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Sample records for antifreeze protein activity

  1. Protein-water dynamics in antifreeze protein III activity

    NASA Astrophysics Data System (ADS)

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

  2. Analysis of antifreeze protein activity using colorimetric gold nanosensors

    NASA Astrophysics Data System (ADS)

    Jing, Xu; Choi, Ho-seok; Park, Ji-In; Kim, Young-Pil

    2015-07-01

    High activity and long stability of antifreeze proteins (AFPs), also known as ice-binding proteins (IBPs), are necessary for exerting their physiological functions in biotechnology and cryomedicine. Here we report a simple analysis of antifreeze protein activity and stability based on self-assembly of gold nanoparticles (AuNPs) via freezing and thawing cycles. While the mercaptosuccinic acid-capped AuNP (MSA-AuNP) was easily self-assembled after a freezing/thawing cycle, due to the mechanical attack of ice crystal on the MSA-AuNP surface, the presence of AFP impeded the self-assembly of MSA-AuNP via the interaction of AFP with ice crystals via freezing and thawing cycles, which led to a strong color in the MSA-AuNP solution. As a result, the aggregation parameter (E520/E650) of MSA-AuNP showed the rapid detection of both activity and stability of AFPs. We suggest that our newly developed method is very suitable for measuring antifreeze activity and stability in a simple and rapid manner with reliable quantification.

  3. Interaction of reduced nicotinamide adenine dinucleotide with an antifreeze protein from Dendroides canadensis: mechanistic implication of antifreeze activity enhancement

    PubMed Central

    Wen, Xin; Wang, Sen; Amornwittawat, Natapol; Houghton, Eric A.; Sacco, Michael A.

    2016-01-01

    Antifreeze proteins (AFPs) found in many organisms can noncolligatively lower the freezing point of water without altering the melting point. The difference between the depressed freezing point and the melting point, termed thermal hysteresis (TH), is usually a measure of the antifreeze activity of AFPs. Certain low molecular mass molecules and proteins can further enhance the antifreeze activity of AFPs. Interaction between an enhancer and arginine is known to play an important role in enhancing the antifreeze activity of an AFP from the beetle Dendroides canadensis (DAFP-1). Here, we examined the enhancement effects of several prevalent phosphate-containing coenzymes on the antifreeze activity of DAFP-1. β-Nicotinamide adenine dinucleotide (reduced) (NADH) is identified as the most efficient enhancer of DAFP-1, which increases the antifreeze activity of DAFP-1 by around 10 times. Examination of the enhancement abilities of a series of NADH analogs and various molecular fragments of NADH reveals that the modifications of nicotinamide generate a series of highly efficient enhancers, though none as effective as NADH itself, and the whole molecular structure of NADH is necessary for its highly efficient enhancement effect. We also demonstrated a 1:1 binding between DAFP-1 and NADH. The binding was characterized by high-performance liquid chromatography (HPLC) using the gel filtration method of Hummel and Dreyer. The data analysis suggests binding between DAFP-1 and NADH with a dissociation constant in the micromolar range. Interactions between DAFP-1 and NADH are discussed along with molecular mechanisms of enhancer action. PMID:22038809

  4. Effects of polyhydroxy compounds on beetle antifreeze protein activity

    PubMed Central

    Amornwittawat, Natapol; Wang, Sen; Banatlao, Joseph; Chung, Melody; Velasco, Efrain; Duman, John G.; Wen, Xin

    2016-01-01

    Antifreeze proteins (AFPs) noncolligatively depress the nonequilibrium freezing point of a solution and produce a difference between the melting and freezing points termed thermal hysteresis (TH). Some low-molecular-mass solutes can affect the TH values. The TH enhancement effects of selected polyhydroxy compounds including polyols and carbohydrates on an AFP from the beetle Dendroides canadensis were systematically investigated using differential scanning calorimetry (DSC). The number of hydroxyl groups dominates the molar enhancement effectiveness of polyhydroxy compounds having one to five hydroxyl groups. However, the above rule does not apply for polyhydroxy compounds having more than five hydroxyl groups. The most efficient polyhydroxy enhancer identified is trehalose. In a combination of enhancers the strongest enhancer plays the major role in determining the TH enhancement. Mechanistic insights into identification of highly efficient AFP enhancers are discussed. PMID:19038370

  5. A two-dimensional adsorption kinetic model for thermal hysteresis activity in antifreeze proteins.

    PubMed

    Li, Q Z; Yeh, Y; Liu, J J; Feeney, R E; Krishnan, V V

    2006-05-28

    Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs), collectively abbreviated as AF(G)Ps, are synthesized by various organisms to enable their cells to survive in subzero environments. Although the AF(G)Ps are markedly diverse in structure, they all function by adsorbing to the surface of embryonic ice crystals to inhibit their growth. This adsorption results in a freezing temperature depression without an appreciable change in the melting temperature. The difference between the melting and freezing temperatures, termed thermal hysteresis (TH), is used to detect and quantify the antifreeze activity. Insights from crystallographic structures of a number of AFPs have led to a good understanding of the ice-protein interaction features. Computational studies have focused either on verifying a specific model of AFP-ice interaction or on understanding the protein-induced changes in the ice crystal morphology. In order to explain the origin of TH, we propose a novel two-dimensional adsorption kinetic model between AFPs and ice crystal surfaces. The validity of the model has been demonstrated by reproducing the TH curve on two different beta-helical AFPs upon increasing the protein concentration. In particular, this model is able to accommodate the change in the TH behavior observed experimentally when the size of the AFPs is increased systematically. Our results suggest that in addition to the specificity of the AFPs for the ice, the coverage of the AFPs on the ice surface is an equally necessary condition for their TH activity. PMID:16774359

  6. Conjugation of type I antifreeze protein to polyallylamine increases thermal hysteresis activity.

    PubMed

    Can, Ozge; Holland, Nolan B

    2011-10-19

    Antifreeze proteins (AFPs) are ice binding proteins found in some plants, insects, and Antarctic fish allowing them to survive at subzero temperatures by inhibiting ice crystal growth. The interaction of AFPs with ice crystals results in a difference between the freezing and melting temperatures, termed thermal hysteresis, which is the most common measure of AFP activity. Creating antifreeze protein constructs that reduce the concentration of protein needed to observe thermal hysteresis activities would be beneficial for diverse applications including cold storage of cells or tissues, ice slurries used in refrigeration systems, and food storage. We demonstrate that conjugating multiple type I AFPs to a polyallylamine chain increases thermal hysteresis activity compared to the original protein. The reaction product is approximately twice as active when compared to the same concentration of free proteins, yielding 0.5 °C thermal hysteresis activity at 0.3 mM protein concentration. More impressively, the amount of protein required to achieve a thermal hysteresis of 0.3 °C is about 100 times lower when conjugated to the polymer (3 μM) compared to free protein (300 μM). Ice crystal morphologies observed in the presence of the reaction product are comparable to those of the protein used in the conjugation reaction. PMID:21905742

  7. The thermal hysteresis activity of the type I antifreeze protein: A statistical mechanics model

    NASA Astrophysics Data System (ADS)

    Li, Li-Fen; Liang, X. X.; Li, Q. Z.

    2009-04-01

    Based on the adsorption-inhibition theory, a statistical mechanics model is proposed to investigate the thermal hysteresis activity of the type I antifreeze protein. The thermal hysteresis activity is evaluated by determining the AFP molecule coverage rate on the ice surface and the Gibbs function of the system. As examples, the calculated results for the thermal hysteresis temperatures of AFP9, HPLC-6(TTTT) and AAAA2kE as functions of the concentration of the AFP solution are obtained and discussed. The theoretical results are in agreement with the experimental data.

  8. Antifreeze proteins of the beetle Dendroides canadensis enhance one another's activities.

    PubMed

    Wang, Lei; Duman, John G

    2005-08-01

    Larvae of the beetle Dendroides canadensis produce a family of 13 antifreeze proteins (DAFPs), four of which are in the hemolymph. Antifreeze proteins lower the noncolligative freezing point of water (in the presence of ice) below the melting point, producing a difference between the freezing and melting points termed thermal hysteresis. This activity (THA) is dependent upon DAFP specific activity, concentration, and the presence of enhancers. Enhancers may be low molecular mass enhancers, such as glycerol, or other proteins. The protein enhancers complex with the DAFPs, thereby blocking a larger surface area of the potential seed ice crystal and consequently lowering the freezing point. A yeast two-hybrid screen was performed using certain hemolymph DAFPs as "bait" in an effort to identify endogenous protein enhancers. Among the positive proteins identified as interacting with the bait DAFPs, and confirmed by co-immunoprecipitation, were other DAFPs. When pure DAFPs were added to one another, those identified by the yeast two-hybrid screen as interacting with one another exhibited a synergistic enhancement of thermal hysteresis activity. In contrast, those DAFPs which the screen indicated did not interact failed to enhance one anothers' activities. DAFPs-1 and -2 interact and enhance one another. Point mutations of one of the interacting DAFPs (DAFP-2) indicated that both of the two amino acid residues that differ between DAFPs-1 and -2 were required for interaction. Glycerol enhanced the THA of the DAFPs only when DAFPs known to interact were present in the test solution. Addition of glycerol to a test solution containing only one DAFP did not produce enhancement. Therefore, glycerol enhances activity by stimulating interactions between DAFPs. PMID:16042407

  9. Re-Evaluation of a Bacterial Antifreeze Protein as an Adhesin with Ice-Binding Activity

    PubMed Central

    Guo, Shuaiqi; Garnham, Christopher P.; Whitney, John C.; Graham, Laurie A.; Davies, Peter L.

    2012-01-01

    A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide MpAFP into five distinct regions, all of which require mM Ca2+ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca2+-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice. PMID:23144980

  10. Re-evaluation of a bacterial antifreeze protein as an adhesin with ice-binding activity.

    PubMed

    Guo, Shuaiqi; Garnham, Christopher P; Whitney, John C; Graham, Laurie A; Davies, Peter L

    2012-01-01

    A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide MpAFP into five distinct regions, all of which require mM Ca(2+) levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca(2+)-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice. PMID:23144980

  11. Antifreeze proteins of teleost fishes.

    PubMed

    Fletcher, G L; Hew, C L; Davies, P L

    2001-01-01

    Marine teleosts at high latitudes can encounter ice-laden seawater that is approximately 1 degrees C colder than the colligative freezing point of their body fluids. They avoid freezing by producing small antifreeze proteins (AFPs) that adsorb to ice and halt its growth, thereby producing an additional non-colligative lowering of the freezing point. AFPs are typically secreted by the liver into the blood. Recently, however, it has become clear that AFP isoforms are produced in the epidermis (skin, scales, fin, and gills) and may serve as a first line of defense against ice propagation into the fish. The basis for the adsorption of AFPs to ice is something of a mystery and is complicated by the extreme structural diversity of the five antifreeze types. Despite the recent acquisition of several AFP three-dimensional structures and the definition of their ice-binding sites by mutagenesis, no common ice-binding motif or even theme is apparent except that surface-surface complementarity is important for binding. The remarkable diversity of antifreeze types and their seemingly haphazard phylogenetic distribution suggest that these proteins might have evolved recently in response to sea level glaciation occurring just 1-2 million years ago in the northern hemisphere and 10-30 million years ago around Antarctica. Not surprisingly, the expression of AFP genes from different origins can also be quite dissimilar. The most intensively studied system is that of the winter flounder, which has a built-in annual cycle of antifreeze expression controlled by growth hormone (GH) release from the pituitary in tune with seasonal cues. The signal transduction pathway, transcription factors, and promoter elements involved in this process are just beginning to be characterized. PMID:11181960

  12. Dendrimer-Linked Antifreeze Proteins Have Superior Activity and Thermal Recovery.

    PubMed

    Stevens, Corey A; Drori, Ran; Zalis, Shiran; Braslavsky, Ido; Davies, Peter L

    2015-09-16

    By binding to ice, antifreeze proteins (AFPs) depress the freezing point of a solution and inhibit ice recrystallization if freezing does occur. Previous work showed that the activity of an AFP was incrementally increased by fusing it to another protein. Even larger increases in activity were achieved by doubling the number of ice-binding sites by dimerization. Here, we have combined the two strategies by linking multiple outward-facing AFPs to a dendrimer to significantly increase both the size of the molecule and the number of ice-binding sites. Using a heterobifunctional cross-linker, we attached between 6 and 11 type III AFPs to a second-generation polyamidoamine (G2-PAMAM) dendrimer with 16 reactive termini. This heterogeneous sample of dendrimer-linked type III constructs showed a greater than 4-fold increase in freezing point depression over that of monomeric type III AFP. This multimerized AFP was particularly effective at ice recrystallization inhibition activity, likely because it can simultaneously bind multiple ice surfaces. Additionally, attachment to the dendrimer has afforded the AFP superior recovery from heat denaturation. Linking AFPs together via polymers can generate novel reagents for controlling ice growth and recrystallization. PMID:26267368

  13. The mechanism by which fish antifreeze proteins cause thermal hysteresis.

    PubMed

    Kristiansen, Erlend; Zachariassen, Karl Erik

    2005-12-01

    Antifreeze proteins are characterised by their ability to prevent ice from growing upon cooling below the bulk melting point. This displacement of the freezing temperature of ice is limited and at a sufficiently low temperature a rapid ice growth takes place. The separation of the melting and freezing temperature is usually referred to as thermal hysteresis, and the temperature of ice growth is referred to as the hysteresis freezing point. The hysteresis is supposed to be the result of an adsorption of antifreeze proteins to the crystal surface. This causes the ice to grow as convex surface regions between adjacent adsorbed antifreeze proteins, thus lowering the temperature at which the crystal can visibly expand. The model requires that the antifreeze proteins are irreversibly adsorbed onto the ice surface within the hysteresis gap. This presupposition is apparently in conflict with several characteristic features of the phenomenon; the absence of superheating of ice in the presence of antifreeze proteins, the dependence of the hysteresis activity on the concentration of antifreeze proteins and the different capacities of different types of antifreeze proteins to cause thermal hysteresis at equimolar concentrations. In addition, there are structural obstacles that apparently would preclude irreversible adsorption of the antifreeze proteins to the ice surface; the bond strength necessary for irreversible adsorption and the absence of a clearly defined surface to which the antifreeze proteins may adsorb. This article deals with these apparent conflicts between the prevailing theory and the empirical observations. We first review the mechanism of thermal hysteresis with some modifications: we explain the hysteresis as a result of vapour pressure equilibrium between the ice surface and the ambient fluid fraction within the hysteresis gap due to a pressure build-up within the convex growth zones, and the ice growth as the result of an ice surface nucleation event at

  14. Hofmeister Effects of Common Monovalent Salts on the Beetle Antifreeze Protein Activity

    PubMed Central

    Wang, Sen; Amornwittawat, Natapol; Banatlao, Joseph; Chung, Melody; Kao, Yu; Wen, Xin

    2009-01-01

    Antifreeze proteins (AFPs) noncolligatively depress the freezing point of a solution and produce a difference between the melting and freezing points termed thermal hysteresis (TH). While the mechanism of the enhancement effect is not well understood, various low-molecular-mass solutes including neutral salts have been identified to enhance the TH activities of AFPs. Here, the effect of monovalent salts on salting out an AFP from the beetle Dendroides canadensis (DAFP-1) on the ice was treated using a simple classical theory and the relationship between the TH activity and the salt concentration was developed. The TH activities of DAFP-1 in the presence of the series of monovalent salts were assessed by differential scanning calorimetry (DSC) and the salting-out constants of DAFP-1 by these salts were determined. This study demonstrates an indirect way to determine the salting-out constants of AFPs by these salts. The results suggest that the Hofmeister effect is a potential mechanism for the TH enhancement effects of some common monovalent salts. PMID:19778062

  15. Perturbation of bacterial ice nucleation activity by a grass antifreeze protein.

    PubMed

    Tomalty, Heather E; Walker, Virginia K

    2014-09-26

    Certain plant-associating bacteria produce ice nucleation proteins (INPs) which allow the crystallization of water at high subzero temperatures. Many of these microbes are considered plant pathogens since the formed ice can damage tissues, allowing access to nutrients. Intriguingly, certain plants that host these bacteria synthesize antifreeze proteins (AFPs). Once freezing has occurred, plant AFPs likely function to inhibit the growth of large damaging ice crystals. However, we postulated that such AFPs might also serve as defensive mechanisms against bacterial-mediated ice nucleation. Recombinant AFP derived from the perennial ryegrass Lolium perenne (LpAFP) was combined with INP preparations originating from the grass epiphyte, Pseudomonas syringae. The presence of INPs had no effect on AFP activity, including thermal hysteresis and ice recrystallization inhibition. Strikingly, the ice nucleation point of the INP was depressed up to 1.9°C in the presence of LpAFP, but a recombinant fish AFP did not lower the INP-imposed freezing point. Assays with mutant LpAFPs and the visualization of bacterially-displayed fluorescent plant AFP suggest that INP and LpAFP can interact. Thus, we postulate that in addition to controlling ice growth, plant AFPs may also function as a defensive strategy against the damaging effects of ice-nucleating bacteria. PMID:25193694

  16. Structural basis for the superior activity of the large isoform of snow flea antifreeze protein.

    PubMed

    Mok, Yee-Foong; Lin, Feng-Hsu; Graham, Laurie A; Celik, Yeliz; Braslavsky, Ido; Davies, Peter L

    2010-03-23

    The snow flea (Hypogastrum harveyi) is protected from freezing at sub-zero temperatures by a glycine-rich antifreeze protein (AFP) that binds to seed ice crystals and prevents them from growing larger. This AFP is hyperactive and comprises two isoforms [Graham, L. A., and Davies, P. L. (2005) Science 310, 461]. The larger isoform (15.7 kDa) exhibits several-fold higher activity than the smaller isoform (6.5 kDa), although it is considerably less abundant. To establish the molecular basis for this difference in activity, we determined the sequence of the large isoform. The primary sequences of these two isoforms are surprisingly divergent. However, both contain tripeptide repeats and turn motifs that enabled us to build a three-dimensional model of the large isoform based upon the six-polyproline helix structure of the small isoform. Our model contains 13 polyproline type II helices connected by proline-containing loops stacked into two flat sheets oriented antiparallel to one another. The structure is strictly amphipathic, with a hydrophilic surface on one side and a hydrophobic, putative ice-binding surface on the other. The putative ice-binding site is approximately twice as large in area as that of the small isoform, providing an explanation for the difference in activity that is consistent with other examples noted. By tagging the recombinant AFP with green fluorescent protein, we observed its binding to multiple planes of ice, especially the basal plane. This finding supports the correlation between AFP hyperactivity and basal plane binding first observed with spruce budworm AFP. PMID:20158269

  17. Coarse grained simulation reveals antifreeze properties of hyperactive antifreeze protein from Antarctic bacterium Colwellia sp.

