chemistry. The cross-linked Trp-cage was successfully photoisomerized and exhibited a strong correlation between protein fold and diazocine isomeric state. As determined by NMR spectroscopy, the cis-isomer stabilized the fold, while the trans-isomer led to complete protein unfolding. The successful switching of the protein fold in principle demonstrates the ability to control protein function, as the activity depends on their structural integrity.

Could Microwave Irradiation Cause Misfolding of Peptides?

Microwaves have been experimentally shown to affect the folding dynamics of peptides and proteins. Using molecular dynamics, we performed all-atom simulations of a model ß-peptide in aqueous solution where individual degrees of freedom of solvent molecules were decoupled to allow for investigation at non-equilibrium microwave-irradiated conditions. An elevated rotational temperature of the water medium was found to significantly affect the conformation of the peptide due to the weakened hydrogen-bonding interactions with the surrounding solvent molecules. Cluster analysis revealed that microwave irradiation can indeed act as a promoter in the formation of new misfolded peptide structures of the hairpin type, which are generally associated with the onset of several neurodegenerative disorders such as Alzheimer's, Parkinson's, Huntington's, and Creutzfeldt-Jakob diseases as well as certain cancer types such as amyloidosis.

The Chlamydomonas deg1c Mutant Accumulates Proteins Involved in High Light Acclimation.

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent Ser endopeptidases that perform key aspects of protein quality control in all domains of life. Here, we characterized Chlamydomonas reinhardtii DEG1C, which together with DEG1A and DEG1B is orthologous to Arabidopsis (Arabidopsis thaliana) Deg1 in the thylakoid lumen. We show that DEG1C is localized to the stroma and the periphery of thylakoid membranes. Purified DEG1C exhibited high proteolytic activity against unfolded model substrates and its activity increased with temperature and pH. DEG1C forms monomers, trimers, and hexamers that are in dynamic equilibrium. DEG1C protein levels increased upon nitrogen, sulfur, and phosphorus starvation; under heat, oxidative, and high light stress; and when Sec-mediated protein translocation was impaired. DEG1C depletion was not associated with any obvious aberrant phenotypes under nonstress conditions, high light exposure, or heat stress. However, quantitative shotgun proteomics revealed differences in the abundance of 307 proteins between a deg1c knock-out mutant and the wild type under nonstress conditions. Among the 115 upregulated proteins are PSII biogenesis factors, FtsH proteases, and proteins normally involved in high light responses, including the carbon dioxide concentrating mechanism, photorespiration, antioxidant defense, and photoprotection. We propose that the lack of DEG1C activity leads to a physiological state of the cells resembling that induced by high light intensities and therefore triggers high light protection responses.

Investigation of the effect of UV-B light on Arabidopsis MYB4 (AtMYB4) transcription factor stability and detection of a putative MYB4-binding motif in the promoter proximal region of AtMYB4.

Here, we have investigated the possible effect of UV-B light on the folding/unfolding properties and stability of Arabidopsis thaliana MYB4 (AtMYB4) transcription factor in vitro by using biophysical approaches. Urea-induced equilibrium unfolding analyses have shown relatively higher stability of the wild-type recombinant AtMYB4 protein than the N-terminal deletion forms after UV-B exposure. However, as compared to wild-type form, AtMYB4Δ2 protein, lacking both the two N-terminal MYB domains, showed appreciable alteration in the secondary structure following UV-B exposure. UV-B irradiated AtMYB4Δ2 also displayed higher propensity of aggregation in light scattering experiments, indicating importance of the N-terminal modules in regulating the stability of AtMYB4 under UV-B stress. DNA binding assays have indicated specific binding activity of AtMYB4 to a putative MYB4 binding motif located about 212 bp upstream relative to transcription start site of AtMYB4 gene promoter, while relatively weak DNA binding activity was detected for another putative MYB4 motif located at -908 bp in AtMYB4 promoter. Gel shift and fluorescence anisotropy studies have shown increased binding affinity of UV-B exposed AtMYB4 to the promoter proximal MYB4 motif. ChIP assay has revealed binding of AtMYB4 to the promoter proximal (-212 position) MYB4 motif (ACCAAAC) in vivo. Docking experiments further revealed mechanistic detail of AtMYB4 interaction with the putative binding motifs. Overall, our results have indicated that the N-terminal 62-116 amino acid residues constituting the second MYB domain plays an important role in maintaining the stability of the C-terminal region and the overall stability of the protein, while a promoter proximal MYB-motif in AtMYB4 promoter may involve in the regulation of its own expression under UV-B light.

