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The effect of previous frozen storage (−18 °C for 6 months) and different coating media (aqueous: water and brine; oily: sunflower, refined olive, and extra-virgin olive oils) on the essential macroelement and trace element content of canned Atlantic mackerel (Scomber scombrus) was studied. Previous frozen storage led to an increased (p < 0.05) content of canned samples of K (oil-coated samples) and Ca (all coating conditions) and to a decreased (p < 0.05) content of P (aqueous-coating samples) and S (water- and oil-coated samples). For trace elements, a content increase (p < 0.05) in Cu and Se (brine-canned samples) and Mn (water- and refined-olive-oil-coated samples) was detected in canned fish muscle with frozen storage. Concerning the coating effect, aqueous-coating samples showed lower (p < 0.05) Mg, P, S, K, and Ca contents than their corresponding oil-coated samples. For trace elements, lower average contents were found for Co, Cu, Mn, Se, and Fe in aqueous-coating fish muscle when compared to their counterparts coated in oily media. Content changes in the different elements in canned fish muscle are discussed based on interactions with other tissue constituents and modifications that such constituents undergo during processing (i.e., protein denaturation, liquor losses from the muscle, lipid changes).

Inflammation is a normal protective reaction against tissue damage caused by physical injury, harmful chemicals, and protein denaturation. Protein denaturation is a process in which proteins lose their tertiary structure and secondary structure due to foreign substances, external compounds, such as strong acids, strong bases, organic salts, organic solvents, and heating. The purpose of this study was to determine the anti-inflammatory activity of ethanol extract and ethyl acetate fraction of the seed coat of Kebiul (Caesalpinia bonduc L.) by calculating the IC50 value of protein denaturation in the sample. The results of the phytochemical test of the ethanol extract of Kebiul seeds contained flavonoids, alkaloids, terpenoids, steroids, saponins, and tannins, while the ethyl acetate fraction only contained tannins and alkaloids. The results of the anti-inflammatory test showed that the highest percent inhibition value of sodium diclofenac, ethanol extract, and ethyl acetate fraction, respectively, at a concentration of 20 ppm was 84.8%; 84.1%; and 50%. The IC50 values "‹"‹of sodium diclofenac, ethanol extract, and ethyl acetate fraction were 5.4 μg/mL; 9.9 μg/mL; and 13.3 μg/mL, respectively. The three samples had percent inhibition values exceeding 20% which indicated that all three can be used as an anti-inflammatory. Keywords: anti-inflammatory, protein denaturation, Caesalpinia bonduc L.

Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E. coli's lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

Passive microwave radiometry (MWR) is a measurement technique based on the detection of passive radiation in the microwave spectrum of different objects. When in equilibrium, this radiation is known to be proportional to the thermodynamic temperature of an emitting body. We hypothesize that living systems feature other mechanisms of emission that are based on protein unfolding and water rotational transitions. To understand the nature of these emissions, microwave radiometry was used in several in vitro experiments. In our study, we performed pilot measurements of microwave emissions from egg whites during denaturation induced by ethanol. Egg whites comprise 10% proteins, such as albumins, mucoproteins, and globulins. We observed a novel phenomenon: microwave emissions changed without a corresponding change in the water’s thermodynamic temperature. We also found striking differences between microwave emissions and thermodynamic temperature kinetics. Therefore, we hypothesize that these two processes are unrelated, contrary to what was thought before. It is known that some pathologies such as stroke or brain trauma feature increased microwave emissions. We hypothesize that this phenomenon originates from protein denaturation and is not related to the thermodynamic temperature. As such, our findings could explain the reason for the increase in microwave emissions after trauma and post mortem for the first time. These findings could be used for the development of novel diagnostics methods. The MWR method is inexpensive and does not require fluorescent or radioactive labels. It can be used in different areas of basic and applied pharmaceutical research, including in kinetics studies in biomedicine., source:https://www.mdpi.com/2075-4418/12/6/1498

Physicochemical, proteomics, and bioinformatics analyses were conducted to investigate protein profiles in Trichiurus haumela under frozen (120 d) and chilled (6 d) storage. Springiness, chewiness, myofibrillar active sulfhydryl content, and Ca2+-ATPase activity significantly decreased, suggesting that cold stress altered muscle proteins. Compared with fresh hairtail (FH), 66 common differentially abundant proteins (DAPs) had lower abundances in chilled (3 d; CSH) and frozen (120 d; FSH) hairtail, including myosin binding proteins, filamins, actinin, troponin, and muscle-restricted coiled-coil protein. Gene Ontology (GO) annotation showed DAPs were mainly involved in cellular process, cellular anatomical entity, intracellular, and binding items. Eukaryotic orthologous group (KOG) analysis revealed that changes in cytoskeleton and energy production and conversion functions dominated during cold storage, degrading the myofibril and connective tissue structures and the physicochemical performance of muscle tissues. This study presents deep insights into the protein alternation mechanisms in hairtail muscle under cold stress

The development of novel and sustainable food products, such as cheese- and meat analogues, requires a better understanding of the use of less refined ingredients. We investigated the distribution of water between the protein and starch phase of doughs and heat-induced gels made from air-classified faba bean fractions by developing a method suited for investigation of such multi-component ingredients. The moisture contents of the protein and starch phases in the dough were determined using a method based on partial sorption isotherms of mixed doughs of protein- and starch-rich fractions at high water activity. Water content of the protein phase is higher than that of the starch phase in dough, showing that protein takes up more water than starch at room temperature. Also, the moisture content of the protein phase in the gels was calculated using a model based on the denaturation temperature of legumin. From the experiments and the modelling, it became evident that the moisture content of the protein phase in the gel is lower than the moisture content of the protein phase in the dough, showing the importance of considering moisture migration from the protein to the starch during heating.