    NASA Astrophysics Data System (ADS)

    Nguyen, Hung; Van, Thanh Dac; Le, Ly

    2015-10-01

    The novel hyperactive antifreeze protein (AFP) of Antarctic sea ice bacterium Colwellia sp. provides a target for studying the protection of psychrophilic microgoranisms against freezing environment. Interestingly, the Colwellia sp. hyperactive antifreeze protein (ColAFP) was crystallized without the structural dynamic characteristics. Here, the result indicated, through coarse grained simulation of ColAFP under various subfreezing temperature, that ColAFP remains active at temperature of equal and greater than 275 K (∼2 °C). Extensive simulation analyses also revealed the adaptive mechanism of ColAFP in subfreezing environment. Our result provides a structural dynamic understanding of the ColAFP.

  18. Structure of solvation water around the active and inactive regions of a type III antifreeze protein and its mutants of lowered activity.

    PubMed

    Grabowska, Joanna; Kuffel, Anna; Zielkiewicz, Jan

    2016-08-21

    Water molecules from the solvation shell of the ice-binding surface are considered important for the antifreeze proteins to perform their function properly. Herein, we discuss the problem whether the extent of changes of the mean properties of solvation water can be connected with the antifreeze activity of the protein. To this aim, the structure of solvation water of a type III antifreeze protein from Macrozoarces americanus (eel pout) is investigated. A wild type of the protein is used, along with its three mutants, with antifreeze activities equal to 54% or 10% of the activity of the native form. The solvation water of the ice-binding surface and the rest of the protein are analyzed separately. To characterize the structure of solvation shell, parameters describing radial and angular characteristics of the mutual arrangement of the molecules were employed. They take into account short-distance (first hydration shell) or long-distance (two solvation shells) effects. The obtained results and the comparison with the results obtained previously for a hyperactive antifreeze protein from Choristoneura fumiferana lead to the conclusion that the structure and amino acid composition of the active region of the protein evolved to achieve two goals. The first one is the modification of the properties of the solvation water. The second one is the geometrical adjustment of the protein surface to the specific crystallographic plane of ice. Both of these goals have to be achieved simultaneously in order for the protein to perform its function properly. However, they seem to be independent from one another in a sense that very small antifreeze activity does not imply that properties of water become different from the ones observed for the wild type. The proteins with significantly lower activity still modify the mean properties of solvation water in a right direction, in spite of the fact that the accuracy of the geometrical match with the ice lattice is lost because of the

  19. Structural basis for antifreeze activity of ice-binding protein from arctic yeast.

    PubMed

    Lee, Jun Hyuck; Park, Ae Kyung; Do, Hackwon; Park, Kyoung Sun; Moh, Sang Hyun; Chi, Young Min; Kim, Hak Jun

    2012-03-30

    Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of ∼25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- and 2.43-Å resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96-115 form a long α-helix that packs along one face of the β-helix), and a C-terminal hydrophobic loop region ((243)PFVPAPEVV(251)). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn(185) provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common β-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared with other known antifreeze proteins and ice-binding proteins. PMID:22303017

  20. Gold Nanoparticle Aggregation as a Probe of Antifreeze (Glyco) Protein-Inspired Ice Recrystallization Inhibition and Identification of New IRI Active Macromolecules

    NASA Astrophysics Data System (ADS)

    Mitchell, Daniel E.; Congdon, Thomas; Rodger, Alison; Gibson, Matthew I.

    2015-10-01

    Antifreeze (glyco)proteins are found in polar fish species and act to slow the rate of growth of ice crystals; a property known as ice recrystallization inhibition. The ability to slow ice growth is of huge technological importance especially in the cryopreservation of donor cells and tissue, but native antifreeze proteins are often not suitable, nor easily available. Therefore, the search for new materials that mimic this function is important, but currently limited by the low-throughout assays associated with the antifreeze properties. Here 30 nm gold nanoparticles are demonstrated to be useful colorimetric probes for ice recrystallization inhibition, giving a visible optical response and is compatible with 96 well plates for high-throughout studies. This method is faster, requires less infrastructure, and has easier interpretation than the currently used ‘splat’ methods. Using this method, a series of serum proteins were identified to have weak, but specific ice recrystallization inhibition activity, which was removed upon denaturation. It is hoped that high-throughput tools such as this will accelerate the discovery of new antifreeze mimics.

  1. Gold Nanoparticle Aggregation as a Probe of Antifreeze (Glyco) Protein-Inspired Ice Recrystallization Inhibition and Identification of New IRI Active Macromolecules

    PubMed Central

    Mitchell, Daniel E.; Congdon, Thomas; Rodger, Alison; Gibson, Matthew I.

    2015-01-01

    Antifreeze (glyco)proteins are found in polar fish species and act to slow the rate of growth of ice crystals; a property known as ice recrystallization inhibition. The ability to slow ice growth is of huge technological importance especially in the cryopreservation of donor cells and tissue, but native antifreeze proteins are often not suitable, nor easily available. Therefore, the search for new materials that mimic this function is important, but currently limited by the low-throughout assays associated with the antifreeze properties. Here 30 nm gold nanoparticles are demonstrated to be useful colorimetric probes for ice recrystallization inhibition, giving a visible optical response and is compatible with 96 well plates for high-throughout studies. This method is faster, requires less infrastructure, and has easier interpretation than the currently used ‘splat’ methods. Using this method, a series of serum proteins were identified to have weak, but specific ice recrystallization inhibition activity, which was removed upon denaturation. It is hoped that high-throughput tools such as this will accelerate the discovery of new antifreeze mimics. PMID:26499135

  2. Freeze resistance in rainbow smelt (Osmerus mordax): seasonal pattern of glycerol and antifreeze protein levels and liver enzyme activity associated with glycerol production.

    PubMed

    Lewis, Johanne M; Ewart, K Vanya; Driedzic, William R

    2004-01-01

    Rainbow smelt (Osmerus mordax) inhabit inshore waters along the North American Atlantic coast. During the winter, these waters are frequently ice covered and can reach temperatures as low as -1.9 degrees C. To prevent freezing, smelt accumulate high levels of glycerol, which lower the freezing point via colligative means, and antifreeze proteins (AFP). The up-regulation of the antifreeze response (both glycerol and AFP) occurs in early fall, when water temperatures are 5 degrees -6 degrees C. The accumulation of glycerol appears to be the main mechanism of freeze resistance in smelt because it contributes more to the lowering of the body's freezing point than the activity of the AFP (0.5 degrees C vs. 0.25 degrees C for glycerol and AFP, respectively) at a water temperature of -1.5 degrees C. Moreover, AFP in smelt appears to be a safeguard mechanism to prevent freezing when glycerol levels are low. Significant increases in activities of the liver enzymes glycerol 3-phosphate dehydrogenase (GPDH), alanine aminotransferase (AlaAT), and phosphoenolpyruvate carboxykinase (PEPCK) during the initiation of glycerol production and significant correlations between enzyme activities and plasma glycerol levels suggest that these enzymes are closely associated with the synthesis and maintenance of elevated glycerol levels for use as an antifreeze. These findings add further support to the concept that carbon for glycerol is derived from amino acids. PMID:15286915

  3. Basal Plane Affinity of an Insect Antifreeze Protein

    NASA Astrophysics Data System (ADS)

    Pertaya, N.; Gauthier, S. Y.; Davies, P. L.; Braslavsky, I.

    2007-03-01

    sbwAFP is a powerful antifreeze protein (AFP) with high thermal hysteresis activity that protects spruce budworm (sbw) from freezing during harsh winters in the spruce and fir forests of USA and Canada. Different types of antifreeze proteins have been found in many other species and have potential applications in cryomedicine and cryopreservation. When an ice crystal is cooled in the presence of AFP below the non-equilibrium freezing point the crystal will suddenly and rapidly grow in specific directions. Hyperactive antifreezes like sbwAFP expand perpendicular to the c-axis (in the plane of the a-axes), whereas moderately active AFPs, like type III from fish, grow in the direction parallel to the c-axis. It has been proposed that the basis for hyperactivity of certain AFPs is that they bind and accumulate on the basal plane to inhibit c-axial growth. By putting fluorescent tags on these two types of AFPs we have been able to directly visualize the binding of different types of AFPs to ice surfaces. We do indeed find that the insect AFP accumulates on the basal plane of an ice crystal while type III AFP does not. Supported by CIHR and BNTI.

  4. [Advances in fish antifreeze protein research].

    PubMed

    Zhong, Qi-Wang; Fan, Ting-Jun

    2002-03-01

    Antifreeze proteins (AFPs) can highly effectively protect cells and embryos from damages in freezing process by lowering the freezing points of their cytoplasmic matrix and body fluids in a noncolligative manner. Based on their origins and properties, AFPs have been classified into four types, i.e. type I, II, III and IV. Each of them possesses rather distinct characteristics both in structure and composition, although all of them have ability of lowering freezing points of fluids. AFPs' genes have been characterized as members of a multigene family and the levels of their mRNA synthesis vary significantly with seasons. Adsorption-inhibition operating at the ice surface is nowadays a hypothesis widely used to interpret the molecular mechanisms of noncolligative lowering of the freezing point, but the details of the mechanism on how the different types of AFP are adsorbed onto ice remain uncertain. Progresses in research on structures, amino acid compositions, genes, antifreeze mechanisms of the 4 distinct types of AFPs, and the application of the AFPs in cryopreservation of cells and embryos are reviewed here. PMID:12007008

  5. NMR structural studies on antifreeze proteins.

    PubMed

    Sönnichsen, F D; Davies, P L; Sykes, B D

    1998-01-01

    Antifreeze proteins (AFPs) are a structurally diverse class of proteins that bind to ice and inhibit its growth in a noncolligative manner. This adsorption-inhibition mechanism operating at the ice surface results in a lowering of the (nonequilibrium) freezing point below the melting point. A lowering of approximately 1 degree C, which is sufficient to prevent fish from freezing in ice-laden seawater, requires millimolar AFP levels in the blood. The solubility of AFPs at these millimolar concentrations and the small size of the AFPs (typically 3-15 kDa) make them ideal subjects for NMR analysis. Although fish AFPs are naturally abundant, seasonal expression, restricted access to polar fishes, and difficulties in separating numerous similar isoforms have made protein expression the method of choice for producing AFPs for structural studies. Expression of recombinant AFPs has also facilitated NMR analysis by permitting isotopic labeling with 15N and 13C and has permitted mutations to be made to help with the interpretation of NMR data. NMR analysis has recently solved two AFP structures and provided valuable information about the disposition of ice-binding side chains in a third. The potential exists to solve other AFP structures, including the newly described insect AFPs, and to use solid-state NMR techniques to address fundamental questions about the nature of the interaction between AFPs and ice. PMID:9923697

  6. Properties, potentials, and prospects of antifreeze proteins.

    PubMed

    Venketesh, S; Dayananda, C

    2008-01-01

    Antifreeze proteins (AFPs) are a group of proteins that protect organisms from deep freezing temperatures and are expressed in vertebrates, invertebrates, plants, bacteria, and fungi. The nuclear magnetic resonance, x-ray structure, and many spectroscopic studies with AFPs have been instrumental in determining the structure-function relationship. Mutational studies have indicated the importance of hydrophobic residues in ice binding. Various studies have pointed out that the mechanism of AFP action is through its adsorption on the ice surface, which leads to a curved surface, preventing further growth of ice by the "Kelvin effect." The AFPs have potential industrial, medical, and agricultural application in different fields, such as food technology, preservation of cell lines, organs, cryosurgery, and cold hardy transgenic plants and animals. However, the applications of AFPs are marred by high cost due to low yield. This review deals with the source and properties of AFPs from an angle of their application and their potential. The possibility of production using different molecular biological techniques, which will help increase the yield, is also dealt with. PMID:18322856

  7. Isolation and characterization of a novel antifreeze protein from carrot (Daucus carota).

    PubMed Central

    Smallwood, M; Worrall, D; Byass, L; Elias, L; Ashford, D; Doucet, C J; Holt, C; Telford, J; Lillford, P; Bowles, D J

    1999-01-01

    A modified assay for inhibition of ice recrystallization which allows unequivocal identification of activity in plant extracts is described. Using this assay a novel, cold-induced, 36 kDa antifreeze protein has been isolated from the tap root of cold-acclimated carrot (Daucus carota) plants. This protein inhibits the recrystallization of ice and exhibits thermal-hysteresis activity. The polypeptide behaves as monomer in solution and is N-glycosylated. The corresponding gene is unique in the carrot genome and induced by cold. The antifreeze protein appears to be localized within the apoplast. PMID:10333479

  8. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins.

    PubMed

    Olijve, Luuk L C; Meister, Konrad; DeVries, Arthur L; Duman, John G; Guo, Shuaiqi; Bakker, Huib J; Voets, Ilja K

    2016-04-01

    Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of commonly used colligative antifreezes such as salts and alcohols, the advantage of using AFPs as an additive is that they do not alter the physicochemical properties of the water-based material. Here, we report the first comprehensive evaluation of thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity of all major classes of AFPs using cryoscopy, sonocrystallization, and recrystallization assays. The results show that TH activities determined by cryoscopy and sonocrystallization differ markedly, and that TH and IRI activities are not correlated. The absence of a distinct correlation in antifreeze activity points to a mechanistic difference in ice growth inhibition by the different classes of AFPs: blocking fast ice growth requires rapid nonbasal plane adsorption, whereas basal plane adsorption is only relevant at long annealing times and at small undercooling. These findings clearly demonstrate that biomimetic analogs of antifreeze (glyco)proteins should be tailored to the specific requirements of the targeted application. PMID:26936953

  9. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins

    PubMed Central

    Olijve, Luuk L. C.; Meister, Konrad; DeVries, Arthur L.; Duman, John G.; Guo, Shuaiqi; Bakker, Huib J.; Voets, Ilja K.

    2016-01-01

    Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of commonly used colligative antifreezes such as salts and alcohols, the advantage of using AFPs as an additive is that they do not alter the physicochemical properties of the water-based material. Here, we report the first comprehensive evaluation of thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity of all major classes of AFPs using cryoscopy, sonocrystallization, and recrystallization assays. The results show that TH activities determined by cryoscopy and sonocrystallization differ markedly, and that TH and IRI activities are not correlated. The absence of a distinct correlation in antifreeze activity points to a mechanistic difference in ice growth inhibition by the different classes of AFPs: blocking fast ice growth requires rapid nonbasal plane adsorption, whereas basal plane adsorption is only relevant at long annealing times and at small undercooling. These findings clearly demonstrate that biomimetic analogs of antifreeze (glyco)proteins should be tailored to the specific requirements of the targeted application. PMID:26936953

  10. Identification and Evaluation of Cryoprotective Peptides from Chicken Collagen: Ice-Growth Inhibition Activity Compared to That of Type I Antifreeze Proteins in Sucrose Model Systems.