Electric field influence on the helical structure of peptides: insights from DFT/PCM computations.

The secondary structure of proteins is of prime importance to their proper functioning and protein misfolding may cause serious disorders in the human body. Here, the electric field influence on the conformational stability of model alpha helical peptides is studied by employing density functional theory calculations combined with continuum dielectric method computations. Our results show that the basic parameters of the electric field - its strength and directionality - are determinative for the alpha helix stability. An electric field strength of 0.005 a.u. (2.5 V nm-1) applied along the X coordinate axis (the long axis of the helix) in the direction of the µx component of the molecular dipole moment does affect the peptide conformation, destroys the helix, and leads to the formation of a cyclic-peptide-like structure. Interestingly, the process of denaturation can be reversible when the electric field is switched-off. The reversibility of the process of the electric field induced disruption of the peptide secondary structure suggests a possible mechanism for the healing of misfolded proteins.

Comparative screening of recombinant antigen thermostability for improved leptospirosis vaccine design.

Recombinant antigens exhibit targeted protectiveproperties and offer important opportunities in the development of therapeutic technologies. Biophysical and structural methods have become important tools for the rational design and engineering of improved antigen-based vaccines. Vaccines containing Leptospira immunoglobulin-like (Lig) protein-derived antigens are currently the most promising candidates for protective immunity against the globally prevalent bacterial pathogen, Leptospira interrogans; however, vaccine trials using these domains have produced inconsistent results. Here, we compare the thermostability of domains from the main immunogenic regions from major leptospiral antigens, LigA and LigB. By measuring temperature-dependent fluorescence decay of the hydrophobic core tryptophan, 17 individual Lig protein immunoglobulin-like (Ig-like) domains were shown to display a broad range of unfolding temperatures. For a majority of the domains, stability issues begin to occur at physiologically relevant temperatures. A set of chimeric Ig-like domains was used to establish the ability of transplanted domain regions to enhance thermostability. Further insights into the determinants for domain stabilization were explored with nuclear magnetic resonance dynamics and mutational analysis. The current study has yielded a set of thermostable Ig-like domain scaffolds for use in engineering antigen-based vaccines and demonstrates the importance of incorporating thermostability screening as a design parameter.

Azobenzene-based small molecular photoswitches for protein modulation.

Molecular photoswitches are a class of chemical structures that can readily isomerize between distinct geometries upon irradiation with light. Molecular photoswitches are utilized to control protein structure and function with temporal and spatial precision. In this review, we summarize the recent progress in the development of azobenzene-based molecular photoswitches and their applications in the photocontrol of protein structure and function. For clarity of discussion, we divide the known photoswitchable proteins into different categories: protein motifs, ion channels, receptors, and enzymes. Basic approaches and considerations for the structure-guided design of photoswitchable ligands are discussed. The applications and limitations of current photoswitches are also discussed.

The effect of microwave on the interaction of flavour compounds with G-actin from grass carp (Catenopharyngodon idella).

BACKGROUND: In order to investigate the influence of non-thermal effects of microwaves on the flavour of fish and meat products, the G-actin of grass carp in ice baths was exposed to different microwave powers (0, 100, 300 or 500 W); the surface hydrophobicity, sulfhydryl contents, secondary structures and adsorption capacity of G-actin to ketones were determined. RESULTS: As microwave power increased from 0 to 300 W, the surface hydrophobicity, total and reactive sulfhydryls increased; α-helix, ß-sheet and random coil fractions turned into ß-turn fractions. As microwave power increased from 300 to 500 W, however, hydrophobicity and sulfhydryl contents decreased; ß-turn and random coil fractions turned into α-helix and ß-sheet fractions. The tendencies of adsorbed capacity of ketones were similar to hydrophobicity and sulfhydryl contents. CONCLUSION: The increased adsorbing of ketones could be attributed to the unfolding of secondary structures by revealing new binding sites, including thiol groups and hydrophobic binding sites. The decreased binding capacity was related to the refolding and aggregation of protein. The results suggested that microwave powers had obvious effects on the flavour retention and proteins structures in muscle foods. © 2017 Society of Chemical Industry.