A growth in consumer preferences for plant-based diets has triggered the utilisation of plant ingredients in a variety of food and beverage applications, including substitutes to dairy products. The majority of commercial plant-based dairy alternative products, however, have reduced protein content compared to dairy products. Such protein plays an important role in dairy products, including yoghurt and cheese, where it imparts functionality, such as gelation and texture. These differences in protein content and properties in plant-based systems, compared to dairy products, may be responsible for the commonly encountered technological challenges, such as the replication of the texture and mouthfeel. With an attempt to fill these gaps, this thesis aimed to explore the potential of almond as an ingredient to develop novel high protein products. First, commercial almond-based yoghurt alternatives were studied to understand the role of almond proteins in these complex food systems and establish the difference between almond and dairy systems. Next, almond proteins were derived in the form of minimally processed extracts or purified isolates to develop almond milk and almond protein isolate. The effect of processing parameters, including temperature and pH, on the fundamental and functional properties of these protein extracts was then studied, with the objective of gaining deeper mechanistic insights into almond protein gelation. First, a range of objective instrumental techniques were used to assess whether commercial almond-based yoghurt alternatives behaved differently to soy and dairy yoghurts. All almond-based yoghurt alternatives contained added stabilisers and were lower in protein compared to dairy yoghurt (less than 2.7 wt percent in almond vs 5 wt percent in dairy). The interconnected protein network, which is known to structure dairy yoghurt, was absent in almond based yoghurts. Instead, these systems appeared to be flocculated and contained swollen starch gra

Newly-translated glycoproteins in the endoplasmic reticulum (ER) often undergo cycles of chaperone binding and release in order to assist in folding. Quality control is required to distinguish between proteins that have completed native folding, those that have yet to fold, and those that have misfolded. Using quantitative modeling, we explore how the design of the quality-control pathway modulates its efficiency. Our results show that an energy-consuming cyclic quality-control process, similar to the observed physiological system, outperforms alternative designs. The kinetic parameters that optimize the performance of this system drastically change with protein production levels, while remaining relatively insensitive to the protein folding rate. Adjusting only the degradation rate, while fixing other parameters, allows the pathway to adapt across a range of protein production levels, aligning with in vivo measurements that implicate the release of degradation-associated enzymes as a rapid-response system for perturbations in protein homeostasis. The quantitative models developed here elucidate design principles for effective glycoprotein quality control in the ER, improving our mechanistic understanding of a system crucial to maintaining cellular health.

Proteins fold on relatively smooth free energy landscapes which are biased toward the native state, but even simple topologies which fold rapidly can experience roughness on their free energy landscape. The details of these interactions are difficult to elucidate experimentally. Closely related to the problem of deciphering the details of the free energy landscape is the problem of defining the interactions in the denatured state ensemble (DSE) which is populated under native conditions, that is, under conditions where the native state is stable. The DSE of many proteins deviates from random coil models, but quantifying and defining the energetics of the transiently populated interactions in this ensemble is extremely challenging. Characterization of the DSE of proteins which fold to compact structures is also relevant to studies of intrinsically disordered proteins (IDPs) since interactions in the dynamic ensemble populated by IDPs can modulate their behavior. Here we show how experimental thermodynamic and pKa measurements can be combined with computational thermodynamic integration to quantify interactions in the DSE. We show that non-native side chain interactions can stabilize native backbone structure in the DSE and demonstrate that that even rapidly folding proteins can form energetically significant non-native interactions in their DSE. As an example, we characterize a non-native salt bridge that stabilizes local native backbone structure in the DSE of a widely studied fast-folding protein, the villin headpiece helical domain. The combined computational experimental approach is applicable to other protein unfolded states and provides insight that is impossible to obtain with either method alone.

The interactions of proteins with surfaces are important in both biological processes and biotechnologies. In contrast to decades of study regarding the biophysics of proteins in bulk solution, however, our mechanistic understanding of the biophysics of proteins interacting with surfaces remains largely qualitative. In response, we have set to explore quantitatively the thermodynamics of protein-surface interactions. In this work, we explore systematically the role of electrostatics in modulating the interaction between proteins and charged surfaces. In particular, we use electrochemistry to explore the extent to which a macroscopic, hydroxyl-coated surface held at a slightly negative potential affects the folding thermodynamics of surface-attached protein variants with different composition of charged amino acids. Doing so, we find that attachment to the surface generally leads to a net stabilization, presumably due to excluded volume effects that reduce the entropy of the unfolded state. The magnitude of this stabilization, however, is strongly correlated with the charged-residue content of the protein. In particular, we find statistically significant correlations with both the net charge of the protein, with greater negative charge leading to less stabilization by the surface, and with the number of arginines, with more arginines leading to greater stabilization. Such findings refine our understanding of protein-surface interactions, providing in turn a guiding rationale to achieve the functional deposition of proteins on artificial surfaces for implementation in, for example, protein-based biotechnologies.

Protein and water modification in pork Longissimus dorsi (LD) pressurized at 200–400 MPa (5 min, 4 °C) followed by storage at −20 °C for up to 84 days were analyzed by complementary methodologies. Changes in proteins depended to a larger extent on storage time than pressure levels. High pressure (HP) improved drip loss by 35% after 84 days of storage. However, the meat's ability to retain water upon mechanical force (expressible moisture) was unaffected by both HP treatment and storage. No free water was present in the meat (due to drip loss), only immobile water and a larger fraction of water entrapped in the myofibrillar network were present. The water mobility after thawing was not affected as such by HP treatment and frozen storage. HP treatment and frozen storage results in a highly dynamic protein-water system, whereby protein and water molecules compete for H-bonding sites, consequently affecting the water binding.

The quality of cooked meat is valuable for consumers and meat industry. The aim of this research was to identify structural determinants of the quality of cooked meat. Muscle type (bovine semitendinosus, psoas major, biceps femoris), cooking temperature (45 C to 85 C), ageing time (14 vs 0 days in beef; prolonged 15 vs conventional 3 days in pork), enzyme inhibition (with/without cathepsin inhibitor) and fibre type (bovine masseter 100 percent type I; cutaneous trunci 93 percent type II) were investigated factors affecting meat quality. Methods for measuring quality (cooking loss and Warner- Bratzler shear force (WBSF)), structural changes (shrinkage) and protein denaturation [Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared (FTIR) microspectroscopy] were used. Cooking loss was higher in semitendinosus compared to biceps femoris and psoas major; and it increased with temperature in beef and pork. Cooking loss increased with ageing of beef independent of temperature and muscle type; and decreased with prolonged ageing compared to conventional ageing, of pork (cooked at 70 C and 80 C). The denaturation enthalpy of masseter and cutaneous trunci explained 58 and 59 percent of the variation in cooking loss, respectively. Reduction of WBSF in bovine muscles rich in collagen (all but psoas major) with cooking at 60 C to 65 C was attributed to collagen denaturation. The increase in WBSF in unaged beef with cooking at 70 C and 80 C was attributed to intact titin denaturation. In relation to shrinkage, 3D laser scanning was compared to caliper measurements, and was found inferior in measuring volume and predicting cooking loss. Cuboids` transverse and longitudinal shrinkage were higher in muscles with higher collagen content and sarcomere length, respectively. Transverse shrinkage started 5 C higher in psoas major fibre fragments, compared to semitendinosus and biceps femoris, likely due to predominant type I fibres. It was proven that transverse and lon