    PubMed

    Du, Lihui; Betti, Mirko

    2016-06-29

    The ability of chicken collagen peptides to inhibit the growth of ice crystals was evaluated and compared to that of fish antifreeze proteins (AFPs). This ice inhibition activity was assessed using a polarized microscope by measuring ice crystal dimensions in a sucrose model system with and without collagen peptides after seven thermal cycles. The system was stabilized at -25 °C and cycled between -16 and -12 °C. Five candidate peptides with ice inhibition activity were identified using liquid chromatography and tandem mass spectrometry and were then synthesized. Their ice inhibition capacity was compared to that of type I AFPs in a 23% sucrose model system. Specific collagen peptides with certain amino acid sequences reduced the extent of ice growth by approximately 70% at a relatively low concentration (1 mg/mL). These results suggest that specific collagen peptides may act in a noncolligative manner, inhibiting ice crystal growth like type I AFPs, but less efficiently. PMID:27293017

  11. Antifreeze Proteins in Winter Rye Leaves Form Oligomeric Complexes1

    PubMed Central

    Yu, Xiao-Ming; Griffith, Marilyn

    1999-01-01

    Antifreeze proteins (AFPs) similar to three pathogenesis-related proteins, a glucanase-like protein (GLP), a chitinase-like protein (CLP), and a thaumatin-like protein (TLP), accumulate during cold acclimation in winter rye (Secale cereale) leaves, where they are thought to modify the growth of intercellular ice during freezing. The objective of this study was to characterize the rye AFPs in their native forms, and our results show that these proteins form oligomeric complexes in vivo. Nine proteins were separated by native-polyacrylamide gel electrophoresis from apoplastic extracts of cold-acclimated winter rye leaves. Seven of these proteins exhibited multiple polypeptides when denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After isolation of the individual proteins, six were shown by immunoblotting to contain various combinations of GLP, CLP, and TLP in addition to other unidentified proteins. Antisera produced against individual cold-induced winter rye GLP, CLP, and TLP all dramatically inhibited glucanase activity in apoplastic extracts from cold-acclimated winter rye leaves, and each antiserum precipitated all three proteins. These results indicate that each of the polypeptides may be exposed on the surface of the protein complexes. By forming oligomeric complexes, AFPs may form larger surfaces to interact with ice, or they may simply increase the mass of the protein bound to ice. In either case, the complexes of AFPs may inhibit ice growth and recrystallization more effectively than the individual polypeptides. PMID:10198095

  12. Antifreeze proteins in winter rye are similar to pathogenesis-related proteins.

    PubMed Central

    Hon, W C; Griffith, M; Mlynarz, A; Kwok, Y C; Yang, D S

    1995-01-01

    The ability to control extracellular ice formation during freezing is critical to the survival of freezing-tolerant plants. Antifreeze proteins, which are proteins that have the ability to retard ice crystal growth, were recently identified as the most abundant apoplastic proteins in cold-acclimated winter rye (Secale cereale L.) leaves. In the experiments reported here, amino-terminal sequence comparisons, immuno-cross-reactions, and enzyme activity assays all indicated that these antifreeze proteins are similar to members of three classes of pathogenesis-related proteins, namely, endochitinases, endo-beta-1,3-glucanases, and thaumatin-like proteins. Apoplastic endochitinases and endo-beta-1,3-glucanases that were induced by pathogens in freezing-sensitive tobacco did not exhibit antifreeze activity. Our findings suggest that subtle structural differences may have evolved in the pathogenesis-related proteins that accumulate at cold temperatures in winter rye to confer upon these proteins the ability to bind to ice. PMID:8552719

  13. Immunolocalization of Antifreeze Proteins in Winter Rye Leaves, Crowns, and Roots by Tissue Printing.

    PubMed Central

    Antikainen, M.; Griffith, M.; Zhang, J.; Hon, W. C.; Yang, DSC.; Pihakaski-Maunsbach, K.

    1996-01-01

    During cold acclimation, antifreeze proteins (AFPs) that are similar to pathogenesis-related proteins accumulate in the apoplast of winter rye (Secale cereale L. cv Musketeer) leaves. AFPs have the ability to modify the growth of ice. To elucidate the role of AFPs in the freezing process, they were assayed and immunolocalized in winter rye leaves, crowns, and roots. Each of the total soluble protein extracts from cold-acclimated rye leaves, crowns, and roots exhibited antifreeze activity, whereas no antifreeze activity was observed in extracts from nonacclimated rye plants. Antibodies raised against three apoplastic rye AFPs, corresponding to a glucanase-like protein (GLP, 32 kD), a chitinase-like protein (CLP, 35 kD), and a thaumatin-like protein (TLP, 25 kD), were used in tissue printing to show that the AFPs are localized in the epidermis and in cells surrounding intercellular spaces in cold-acclimated plants. Although GLPs, CLPs, and TLPs were present in nonacclimated plants, they were found in different locations and did not exhibit antifreeze activity, which suggests that different isoforms of pathogenesis-related proteins are produced at low temperature. The location of rye AFPs may prevent secondary nucleation of cells by epiphytic ice or by ice propagating through the xylem. The distributions of pathogenesis-induced and cold-accumulated GLPs, CLPs, and TLPs are similar and may reflect the common pathways by which both pathogens and ice enter and propagate through plant tissues. PMID:12226223

  14. Interfacial adsorption of antifreeze proteins: a neutron reflection study.

    PubMed

    Xu, Hai; Perumal, Shiamalee; Zhao, Xiubo; Du, Ning; Liu, Xiang-Yang; Jia, Zongchao; Lu, Jian R

    2008-06-01

    Interfacial adsorption from two antifreeze proteins (AFP) from ocean pout (Macrozoarces americanus, type III AFP, AFP III, or maAFP) and spruce budworm (Choristoneura fumiferana, isoform 501, or cfAFP) were studied by neutron reflection. Hydrophilic silicon oxide was used as model substrate to facilitate the solid/liquid interfacial measurement so that the structural features from AFP adsorption can be examined. All adsorbed layers from AFP III could be modeled into uniform layer distribution assuming that the protein molecules were adsorbed with their ice-binding surface in direct contact with the SiO(2) substrate. The layer thickness of 32 A was consistent with the height of the molecule in its crystalline form. With the concentration decreasing from 2 mg/ml to 0.01 mg/ml, the volume fraction of the protein packed in the monolayer decreased steadily from 0.4 to 0.1, consistent with the concentration-dependent inhibition of ice growth observed over the range. In comparison, insect cfAFP showed stronger adsorption over the same concentration range. Below 0.1 mg/ml, uniform layers were formed. But above 1 mg/ml, the adsorbed layers were characterized by a dense middle layer and two outer diffuse layers, with a total thickness around 100 A. The structural transition indicated the responsive changes of conformational orientation to increasing surface packing density. As the higher interfacial adsorption of cfAFP was strongly correlated with the greater thermal hysteresis of spruce budworm, our results indicated the important relation between protein adsorption and antifreeze activity. PMID:18234809

  15. Unusual structural properties of water within the hydration shell of hyperactive antifreeze protein.

    PubMed

    Kuffel, Anna; Czapiewski, Dariusz; Zielkiewicz, Jan

    2014-08-01

    Many hypotheses can be encountered explaining the mechanism of action of antifreeze proteins. One widespread theory postulates that the similarity of structural properties of solvation water of antifreeze proteins to ice is crucial to the antifreeze activity of these agents. In order to investigate this problem, the structural properties of solvation water of the hyperactive antifreeze protein from Choristoneura fumiferana were analyzed and compared with the properties of solvation water present at the surface of ice. The most striking observations concerned the temperature dependence of changes in water structure. In the case of solvation water of the ice-binding plane, the difference between the overall structural ordering of solvation water and bulk water diminished with increasing temperature; in the case of solvation water of the rest of the protein, the trend was opposite. In this respect, the solvation water of the ice-binding plane roughly resembled the hydration layer of ice. Simultaneously, the whole solvation shell of the protein displayed some features that are typical for solvation shells of many other proteins and are not encountered in the solvation water of ice. In the first place, this is an increase in density of water around the protein. The opposite is true for the solvation water of ice - it is less dense than bulk water. Therefore, even though the structure of solvation water of ice-binding plane and the structure of solvation water of ice seem to share some similarities, densitywise they differ. PMID:25106616

  16. Anti-virulence properties of an antifreeze protein

    PubMed Central

    Heisig, Martin; Abraham, Nabil M.; Liu, Lei; Neelakanta, Girish; Mattessich, Sarah; Sultana, Hameeda; Shang, Zhengling; Ansari, Juliana M.; Killiam, Charlotte; Walker, Wendy; Cooley, Lynn; Flavell, Richard A.; Agaisse, Herve; Fikrig, Erol

    2014-01-01

    Summary As microbial drug-resistance increases, there is a critical need for new classes of compounds to combat infectious diseases. The Ixodes scapularis tick antifreeze glycoprotein, IAFGP, functions as an anti-virulence agent against diverse bacteria including methicillin-resistant Staphylococcus aureus. Recombinant IAFGP and a peptide, P1, derived from this protein bind to microbes and alter biofilm formation. Transgenic iafgp-expressing flies and mice challenged with bacteria, as well as wild-type animals administered P1, were resistant to infection, septic shock, or biofilm development on implanted catheter tubing. These data show that an antifreeze protein facilitates host control of bacterial infections and suggest new therapeutic strategies to counter pathogens. PMID:25373896

  17. Function of the hydration layer around an antifreeze protein revealed by atomistic molecular dynamics simulations

    SciTech Connect

    Nutt, David; Smith, Jeremy C

    2008-10-01

    Atomistic molecular dynamics simulations are used to investigate the mechanism by which the antifreeze protein from the spruce budworm, Choristoneura fumiferana, binds to ice. Comparison of structural and dynamic properties of the water around the three faces of the triangular prism-shaped protein in aqueous solution reveals that at low temperature the water structure is ordered and the dynamics slowed down around the ice-binding face of the protein, with a disordering effect observed around the other two faces. These results suggest a dual role for the solvation water around the protein. The preconfigured solvation shell around the ice-binding face is involved in the initial recognition and binding of the antifreeze protein to ice by lowering the barrier for binding and consolidation of the protein:ice interaction surface. Thus, the antifreeze protein can bind to the molecularly rough ice surface by becoming actively involved in the formation of its own binding site. Also, the disruption of water structure around the rest of the protein helps prevent the adsorbed protein becoming covered by further ice growth.

  18. Dynamical mechanism of antifreeze proteins to prevent ice growth

    NASA Astrophysics Data System (ADS)

    Kutschan, B.; Morawetz, K.; Thoms, S.

    2014-08-01

    The fascinating ability of algae, insects, and fishes to survive at temperatures below normal freezing is realized by antifreeze proteins (AFPs). These are surface-active molecules and interact with the diffusive water-ice interface thus preventing complete solidification. We propose a dynamical mechanism on how these proteins inhibit the freezing of water. We apply a Ginzburg-Landau-type approach to describe the phase separation in the two-component system (ice, AFP). The free-energy density involves two fields: one for the ice phase with a low AFP concentration and one for liquid water with a high AFP concentration. The time evolution of the ice reveals microstructures resulting from phase separation in the presence of AFPs. We observed a faster clustering of pre-ice structure connected to a locking of grain size by the action of AFP, which is an essentially dynamical process. The adsorption of additional water molecules is inhibited and the further growth of ice grains stopped. The interfacial energy between ice and water is lowered allowing the AFPs to form smaller critical ice nuclei. Similar to a hysteresis in magnetic materials we observe a thermodynamic hysteresis leading to a nonlinear density dependence of the freezing point depression in agreement with the experiments.

  19. Dynamical mechanism of antifreeze proteins to prevent ice growth.

    PubMed

    Kutschan, B; Morawetz, K; Thoms, S

    2014-08-01

    The fascinating ability of algae, insects, and fishes to survive at temperatures below normal freezing is realized by antifreeze proteins (AFPs). These are surface-active molecules and interact with the diffusive water-ice interface thus preventing complete solidification. We propose a dynamical mechanism on how these proteins inhibit the freezing of water. We apply a Ginzburg-Landau-type approach to describe the phase separation in the two-component system (ice, AFP). The free-energy density involves two fields: one for the ice phase with a low AFP concentration and one for liquid water with a high AFP concentration. The time evolution of the ice reveals microstructures resulting from phase separation in the presence of AFPs. We observed a faster clustering of pre-ice structure connected to a locking of grain size by the action of AFP, which is an essentially dynamical process. The adsorption of additional water molecules is inhibited and the further growth of ice grains stopped. The interfacial energy between ice and water is lowered allowing the AFPs to form smaller critical ice nuclei. Similar to a hysteresis in magnetic materials we observe a thermodynamic hysteresis leading to a nonlinear density dependence of the freezing point depression in agreement with the experiments. PMID:25215762

  20. Ice-binding mechanism of winter flounder antifreeze proteins.

    PubMed Central

    Cheng, A; Merz, K M

    1997-01-01

    We have studied the winter flounder antifreeze protein (AFP) and two of its mutants using molecular dynamics simulation techniques. The simulations were performed under four conditions: in the gas phase, solvated by water, adsorbed on the ice (2021) crystal plane in the gas phase and in aqueous solution. This study provided details of the ice-binding pattern of the winter flounder AFP. Simulation results indicated that the Asp, Asn, and Thr residues in the AFP are important in ice binding and that Asn and Thr as a group bind cooperatively to the ice surface. These ice-binding residues can be collected into four distinct ice-binding regions: Asp-1/Thr-2/Asp-5, Thr-13/Asn-16, Thr-24/Asn-27, and Thr-35/Arg-37. These four regions are 11 residues apart and the repeat distance between them matches the ice lattice constant along the (1102) direction. This match is crucial to ensure that all four groups can interact with the ice surface simultaneously, thereby, enhancing ice binding. These Asx (x = p or n)/Thr regions each form 5-6 hydrogen bonds with the ice surface: Asn forms about three hydrogen bonds with ice molecules located in the step region while Thr forms one to two hydrogen bonds with the ice molecules in the ridge of the (2021) crystal plane. Both the distance between Thr and Asn and the ordering of the two residues are crucial for effective ice binding. The proper sequence is necessary to generate a binding surface that is compatible with the ice surface topology, thus providing a perfect "host/guest" interaction that simultaneously satisfies both hydrogen bonding and van der Waals interactions. The results also show the relation among binding energy, the number of hydrogen bonds, and the activity. The activity is correlated to the binding energy, and in the case of the mutants we have studied the number of hydrogen bonds. The greater the number of the hydrogen bonds the greater the antifreeze activity. The roles van der Waals interactions and the hydrophobic

  1. Variation in blood serum antifreeze activity of Antarctic Trematomus fishes across habitat temperature and depth.

    PubMed

    Fields, Lauren G; DeVries, Arthur L

    2015-07-01

    High latitude waters in the Southern Ocean can be near their freezing point and remain ice-covered throughout the year whereas lower latitude Southern Ocean waters have seasonal ice coverage and comparatively large (6 °C) annual temperature changes. The genus Trematomus (suborder Notothenioidei) is regarded primarily as a high latitude group because of its abundance there, they also inhabit the warmer regions in smaller numbers. Freeze avoidance in the notothenioids is linked to the presence of two antifreeze proteins (AFPs); the antifreeze glycoproteins (AFGPs) and antifreeze potentiating protein (AFPP), both of which adsorb to internal ice crystals inhibiting growth. Both high and low latitude trematomids possess sufficient AFP to lower their blood freezing point below that of seawater (-1.9 °C). We investigated the contributions of AFGPs and AFPP to the blood freezing point depression to determine how they varied with depth, water temperature, and the presence of ice. High latitude trematomids had lower blood freezing points than those inhabiting lower latitude waters indicating differences in their freeze avoidance capacities. Lower freezing points were associated with higher levels of antifreeze activity due to higher levels of both AFGP and AFPP. Populations of Trematomus hansoni and Trematomus bernacchii from shallow depths appear more freeze avoidant than populations inhabiting deep, ice-free water based on their lower freezing points and higher antifreeze activities. Gel electrophoresis of the trichloroacetic acid-soluble AFGPs indicates that only high molecular weight isoforms, which contribute more to AFGP activity, vary across species as well as between individuals of a species. PMID:25770668

  2. Local water dynamics around antifreeze protein residues in the presence of osmolytes: the importance of hydroxyl and disaccharide groups.

    PubMed

    Krishnamoorthy, Anand Narayanan; Holm, Christian; Smiatek, Jens

    2014-10-01

    Antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP) are synthesized by various organisms to enable their cells to survive low temperature environments like in the polar regions. The presence of antifreeze proteins leads to a temperature difference between the melting and freezing point of the solution known as thermal hysteresis. It is nowadays common knowledge that the antifreeze activity of AFPs is mainly determined by a short-range effect which includes a direct binding to the ice phase. Recently, experimental findings also revealed a long-range effect which implies a significant retardation of the water dynamics to facilitate the ice-binding process specifically for AFGPs. The aim of this work is to examine the dynamics of water molecules around different antifreeze protein residues by using atomistic molecular dynamics simulations. A prototype of AFP from antarctic notothenioids with the main subunit alanine-alanine-threonine (AAT) and a mutant (polyalanine) together with the residues of an antifreeze glycoprotein (AFGP) were simulated and compared with respect to their influence on the local water shell. The analysis of the water hydrogen bond characteristics and the dipolar relaxation times reveals a strong retardation effect of the water dynamics around the AFGP prototype. Our numerical results reveal the significant importance of polar units like threonine and disaccharides for the direct binding of water molecules in terms of hydrogen bonds and a significant retardation of water dynamics. In addition, a considerable change of the hydration dynamics is additionally observed in the presence of osmolytes like urea and hydroxyectoine. Our findings indicate that this effect is even more pronounced in the presence of kosmotropic osmolytes. PMID:25207443

  3. Unusual dynamic properties of water near the ice-binding plane of hyperactive antifreeze protein

    SciTech Connect

    Kuffel, Anna; Czapiewski, Dariusz; Zielkiewicz, Jan

    2015-10-07

    The dynamical properties of solvation water of hyperactive antifreeze protein from Choristoneura fumiferana (CfAFP) are analyzed and discussed in context of its antifreeze activity. The protein comprises of three well-defined planes and one of them binds to the surface of ice. The dynamical properties of solvation water around each of these planes were analyzed separately; the results are compared with the dynamical properties of solvation water of ice around its two crystallographic planes: basal and prism. Three main conclusions are inferred from our investigations. The first one is that the solvation shell of CfAFP does not seem to be particularly far-ranged, at least not beyond what is usually observed for proteins that do not interact with ice. Therefore, it does not appear to us that the antifreeze activity is enhanced by a long-ranged retardation of water mobility. Also the correlation between the collective mobility of water and the collective mobility of protein atoms highly resembles the one measured for the protein that does not interact with ice. Our second conclusion is that the dynamical properties of solvation water of CfAFP are non-uniform. The dynamics of solvation water of ice-binding plane is, in some respects, different from the dynamics of solvation water of the two remaining planes. The feature that distinguishes the dynamics of solvation water of the three planes is the activation energy of diffusion process. The third conclusion is that—from the three analyzed solvation shells of CfAFP—the dynamical properties of solvation water of the ice-binding plane resemble the most the properties of solvation water of ice; note, however, that these properties still clearly differ from the dynamic properties of solvation water of ice.