Reversible thermal unfolding of a yfdX protein with chaperone-like activity.

yfdX proteins are ubiquitously present in a large number of virulent bacteria. A member of this family of protein in E. coli is known to be up-regulated by the multidrug response regulator. Their abundance in such bacteria suggests some important yet unidentified functional role of this protein. Here, we study the thermal response and stability of yfdX protein STY3178 from Salmonella Typhi using circular dichroism, steady state fluorescence, dynamic light scattering and nuclear magnetic resonance experiments. We observe the protein to be stable up to a temperature of 45 °C. It folds back to the native conformation from unfolded state at temperature as high as 80 °C. The kinetic measurements of unfolding and refolding show Arrhenius behavior where the refolding involves less activation energy barrier than that of unfolding. We propose a homology model to understand the stability of the protein. Our molecular dynamic simulation studies on this model structure at high temperature show that the structure of this protein is quite stable. Finally, we report a possible functional role of this protein as a chaperone, capable of preventing DTT induced aggregation of insulin. Our studies will have broader implication in understanding the role of yfdX proteins in bacterial function and virulence.

Aggregation of Trp > Glu point mutants of human gamma-D crystallin provides a model for hereditary or UV-induced cataract.

Numerous mutations and covalent modifications of the highly abundant, long-lived crystallins of the eye lens cause their aggregation leading to progressive opacification of the lens, cataract. The nature and biochemical mechanisms of the aggregation process are poorly understood, as neither amyloid nor native-state polymers are commonly found in opaque lenses. The ßγ-crystallin fold contains four highly conserved buried tryptophans, which can be oxidized to more hydrophilic products, such as kynurenine, upon UV-B irradiation. We mimicked this class of oxidative damage using Trp→Glu point mutants of human γD-crystallin. Such substitutions may represent a model of UV-induced photodamage-introduction of a charged group into the hydrophobic core generating "denaturation from within." The effects of Trp→Glu substitutions were highly position dependent. While each was destabilizing, only the two located in the bottom of the double Greek key fold-W42E and W130E-yielded robust aggregation of partially unfolded intermediates at 37°C and pH 7. The αB-crystallin chaperone suppressed aggregation of W130E, but not W42E, indicating distinct aggregation pathways from damage in the N-terminal vs C-terminal domain. The W130E aggregates had loosely fibrillar morphology, yet were nonamyloid, noncovalent, showed little surface hydrophobicity, and formed at least 20°C below the melting temperature of the native ß-sheets. These features are most consistent with domain-swapped polymerization. Aggregation of partially destabilized crystallins under physiological conditions, as occurs in this class of point mutants, could provide a simple in vitro model system for drug discovery and optimization.

Molecular mechanism of thermosensory function of human heat shock transcription factor Hsf1.

The heat shock response is a universal homeostatic cell autonomous reaction of organisms to cope with adverse environmental conditions. In mammalian cells, this response is mediated by the heat shock transcription factor Hsf1, which is monomeric in unstressed cells and upon activation trimerizes, and binds to promoters of heat shock genes. To understand the basic principle of Hsf1 activation we analyzed temperature-induced alterations in the conformational dynamics of Hsf1 by hydrogen exchange mass spectrometry. We found a temperature-dependent unfolding of Hsf1 in the regulatory region happening concomitant to tighter packing in the trimerization region. The transition to the active DNA binding-competent state occurred highly cooperative and was concentration dependent. Surprisingly, Hsp90, known to inhibit Hsf1 activation, lowered the midpoint temperature of trimerization and reduced cooperativity of the process thus widening the response window. Based on our data we propose a kinetic model of Hsf1 trimerization.

Voltage-Induced Misfolding of Zinc-Replete ALS Mutant Superoxide Dismutase-1.