Much of our current knowledge of biological chemistry is founded in the structure-function relationship, whereby sequence determines structure that determines function. Thus, the discovery that a large fraction of the proteome is intrinsically disordered, while being functional, has revolutionized our understanding of proteins and raised new and interesting questions. Many intrinsically disordered proteins (IDPs) have been determined to undergo a disorder-to-order transition when recognizing their physiological partners, suggesting that their mechanisms of folding are intrinsically different from those observed in globular proteins. However, IDPs also follow some of the classic paradigms established for globular proteins, pointing to important similarities in their behavior. In this review, we compare and contrast the folding mechanisms of globular proteins with the emerging features of binding-induced folding of intrinsically disordered proteins. Specifically, whereas disorder-to-order transitions of intrinsically disordered proteins appear to follow rules of globular protein folding, such as the cooperative nature of the reaction, their folding pathways are remarkably more malleable, due to the heterogeneous nature of their folding nuclei, as probed by analysis of linear free-energy relationship plots. These insights have led to a new model for the disorder-to-order transition in IDPs termed ?templated folding,? whereby the binding partner dictates distinct structural transitions en route to product, while ensuring a cooperative folding.

Objective: The use of naturally occurring medicines dependent on essential oils (EO) is now a days of great interest. In addition, within the human body, EO shows high efficacy as antioxidants and anti-inflammatory drugs. The present experiment was conducted to access the anti-inflammatory activity of essential oil obtained from the fruit peels of Citrus macroptera Montr. (Rutaceae) against the denaturation of protein in-vitro model. Materials and Methods: The test sample (essential oil) was incubated under controlled laboratory conditions at varying concentrations with egg albumin and was subjected to absorbance determination for the anti-inflammatory property analysis. Diclofenac sodium was used as the standard reference drug for the experiment. Results: The results show a concentration dependent inhibition of protein (albumin) denaturation by the test oil. This was concluded by comparing their IC50 average values. Citrus macroptera Montr. essential oil possessed IC50 average value 54.6+0.07 ?g/mL whereas that of diclofenac sodium was found to be 52.89+0.06 ?g/ml. The result shows that the test oil is more effective than the standard drug. Conclusion: From the above experimental finding it can be concluded that the Citrus macroptera Montr. essential oil has significance anti-inflammatory effect against the denaturation of the protein in-vitro model. The activity may be due to the presence of terpene polyphenolic component or some other active compound present in the oil. The provided information was first of its kind of knowledge to keep the scientific data for future reference.

Proteins are molecular machines whose function depends on their ability to achieve complex folds with precisely defined structural and dynamic properties. The rational design of proteins from first-principles, or de novo, was once considered to be impossible, but today proteins with a variety of folds and functions have been realized. We review the evolution of the field from its earliest days, placing particular emphasis on how this endeavor has illuminated our understanding of the principles underlying the folding and function of natural proteins, and is informing the design of macromolecules with unprecedented structures and properties. An initial set of milestones in de novo protein design focused on the construction of sequences that folded in water and membranes to adopt folded conformations. The first proteins were designed from first-principles using very simple physical models. As computers became more powerful, the use of the rotamer approximation allowed one to discover amino acid sequences that stabilize the desired fold. As the crystallographic database of protein structures expanded in subsequent years, it became possible to construct proteins by assembling short backbone fragments that frequently recur in Nature. The second set of milestones in de novo design involves the discovery of complex functions. Proteins have been designed to bind a variety of metals, porphyrins, and other cofactors. The design of proteins that catalyze hydrolysis and oxygen-dependent reactions has progressed significantly. However, de novo design of catalysts for energetically demanding reactions, or even proteins that bind with high affinity and specificity to highly functionalized complex polar molecules remains an importnant challenge that is now being achieved. Finally, the protein design contributed significantly to our understanding of membrane protein folding and transport of ions across membranes. The area of membrane protein design, or more generally of biomimetic po

This study analysed the effect of prior high-pressure processing (HPP; 200–600 MPa, 2 min), freezing (−30 °C, 48 h), and frozen storage (−18 °C, 6 months) on the macroelement and trace element content in brine-canned mackerel (Scomber colias). Most elements (Na, Ca, Ba, Mn, Fe, Cu, Cd, Sn, As, S, and Se) showed an increased (p < 0.05) presence in mackerel muscle canned after freezing. A content increase (p < 0.05) was also observed for Na and Sn if prior frozen storage was also applied; on the contrary, Ca, Ba, Mn, Fe, Cd, S, and Se showed a content decrease (p < 0.05) as a result of such storage. Freezing, frozen storage, and canning led to lower values (p < 0.05) in canned fish for K, Mg, Pb, and P. Prior HPP led to relevant content decreases (p < 0.05) for K, Mg, Ca, Ba, Mn, Fe, Pb, and P contents in fish canned after the freezing step; HPP provoked additional decreases (p < 0.05) in Ca, Ba, and Mn levels in samples corresponding to 6-month frozen storage. On the contrary, prior HPP led to marked increases (p < 0.05) for Cd, S, and Se contents in all canned samples. Content changes are explained on the basis of modifications of other constituents and liquor losses from muscle

Low levels of salt are frequently used to increase flavor and water retention in cod. This alters the biochemical properties of cod during heating. In this paper, properties needed to mathematically model moisture transfer during cooking – water holding capacity and storage modulus – were determined for cod containing 0.06, 1 and 3 g/100 g NaCl. Protein denaturation and microstructure was also investigated to increase the understanding of quality effects of salt during cooking. A model was established to investigate the effect of the measured storage modulus and water holding capacity on the predicted moisture retention during heating. Salting lead to a higher water holding capacity, less hardening of the muscle tissue during heating, diffused protein denaturation peaks and caused swelling of the muscle fibers. By interchanging the acquired variables in the model of coupled heat and moisture transfer, we found a higher predicted water retention during cooking of brined cod compared to unsalted cod. This knowledge may be utilized in creating modeling tools for optimization of cooking processes, which may support chefs and ready-to-eat meal producers in reducing weight loss and improving the texture and juiciness of their products.