  4. Animal ice-binding (antifreeze) proteins and glycolipids: an overview with emphasis on physiological function.

    PubMed

    Duman, John G

    2015-06-01

    Ice-binding proteins (IBPs) assist in subzero tolerance of multiple cold-tolerant organisms: animals, plants, fungi, bacteria etc. IBPs include: (1) antifreeze proteins (AFPs) with high thermal hysteresis antifreeze activity; (2) low thermal hysteresis IBPs; and (3) ice-nucleating proteins (INPs). Several structurally different IBPs have evolved, even within related taxa. Proteins that produce thermal hysteresis inhibit freezing by a non-colligative mechanism, whereby they adsorb onto ice crystals or ice-nucleating surfaces and prevent further growth. This lowers the so-called hysteretic freezing point below the normal equilibrium freezing/melting point, producing a difference between the two, termed thermal hysteresis. True AFPs with high thermal hysteresis are found in freeze-avoiding animals (those that must prevent freezing, as they die if frozen) especially marine fish, insects and other terrestrial arthropods where they function to prevent freezing at temperatures below those commonly experienced by the organism. Low thermal hysteresis IBPs are found in freeze-tolerant organisms (those able to survive extracellular freezing), and function to inhibit recrystallization - a potentially damaging process whereby larger ice crystals grow at the expense of smaller ones - and in some cases, prevent lethal propagation of extracellular ice into the cytoplasm. Ice-nucleator proteins inhibit supercooling and induce freezing in the extracellular fluid at high subzero temperatures in many freeze-tolerant species, thereby allowing them to control the location and temperature of ice nucleation, and the rate of ice growth. Numerous nuances to these functions have evolved. Antifreeze glycolipids with significant thermal hysteresis activity were recently identified in insects, frogs and plants. PMID:26085662

  5. Perturbation of long-range water dynamics as the mechanism for the antifreeze activity of antifreeze glycoprotein.

    PubMed

    Mallajosyula, Sairam S; Vanommeslaeghe, Kenno; MacKerell, Alexander D

    2014-10-01

    Very little is known about the mechanism of antifreeze action of antifreeze glycoproteins (AFGPs) present in Antarctic teleost fish. Recent NMR and CD studies assisted with total synthesis of synthetic AFGP variants have provided insight into the structure of short AFGP glycopeptides, though the observations did not yield information on the antifreeze mechanism of action. In this study, we use Hamiltonian replica exchange (HREX) molecular dynamics simulations to probe the structure and surrounding aqueous environments of both the natural (AFGP8) and synthetic (s-AFGP4) AFGPs. AFGPs can adopt both amphiphilic and pseudoamphiphilic conformations, the preference of which is related to the proline content of the peptide. The arrangement of carbohydrates allows the hydroxyl groups on terminal galactose units to form stable water bridges which in turn influence the hydrogen-bond network, structure, and dynamics of the surrounding solvent. Interestingly, these local effects lead to the perturbation of the tetrahedral environment for water molecules in hydration layers far (10.0-12.0 Å) from the AFGPs. This structure-induced alteration of long-range hydration dynamics is proposed to be the major contributor to antifreeze activity, a conclusion that is in line with terahertz spectroscopy experiments. The detailed structure-mechanism correlation provided in this study could lead to the design of better synthetic AFGP variants. PMID:25137353

  6. When are antifreeze proteins in solution essential for ice growth inhibition?

    PubMed

    Drori, Ran; Davies, Peter L; Braslavsky, Ido

    2015-06-01

    Antifreeze proteins (AFPs) are a widespread class of proteins that bind to ice and facilitate the survival of organisms under freezing conditions. AFPs have enormous potential in applications that require control over ice growth. However, the nature of the binding interaction between AFPs and ice remains the subject of debate. Using a microfluidics system developed in-house we previously showed that hyperactive AFP from the Tenebrio molitor beetle, TmAFP, remains bound to an ice crystal surface after exchanging the solution surrounding the ice crystal to an AFP-free solution. Furthermore, these surface-adsorbed TmAFP molecules sufficed to prevent ice growth. These experiments provided compelling evidence for the irreversible binding of hyperactive AFPs to ice. Here, we tested a moderately active type III AFP (AFPIII) from a fish in a similar microfluidics system. We found, in solution exchange experiments that the AFPIIIs were also irreversibly bound to the ice crystals. However, some crystals displayed "burst" growth during the solution exchange. AFPIII, like other moderately active fish AFPs, is unable to bind to the basal plane of an ice crystal. We showed that although moderate AFPs bound to ice irreversibly, moderate AFPs in solution were needed to inhibit ice growth from the bipyramidal crystal tips. Instead of binding to the basal plane, these AFPs minimized the basal face size by stabilizing other crystal planes that converge to form the crystal tips. Furthermore, when access of solution to the basal plane was physically blocked by the microfluidics device walls, we observed enhancement of the antifreeze activity. These findings provide direct evidence that the weak point of ice growth inhibition by fish AFPs is the basal plane, whereas insect AFPs, which can bind to the basal plane, are able to inhibit its growth and thereby increase antifreeze activity. PMID:25946514

  7. Solvation structure of ice-binding antifreeze proteins

    NASA Astrophysics Data System (ADS)

    Hansen-Goos, Hendrik; Wettlaufer, John

    2009-03-01

    Antifreeze proteins (AFPs) can be found in organisms which survive at subzero temperatures. They were first discovered in polar fishes since the 1950's [1] and have been isolated meanwhile also from insects, plants, and bacteria. While AFPs shift the freezing point of water below the bulk melting point and hence can prevent recrystallization; the effect is non-colligative and there is a pronounced hysteresis between freezing and melting. For many AFPs it is generally accepted that they function through an irreversible binding to the ice-water interface which leads to a piecewise convex growth front with a lower nonequilibrium freezing point due to the Kelvin effect. Recent molecular dynamics simulations of the AFP from Choristoneura fumiferana reveal that the solvation structures of water at ice-binding and non-ice-binding faces of the protein are crucial for understanding how the AFP binds to the ice surface and how it is protected from being overgrown [2]. We use density functional theory of classical fluids in order to assess the microscopic solvent structure in the vicinity of protein faces with different surface properties. With our method, binding energies of different protein faces to the water-ice-interface can be computed efficiently in a simplified model. [1] Y. Yeh and R.E. Feeney, Chem. Rev. 96, 601 (1996). [2] D.R. Nutt and J.C. Smith, J. Am. Chem. Soc. 130, 13066 (2008).

  8. Use of proline mutants to help solve the NMR solution structure of type III antifreeze protein.

    PubMed Central

    Chao, H.; Davies, P. L.; Sykes, B. D.; Sönnichsen, F. D.

    1993-01-01

    To help understand the structure/function relationships in antifreeze proteins (AFP), and to define the motifs required for ice binding, a Type III AFP suitable for two-dimensional (2D) NMR studies was produced in Escherichia coli. A synthetic gene for one of the Type III AFP isoforms was assembled in a T7 polymerase-directed expression vector. The 67-amino acid-long gene product differed from the natural AFP by inclusion of an N-terminal methionine but was indistinguishable in activity. The NMR spectra of this AFP were complicated by cis-trans proline isomerization from the C-terminal sequence YPPA. Substitution of this sequence by YAA eliminated isomer signals without altering the activity or structure of the mutant AFP. This variant (rQAE m1.1) was selected for sequential assignment and the secondary structure determination using 2D 1H NMR spectroscopy. Nine beta-strands are paired to form two triple-stranded antiparallel sheets and one double-stranded antiparallel sheet. Two further proline replacements, P29A and P33A, were made to delineate the role of conserved prolines in Type III AFP. These mutants were valuable in clarifying ambiguous NMR spectral assignments amongst the remaining six prolines of rQAE m1.1. In contrast to the replacement of the C-terminal prolyl residues, the exchange of P29 and P33 caused some structural changes and significantly decreased protein solubility and antifreeze activity. PMID:8401227

  9. Anchored Clathrate Waters Bind Antifreeze Proteins to Ice

    SciTech Connect

    C Garnham; R Campbell; P Davies

    2011-12-31

    The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, the largest AFP structure determined to date, folds as a Ca{sup 2+}-bound parallel beta-helix with an extensive array of ice-like surface waters that are anchored via hydrogen bonds directly to the polypeptide backbone and adjacent side chains. These bound waters make an excellent three-dimensional match to both the primary prism and basal planes of ice and in effect provide an extensive X-ray crystallographic picture of the AFP{vert_ellipsis}ice interaction. This unobstructed view, free from crystal-packing artefacts, shows the contributions of both the hydrophobic effect and hydrogen bonding during AFP adsorption to ice. We term this mode of binding the 'anchored clathrate' mechanism of AFP action.

  10. Investigations of structure and dynamics of water solvation of the type I antifreeze protein

    NASA Astrophysics Data System (ADS)

    Cui, Jun; Battle, Keith; Wierzbicki, Andrzej; Madura, Jeffry D.

    We use molecular dynamics simulations to study the water structure and dynamics around the winter flounder antifreeze protein (AFP) and its two mutant forms in which the four key threonine residues of the winter flounder AFP are mutated to alanines and serines, respectively. The TIP4P-Ew water model is used to better describe the water interactions and water structure; all simulations are performed at 245.5 K, a temperature near the freezing point of the TIP4P-Ew water model. Analysis of structural and dynamic properties of the water around the threonines in the winter flounder AFP reveals that the water structure is ordered around the threonine residues, especially in the second-solvation shell. Alanine and serine mutations instead promote water hydration in the first-solvation shell. Also our calculations show that in the close vicinity of the threonine residues of the wild-type AFP, the mobility of water molecules is substantially decreased. A smaller effect is observed for the weakly active alanine-substituted mutant, and no effect is observed for the inactive serine-substituted mutant. The results of this study suggest that water ordering and immobilization play important roles in the recognition and adsorption of the antifreeze protein to ice.

  11. Induced ice melting by the snow flea antifreeze protein from molecular dynamics simulations.

    PubMed

    Todde, Guido; Whitman, Christopher; Hovmöller, Sven; Laaksonen, Aatto

    2014-11-26

    Antifreeze proteins (AFP) allow different life forms, insects as well as fish and plants, to survive in subzero environments. AFPs prevent freezing of the physiological fluids. We have studied, through molecular dynamics simulations, the behavior of the small isoform of the AFP found in the snow flea (sfAFP), both in water and at the ice/water interface, of four different ice planes. In water at room temperature, the structure of the sfAFP is found to be slightly unstable. The loop between two polyproline II helices has large fluctuations as well as the C-terminus. Torsional angle analyses show a decrease of the polyproline II helix area in the Ramachandran plots. The protein structure instability, in any case, should not affect its antifreeze activity. At the ice/water interface the sfAFP triggers local melting of the ice surface. Bipyramidal, secondary prism, and prism ice planes melt in the presence of AFP at temperatures below the melting point of ice. Only the basal plane is found to be stable at the same temperatures, indicating an adsorption of the sfAFP on this ice plane as confirmed by experimental evidence. PMID:25353109

  12. The biological function of an insect antifreeze protein simulated by molecular dynamics

    PubMed Central

    Kuiper, Michael J; Morton, Craig J; Abraham, Sneha E; Gray-Weale, Angus

    2015-01-01

    Antifreeze proteins (AFPs) protect certain cold-adapted organisms from freezing to death by selectively adsorbing to internal ice crystals and inhibiting ice propagation. The molecular details of AFP adsorption-inhibition is uncertain but is proposed to involve the Gibbs–Thomson effect. Here we show by using unbiased molecular dynamics simulations a protein structure-function mechanism for the spruce budworm Choristoneura fumiferana AFP, including stereo-specific binding and consequential melting and freezing inhibition. The protein binds indirectly to the prism ice face through a linear array of ordered water molecules that are structurally distinct from the ice. Mutation of the ice binding surface disrupts water-ordering and abolishes activity. The adsorption is virtually irreversible, and we confirm the ice growth inhibition is consistent with the Gibbs–Thomson law. DOI: http://dx.doi.org/10.7554/eLife.05142.001 PMID:25951514

  13. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    PubMed

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-01

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters. PMID:26371748

  14. Creating Anti-icing Surfaces via the Direct Immobilization of Antifreeze Proteins on Aluminum

    PubMed Central

    Gwak, Yunho; Park, Ji-in; Kim, Minjae; Kim, Hong Suk; Kwon, Myong Jong; Oh, Seung Jin; Kim, Young-Pil; Jin, EonSeon

    2015-01-01

    Cryoprotectants such as antifreeze proteins (AFPs) and sugar molecules may provide a solution for icing problems. These anti-icing substances protect cells and tissues from freezing by inhibiting ice formation. In this study, we developed a method for coating an industrial metal material (aluminum, Al) with AFP from the Antarctic marine diatom, Chaetoceros neogracile (Cn-AFP), to prevent or delay ice formation. To coat Al with Cn-AFP, we used an Al-binding peptide (ABP) as a conjugator and fused it with Cn-AFP. The ABP bound well to the Al and did not considerably change the functional properties of AFP. Cn-AFP-coated Al (Cn-AFP-Al) showed a sufficiently low supercooling point. Additional trehalose coating of Cn-AFP-Al considerably delayed AFP denaturation on the Al without affecting its antifreeze activity. This metal surface–coating method using trehalose-fortified AFP can be applied to other metals important in the aircraft and cold storage fields where anti-icing materials are critical. PMID:26153855

  15. Fluorescence microscopy studies of the hyperactive antifreeze protein from an insect

    NASA Astrophysics Data System (ADS)

    Pertaya, N.; di Prinzio, C. L.; Wilen, L.; Thomson, E.; Wettlaufer, J. S.; Marshall, C. B.; Davies, P. L.; Braslavsky, I.

    2006-03-01

    Antifreeze proteins (AFPs) protect animals from freezing by binding to extracellular ice and inhibiting its growth. Since the initial discovery of AFPs in fish, non-homologous types have been found in insects, plants, bacteria, fungi, and vertebrates. Different AFP types have diverse structures and varied activities. For example, AFPs produced by insects are much more active in inhibiting ice crystal growth compared to most AFPs found in fish or plants. By putting a fluorescent tag on an insect AFP we were able to visualize AFP binding to ice, to determine the ice crystal surfaces to which the AFP adheres, and to follow the kinetics of AFP binding to ice. We expect this approach will contribute to a better understanding of the mechanism of AFP activity and in particular the hyperactivity of insect AFPs.

  16. A study of the growth rates and growth habits of ice crystals in a solution of antifreeze (glyco) proteins

    NASA Astrophysics Data System (ADS)

    Li, Qianzhong; Luo, Liaofu

    1996-12-01

    The mechanism of the antifreeze glycoprotein/antifreeze protein interaction on the surface of ice is analyzed. The theory of ice crystal growth in an AF(G)P solution is presented. A quantitative calculation of the growth rates for gain growth has been obtained. The anisotropic growth habits and growth rates of ice crystals in an AF(G)P solution are explained.

  17. Differential expression of two antifreeze proteins in the desert beetle Anatolica polita (Coleoptera: Tenebriondae): seasonal variation and environmental effects.