The monomerization of Cu, Zn superoxide dismutase (SOD1) is an early step along pathways of misfolding linked to amyotrophic lateral sclerosis (ALS). Monomerization requires the reversal of two post-translational modifications that are thermodynamically favorable: (i) dissociation of active-site metal ions and (ii) reduction of intramolecular disulfide bonds. This study found, using amide hydrogen/deuterium (H/D) exchange, capillary electrophoresis, and lysine-acetyl protein charge ladders, that ALS-linked A4V SOD1 rapidly monomerizes and partially unfolds in an external electric field (of physiological strength), without loss of metal ions, exposure to disulfide-reducing agents, or Joule heating. Voltage-induced monomerization was not observed for metal-free A4V SOD1, metal-free WT SOD1, or metal-loaded WT SOD1. Computational modeling suggested a mechanism for this counterintuitive effect: subunit macrodipoles of dimeric SOD1 are antiparallel and amplified 2-fold by metal coordination, which increases torque at the dimer interface as subunits rotate to align with the electric field.

NMR illuminates the pathways to ALS.

A combination of NMR techniques is able to explore the structure of short-lived protein conformations.

Thermal fluctuations of immature SOD1 lead to separate folding and misfolding pathways.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease involving cytotoxic conformations of Cu, Zn superoxide dismutase (SOD1). A major challenge in understanding ALS disease pathology has been the identification and atomic-level characterization of these conformers. Here, we use a combination of NMR methods to detect four distinct sparsely populated and transiently formed thermally accessible conformers in equilibrium with the native state of immature SOD1 (apoSOD1(2SH)). Structural models of two of these establish that they possess features present in the mature dimeric protein. In contrast, the other two are non-native oligomers in which the native dimer interface and the electrostatic loop mediate the formation of aberrant intermolecular interactions. Our results show that apoSOD1(2SH) has a rugged free energy landscape that codes for distinct kinetic pathways leading to either maturation or non-native association and provide a starting point for a detailed atomic-level understanding of the mechanisms of SOD1 oligomerization.

Supersaturation-limited amyloid fibrillation of insulin revealed by ultrasonication.

Amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. We proposed that ultrasonication may be an effective agitation to trigger nucleation that would otherwise not occur under the persistent metastability of supersaturation. However, the roles of supersaturation and effects of ultrasonication have not been elucidated in detail except for limited cases. Insulin is an amyloidogenic protein that is useful for investigating the mechanisms underlying amyloid fibrillation with biological relevance. We studied the alcohol-induced amyloid fibrillation of insulin using various concentrations of 2,2,2-trifluoroethanol and 1,1,1,3,3,3-hexafluoro-2-propanol at pH 2.0 and 4.8. Ultrasonic irradiation effectively triggered fibrillation under conditions in which insulin retained persistent supersaturation. Structural analyses by circular dichroism, Fourier transform infrared spectroscopy, transmission electron microscopy, and atomic force microscopy revealed that the dominant structures of fibrils varied between parallel and antiparallel ß-sheets depending on the solvent conditions. pH and alcohol concentration-dependent phase diagrams showed a marked difference before and after the ultrasonic treatment, which indicated that the persistent metastability of supersaturation determined the conformations of insulin. These results indicate the importance of an alternative view of amyloid fibrils as supersaturation-limited crystal-like aggregates formed above the solubility limit.

The extracellular protein VlsE is destabilized inside cells.

We use U2OS cells as in vivo "test tubes" to study how the same cytoplasmic environment has opposite effects on the stability of two different proteins. Protein folding stability and kinetics were compared by fast relaxation imaging, which combines a temperature jump with fluorescence microscopy of FRET (Förster resonance energy transfer)-labeled proteins. While the stability of the cytoplasmic enzyme PGK (phosphoglycerate kinase) increases in cells, the stability of the cell surface antigen VlsE, which presumably did not evolve for stability inside cells, decreases. VlsE folding also slows down more than PGK folding in cells, relative to their respective aqueous buffer kinetics. Our FRET measurements provide evidence that VlsE is more compact inside cells than in aqueous buffer. Two kinetically distinct protein populations exist inside cells, making a connection with previous in vitro crowding studies. In addition, we confirm previous studies showing that VlsE is stabilized by 150mg/mL of the carbohydrate crowder Ficoll, even though it is destabilized in the cytoplasm relative to aqueous buffer. We propose two mechanisms for the observed destabilization of VlsE in U2OS cells: long-range interactions competing with crowding or shape-dependent crowding favoring more compact states inside the cell over the elongated aqueous buffer native state.

Ultraviolet a induces endoplasmic reticulum stress response in human dermal fibroblasts.