This study analysed the effect of prior high-pressure processing (HPP; 200–600 MPa, 2 min), freezing (−30 °C, 48 h), and frozen storage (−18 °C, 6 months) on the macroelement and trace element content in brine-canned mackerel (Scomber colias). Most elements (Na, Ca, Ba, Mn, Fe, Cu, Cd, Sn, As, S, and Se) showed an increased (p < 0.05) presence in mackerel muscle canned after freezing. A content increase (p < 0.05) was also observed for Na and Sn if prior frozen storage was also applied; on the contrary, Ca, Ba, Mn, Fe, Cd, S, and Se showed a content decrease (p < 0.05) as a result of such storage. Freezing, frozen storage, and canning led to lower values (p < 0.05) in canned fish for K, Mg, Pb, and P. Prior HPP led to relevant content decreases (p < 0.05) for K, Mg, Ca, Ba, Mn, Fe, Pb, and P contents in fish canned after the freezing step; HPP provoked additional decreases (p < 0.05) in Ca, Ba, and Mn levels in samples corresponding to 6-month frozen storage. On the contrary, prior HPP led to marked increases (p < 0.05) for Cd, S, and Se contents in all canned samples. Content changes are explained on the basis of modifications of other constituents and liquor losses from muscle.

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

The widespread use of synthetic transvaginal polypropylene mesh for treating Pelvic Organ Prolapse (POP) has been curtailed due to serious adverse effects highlighted in 2008 and 2011 FDA warnings and subsequent legal action. We are developing new synthetic mesh to deliver endometrial mesenchymal stem cells (eMSC) to improve mesh biocompatibility and restore strength to prolapsed vaginal tissue. Here we evaluated knitted polyamide (PA) mesh in an ovine multiparous model using transvaginal implantation and matched for the degree of POP. Polyamide mesh dip-coated in gelatin and stabilised with 0.5% glutaraldehyde (PA/G) were used either alone or seeded with autologous ovine eMSC (eMSC/PA/G), which resulted in substantial mesh folding, poor tissue integration and 42% mesh exposure in the ovine model. In contrast, a two-step insertion protocol, whereby the uncoated PA mesh was inserted transvaginally followed by application of autologous eMSC in a gelatin hydrogel onto the mesh and crosslinked with blue light (PA + eMSC/G), integrated well with little folding and no mesh exposure. The autologous ovine eMSC survived 30 days in vivo but had no effect on mesh integration. The stiff PA/G constructs provoked greater myofibroblast and inflammatory responses in the vaginal wall, disrupted the muscularis layer and reduced elastin fibres compared to PA + eMSC/G constructs. This study identified the superiority of a two-step protocol for implanting synthetic mesh in cellular compatible composite constructs and simpler surgical application, providing additional translational value.Copyright © 2019 Elsevier Ltd

The scavenging capacity of glycoprotein DMBT1 helps defend mucosal epithelia against microbes. DMBT1 binding to multiple bacterial species involves its conserved Scavenger Receptor Cysteine-Rich (SRCR) domains, localized to a 16-mer consensus sequence peptide, SRCRP2. Previously, we showed that DMBT1 bound Pseudomonas aeruginosa pili, and inhibited twitching motility, a pilus-mediated movement important for virulence. Here, we determined molecular characteristics required for twitching motility inhibition. Heat-denatured DMBT1 lost capacity to inhibit twitching motility and showed reduced pili binding (~40%). Size-exclusion chromatography of Lys-C-digested native DMBT1 showed that only high-Mw fractions retained activity, suggesting involvement of the N-terminal containing repeated SRCR domains with glycosylated SRCR-Interspersed Domains (SIDs). However, individual or pooled consensus sequence peptides (SRCRPs 1 to 7) showed no activity and did not bind P. aeruginosa pili; nor did recombinant DMBT1 (aa 1-220) or another SRCR-rich glycoprotein, CD163. Enzymatic de-N-glycosylation of DMBT1, but not de-O-glycosylation, reduced its capacity to inhibit twitching motility (~57%), without reducing pili binding. Therefore, DMBT1 inhibition of P. aeruginosa twitching motility involves its N-glycosylation, its pili-binding capacity is insufficient, and it cannot be conferred by the SRCR bacteria-binding peptide domain, either alone or mixed with other unlinked SRCRPs, suggesting an additional mechanism for DMBT1-mediated mucosal defense.

The dimensions that unfolded proteins, including intrinsically disordered proteins (IDPs), adopt in the absence of denaturant remain controversial. We developed an analysis procedure for small-angle X-ray scattering (SAXS) profiles and used it to demonstrate that even relatively hydrophobic IDPs remain nearly as expanded in water as they are in high denaturant concentrations. In contrast, as demonstrated here, most fluorescence resonance energy transfer (FRET) measurements have indicated that relatively hydrophobic IDPs contract significantly in the absence of denaturant. We use two independent approaches to further explore this controversy. First, using SAXS we show that fluorophores employed in FRET can contribute to the observed discrepancy. Specifically, we find that addition of Alexa-488 to a normally expanded IDP causes contraction by an additional 15%, a value in reasonable accord with the contraction reported in FRET-based studies. Second, using our simulations and analysis procedure to accurately extract both the radius of gyration (Rg) and end-to-end distance (Ree) from SAXS profiles, we tested the recent suggestion that FRET and SAXS results can be reconciled if the Rg and Ree are "uncoupled" (i.e., no longer simply proportional), in contrast to the case for random walk homopolymers. We find, however, that even for unfolded proteins, these two measures of unfolded state dimensions remain proportional. Together, these results suggest that improved analysis procedures and a correction for significant, fluorophore-driven interactions are sufficient to reconcile prior SAXS and FRET studies, thus providing a unified picture of the nature of unfolded polypeptide chains in the absence of denaturant.