    PubMed

    Ma, J; Wang, J; Mao, X F; Wang, Y

    2012-01-01

    Antifreeze proteins (AFPs) can inhibit and modify the growth of ice crystals. Two antifreeze protein genes, Apafp752 and Apafp914, were cloned from the desert beetle Anatolica polita (Coleoptera: Tenebriondae), and they shared 61.3 percent similarity at the amino acid level. Apafp752 also contained one variation in the most conserved TCT motif of beetle AFPs. Apafp752 and Apafp914 mRNAs had similar seasonal expression pattern. Both were stimulated by cold stress, but they expressed slightly differentially with Apafp752 being more sensitive to cold stress than Apafp914, and no more sensitive to desiccation stress than Apafp914. The thermal hysteresis activity (THA) in the beetle's hemolymph followed approximately the patterns of mRNA seasonal expression and expression upon environmental stress, with a time lag. Summer adults of the desert beetle also express mRNA of Apafp752 and Apafp914, and exhibit some hemolymph THA, suggesting other likely function of these proteins beyond antifreeze. PMID:23224367

  18. Functional diversification and evolution of antifreeze proteins in the antarctic fish Lycodichthys dearborni.

    PubMed

    Kelley, Joanna L; Aagaard, Jan E; MacCoss, Michael J; Swanson, Willie J

    2010-08-01

    Antifreeze proteins (AFPs) have independently evolved in many organisms. AFPs act by binding to ice crystals, effectively lowering the freezing point. AFPs are often at high copy number in a genome and diversity exists between copies. Type III antifreeze proteins are found in Arctic and Antarctic eel pouts, and have previously been shown to evolve under positive selection. Here we combine molecular and proteomic techniques to understand the molecular evolution and diversity of Type III antifreeze proteins in a single individual Antarctic fish Lycodichthys dearborni. Our expressed sequence tag (EST) screen reveals that at least seven different AFP variants are transcribed, which are ultimately translated into five different protein isoforms. The isoforms have identical 66 base pair signal sequences and different numbers of subsequent ice-binding domains followed by a stop codon. Isoforms with one ice-binding unit (monomer), two units (dimer), and multiple units (multimer) were present in the EST library. We identify a previously uncharacterized protein dimer, providing further evidence that there is diversity between Type III AFP isoforms, perhaps driven by positive selection for greater thermal hysteresis. Proteomic analysis confirms that several of these isoforms are translated and present in the liver. Our molecular evolution study shows that paralogs have diverged under positive selection. We hypothesize that antifreeze protein diversity is an important contributor to depressing the serum freezing point. PMID:20686757

  19. Molecular Recognition of Methyl α-d-Mannopyranoside by Antifreeze (Glyco)Proteins

    PubMed Central

    2015-01-01

    Antifreeze proteins and glycoproteins [AF(G)Ps] have been well-known for more than three decades for their ability to inhibit the growth and recrystallization of ice through binding to specific ice crystal faces, and they show remarkable structural compatibility with specific ice crystal faces. Here, we show that the crystal growth faces of methyl α-d-mannopyranoside (MDM), a representative pyranose sugar, also show noteworthy structural compatibility with the known periodicities of AF(G)Ps. We selected fish AFGPs (AFGP8, AFGP1–5), and a beetle AFP (DAFP1) with increasing antifreeze activity as potential additives for controlling MDM crystal growth. Similar to their effects on ice growth, the AF(G)Ps can inhibit MDM crystal growth and recrystallization, and more significantly, the effectiveness for the AF(G)Ps are well correlated with their antifreeze activity. MDM crystals grown in the presence of AF(G)Ps are smaller and have better defined shapes and are of higher quality as indicated by single crystal X-ray diffraction and polarized microscopy than control crystals, but no new polymorphs of MDM were identified by single crystal X-ray diffraction, solid-state NMR, and attenuated total reflectance infrared spectroscopy. The observed changes in the average sizes of the MDM crystals can be related to the changes in the number of the MDM nuclei in the presence of the AF(G)Ps. The critical free energy change differences of the MDM nucleation in the absence and presence of the additives were calculated. These values are close to those of the ice nucleation in the presence of AF(G)Ps suggesting similar interactions are involved in the molecular recognition of MDM by the AF(G)Ps. To our knowledge this is the first report where AF(G)Ps have been used to control crystal growth of carbohydrates and on AFGPs controlling non-ice-like crystals. Our finding suggests MDM might be a possible alternative to ice for studying the detailed mechanism of AF

  20. Antifreeze proteins govern the precipitation of trehalose in a freezing-avoiding insect at low temperature.

    PubMed

    Wen, Xin; Wang, Sen; Duman, John G; Arifin, Josh Fnu; Juwita, Vonny; Goddard, William A; Rios, Alejandra; Liu, Fan; Kim, Soo-Kyung; Abrol, Ravinder; DeVries, Arthur L; Henling, Lawrence M

    2016-06-14

    The remarkable adaptive strategies of insects to extreme environments are linked to the biochemical compounds in their body fluids. Trehalose, a versatile sugar molecule, can accumulate to high levels in freeze-tolerant and freeze-avoiding insects, functioning as a cryoprotectant and a supercooling agent. Antifreeze proteins (AFPs), known to protect organisms from freezing by lowering the freezing temperature and deferring the growth of ice, are present at high levels in some freeze-avoiding insects in winter, and yet, paradoxically are found in some freeze-tolerant insects. Here, we report a previously unidentified role for AFPs in effectively inhibiting trehalose precipitation in the hemolymph (or blood) of overwintering beetle larvae. We determine the trehalose level (29.6 ± 0.6 mg/mL) in the larval hemolymph of a beetle, Dendroides canadensis, and demonstrate that the hemolymph AFPs are crucial for inhibiting trehalose crystallization, whereas the presence of trehalose also enhances the antifreeze activity of AFPs. To dissect the molecular mechanism, we examine the molecular recognition between AFP and trehalose crystal interfaces using molecular dynamics simulations. The theory corroborates the experiments and shows preferential strong binding of the AFP to the fast growing surfaces of the sugar crystal. This newly uncovered role for AFPs may help explain the long-speculated role of AFPs in freeze-tolerant species. We propose that the presence of high levels of molecules important for survival but prone to precipitation in poikilotherms (their body temperature can vary considerably) needs a companion mechanism to prevent the precipitation and here present, to our knowledge, the first example. Such a combination of trehalose and AFPs also provides a novel approach for cold protection and for trehalose crystallization inhibition in industrial applications. PMID:27226297

  1. Interaction of Tenebrio Molitor Antifreeze Protein with Ice Crystal: Insights from Molecular Dynamics Simulations.

    PubMed

    Ramya, L; Ramakrishnan, Vigneshwar

    2016-07-01

    Antifreeze proteins (AFP) observed in cold-adapting organisms bind to ice crystals and prevent further ice growth. However, the molecular mechanism of AFP-ice binding and AFP-inhibited ice growth remains unclear. Here we report the interaction of the insect antifreeze protein (Tenebrio molitor, TmAFP) with ice crystal by molecular dynamics simulation studies. Two sets of simulations were carried out at 263 K by placing the protein near the primary prism plane (PP) and basal plane (BL) of the ice crystal. To delineate the effect of temperatures, both the PP and BL simulations were carried out at 253 K as well. The analyses revealed that the protein interacts strongly with the ice crystal in BL simulation than in PP simulation both at 263 K and 253 K. Further, it was observed that the interactions are primarily mediated through the interface waters. We also observed that as the temperature decreases, the interaction between the protein and the ice increases which can be attributed to the decreased flexibility and the increased structuring of the protein at low temperature. In essence, our study has shed light on the interaction mechanism between the TmAFP antifreeze protein and the ice crystal. PMID:27492241

  2. Antifreeze Protein (AFP) and Antifreeze Glycoprotein (AFGP) Kinetics at the Ice/Solution Interface

    NASA Astrophysics Data System (ADS)

    Zepeda, Salvador; Nakaya, Hiroyuki; Uda, Yukihiro; Yokoyama, Etsuro; Furukawa, Yoshinori

    2007-03-01

    AFPs and AFGPs found in some fish, plants and insects are a necessary tool for surviving sub-freezing environments. They occur in a wide range of compositions and structure, but to some extent they all accomplish the same functions: they suppress the freezing temperature, inhibit recrystallization, and modify ice crystal growth. Here, we observe the exact location of AFGPs, Type I and Type III AFPs by 1-directional growth experiments using fluorescence and phase contrast microscopy as well as free growth experiments using 3-d confocal microscopy. In all cases, the proteins clearly adsorb at the interface. By comparing the fluorescent image with the corresponding phase contrast image we find that AFGPs incorporate only into the solid in veins and not into the ice lattice structure. Type I AFPs show similar behavior as AFGPs, but type III AFPs adsorb to specific planes within the ice lattice. We have also calculated the diffusion constants and the surface adsorption concentration from both types of experiments. Our results indicated that the different types of AFPs or AFGPs accomplish essentially the same function in slightly different ways and that it is not necessary for the protein adsorption to the ice interface to be as rigid as once thought.

  3. Rapid amyloid fibril formation by a winter flounder antifreeze protein requires specific interaction with ice.

    PubMed

    Dubé, André; Leggiadro, Cindy; Ewart, Kathryn Vanya

    2016-05-01

    A typically α-helical antifreeze protein (wflAFP-6) from winter flounder, Pseudopleuronectes americanus, forms amyloid fibrils during freezing. In this study, the effects of distinct components of the freezing process were examined. Freezing of wflAFP-6 in the presence of template ice was shown to be necessary for rapid conversion to an amyloid conformation. Neither subfreezing temperature nor phase change was sufficient. Thus, specific interaction with the ice surface was essential. The ice-induced formation of amyloid appeared to be unique to this helical antifreeze, it required high concentrations of protein and it occurred over a range of pH values. These results define a method for rapid formation of amyloid by wflAFP-6 on demand under physiological conditions. PMID:27086686

  4. Ice Shaping Properties, Similar to That of Antifreeze Proteins, of a Zirconium Acetate Complex

    PubMed Central

    Deville, Sylvain; Viazzi, Céline; Leloup, Jérôme; Lasalle, Audrey; Guizard, Christian; Maire, Eric; Adrien, Jérôme; Gremillard, Laurent

    2011-01-01

    The control of the growth morphologies of ice crystals is a critical issue in fields as diverse as biomineralization, medicine, biology, civil or food engineering. Such control can be achieved through the ice-shaping properties of specific compounds. The development of synthetic ice-shaping compounds is inspired by the natural occurrence of such properties exhibited by antifreeze proteins. We reveal how a particular zirconium acetate complex is exhibiting ice-shaping properties very similar to that of antifreeze proteins, albeit being a radically different compound. We use these properties as a bioinspired approach to template unique faceted pores in cellular materials. These results suggest that ice-structuring properties are not exclusive to long organic molecules and should broaden the field of investigations and applications of such substances. PMID:22028886

  5. Adsorption thermodynamics of two-domain antifreeze proteins: theory and Monte Carlo simulations.

    PubMed

    Narambuena, Claudio F; Sanchez Varretti, Fabricio O; Ramirez-Pastor, Antonio J

    2016-09-21

    In this paper we develop the statistical thermodynamics of two-domain antifreeze proteins adsorbed on ice. We use a coarse-grained model and a lattice network in order to represent the protein and ice, respectively. The theory is obtained by combining the exact analytical expression for the partition function of non-interacting linear k-mers adsorbed in one dimension, and its extension to higher dimensions. The total and partial adsorption isotherms, and the coverage and temperature dependence of the Helmholtz free energy and configurational entropy are given. The formalism reproduces the classical Langmuir equation, leads to the exact statistical thermodynamics of molecules adsorbed in one dimension, and provides a close approximation for two-dimensional systems. Comparisons with analytical data obtained using the modified Langmuir model (MLM) and Monte Carlo simulations in the grand canonical ensemble were performed in order to test the validity of the theoretical predictions. In the MC calculations, the different mechanisms proposed in the literature to describe the adsorption of two-domain antifreeze proteins on ice were analyzed. Indistinguishable results were obtained in all cases, which verifies the thermodynamic equivalence of these mechanisms and allows the choice of the most suitable mechanism for theoretical studies of equilibrium properties. Even though a good qualitative agreement is obtained between MLM and MC data, it is found that the new theoretical framework offers a more accurate description of the phenomenon of adsorption of two-domain antifreeze proteins. PMID:27539563

  6. Arginine, a key residue for the enhancing ability of an antifreeze protein of the beetle Dendroides canadensis†

    PubMed Central

    Wang, Sen; Amornwittawat, Natapol; Juwita, Vonny; Kao, Yu; Duman, John G.; Pascal, Tod A.; Goddard, William A.; Wen, Xin

    2009-01-01

    Antifreeze proteins (AFPs) can produce a difference between the nonequilibrium freezing point and the melting point is termed thermal hysteresis (TH). The TH activity of an antifreeze protein (AFP) depends on the specific AFP, its concentration as well as the presence of co-solutes including low-molecular-mass solutes and/or proteins. We recently identified series of carboxylates and polyols as efficient enhancers for an AFP from the beetle Dendroides canadensis. In this study, we chemically modified DAFP-1 using the arginine-specific reagent 1,2-cyclohexanedione. We demonstrated that 1,2-cyclohexanedione specifically modifies one arginine residue and the modified DAFP-1 loses its enhancing ability completely or partially in the presence of previously identified enhancers. The stronger the enhancement ability of the enhancer on the native DAFP-1, the stronger the enhancement effect of the enhancer has on the modified DAFP-1. The weaker enhancers (e.g., glycerol) completely lose their enhancement effect on the modified DAFP-1 due to their inability to compete with 1,2-cyclohexanedione for the arginine residue. Regeneration of the arginine residue using hydroxylamine fully restored the enhancing ability of DAFP-1. These studies indicated that an arginine residue is critical for the enhancing ability of DAFP-1 and the guanidinium group of the arginine residue is important for its interaction with the enhancers, where the general mechanism of arginine-ligand interaction is borne. This work may initiate a complete mechanistic study of the enhancement effect in AFPs. PMID:19746966

  7. A low molecular weight peptide from snow mold with epitopic homology to the winter flounder antifreeze protein.

    PubMed

    Newsted, W J; Polvi, S; Papish, B; Kendall, E; Saleem, M; Koch, M; Hussain, A; Cutler, A J; Georges, F

    1994-01-01

    Evidence for a small size protein (ca. 3500 kDa) exhibiting epitopic homology to the Atlantic winter flounder antifreeze protein (AFP) is found in the snow molds Coprinus psychromorbidus, Myriosclerotinia borealis, and Typhula incarnata. The protein shows strong cross-reactivity with antisera specific for the flounder AFP. Preliminary studies suggest that the protein is synthesized in response to lowering the culture temperature, and that it is membrane associated and, therefore, may function in an analogous capacity to the fish AFP. Also, the protein is shown to have antifreeze properties as determined by nuclear magnetic resonance microimaging experiments. PMID:7818849

  8. A diminished role for hydrogen bonds in antifreeze protein binding to ice.

    PubMed

    Chao, H; Houston, M E; Hodges, R S; Kay, C M; Sykes, B D; Loewen, M C; Davies, P L; Sönnichsen, F D

    1997-12-01

    The most abundant isoform (HPLC-6) of type I antifreeze protein (AFP1) in winter flounder is a 37-amino-acid-long, alanine-rich, alpha-helical peptide, containing four Thr spaced 11 amino acids apart. It is generally assumed that HPLC-6 binds ice through a hydrogen-bonding match between the Thr and neighboring Asx residues to oxygens atoms on the {2021} plane of the ice lattice. The result is a lowering of the nonequilibrium freezing point below the melting point (thermal hysteresis). HPLC-6, and two variants in which the central two Thr were replaced with either Ser or Val, were synthesized. The Ser variant was virtually inactive, while only a minor loss of activity was observed in the Val variant. CD, ultracentrifugation, and NMR studies indicated no significant structural changes or aggregation of the variants compared to HPLC-6. These results call into question the role of hydrogen bonds and suggest a much more significant role for entropic effects and van der Waals interactions in binding AFP to ice. PMID:9398184

  9. An open source cryostage and software analysis method for detection of antifreeze activity.

    PubMed

    Buch, J L; Ramløv, H

    2016-06-01

    The aim of this study is to provide the reader with a simple setup that can detect antifreeze proteins (AFP) by inhibition of ice recrystallisation in very small sample sizes. This includes an open source cryostage, a method for preparing and loading samples as well as a software analysis method. The entire setup was tested using hyperactive AFP from the cerambycid beetle, Rhagium mordax. Samples containing AFP were compared to buffer samples, and the results are visualised as crystal radius evolution over time and in absolute change over 30 min. Statistical analysis showed that samples containing AFP could reliably be told apart from controls after only two minutes of recrystallisation. The goal of providing a fast, cheap and easy method for detecting antifreeze proteins in solution was met, and further development of the system can be followed at https://github.com/pechano/cryostage. PMID:27041219

  10. Recombinant Dendroides canadensis antifreeze proteins as potential ingredients in cryopreservation solutions.

    PubMed

    Halwani, Dina O; Brockbank, Kelvin G M; Duman, John G; Campbell, Lia H

    2014-06-01

    Expanding cryopreservation methods to include a wider range of cell types, such as those sensitive to freezing, is needed for maintaining the viability of cell-based regenerative medicine products. Conventional cryopreservation protocols, which include use of cryoprotectants such as dimethylsulfoxide (Me2SO), have not prevented ice-induced damage to cell and tissue matrices during freezing. A family of antifreeze proteins (AFPs) produced in the larvae of the beetle, Dendroides canadensis allow this insect to survive subzero temperatures as low as -26°C. This study is an assessment of the effect of the four hemolymph D. canadensis AFPs (DAFPs) on the supercooling (nucleating) temperature, ice structure patterns and viability of the A10 cell line derived from the thoracic aorta of embryonic rat. Cryoprotectant solution cocktails containing combinations of DAFPs in concentrations ranging from 0 to 3mg/mL in Unisol base mixed with 1M Me2SO were first evaluated by cryomicroscopy. Combining multiple DAFPs demonstrated significant supercooling point depressing activity (∼9°C) when compared to single DAFPs and/or conventional 1M Me2SO control solutions. Concentrations of DAFPs as low as 1 μg/mL were sufficient to trigger this effect. In addition, significantly improved A10 smooth muscle cell viability was observed in cryopreservation experiments with low DAFP-6 and DAFP-2 concentrations in combination with Me2SO. No significant improvement in viability was observed with either DAFP-1 or DAFP-4. Low and effective DAFP concentrations are advantageous because they minimize concerns regarding cell cytotoxicity and manufacturing cost. These findings support the potential of incorporating DAFPs in solutions used to cryopreserve cells and tissues. PMID:24662031

  11. Analysis of ice-binding sites in fish type II antifreeze protein by quantum mechanics.

    PubMed Central

    Cheng, Yuhua; Yang, Zuoyin; Tan, Hongwei; Liu, Ruozhuang; Chen, Guangju; Jia, Zongchao

    2002-01-01

    Many organisms living in cold environments can survive subzero temperatures by producing antifreeze proteins (AFPs) or antifreeze glycoproteins. In this paper we investigate the ice-binding surface of type II AFP by quantum mechanical methods, which, to the best of our knowledge, represents the first time that molecular orbital computational approaches have been applied to AFPs. Molecular mechanical approaches, including molecular docking, energy minimization, and molecular dynamics simulation, were used to obtain optimal systems for subsequent quantum mechanical analysis. We selected 17 surface patches covering the entire surface of the type II AFP and evaluated the interaction energy between each of these patches and two different ice planes using semi-empirical quantum mechanical methods. We have demonstrated the weak orbital overlay phenomenon and the change of bond orders in ice. These results consistently indicate that a surface patch containing 19 residues (K37, L38, Y20, E22, Y21, I19, L57, T56, F53, M127, T128, F129, R17, C7, N6, P5, G10, Q1, and W11) is the most favorable ice-binding site for both a regular ice plane and an ice plane where water O atoms are randomly positioned. Furthermore, for the first time the computation results provide new insights into the weakening of the ice lattice upon AFP binding, which may well be a primary factor leading to AFP-induced ice growth inhibition. PMID:12324437

  12. An Effective Antifreeze Protein Predictor with Ensemble Classifiers and Comprehensive Sequence Descriptors

    PubMed Central

    Yang, Runtao; Zhang, Chengjin; Gao, Rui; Zhang, Lina

    2015-01-01

    Antifreeze proteins (AFPs) play a pivotal role in the antifreeze effect of overwintering organisms. They have a wide range of applications in numerous fields, such as improving the production of crops and the quality of frozen foods. Accurate identification of AFPs may provide important clues to decipher the underlying mechanisms of AFPs in ice-binding and to facilitate the selection of the most appropriate AFPs for several applications. Based on an ensemble learning technique, this study proposes an AFP identification system called AFP-Ensemble. In this system, random forest classifiers are trained by different training subsets and then aggregated into a consensus classifier by majority voting. The resulting predictor yields a sensitivity of 0.892, a specificity of 0.940, an accuracy of 0.938 and a balanced accuracy of 0.916 on an independent dataset, which are far better than the results obtained by previous methods. These results reveal that AFP-Ensemble is an effective and promising predictor for large-scale determination of AFPs. The detailed feature analysis in this study may give useful insights into the molecular mechanisms of AFP-ice interactions and provide guidance for the related experimental validation. A web server has been designed to implement the proposed method. PMID:26370959

  13. Transcriptomic and proteomic analyses on the supercooling ability and mining of antifreeze proteins of the Chinese white wax scale insect.