The endoplasmic reticulum (ER) stress response is a cytoprotective mechanism against the accumulation of unfolded proteins in the ER (ER stress) that consists of three response pathways (the ATF6, IRE1 and PERK pathways) in mammals. These pathways regulate the transcription of ER-related genes through specific cis-acting elements, ERSE, UPRE and AARE, respectively. Because the mammalian ER stress response is markedly activated in professional secretory cells, its main function was thought to be to upregulate the capacity of protein folding in the ER in accordance with the increased synthesis of secretory proteins. Here, we found that ultraviolet A (UVA) irradiation induced the conversion of an ER-localized sensor pATF6α(P) to an active transcription factor pATF6α(N) in normal human dermal fibroblasts (NHDFs). UVA also induced IRE1-mediated splicing of XBP1 mRNA as well as PERK-mediated phosphorylation of an α subunit of eukaryotic initiation factor 2. Consistent with these observations, we found that UVA increased transcription from ERSE, UPRE and AARE elements. From these results, we concluded that UVA irradiation activates all branches of the mammalian ER stress response in NHDFs. This suggests that the mammalian ER stress response is activated by not only intrinsic stress but also environmental stress.

Mechanistic insight of photo-induced aggregation of chicken egg white lysozyme: the interplay between hydrophobic interactions and formation of intermolecular disulfide bonds.

Recently, it was reported that ultraviolet (UV) illumination could trigger the unfolding of proteins by disrupting the buried disulfide bonds. However, the consequence of such unfolding has not been adequately evaluated. Here, we report that unfolded chicken egg white lysozyme (CEWL) triggered by UV illumination can form uniform globular aggregates as confirmed by dynamic light scattering, atomic force microscopy, and transmission electron microscopy. The assembling process of such aggregates was also monitored by several other methods, such as circular dichroism, fluorescence spectroscopy, mass spectrometry based on chymotrypsin digestion, ANS-binding assay, Ellman essay, and SDS-PAGE. Our finding is that due to the dissociation of the native disulfide bonds by UV illumination, CEWL undergoes drastic conformational changes resulting in the exposure of some hydrophobic residues and free thiols. Subsequently, these partially unfolded molecules self-assemble into small granules driven by intermolecular hydrophobic interaction. With longer UV illumination or longer incubation time, these granules can further self-assemble into larger globular aggregates. The combined effects from both the hydrophobic interaction and the formation of intermolecular disulfide bonds dominate this process. Additionally, similar aggregation behavior can also be found in other three typical disulfide-bonded proteins, that is, α-lactalbumin, RNase A, and bovine serum albumin. Thus, we propose that such aggregation behavior might be a general mechanism for some disulfide-bonded proteins under UV irradiation.

Biochemical analysis and kinetic modeling of the thermal inactivation of MBP-fused heparinase I: implications for a comprehensive thermostabilization strategy.

Enzymatic degradation of heparin by heparin lyases has not only largely facilitated heparin structural analysis and contamination detection, but also showed great potential to be a green and cost-effective way to produce low molecular weight heparin (LMWH). However, the commercial use of heparinase I (HepI), one of the most studied heparin lyases, has been largely hampered by its low productivity and extremely poor thermostability. Here we report the thermal inactivation mechanism and strategic thermal stabilization of maltose-binding protein (MBP)-HepI, a fusion HepI produced in E. coli with high yield, solubility and activity. Biochemical studies demonstrated that the thermal inactivation of MBP-HepI involves an unfolding step that is temperature-dependently reversible, followed by an irreversible dimerization step induced by intermolecular disulfide bonds. A good consistency between the kinetic modeling and experimental data of the inactivation was obtained within a wide range of temperature and enzyme concentration, confirming the adequacy of the proposed inactivation model. Based on the inactivation mechanism, a comprehensive strategy was proposed for the thermal stabilization of MBP-HepI, in which Ca(2+) and Tween 80 were used to inhibit unfolding while site mutation at Cys297 and DTT were employed to suppress dimerization. The engineered enzyme exhibits remarkably improved storage and operational thermostability, for example, 16-fold increase in half-life at its optimum temperature of 30 °C and 8-fold increase in remaining activity of 95% after 1-week storage at 4 °C, and therefore shows great potential as a commercial biocatalyst for heparin degradation in the pharmaceutical industry.