It has become clear that the standard cartoon, in which macromolecular particles prepared for electron cryo-microscopy are shown to be surrounded completely by vitreous ice, often is not accurate. In particular, the standard picture does not include the fact that diffusion to the air-water interface, followed by adsorption and possibly denaturation, can occur on the time scale that normally is required to make thin specimens. The extensive literature on interaction of proteins with the air-water interface suggests that many proteins can bind to the interface, either directly or indirectly via a sacrificial layer of already-denatured protein. In the process, the particles of interest can, in some cases, become preferentially oriented, and in other cases they can be damaged and/or aggregated at the surface. Thus, although a number of methods and recipes have evolved for dealing with protein complexes that prove to be difficult, making good cryo-grids can still be a major challenge for each new type of specimen. Recognition that the air-water interface is a very dangerous place to be has inspired work on some novel approaches for preparing cryo-grids. At the moment, two of the most promising ones appear to be: (1) thin and vitrify the specimen much faster than is done currently or (2) immobilize the particles onto a structure-friendly support film so that they cannot diffuse to the air-water interface.

ClC chloride channels and transporters are important for chloride homeostasis in species from bacteria to human. Mutations in ClC proteins cause genetically inherited diseases, some of which are likely to involve folding defects. The ClC proteins present a challenging and unusual biological folding problem because they are large membrane proteins possessing a complex architecture, with many reentrant helices that go only partway through membrane and loop back out. Here we were able to examine the unfolding of the Escherichia coli ClC transporter, ClC-ec1, using single-molecule forced unfolding methods. We found that the protein could be separated into two stable halves that unfolded independently. The independence of the two domains is consistent with an evolutionary model in which the two halves arose from independently folding subunits that later fused together. Maintaining smaller folding domains of lesser complexity within large membrane proteins may be an advantageous strategy to avoid misfolding traps.

The functionality of the tumor suppressor p53 is altered in more than 50% of human cancers, and many individuals with cancer exhibit amyloid-like buildups of aggregated p53. An understanding of what triggers the pathogenic amyloid conversion of p53 is required for the further development of cancer therapies. Here, perturbation of the p53 core domain (p53C) with subdenaturing concentrations of guanidine hydrochloride and high hydrostatic pressure revealed native-like molten globule (MG) states, a subset of which were highly prone to amyloidogenic aggregation. We found that MG conformers of p53C, probably representing population-weighted averages of multiple states, have different volumetric properties, as determined by pressure perturbation and size-exclusion chromatography. We also found that they bind the fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and have a native-like tertiary structure that occludes the single Trp residue in p53. Fluorescence experiments revealed conformational changes of the single Trp and Tyr residues before p53 unfolding and the presence of MG conformers, some of which were highly prone to aggregation. p53C exhibited marginal unfolding cooperativity, which could be modulated from unfolding to aggregation pathways with chemical or physical forces. We conclude that trapping amyloid precursor states in solution is a promising approach for understanding p53 aggregation in cancer. Our findings support the use of single-Trp fluorescence as a probe for evaluating p53 stability, effects of mutations, and the efficacy of therapeutics designed to stabilize p53.

While it is widely appreciated that the denatured state of a protein is a heterogeneous conformational ensemble, there is still debate over how this ensemble changes with environmental conditions. Here, we use single-molecule chemo-mechanical unfolding, which combines force and urea using the optical tweezers, together with traditional protein unfolding studies to explore how perturbants commonly used to unfold proteins (urea, force, and temperature) affect the denatured-state ensemble. We compare the urea m-values, which report on the change in solvent accessible surface area for unfolding, to probe the denatured state as a function of force, temperature, and urea. We find that while the urea- and force-induced denatured states expose similar amounts of surface area, the denatured state at high temperature and low urea concentration is more compact. To disentangle these two effects, we use destabilizing mutations that shift the Tm and Cm. We find that the compaction of the denatured state is related to changing temperature as the different variants of acyl-coenzyme A binding protein have similar m-values when they are at the same temperature but different urea concentration. These results have important implications for protein folding and stability under different environmental conditions.

Effects of freezing assisted with magnetic fields (MFs) in a commercial Cell Alive System (CAS) unit at − 50 °C, with a static MF only (0% CAS) and with a static MF plus oscillating MF (10% CAS), on egg yolk (Y) was investigated. Y samples were obtained from commercial eggs laid by three hen strains (two of them in fortified cages and the third one free outdoors). The main goal was to study the thermal denaturation of protein involved in MF processing. Results showed that freezing treatment was the factor with the highest influence. Thermal denaturation enthalpy of Y was markedly affected (~ 45% total protein in comparison with Fresh sample), but similar for the two MF processes, where 0% CAS was taken as the Control freezing treatment. MF effects were predominantly thermal in nature and were the factor with the highest influence on the thermal behavior (which ran parallel to that of egg white, as described in Part 1), as well as on most of the functional properties exhibited by the MF samples. Rheology and free Sulfhydryl content were studied complementarily to Differential Scanning Calorimetry (DSC) data. Some important functional properties such as Emulsion Ability and Emulsion Stability were also determined. The hen strain feeding factor had practically no influence on the physical or functional behavior of both untreated and processed Y samples, except on their color parameters.

Essentially all biochemistry and most molecular biology experiments require recombinant proteins. However, large, hydrophobic proteins typically aggregate into insoluble and misfolded species, and are directed into inclusion bodies. Current techniques to fold proteins recovered from inclusion bodies rely on denaturation followed by dialysis or rapid dilution. Such approaches can be time consuming, wasteful, and inefficient. Here, we describe rapid protein folding using a vortex fluidic device (VFD). This process uses mechanical energy introduced into thin films to rapidly and efficiently fold proteins. With the VFD in continuous flow mode, large volumes of protein solution can be processed per day with 100-fold reductions in both folding times and buffer volumes.

The increasing availability of high-quality experimental data and first-principles calculations creates opportunities for developing more accurate empirical force fields for simulation of proteins. We developed the AMBER-FB15 protein force field by building a high-quality quantum chemical data set consisting of comprehensive potential energy scans and employing the ForceBalance software package for parameter optimization. The optimized potential surface allows for more significant thermodynamic fluctuations away from local minima. In validation studies where simulation results are compared to experimental measurements, AMBER-FB15 in combination with the updated TIP3P-FB water model predicts equilibrium properties with equivalent accuracy, and temperature dependent properties with significantly improved accuracy, in comparison with published models. We also discuss the effect of changing the protein force field and water model on the simulation results.