    PubMed

    Yu, Shu-Hui; Yang, Pu; Sun, Tao; Qi, Qian; Wang, Xue-Qing; Chen, Xiao-Ming; Feng, Ying; Liu, Bo-Wen

    2016-06-01

    The Chinese white wax scale insect, Ericerus pela, can survive at extremely low temperatures, and some overwintering individuals exhibit supercooling at temperatures below -30°C. To investigate the deep supercooling ability of E. pela, transcriptomic and proteomic analyses were performed to delineate the major gene and protein families responsible for the deep supercooling ability of overwintering females. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that genes involved in the mitogen-activated protein kinase, calcium, and PI3K-Akt signaling pathways and pathways associated with the biosynthesis of soluble sugars, sugar alcohols and free amino acids were dominant. Proteins responsible for low-temperature stress, such as cold acclimation proteins, glycerol biosynthesis-related enzymes and heat shock proteins (HSPs) were identified. However, no antifreeze proteins (AFPs) were identified through sequence similarity search methods. A random forest approach identified 388 putative AFPs in the proteome. The AFP gene ep-afp was expressed in Escherichia coli, and the expressed protein exhibited a thermal hysteresis activity of 0.97°C, suggesting its potential role in the deep supercooling ability of E. pela. PMID:26799455

  14. Why ice-binding type I antifreeze protein acts as a gas hydrate crystal inhibitor.

    PubMed

    Bagherzadeh, S Alireza; Alavi, Saman; Ripmeester, John A; Englezos, Peter

    2015-04-21

    Antifreeze proteins (AFPs) prevent ice growth by binding to a specific ice plane. Some AFPs have been found to inhibit the formation of gas hydrates which are a serious safety and operational challenge for the oil and gas industry. Molecular dynamics simulations are used to determine the mechanism of action of the winter flounder AFP (wf-AFP) in inhibiting methane hydrate growth. The wf-AFP adsorbs onto the methane hydrate surface via cooperative binding of a set of hydrophobic methyl pendant groups to the empty half-cages at the hydrate/water interface. Each binding set is composed of the methyl side chain of threonine and two alanine residues, four and seven places further down in the sequence of the protein. Understanding the principle of action of AFPs can lead to the rational design of green hydrate inhibitor molecules with potential superior performance. PMID:25786071

  15. Experimental investigation of the interactions of hyperactive antifreeze proteins with ice crystals

    NASA Astrophysics Data System (ADS)

    Celik, Yeliz

    Antifreeze proteins (AFPs) evolved in cold-adapted organisms and serve to protect them against freezing cold conditions by arresting ice crystal growth and inhibiting ice recrystallization. The freezing point depression by AFPs is defined as thermal hysteresis (TH) and AFPs are classified as hyperactive (hypAFPs) and moderate according to their TH activities. The mechanism of action of AFPs is not well understood. In particular, it is not clear what determines the concentration dependence of TH and whether the binding of AFP to ice is irreversible. Additionally, it is not known why some types of AFP are hyperactive compared to others and it was suggested that hyperactivity might be related to basal plane affinity of hypAFP to ice. The present study utilizes the techniques of microfluidic devices and fluorescence microscopy to study the interaction of AFPs with ice crystals. With novel temperature controlled microfluidic devices, we showed the accumulation and affinity of hypAFPs on the basal plane of ice. This supports the view that hypAFPs adhere to the basal plane. Additionally, for the first time in literature, small ice crystals of 30-50 mum sizes covered with adsorbed GFP tagged hypAFPs were stabilized in supercooled non-AFP solutions for hours with no observed ice growth in temperature controlled microfluidic devices. Repeated TH experiments of ice crystals incubated in AFP solutions before and after the exchange of liquids in microfluidic devices gave the same TH activity. This finding clarifies our understanding of concentration dependence of TH. Furthermore, we found that hypAFPs protect ice against melting as well as freezing, resulting in superheated ice. Ice crystals were superheated up to 0.5°C above their equilibrium melting temperatures and remained stable in this superheated state for hours. Measurements of fast melting velocities added additional evidence to the observed superheating of ice in AFP solutions. The experimental results of the current

  16. Antifreeze poisoning

    MedlinePlus

    The poisonous ingredients in antifreeze are: Ethylene glycol Methanol Propylene glycol ... For ethylene glycol: Death may occur within the first 24 hours. If the patient survives, there may be little ...

  17. FAST TRACK COMMUNICATION: Growth melt asymmetry in ice crystals under the influence of spruce budworm antifreeze protein

    NASA Astrophysics Data System (ADS)

    Pertaya, Natalya; Celik, Yeliz; Di Prinzio, Carlos L.; Wettlaufer, J. S.; Davies, Peter L.; Braslavsky, Ido

    2007-10-01

    Here we describe studies of the crystallization behavior of ice in an aqueous solution of spruce budworm antifreeze protein (sbwAFP) at atmospheric pressure. SbwAFP is an ice binding protein with high thermal hysteresis activity, which helps protect Choristoneura fumiferana (spruce budworm) larvae from freezing as they overwinter in the spruce and fir forests of the north eastern United States and Canada. Different types of ice binding proteins have been found in many other species. They have a wide range of applications in cryomedicine and cryopreservation, as well as the potential to protect plants and vegetables from frost damage through genetic engineering. However, there is much to learn regarding the mechanism of action of ice binding proteins. In our experiments, a solution containing sbwAFP was rapidly frozen and then melted back, thereby allowing us to produce small single crystals. These maintained their hexagonal shapes during cooling within the thermal hysteresis gap. Melt-growth-melt sequences in low concentrations of sbwAFP reveal the same shape transitions as are found in pure ice crystals at low temperature (-22 °C) and high pressure (2000 bar) (Cahoon et al 2006 Phys. Rev. Lett. 96 255502) while both growth and melt shapes display faceted hexagonal morphology, they are rotated 30° relative to one another. Moreover, the initial melt shape and orientation is recovered in the sequence. To visualize the binding of sbwAFP to ice, we labeled the antifreeze protein with enhanced green fluorescent protein (eGFP) and observed the sbwAFP-GFP molecules directly on ice crystals using confocal microscopy. When cooling the ice crystals, facets form on the six primary prism planes (slowest growing planes) that are evenly decorated with sbwAFP-GFP. During melting, apparent facets form on secondary prism planes (fastest melting planes), leaving residual sbwAFP at the six corners of the hexagon. Thus, the same general growth-melt behavior of an apparently rotated

  18. Helical Antifreeze Proteins Have Independently Evolved in Fishes on Four Occasions

    PubMed Central

    Graham, Laurie A.; Hobbs, Rod S.; Fletcher, Garth L.; Davies, Peter L.

    2013-01-01

    Alanine-rich α-helical (type I) antifreeze proteins (AFPs) are produced by a variety of fish species from three different orders to protect against freezing in icy seawater. Interspersed amongst and within these orders are fishes making AFPs that are completely different in both sequence and structure. The origin of this variety of types I, II, III and antifreeze glycoproteins (AFGPs) has been attributed to adaptation following sea-level glaciations that occurred after the divergence of most of the extant families of fish. The presence of similar types of AFPs in distantly related fishes has been ascribed to lateral gene transfer in the case of the structurally complex globular type II lectin-like AFPs and to convergent evolution for the AFGPs, which consist of a well-conserved tripeptide repeat. In this paper, we examine the genesis of the type I AFPs, which are intermediate in complexity. These predominantly α-helical peptides share many features, such as putative capping structures, Ala-richness and amphipathic character. We have added to the type I repertoire by cloning additional sequences from sculpin and have found that the similarities between the type I AFPs of the four distinct groups of fishes are not borne out at the nucleotide level. Both the non-coding sequences and the codon usage patterns are strikingly different. We propose that these AFPs arose via convergence from different progenitor helices with a weak affinity for ice and that their similarity is dictated by the propensity of specific amino acids to form helices and to align water on one side of the helix into an ice-like pattern. PMID:24324684

  19. Structure-function relationship in the globular type III antifreeze protein: identification of a cluster of surface residues required for binding to ice.

    PubMed Central

    Chao, H.; Sönnichsen, F. D.; DeLuca, C. I.; Sykes, B. D.; Davies, P. L.

    1994-01-01

    Antifreeze proteins (AFPs) depress the freezing point of aqueous solutions by binding to and inhibiting the growth of ice. Whereas the ice-binding surface of some fish AFPs is suggested by their linear, repetitive, hydrogen bonding motifs, the 66-amino-acid-long Type III AFP has a compact, globular fold without any obvious periodicity. In the structure, 9 beta-strands are paired to form 2 triple-stranded antiparallel sheets and 1 double-stranded antiparallel sheet, with the 2 triple sheets arranged as an orthogonal beta-sandwich (Sönnichsen FD, Sykes BD, Chao H, Davies PL, 1993, Science 259:1154-1157). Based on its structure and an alignment of Type III AFP isoform sequences, a cluster of conserved, polar, surface-accessible amino acids (N14, T18, Q44, and N46) was noted on and around the triple-stranded sheet near the C-terminus. At 3 of these sites, mutations that switched amide and hydroxyl groups caused a large decrease in antifreeze activity, but amide to carboxylic acid changes produced AFPs that were fully active at pH 3 and pH 6. This is consistent with the observation that Type III AFP is optimally active from pH 2 to pH 11. At a concentration of 1 mg/mL, Q44T, N14S, and T18N had 50%, 25%, and 10% of the activity of wild-type antifreeze, respectively. The effects of the mutations were cumulative, such that the double mutant N14S/Q44T had 10% of the wild-type activity and the triple mutant N14S/T18N/Q44T had no activity. All mutants with reduced activity were shown to be correctly folded by NMR spectroscopy. Moreover, a complete characterization of the triple mutant by 2-dimensional NMR spectroscopy indicated that the individual and combined mutations did not significantly alter the structure of these proteins. These results suggest that the C-terminal beta-sheet of Type III AFP is primarily responsible for antifreeze activity, and they identify N14, T18, and Q44 as key residues for the AFP-ice interaction. PMID:7849594

  20. Antifreeze and cryoprotective activities of ice-binding collagen peptides from pig skin.

    PubMed

    Cao, Hui; Zhao, Ying; Zhu, Yu Bing; Xu, Fei; Yu, Jing Song; Yuan, Min

    2016-03-01

    A novel "hyperactive" ice-binding peptide from porcine collagen was prepared by alkaline protease hydrolysis and a series of column chromatography separations, and then its antifreeze and cryoprotective properties were reported. Using differential scanning calorimetry (DSC), the thermal hysteresis (TH) of ice-binding collagen peptides was closely related to their concentration and crystal fraction. Collagen hydrolysates with maximal TH were obtained by hydrolysis at pH 8.0, DH 15.0%, and 5% alkaline protease at 55°C. After purification by column chromatography, the AP-3 ice-binding collagen peptide (GLLGPLGPRGLL) with 1162.8Da molecular weights exhibited the highest TH (5.28°C), which can be classified as "hyperactive". Recrystallisation and melt-resistance of ice cream were improved by AP-3 ice-binding collagen peptide at 0.2% (w/v) in a similar manner to natural antifreeze proteins. Moreover, the addition of AP-3 collagen peptides in ice cream greatly elevated the glass transition temperature (Tg) to -17.64°C. PMID:26471678

  1. Functional Analysis of a Bacterial Antifreeze Protein Indicates a Cooperative Effect between Its Two Ice-Binding Domains.

    PubMed

    Wang, Chen; Oliver, Erin E; Christner, Brent C; Luo, Bing-Hao

    2016-07-19

    Antifreeze proteins make up a class of ice-binding proteins (IBPs) that are possessed and expressed by certain cold-adapted organisms to enhance their freezing tolerance. Here we report the biophysical and functional characterization of an IBP discovered in a bacterium recovered from a deep glacial ice core drilled at Vostok Station, Antarctica (IBPv). Our study showed that the recombinant protein rIBPv exhibited a thermal hysteresis of 2 °C at concentrations of >50 μM, effectively inhibited ice recrystallization, and enhanced bacterial viability during freeze-thaw cycling. Circular dichroism scans indicated that rIBPv mainly consists of β strands, and its denaturing temperature was 53.5 °C. Multiple-sequence alignment of homologous IBPs predicted that IBPv contains two ice-binding domains, a feature unique among known IBPs. To examine functional differences between the IBPv domains, each domain was cloned, expressed, and purified. The second domain (domain B) expressed greater ice binding activity. Data from thermal hysteresis and gel filtration assays supported the idea that the two domains cooperate to achieve a higher ice binding effect by forming heterodimers. However, physical linkage of the domains was not required for this effect. PMID:27359086

  2. Investigation of the Ice-Binding Site of an Insect Antifreeze Protein Using Sum-Frequency Generation Spectroscopy.

    PubMed

    Meister, Konrad; Lotze, Stephan; Olijve, Luuk L C; DeVries, Arthur L; Duman, John G; Voets, Ilja K; Bakker, Huib J

    2015-04-01

    We study the ice-binding site (IBS) of a hyperactive antifreeze protein from the beetle Dendroides canadensis (DAFP-1) using vibrational sum-frequency generation spectroscopy. We find that DAFP-1 accumulates at the air-water interface due to the hydrophobic character of its threonine-rich IBS while retaining its highly regular β-helical fold. We observe a narrow band at 3485 cm(-1) that we assign to the O-H stretch vibration of threonine hydroxyl groups of the IBS. The narrow character of the 3485 cm(-1) band suggests that the hydrogen bonds between the threonine residues at the IBS and adjacent water molecules are quite similar in strength, indicating that the IBS of DAFP-1 is extremely well-ordered, with the threonine side chains showing identical rotameric confirmations. The hydrogen-bonded water molecules do not form an ordered ice-like layer, as was recently observed for the moderate antifreeze protein type III. It thus appears that the antifreeze action of DAFP-1 does not require the presence of ordered water but likely results from the direct binding of its highly ordered array of threonine residues to the ice surface. PMID:26262966

  3. Structural Basis for the Inhibition of Gas Hydrates by α-Helical Antifreeze Proteins.

    PubMed

    Sun, Tianjun; Davies, Peter L; Walker, Virginia K

    2015-10-20

    Kinetic hydrate inhibitors (KHIs) are used commercially to inhibit gas hydrate formation and growth in pipelines. However, improvement of these polymers has been constrained by the lack of verified molecular models. Since antifreeze proteins (AFPs) act as KHIs, we have used their solved x-ray crystallographic structures in molecular modeling to explore gas hydrate inhibition. The internal clathrate water network of the fish AFP Maxi, which extends to the protein's outer surface, is remarkably similar to the {100} planes of structure type II (sII) gas hydrate. The crystal structure of this water web has facilitated the construction of in silico models for Maxi and type I AFP binding to sII hydrates. Here, we have substantiated our models with experimental evidence of Maxi binding to the tetrahydrofuran sII model hydrate. Both in silico and experimental evidence support the absorbance-inhibition mechanism proposed for KHI binding to gas hydrates. Based on the Maxi crystal structure we suggest that the inhibitor adsorbs to the gas hydrate lattice through the same anchored clathrate water mechanism used to bind ice. These results will facilitate the rational design of a next generation of effective green KHIs for the petroleum industry to ensure safe and efficient hydrocarbon flow. PMID:26488661

  4. Structure and Evolutionary Origin of Ca2+-Dependent Herring Type II Antifreeze Protein

    SciTech Connect

    Liu,Y.; Li, Z.; Lin, Q.; Kosinski, J.; Seetharaman, J.; Bujnicki, J.; Sivaraman, J.; Hew, C.