Samples prepared for single-particle electron cryo-microscopy (cryo-EM) necessarily have a very high surface-to-volume ratio during the short period of time between thinning and vitrification. During this time, there is an obvious risk that macromolecules of interest may adsorb to the air-water interface with a preferred orientation, or that they may even become partially or fully unfolded at the interface. In addition, adsorption of macromolecules to an air-water interface may occur even before thinning. This paper addresses the question whether currently used methods of sample preparation might be improved if one could avoid such interfacial interactions. One possible way to do so might be to preemptively form a surfactant monolayer over the air-water interfaces, to serve as a structure-friendly slide and coverslip. An alternative is to immobilize particles of interest by binding them to some type of support film, which-to continue using the analogy-thus serves as a slide. In this case, the goal is not only to prevent the particles of interest from diffusing into contact with the air-water interface but also to increase the number of particles seen in each image. In this direction, it is natural to think of developing various types of affinity grids as structure-friendly alternatives to thin carbon films. Perhaps ironically, if precautions are not taken against adsorption of particles to air-water interfaces, sacrificial monolayers of denatured protein may take the roles of slide, coverslip, or even both.

The protein-folding mechanism remains a major puzzle in life science. Purified soluble activation-induced cytidine deaminase (AID) is one of the most difficult proteins to obtain. Starting from inclusion bodies containing a C-terminally truncated version of AID (residues 1-153; AID153), an optimized in vitro folding procedure was derived to obtain large amounts of AID153, which led to crystals with good quality and to final structural determination. Interestingly, it was found that the final refolding yield of the protein is proline residue-dependent. The difference in the distribution of cis and trans configurations of proline residues in the protein after complete denaturation is a major determining factor of the final yield. A point mutation of one of four proline residues to an asparagine led to a near-doubling of the yield of refolded protein after complete denaturation. It was concluded that the driving force behind protein folding could not overcome the cis-to-trans proline isomerization, or vice versa, during the protein-folding process. Furthermore, it was found that successful refolding of proteins optimally occurs at high pH values, which may mimic protein folding in vivo. It was found that high pH values could induce the polarization of peptide bonds, which may trigger the formation of protein secondary structures through hydrogen bonds. It is proposed that a hydrophobic environment coupled with negative charges is essential for protein folding. Combined with our earlier discoveries on protein-unfolding mechanisms, it is proposed that hydrogen bonds are a primary driving force for de novo protein folding.

Dystroglycan (DG) is a highly glycosylated protein complex that links the cytoskeleton with the extracellular matrix, mediating fundamental physiological functions such as mechanical stability of tissues, matrix organization and cell polarity. A crucial role in the glycosylation of the DG α subunit is played by its own N-terminal region that is required by the glycosyltransferase LARGE. Alteration in this O-glycosylation deeply impairs the high affinity binding to other extracellular matrix proteins such as laminins. Recently, three missense mutations in the gene encoding DG, mapped in the α-DG N-terminal region, were found to be responsible for hypoglycosylated states, causing congenital diseases of different severity referred as primary dystroglycanopaties.To gain insight on the molecular basis of these disorders, we investigated the crystallographic and solution structures of these pathological point mutants, namely V72I, D109N and T190M. Small Angle X-ray Scattering analysis reveals that these mutations affect the structures in solution, altering the distribution between compact and more elongated conformations. These results, supported by biochemical and biophysical assays, point to an altered structural flexibility of the mutant α-DG N-terminal region that may have repercussions on its interaction with LARGE and/or other DG-modifying enzymes, eventually reducing their catalytic efficiency.

Effects of freezing assisted with magnetic fields (MFs) in a Cell Alive System (CAS) commercial unit at –50 ºC, with a static MF only (0% CAS) and with a static MF plus oscillating MF (10% CAS) on egg white (W) was investigated. W samples were from commercial eggs from three strains of laying hens (two of them living in fortified cages and the third one in liberty at outdoor). The main goal was to follow the protein thermal denaturation involved in W processing. Thermal denaturation enthalpy was markedly affected (~50% total protein in comparison with fresh sample) but similar for both MFs processes, where 0% CAS was taken as Control freezing treatment. MFs effects were predominantly thermal in nature and constituted the factor with the highest influence on thermal behavior, as well as on the majority of the functional properties exhibited by MFs samples. Rheology and free Sulfhydryl content were studied complementarily to Differential Scanning Calorimetry (DSC) data. Some important functional properties as Foaming parameters and Texture of W gel were also determined. The hen strains-feeding factor did not practically influence the physical or functional behaviors of both untreated and processed W samples.

Trypsin is a serine protease with widespread applications, including protein sequencing and trypsin mass fingerprinting. In the present study, the storage of trypsin in acidic conditions significantly affected the recovery of activity (40%) after 7 freeze–thaw cycles. Further, trypsin lost parts of its native secondary structure elements, which resulted in a 10% increase in β-sheet content (band maximum detected at a frequency of 1634 cm−1 in the Fourier transform infrared (FT-IR) spectrum) indicative of freezing-induced denaturation of the protein. The cold storage of trypsin in ammonium bicarbonate (pH 8.2) with the addition of cryoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery (>90%) and modest autolysis (<10%). High activity recovery (>90%) was also detected with the addition of propylene glycol and polyethylene glycol, saccharides and arginine. Nevertheless, trypsin stored at pH 8.2 with the addition of glycerol or lysine was as efficient as untreated trypsin in the trypsin mass fingerprinting analysis of BSA, suggesting that the cold storage of trypsin in slightly alkaline conditions with the addition of cryoprotectants could prolong its shelf life.

Endocytic sorting and lysosomal degradation are integral to the regulation of G protein-coupled receptor (GPCR) function. Upon ligand binding, classical GPCRs are activated, internalized and recycled or sorted to lysosomes for degradation, a process that requires receptor ubiquitination. However, recent studies have demonstrated that numerous GPCRs are sorted to lysosomes independent of receptor ubiquitination. Here, we describe an ubiquitin-independent lysosomal sorting pathway for the purinergic GPCR P2Y1. After activation, P2Y1 sorts to lysosomes for degradation independent of direct ubiquitination that is mediated by a YPX3L motif within the second intracellular loop that serves as a binding site for the adaptor protein ALIX. Depletion of ALIX or site-directed mutation of the YPX3L motif inhibits P2Y1 sorting into the lumen of multivesicular endosomes/lysosomes and degradation. These findings confirm the function of YPX3L motifs as lysosomal targeting sequences for GPCRs and demonstrate that ALIX mediates the ubiquitin-independent degradation of certain GPCRs.

ETHNOPHARMACOLOGICAL RELEVANCE: Boldoa purpurascens Cav. (Nyctaginaceae) is a plant species used in traditional medicine in Cuba as a diuretic. AIM OF THE STUDY: The aim of the present investigation was to evaluate the safety profile of a hydroalcoholic extract from leaves of Boldoa purpurascens. MATERIALS AND METHODS: First, an experimental study to assess the oral acute toxicity at a dose of 2000mg/kg body weight of the extract was carried out. Potential genotoxicity of the extract was evaluated using the Ames test and the micronucleus induction assay in mouse bone marrow. In the Ames test a concentration range of 50, 100, 150, 300 and 500µg/plate was tested. In the micronucleus induction assay, doses of 500, 1000 and 2000mg/kg of body weight were tested. For completeness, since the extract contains saponins, the evaluation of the hemolytic activity, ocular and skin irritation were included. RESULTS: No signs or symptoms of toxicity were observed in the oral acute toxicity test (body weight at baseline, seven days and end of the experiment of 236.41±20.07, 256.81±30.44 and 240.02±26.16 respectively for the treated group). The hydroalcoholic extract from the leaves was not mutagenic in the Ames test, and no genotoxicity was observed in the micronucleus assay. A hemolysis test at concentration of 1mg/mL confirmed hemolytic activity, which is not a safety concern since saponins are not absorbed after oral administration. In order to evaluate the percentage of protein denaturation, the ocular irritability index was calculated. The extract was found to be irritating. Finally, skin irritability was evaluated and the irritation index was equal to zero. CONCLUSIONS: Based on the toxicological evaluation of a traditionally used hydroalcoholic extract from the leaves of Boldoa purpurascens we can confirm the safety of its oral use.

Fibronectin (Fn) forms a fibrillar network that controls cell behavior in both physiological and diseased conditions including cancer. Indeed, breast cancer-associated stromal cells not only increase the quantity of deposited Fn but also modify its conformation. However, (i) the interplay between mechanical and conformational properties of early tumor-associated Fn networks and (ii) its effect on tumor vascularization remain unclear. Here, we first used the Surface Forces Apparatus to reveal that 3T3-L1 preadipocytes exposed to tumor-secreted factors generate a stiffer Fn matrix relative to control cells. We then show that this early matrix stiffening correlates with increased molecular unfolding in Fn fibers, as determined by Förster Resonance Energy Transfer. Finally, we assessed the resulting changes in adhesion and proangiogenic factor (VEGF) secretion of newly seeded 3T3-L1s, and we examined altered integrin specificity as a potential mechanism of modified cell-matrix interactions through integrin blockers. Our data indicate that tumor-conditioned Fn decreases adhesion while enhancing VEGF secretion by preadipocytes, and that an integrin switch is responsible for such changes. Collectively, our findings suggest that simultaneous stiffening and unfolding of initially deposited tumor-conditioned Fn alters both adhesion and proangiogenic behavior of surrounding stromal cells, likely promoting vascularization and growth of the breast tumor. This work enhances our knowledge of cell - Fn matrix interactions that may be exploited for other biomaterials-based applications, including advanced tissue engineering approaches.

Thermal exposure is a serious and growing challenge facing fish species worldwide. Chinook salmon (Oncorhynchus tshawytscha) living in the southern portion of their native range are particularly likely to encounter warmer water due to a confluence of factors. River alterations have increased the likelihood that juveniles will be exposed to warm water temperatures during their freshwater life stage, which can negatively impact survival, growth, and development and pose a threat to dwindling salmon populations. To better understand how acute thermal exposure affects the biology of salmon, we performed a transcriptional analysis of gill tissue from Chinook salmon juveniles reared at 12° and exposed acutely to water temperatures ranging from ideal to potentially lethal (12° to 25°). Reverse-transcribed RNA libraries were sequenced on the Illumina HiSeq2000 platform and a de novo reference transcriptome was created. Differentially expressed transcripts were annotated using Blast2GO and relevant gene clusters were identified. In addition to a high degree of downregulation of a wide range of genes, we found upregulation of genes involved in protein folding/rescue, protein degradation, cell death, oxidative stress, metabolism, inflammation/immunity, transcription/translation, ion transport, cell cycle/growth, cell signaling, cellular trafficking, and structure/cytoskeleton. These results demonstrate the complex multi-modal cellular response to thermal stress in juvenile salmon.

Application of hydrostatic pressure shifts protein conformational equilibria in a direction to reduce the volume of the system. A current view is that the volume reduction is dominated by elimination of voids or cavities in the protein interior via cavity hydration, although an alternative mechanism wherein cavities are filled with protein side chains resulting from a structure relaxation has been suggested [López CJ, Yang Z, Altenbach C, Hubbell WL (2013) Proc Natl Acad Sci USA 110(46):E4306-E4315]. In the present study, mechanisms for elimination of cavities under high pressure are investigated in the L99A cavity mutant of T4 lysozyme and derivatives thereof using site-directed spin labeling, pressure-resolved double electron-electron resonance, and high-pressure circular dichroism spectroscopy. In the L99A mutant, the ground state is in equilibrium with an excited state of only ∼ 3% of the population in which the cavity is filled by a protein side chain [Bouvignies et al. (2011) Nature 477(7362):111-114]. The results of the present study show that in L99A the native ground state is the dominant conformation to pressures of 3 kbar, with cavity hydration apparently taking place in the range of 2-3 kbar. However, in the presence of additional mutations that lower the free energy of the excited state, pressure strongly populates the excited state, thereby eliminating the cavity with a native side chain rather than solvent. Thus, both cavity hydration and structure relaxation are mechanisms for cavity elimination under pressure, and which is dominant is determined by details of the energy landscape.

Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant.