    2007-01-01

    In order to survive under extremely cold environments, many organisms produce antifreeze proteins (AFPs). AFPs inhibit the growth of ice crystals and protect organisms from freezing damage. Fish AFPs can be classified into five distinct types based on their structures. Here we report the structure of herring AFP (hAFP), a Ca2+-dependent fish type II AFP. It exhibits a fold similar to the C-type (Ca2+-dependent) lectins with unique ice-binding features. The 1.7 Angstroms crystal structure of hAFP with bound Ca2+ and site-directed mutagenesis reveal an ice-binding site consisting of Thr96, Thr98 and Ca2+-coordinating residues Asp94 and Glu99, which initiate hAFP adsorption onto the [10-10] prism plane of the ice lattice. The hAFP-ice interaction is further strengthened by the bound Ca2+ through the coordination with a water molecule of the ice lattice. This Ca2+-coordinated ice-binding mechanism is distinct from previously proposed mechanisms for other AFPs. However, phylogenetic analysis suggests that all type II AFPs evolved from the common ancestor and developed different ice-binding modes. We clarify the evolutionary relationship of type II AFPs to sugar-binding lectins.

  5. Comparative modeling of the three-dimensional structure of type II antifreeze protein.

    PubMed Central

    Sönnichsen, F. D.; Sykes, B. D.; Davies, P. L.

    1995-01-01

    Type II antifreeze proteins (AFP), which inhibit the growth of seed ice crystals in the blood of certain fishes (sea raven, herring, and smelt), are the largest known fish AFPs and the only class for which detailed structural information is not yet available. However, a sequence homology has been recognized between these proteins and the carbohydrate recognition domain of C-type lectins. The structure of this domain from rat mannose-binding protein (MBP-A) has been solved by X-ray crystallography (Weis WI, Drickamer K, Hendrickson WA, 1992, Nature 360:127-134) and provided the coordinates for constructing the three-dimensional model of the 129-amino acid Type II AFP from sea raven, to which it shows 19% sequence identity. Multiple sequence alignments between Type II AFPs, pancreatic stone protein, MBP-A, and as many as 50 carbohydrate-recognition domain sequences from various lectins were performed to determine reliably aligned sequence regions. Successive molecular dynamics and energy minimization calculations were used to relax bond lengths and angles and to identify flexible regions. The derived structure contains two alpha-helices, two beta-sheets, and a high proportion of amino acids in loops and turns. The model is in good agreement with preliminary NMR spectroscopic analyses. It explains the observed differences in calcium binding between sea raven Type II AFP and MBP-A. Furthermore, the model proposes the formation of five disulfide bridges between Cys 7 and Cys 18, Cys 35 and Cys 125, Cys 69 and Cys 100, Cys 89 and Cys 111, and Cys 101 and Cys 117.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7540906

  6. Distinct molecular features facilitating ice-binding mechanisms in hyperactive antifreeze proteins closely related to an Antarctic sea ice bacterium.

    PubMed

    Banerjee, Rachana; Chakraborti, Pratim; Bhowmick, Rupa; Mukhopadhyay, Subhasish

    2015-01-01

    Antifreeze proteins or ice-binding proteins (IBPs) facilitate the survival of certain cellular organisms in freezing environment by inhibiting the growth of ice crystals in solution. Present study identifies orthologs of the IBP of Colwellia sp. SLW05, which were obtained from a wide range of taxa. Phylogenetic analysis on the basis of conserved regions (predicted as the 'ice-binding domain' [IBD]) present in all the orthologs, separates the bacterial and archaeal orthologs from that of the eukaryotes'. Correspondence analysis pointed out that the bacterial and archaeal IBDs have relatively higher average hydrophobicity than the eukaryotic members. IBDs belonging to bacterial as well as archaeal AFPs contain comparatively more strands, and therefore are revealed to be under higher evolutionary selection pressure. Molecular docking studies prove that the ice crystals form more stable complex with the bacterial as well as archaeal proteins than the eukaryotic orthologs. Analysis of the docked structures have traced out the ice-binding sites (IBSs) in all the orthologs which continue to facilitate ice-binding activity even after getting mutated with respect to the well-studied IBSs of Typhula ishikariensis and notably, all these mutations performing ice-binding using 'anchored clathrate mechanism' have been found to prefer polar and hydrophilic amino acids. Horizontal gene transfer studies point toward a strong selection pressure favoring independent evolution of the IBPs in some polar organisms including prokaryotes as well as eukaryotes because these proteins facilitate the polar organisms to acclimatize to the adversities in their niche, thus safeguarding their existence. PMID:25190099

  7. X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers.

    PubMed

    Pentelute, Brad L; Gates, Zachary P; Tereshko, Valentina; Dashnau, Jennifer L; Vanderkooi, Jane M; Kossiakoff, Anthony A; Kent, Stephen B H

    2008-07-30

    Chemical protein synthesis and racemic protein crystallization were used to determine the X-ray structure of the snow flea antifreeze protein (sfAFP). Crystal formation from a racemic solution containing equal amounts of the chemically synthesized proteins d-sfAFP and l-sfAFP occurred much more readily than for l-sfAFP alone. More facile crystal formation also occurred from a quasi-racemic mixture of d-sfAFP and l-Se-sfAFP, a chemical protein analogue that contains an additional -SeCH2- moiety at one residue and thus differs slightly from the true enantiomer. Multiple wavelength anomalous dispersion (MAD) phasing from quasi-racemate crystals was then used to determine the X-ray structure of the sfAFP protein molecule. The resulting model was used to solve by molecular replacement the X-ray structure of l-sfAFP to a resolution of 0.98 A. The l-sfAFP molecule is made up of six antiparallel left-handed PPII helixes, stacked in two sets of three, to form a compact brick-like structure with one hydrophilic face and one hydrophobic face. This is a novel experimental protein structure and closely resembles a structural model proposed for sfAFP. These results illustrate the utility of total chemical synthesis combined with racemic crystallization and X-ray crystallography for determining the unknown structure of a protein. PMID:18598029

  8. X-ray Structure of Snow Flea Antifreeze Protein Determined by Racemic Crystallization of Synthetic Protein Enantiomers

    SciTech Connect

    Pentelute, Brad L.; Gates, Zachary P.; Tereshko, Valentina; Dashnau, Jennifer L.; Vanderkooi, Jane M.; Kossiakoff, Anthony A.; Kent, Stephen B.H.

    2008-08-20

    Chemical protein synthesis and racemic protein crystallization were used to determine the X-ray structure of the snow flea antifreeze protein (sfAFP). Crystal formation from a racemic solution containing equal amounts of the chemically synthesized proteins d-sfAFP and l-sfAFP occurred much more readily than for l-sfAFP alone. More facile crystal formation also occurred from a quasi-racemic mixture of d-sfAFP and l-Se-sfAFP, a chemical protein analogue that contains an additional -SeCH2- moiety at one residue and thus differs slightly from the true enantiomer. Multiple wavelength anomalous dispersion (MAD) phasing from quasi-racemate crystals was then used to determine the X-ray structure of the sfAFP protein molecule. The resulting model was used to solve by molecular replacement the X-ray structure of l-sfAFP to a resolution of 0.98 {angstrom}. The l-sfAFP molecule is made up of six antiparallel left-handed PPII helixes, stacked in two sets of three, to form a compact brick-like structure with one hydrophilic face and one hydrophobic face. This is a novel experimental protein structure and closely resembles a structural model proposed for sfAFP. These results illustrate the utility of total chemical synthesis combined with racemic crystallization and X-ray crystallography for determining the unknown structure of a protein.

  9. Crystal structure and mutational analysis of Ca2+-independent type II antifreeze protein from longsnout poacher, Brachyopsis rostratus.

    PubMed

    Nishimiya, Yoshiyuki; Kondo, Hidemasa; Takamichi, Manabu; Sugimoto, Hiroshi; Suzuki, Mamoru; Miura, Ai; Tsuda, Sakae

    2008-10-10

    We recently found that longsnout poacher (Brachyosis rostratus) produces a Ca(2+)-independent type II antifreeze protein (lpAFP) and succeeded in expressing recombinant lpAFP using Phichia pastoris. Here, we report, for the first time, the X-ray crystal structure of lpAFP at 1.34 A resolution. The lpAFP structure displayed a relatively planar surface, which encompasses two loop regions (Cys86-Lys89 and Asn91-Cys97) and a short beta-strand (Trp109-Leu112) with three unstructured segments (Gly57-Ile58, Ala103-Ala104, and Pro113-His118). Electrostatic calculation of the protein surface showed that the relatively planar surface was divided roughly into a hydrophobic area (composed of the three unstructured segments lacking secondary structure) and a hydrophilic area (composed of the loops and beta-strand). Site-directed mutation of Ile58 with Phe at the center of the hydrophobic area decreased activity significantly, whereas mutation of Leu112 with Phe at an intermediate area between the hydrophobic and hydrophilic areas retained complete activity. In the hydrophilic area, a peptide-swap mutant in the loops retained 60% activity despite simultaneous mutations of eight residues. We conclude that the epicenter of the ice-binding site of lpAFP is the hydrophobic region, which is centered by Ile58, in the relatively planar surface. We built an ice-binding model for lpAFP on the basis of a lattice match of ice and constrained water oxygen atoms surrounding the hydrophobic area in the lpAFP structure. The model in which lpAFP has been docked to a secondary prism (2-1-10) plane, which is different from the one determined for Ca(2+)-independent type II AFP from sea raven (11-21), appears to explain the results of the mutagenesis analysis. PMID:18674542

  10. Effect of Antifreeze Protein on Mouse Ovarian Tissue Cryopreservation and Transplantation

    PubMed Central

    Lee, Jung Ryeol; Youm, Hye Won; Lee, Hee Jun; Jee, Byung Chul; Suh, Chang Suk

    2015-01-01

    Purpose To investigate the effect of antifreeze protein (AFP) supplementation on ovarian vitrification and transplantation. Materials and Methods In this experimental study, we researched a total of 182 ovaries from 4-week-old ICR mice. The equilibration solution included 20% ethylene glycol (EG), and the vitrification solution included 40% EG, 18% Ficoll, and 0.3 M sucrose. Intact ovaries were first suspended in 1 mL of equilibration solution for 10 min, and then mixed with 0.5 mL of vitrification solution for 5 min. Ovaries were randomly assigned to 3 groups and 0, 5, or 20 mg/mL of type III AFP was added into the vitrification solution (control, AFP5, and AFP20 groups, respectively). The vitrified ovaries were evaluated after warming and 2 weeks after autotransplantation. The main outcome measurements are follicular morphology and apoptosis assessed by histology and the TUNEL assay. Results A significantly higher intact follicle ratio was shown in the AFP treated groups (control, 28.9%; AFP5, 42.3%; and AFP20, 44.7%). The rate of apoptotic follicles was significantly lower in the AFP treated groups (control, 26.6%; AFP5, 18.7%; and AFP20, 12.6%). After transplantation of the vitrified-warmed ovaries, a significantly higher intact follicle ratio was shown in the AFP20 group. The rate of apoptotic follicles was similar among the groups. Conclusion The results of the present study suggest that supplementing AFP in the vitrification solution has beneficial effects on the survival of ovarian tissue during cryopreservation and transplantation. PMID:25837185

  11. Antifreeze protein-induced superheating of ice inside Antarctic notothenioid fishes inhibits melting during summer warming.

    PubMed

    Cziko, Paul A; DeVries, Arthur L; Evans, Clive W; Cheng, Chi-Hing Christina

    2014-10-01

    Antifreeze proteins (AFPs) of polar marine teleost fishes are widely recognized as an evolutionary innovation of vast adaptive value in that, by adsorbing to and inhibiting the growth of internalized environmental ice crystals, they prevent death by inoculative freezing. Paradoxically, systemic accumulation of AFP-stabilized ice could also be lethal. Whether or how fishes eliminate internal ice is unknown. To investigate if ice inside high-latitude Antarctic notothenioid fishes could melt seasonally, we measured its melting point and obtained a decadal temperature record from a shallow benthic fish habitat in McMurdo Sound, Antarctica. We found that AFP-stabilized ice resists melting at temperatures above the expected equilibrium freezing/melting point (eqFMP), both in vitro and in vivo. Superheated ice was directly observed in notothenioid serum samples and in solutions of purified AFPs, and ice was found to persist inside live fishes at temperatures more than 1 °C above their eqFMP for at least 24 h, and at a lower temperature for at least several days. Field experiments confirmed that superheated ice occurs naturally inside wild fishes. Over the long-term record (1999-2012), seawater temperature surpassed the fish eqFMP in most summers, but never exceeded the highest temperature at which ice persisted inside experimental fishes. Thus, because of the effects of AFP-induced melting inhibition, summer warming may not reliably eliminate internal ice. Our results expose a potentially antagonistic pleiotropic effect of AFPs: beneficial freezing avoidance is accompanied by melting inhibition that may contribute to lifelong accumulation of detrimental internal ice crystals. PMID:25246548

  12. Antifreeze protein-induced superheating of ice inside Antarctic notothenioid fishes inhibits melting during summer warming

    PubMed Central

    Cziko, Paul A.; DeVries, Arthur L.; Evans, Clive W.; Cheng, Chi-Hing Christina

    2014-01-01

    Antifreeze proteins (AFPs) of polar marine teleost fishes are widely recognized as an evolutionary innovation of vast adaptive value in that, by adsorbing to and inhibiting the growth of internalized environmental ice crystals, they prevent death by inoculative freezing. Paradoxically, systemic accumulation of AFP-stabilized ice could also be lethal. Whether or how fishes eliminate internal ice is unknown. To investigate if ice inside high-latitude Antarctic notothenioid fishes could melt seasonally, we measured its melting point and obtained a decadal temperature record from a shallow benthic fish habitat in McMurdo Sound, Antarctica. We found that AFP-stabilized ice resists melting at temperatures above the expected equilibrium freezing/melting point (eqFMP), both in vitro and in vivo. Superheated ice was directly observed in notothenioid serum samples and in solutions of purified AFPs, and ice was found to persist inside live fishes at temperatures more than 1 °C above their eqFMP for at least 24 h, and at a lower temperature for at least several days. Field experiments confirmed that superheated ice occurs naturally inside wild fishes. Over the long-term record (1999–2012), seawater temperature surpassed the fish eqFMP in most summers, but never exceeded the highest temperature at which ice persisted inside experimental fishes. Thus, because of the effects of AFP-induced melting inhibition, summer warming may not reliably eliminate internal ice. Our results expose a potentially antagonistic pleiotropic effect of AFPs: beneficial freezing avoidance is accompanied by melting inhibition that may contribute to lifelong accumulation of detrimental internal ice crystals. PMID:25246548

  13. Biomimetic peptoid oligomers as dual-action antifreeze agents.

    PubMed

    Huang, Mia L; Ehre, David; Jiang, Qi; Hu, Chunhua; Kirshenbaum, Kent; Ward, Michael D

    2012-12-01

    The ability of natural peptides and proteins to influence the formation of inorganic crystalline materials has prompted the design of synthetic compounds for the regulation of crystal growth, including the freezing of water and growth of ice crystals. Despite their versatility and ease of structural modification, peptidomimetic oligomers have not yet been explored extensively as crystallization modulators. This report describes a library of synthetic N-substituted glycine peptoid oligomers that possess "dual-action" antifreeze activity as exemplified by ice crystal growth inhibition concomitant with melting temperature reduction. We investigated the structural features responsible for these phenomena and observed that peptoid antifreeze activities depend both on oligomer backbone structure and side chain chemical composition. These studies reveal the capability of peptoids to act as ice crystallization regulators, enabling the discovery of a unique and diverse family of synthetic oligomers with potential as antifreeze agents in food production and biomedicine. PMID:23169638

  14. Structure and Dynamics of Antifreeze Protein--Model Membrane Interactions: A Combined Spectroscopic and Molecular Dynamics Study.

    PubMed

    Kar, Rajiv K; Mroue, Kamal H; Kumar, Dinesh; Tejo, Bimo A; Bhunia, Anirban

    2016-02-11

    Antifreeze proteins (AFPs) are the key biomolecules that enable species to survive under subzero temperature conditions. The physiologically relevant activities of AFPs are based on the adsorption to ice crystals, followed by the inhibition of subsequent crystal layer growth of ice, routed with depression in freezing point in a noncolligative manner. The functional attributes governing the mechanism by which AFPs inhibit freezing of body fluids in bacteria, fungi, plants, and fishes are mainly attributed to their adsorption onto the surface of ice within the physiological system. Importantly, AFPs are also known for their application in cryopreservation of biological samples that might be related to membrane interaction. To date, there is a paucity of information detailing the interaction of AFPs with membrane structures. Here, we focus on elucidating the biophysical properties of the interactions between AFPs and micelle models that mimic the membrane system. Micelle model systems of zwitterionic DPC and negatively charged SDS were utilized in this study, against which a significant interaction is experienced by two AFP molecules, namely, Peptide 1m and wfAFP (the popular AFP sourced from winter flounder). Using low- and high-resolution biophysical characterization techniques, such as circular dichroism (CD) and NMR spectroscopy, a strong evidence for the interactions of these AFPs with the membrane models is revealed in detail and is corroborated by in-depth residue-specific information derived from molecular dynamics simulation. Altogether, these results not only strengthen the fact that AFPs interact actively with membrane systems, but also demonstrate that membrane-associated AFPs are dynamic and capable of adopting a number of conformations rendering fluidity to the system. PMID:26785292

  15. Effect of the antifreeze protein from the arctic yeast Leucosporidium sp. AY30 on cryopreservation of the marine diatom Phaeodactylum tricornutum.