The crosslinking agent genipin is increasingly invoked for the mechanical augmentation of collagen tissues and implants, and has previously been demonstrated to arrest mechanical damage accumulation in various tissues. This study established an in vitro dose-response baseline for the effects of genipin treatment on tendon cells and their matrix, with a view to in vivo application to the repair of partial tendon tears. Regression models based on a broad range of experimental data were used to delineate the range of concentrations that are likely to achieve functionally effective crosslinking, and predict the corresponding degree of cell loss and diminished metabolic activity that can be expected. On these data, it was concluded that rapid mechanical augmentation of tissue properties can only be achieved by accepting some degree of cytotoxicity, yet that post-treatment cell survival may be adequate to eventually repopulate and stabilize the tissue. On this basis, development of delivery strategies and subsequent in vivo study seems warranted.

Kinetics of thermal inactivation of acrylodan-labeled cAMP dependent protein kinase catalytic subunit, its binary complexes with ATP and peptide inhibitor PKI[5-24], respectively, and the ternary complex involving both of these ligands were studied at different temperatures (5-50 A degrees C). The thermodynamic parameters Delta H and Delta S for ligand binding equilibria as well as for the allosteric interaction between the binding sites of these ligands were obtained by using the Van't Hoff analysis. The results indicated that more inter- and intra-molecular non-covalent bonds were involved in ATP binding with the protein when compared to the peptide binding. Similarly, nucleotide and peptide binding steps were accompanied with different entropy effects, while almost no entropy change accompanied PKI[5-24] binding, suggesting that the protein flexibility was not affected in this case. Differently from the binary complex formation the ternary complex formation was accompanied by a significant entropy change and with intensive formation of new non-covalent interactions (Delta H). At the same time both ligand binding steps as well as the allosteric interaction between ligand binding sites could be described by a common entropy-enthalpy compensation plot, pointing to a similar mechanism of these phenomena. It was concluded that numerous weak interactions govern the allostery of cAMP dependent protein kinase catalytic subunit.

Glucose oxidase (GOx) is a flavoenzyme having applications in food and medical industries. However, GOx, as many other enzymes when extracted from the cells, has relatively short operational lifetimes. Several recent studies (both experimental and theoretical), carried out on small proteins (or small fractions of large proteins), show that a detailed knowledge of how the breakdown process starts and proceeds on molecular level could be of significant help to artificially improve the stability of fragile proteins. We have performed extended molecular dynamics (MD) simulations to study the denaturation of GOx (a protein dimer containing nearly 1200 amino acids) to identify weak points in its structure and in this way gather information to later make it more stable, for example, by mutations. A denaturation of a protein can be simulated by increasing the temperature far above physiological temperature. We have performed a series of MD simulations at different temperatures (300, 400, 500, and 600 K). The exit from the protein's native state has been successfully identified with the clustering method and supported by other methods used to analyze the simulation data. A common set of amino acids is regularly found to initiate the denaturation, suggesting a moiety where the enzyme could be strengthened by a suitable amino acid based modification., AuthorCount:4

The thermodynamic stability of proteins is typically measured at high denaturant concentrations and then extrapolated back to zero denaturant conditions to obtain unfolding free energies under native conditions. For membrane proteins, the extrapolations are fraught with considerable uncertainty as the denaturants may have complex effects on the membrane or micellar structure. We therefore sought to measure stability under native conditions, using a method that does not perturb the properties of the membrane or membrane mimetics. We use a technique called steric trapping to measure the thermodynamic stability of bacteriorhodopsin in bicelles and micelles. We find that bacteriorhodopsin has a high thermodynamic stability, with an unfolding free energy of ∼11 kcal/mol in dimyristoyl phosphatidylcholine bicelles. Nevertheless, the stability is much lower than predicted by extrapolation of measurements made at high denaturant concentrations. We investigated the discrepancy and found that unfolding free energy is not linear with denaturant concentration. Apparently, long extrapolations of helical membrane protein unfolding free energies must be treated with caution. Steric trapping, however, provides a method for making these measurements.

Chlamydia trachomatis is a major bacterial pathogen throughout the world. Although antibiotic therapy can be implemented in the case of early detection, a majority of the infections are asymptomatic, requiring the development of preventive measures. Efforts have focused on the production of a vaccine using the C. trachomatis major outer membrane protein (MOMP). MOMP is purified in its native (n) trimeric form using the zwitterionic detergent Z3-14, but its stability in detergent solutions is limited. Amphipols (APols) are synthetic polymers that can stabilize membrane proteins (MPs) in detergent-free aqueous solutions. Preservation of protein structure and optimization of exposure of the most effective antigenic regions can avoid vaccination with misfolded, poorly protective protein. Previously, we showed that APols maintain nMOMP secondary structure and that nMOMP/APol vaccine formulations elicit better protection than formulations using either recombinant or nMOMP solubilized in Z3-14. To achieve a greater understanding of the structural behavior and stability of nMOMP in APols, we have used several spectroscopic techniques to characterize its secondary structure (circular dichroism), tertiary and quaternary structures (immunochemistry and gel electrophoresis) and aggregation state (light scattering) as a function of temperature and time. We have also recorded NMR spectra of (15)N-labeled nMOMP and find that the exposed loops are detectable in APols but not in detergent. Our analyses show that APols protect nMOMP much better than Z3-14 against denaturation due to continuous heating, repeated freeze/thaw cycles, or extended storage at room temperature. These results indicate that APols can help improve MP-based vaccine formulations.

Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native proteins from samples containing small numbers of cells. Our approach utilizes oligodeoxythymidylate [oligo(dT)25]-coated paramagnetic beads in an optimized reaction buffer to isolate mRNA comparable in quantity and quality to mRNA isolated with existing methods, while maintaining the proteins in their native state for traditional protein assays. We validated the procedure using neonatal rat ovaries and small numbers of human granulosa cells, demonstrating the extraction of mRNA suitable for gene expression analysis with simultaneous isolation of native proteins suitable for downstream characterization using different protein assays.

Upon biological self-assembly, the number of accessible translational configurations of water in the system increases considerably, leading to a large gain in water entropy. It is important to calculate the solvation entropy of a biomolecule with a prescribed structure by accounting for the change in water–water correlations caused by solute insertion. Modeling water as a dielectric continuum is not capable of capturing the physical essence of the water entropy effect. As a reliable tool, we propose a hybrid of the angle-dependent integral equation theory combined with a multipolar water model and a morphometric approach. Using our methods wherein the water entropy effect is treated as the key factor, we can elucidate a variety of processes such as protein folding, cold, pressure, and heat denaturating of a protein, molecular recognition, ordered association of proteins such as amyloid fibril formation, and functioning of ATP-driven proteins.