    PubMed

    Koh, Hye Yeon; Lee, Jun Hyuck; Han, Se Jong; Park, Hyun; Lee, Sung Gu

    2015-01-01

    Antifreeze proteins are a group of proteins that allow organisms to survive in subzero environments. These proteins possess thermal hysteresis and ice recrystallization inhibition activities. In the present study, we demonstrated the efficiency of a recombinant antifreeze protein from the Arctic yeast Leucosporidium sp. AY30, LeIBP, in cryopreservation of the marine diatom Phaeodactylum tricornutum, which is one of the classical model diatoms and has most widely been studied with regard to its ecology, physiology, biochemistry, and molecular biology. P. tricornutum cells were frozen by either a fast or two-step freezing method in freezing medium containing 10 % dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol, respectively, with or without LeIBP supplement. When cells were frozen using the two-step freezing method, cell survival was significantly increased and statistically the same as that of unfrozen native cells in the presence of 0.1 mg/ml LeIBP in 10 % propylene glycol or 10 % ethylene glycol at day 11 of post-thaw culture. In the presence of LeIBP, the concentration of chlorophyll a was dramatically increased to 14-, 48-, 1.6-, and 8.8-fold when cells were frozen in freezing medium containing dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), and ethylene glycol (EG), respectively. Scanning electron microscopy observations demonstrated that the cells were also successfully preserved and epitheca or hypotheca were not deformed. These results demonstrate that LeIBP was successfully applied to improve cryopreservation of the marine diatom P. tricornutum. PMID:25342270

  16. Heterologous expression of antifreeze protein gene AnAFP from Ammopiptanthus nanus enhances cold tolerance in Escherichia coli and tobacco.

    PubMed

    Deng, Long-Qun; Yu, Hao-Qiang; Liu, Yan-Ping; Jiao, Pei-Pei; Zhou, Shu-Feng; Zhang, Su-Zhi; Li, Wan-Chen; Fu, Feng-Ling

    2014-04-10

    Antifreeze proteins are a class of polypeptides produced by certain animals, plants, fungi and bacteria that permit their survival under the subzero environments. Ammopiptanthus nanus is the unique evergreen broadleaf bush endemic to the Mid-Asia deserts. It survives at the west edge of the Tarim Basin from the disappearance of the ancient Mediterranean in the Tertiary Period. Its distribution region is characterized by the arid climate and extreme temperatures, where the extreme temperatures range from -30 °C to 40 °C. In the present study, the antifreeze protein gene AnAFP of A. nanus was used to transform Escherichia coli and tobacco, after bioinformatics analysis for its possible function. The transformed E. coli strain expressed the heterologous AnAFP gene under the induction of isopropyl β-D-thiogalactopyranoside, and demonstrated significant enhancement of cold tolerance. The transformed tobacco lines expressed the heterologous AnAFP gene in response to cold stress, and showed a less change of relative electrical conductivity under cold stress, and a less wilting phenotype after 16 h of -3 °C cold stress and thawing for 1h than the untransformed wild-type plants. All these results imply the potential value of the AnAFP gene to be used in genetic modification of commercially important crops for improvement of cold tolerance. PMID:24502990

  17. Frostbite Protection in Mice Expressing an Antifreeze Glycoprotein

    PubMed Central

    Heisig, Martin; Mattessich, Sarah; Rembisz, Alison; Acar, Ali; Shapiro, Martin; Booth, Carmen J.; Neelakanta, Girish; Fikrig, Erol

    2015-01-01

    Ectotherms in northern latitudes are seasonally exposed to cold temperatures. To improve survival under cold stress, they use diverse mechanisms to increase temperature resistance and prevent tissue damage. The accumulation of anti-freeze proteins that improve cold hardiness occurs in diverse species including plants, arthropods, fish, and amphibians. We previously identified an Ixodes scapularis anti-freeze glycoprotein, named IAFGP, and demonstrated its cold protective function in the natural tick host and in a transgenic Drosophila model. Here we show, in a transgenic mouse model expressing an anti-freeze glycoprotein, that IAFGP protects mammalian cells and mice from cold shock and frostbite respectively. Transgenic skin samples showed reduced cell death upon cold storage ex vivo and transgenic mice demonstrated increased resistance to frostbite injury in vivo. IAFGP actively protects mammalian tissue from freezing, suggesting its application for the prevention of frostbite, and other diseases associated with cold exposure. PMID:25714402

  18. Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit.

    PubMed

    Nishijima, Kazutoshi; Tanaka, Mai; Sakai, Yusuke; Koshimoto, Chihiro; Morimoto, Masatoshi; Watanabe, Teruo; Fan, Jianglin; Kitajima, Shuji

    2014-08-01

    We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN2) and then preserved in the LN2. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN2. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos. PMID:24809634

  19. The influence of adsorption orientation on the statistical mechanics model of type I antifreeze protein: The coverage rate

    NASA Astrophysics Data System (ADS)

    Li, Li-Fen; Liang, X. X.

    2015-03-01

    Based on the statistical mechanics model, the coverage rate of the type I antifreeze protein (AFP) on the surface of ice-crystal in a protein solution is studied by including the adsorption orientation, as well as both the AFP-ice and AFP-water hydrophobic interactions. As an example, the computed results for HPLC-6 are given and discussed. It is shown that the coverage rate increases with the AFP concentration for the dilute protein solutions. The effect of the adsorption orientation of AFP molecules on the coverage rate cannot be ignored. Including the adsorption orientation enhances the coverage rate when the AFP solution is very dilute, but reduces it for the slightly thick solution. The stronger the AFP-ice and AFP-water hydrophobic interactions are, the larger the coverage rate is.

  20. Will It Be Beneficial To Simulate the Antifreeze Proteins at Ice Freezing Condition or at Lower Temperature?

    PubMed

    Kar, Rajiv K; Bhunia, Anirban

    2015-09-01

    Antifreeze proteins (AFPs) enable the polar living species to survive subzero temperature conditions through effective lowering of the freezing point of body fluids. At the molecular level, AFPs directly interact with the growing seeds of ice crystals to inhibit their formation. To understand the structural and dynamic aspects of this interaction at the atomistic level, molecular dynamics (MD) simulations were carried out on several type I AFPs at multiple temperatures, including the physiologically relevant temperature of 273 K, a lower temperature of 227 K, and the conventional 300 K. A comparison of the principal component analysis (PCA) and mean squared deviation plots for Winter flounder AFP, HPLC6 (mutant of winter flounder AFP), Sculpin, and peptide 1m AFPs reveals that simulations at 273 and 227 K result in the formation of more conserved metastable states than at 300 K. Other parameters such as root-mean-square deviation (rmsd), solvent accessibility surface area (SASA), H-bonding and residual density function (RDF) also suggest the same. MD simulations with ice crystal, where AFPs are complexed to ice plane with TIP4P/ice water model, help in finding relevance of dynamic behavior, and physiological temperature becomes more pronounced. Additionally, a control study on a nonantifreeze protein (LL37) is included, which aids in exploring significant information. On the basis of this approach, it was found that AFPs at 273 and 227 K display relevant dynamic properties that appear at 300 K for nonantifreeze proteins. The present study hence emphasizes the importance of performing computational simulations for antifreeze proteins at the physiologically relevant temperature (273 K), and even at lower temperatures (like 227 K), rather than at room temperatures (300 K). PMID:26287639

  1. Effects of Three Different Types of Antifreeze Proteins on Mouse Ovarian Tissue Cryopreservation and Transplantation

    PubMed Central

    Youm, Hye Won; Kim, Hak Jun; Lee, Jung Ryeol; Suh, Chang Suk; Kim, Seok Hyun

    2015-01-01

    Background Ovarian tissue (OT) cryopreservation is effective in preserving fertility in cancer patients who have concerns about fertility loss due to cancer treatment. However, the damage incurred at different steps during the cryopreservation procedure may cause follicular depletion; hence, preventing chilling injury would help maintain ovarian function. Objective This study was designed to investigate the beneficial effects of different antifreeze proteins (AFPs) on mouse ovarian tissue cryopreservation and transplantation. Methodology Ovaries were obtained from 5-week-old B6D2F1 mice, and each ovary was cryopreserved using two-step vitrification and four-step warming procedures. In Experiment I, ovaries were randomly allocated into fresh, vitrification control, and nine experimental groups according to the AFP type (FfIBP, LeIBP, type III) and concentration (0.1, 1, 10 mg/mL) used. After vitrification and warming, 5,790 ovarian follicles were evaluated using histology and TUNEL assays, and immunofluorescence for τH2AX and Rad51 was used to detect DNA double-strand breaks (DSBs) and repair (DDR), respectively. In Experiment II, 20 mice were randomly divided into two groups: one where the vitrification and warming media were supplemented with 10 mg/mL LeIBP, and the other where media alone were used (control). Ovaries were then autotransplanted under both kidney capsules 7 days after vitrification together with the addition of 10 mg/mL LeIBP in the vitrification-warming media. After transplantation, the ovarian follicles, the percentage of apoptotic follicles, the extent of the CD31-positive area, and the serum FSH levels of the transplanted groups were compared. Principal Findings In Experiment I, the percentage of total grade 1 follicles was significantly higher in the 10 mg/mL LeIBP group than in the vitrification control, while all AFP-treated groups had significantly improved grade 1 primordial follicle numbers compared with those of the vitrification

  2. Potential of mean force calculation of the free energy of adsorption of Type I winter flounder antifreeze protein on ice

    NASA Astrophysics Data System (ADS)

    Battle, Keith; Alan Salter, E.; Wesley Edmunds, R.; Wierzbicki, Andrzej

    2010-04-01

    Antifreeze proteins (AFPs) are a unique class of proteins that inhibit ice growth without changing the melting point of ice. In this work, we study the detailed molecular mechanism of interactions between the hydrophobic side of the winter flounder (WF) AFP and two mutants, AAAA and SSSS, in which threonine residues are substituted by serines and alanines, respectively. Umbrella sampling molecular dynamics simulations of the separation of the proteins from the (2 0 1) surface in an explicit water box is carried out to calculate the potential of mean force free energies of adsorption using AMBER10i. We estimate wild-type WF's free energy of adsorption to ice to be about -12.0 kcal/mol. Gas-phase pseudopotential plane-wave calculations of methane adsorption onto select surfaces of ice are also carried out under periodic boundary conditions to address the possible enthalpic role of WF's methyl groups in binding. The contributions of hydrophobic residues to the free energy of adsorption are discussed.

  3. Self-oscillatory ice crystal growth in antifreeze protein (AFP) and glycoprotein (AFGP) solutions

    NASA Astrophysics Data System (ADS)

    Zepeda, Salvador; Nakaya, Hiroyuki; Uda, Yukihiro; Yokoyama, Etsuro; Furukawa, Yoshinori

    2006-03-01

    AFPs and AFGPs allow many organisms including fish, plants and insects to survive sub-freezing environments. They occur in a wide range of compositions and structure, but to some extent they all accomplish the same functions: they suppress the freezing temperature, inhibit recrystallization, and modify ice crystal growth. A complete description of the AFGP/AFP surface mechanism as well as other ice surface phenomenon has eluded scientists primarily due to a lack of direct surface studies. We study ice crystal growth in AFGP and AFP solutions with phase contrast microscopy during free solution growth under various conditions including microgravity. Free-solution growth experiments show an anisotropic self-oscillatory growth mode of the steps and interface near the freezing temperature and enhancement of the growth rates in the c-axis. These results contradict the previous ?tight-binding? mechanism thought to be responsible for antifreeze function. To study the effects of temperature driven convective flows on the interface kinetics, microgravity experiments were performed in a jet airplane during a parabolic flight path. Step propagation on the basal plane slows down considerably when entering the microgravity condition and reaches a critical condition just below 0.2g.

  4. Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation

    PubMed Central

    Kondo, Hidemasa; Hanada, Yuichi; Sugimoto, Hiroshi; Hoshino, Tamotsu; Garnham, Christopher P.; Davies, Peter L.; Tsuda, Sakae

    2012-01-01

    Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host. Here we report the 0.95-Å high-resolution crystal structure of a 223-residue secreted AFP from the snow mold fungus Typhula ishikariensis. Its main structural element is an irregular β-helix with six loops of 18 or more residues that lies alongside an α-helix. β-Helices have independently evolved as AFPs on several occasions and seem ideally structured to bind to several planes of ice, including the basal plane. A novelty of the β-helical fold is the nonsequential arrangement of loops that places the N- and C termini inside the solenoid of β-helical coils. The ice-binding site (IBS), which could not be predicted from sequence or structure, was located by site-directed mutagenesis to the flattest surface of the protein. It is remarkable for its lack of regularity and its poor conservation in homologs from psychrophilic diatoms and bacteria and other fungi. PMID:22645341

  5. A unique capsular polysaccharide structure from the psychrophilic marine bacterium Colwellia psychrerythraea 34H that mimics antifreeze (glyco)proteins.

    PubMed

    Carillo, Sara; Casillo, Angela; Pieretti, Giuseppina; Parrilli, Ermenegilda; Sannino, Filomena; Bayer-Giraldi, Maddalena; Cosconati, Sandro; Novellino, Ettore; Ewert, Marcela; Deming, Jody W; Lanzetta, Rosa; Marino, Gennaro; Parrilli, Michelangelo; Randazzo, Antonio; Tutino, Maria L; Corsaro, M Michela

    2015-01-14

    The low temperatures of polar regions and high-altitude environments, especially icy habitats, present challenges for many microorganisms. Their ability to live under subfreezing conditions implies the production of compounds conferring cryotolerance. Colwellia psychrerythraea 34H, a γ-proteobacterium isolated from subzero Arctic marine sediments, provides a model for the study of life in cold environments. We report here the identification and detailed molecular primary and secondary structures of capsular polysaccharide from C. psychrerythraea 34H cells. The polymer was isolated in the water layer when cells were extracted by phenol/water and characterized by one- and two-dimensional NMR spectroscopy together with chemical analysis. Molecular mechanics and dynamics calculations were also performed. The polysaccharide consists of a tetrasaccharidic repeating unit containing two amino sugars and two uronic acids bearing threonine as substituent. The structural features of this unique polysaccharide resemble those present in antifreeze proteins and glycoproteins. These results suggest a possible correlation between the capsule structure and the ability of C. psychrerythraea to colonize subfreezing marine environments. PMID:25525681

  6. Thermodynamic Analysis of Thermal Hysteresis: Mechanistic Insights into Biological Antifreezes

    PubMed Central

    Wang, Sen; Amornwittawat, Natapol; Wen, Xin

    2012-01-01

    Antifreeze proteins (AFPs) bind to ice crystal surfaces and thus inhibit the ice growth. The mechanism for how AFPs suppress freezing is commonly modeled as an adsorption-inhibition process by the Gibbs-Thomson effect. Here we develop an improved adsorption-inhibition model for AFP action based on the thermodynamics of impurity adsorption on the crystal surfaces. We demonstrate the derivation of a realistic relationship between surface protein coverage and the protein concentration. We show that the improved model provides a quantitatively better fit to the experimental antifreeze activities of AFPs from distinct structural classes, including fish and insect AFPs, in a wide range of concentrations. Our theoretical results yielded the adsorption coefficients of the AFPs on ice, suggesting that, despite the distinct difference in their antifreeze activities and structures, the affinities of the AFPs to ice are very close and the mechanism of AFP action is a kinetically controlled, reversible process. The applications of the model to more complex systems along with its potential limitations are also discussed. PMID:22822266

  7. Solution structures, dynamics, and ice growth inhibitory activity of peptide fragments derived from an antarctic yeast protein.

    PubMed

    Shah, Syed Hussinien H; Kar, Rajiv K; Asmawi, Azren A; Rahman, Mohd Basyaruddin A; Murad, Abdul Munir A; Mahadi, Nor M; Basri, Mahiran; Rahman, Raja Noor Zaliha A; Salleh, Abu B; Chatterjee, Subhrangsu; Tejo, Bimo A; Bhunia, Anirban

    2012-01-01

    Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities. PMID:23209600

  8. Epithelial dominant expression of antifreeze proteins in cunner suggests recent entry into a high freeze-risk ecozone.

    PubMed

    Hobbs, Rod S; Fletcher, Garth L

    2013-01-01

    Most marine teleost fishes residing in a high freeze-risk ecozone, such as the coastal waters of Newfoundland during winter, avoid freezing by secreting high concentrations of antifreeze proteins (AFP) into their blood plasma where they can bind to and prevent the growth of ice that enter the fish. Cunner (Tautogolabrus adspersus), which overwinter in such shallow waters are the only known exception. Although this species does produce type I AFP, the plasma levels are too low to be of value as a freeze protectant. Southern and Northern blot analyses carried out in this study establish that the cunner AFP genes belong to a multigene family that is predominantly expressed in external epithelia (skin and gill filaments). These results support the hypothesis that the survival of cunner in icy waters is attributable in part to epithelial AFP that help block ice propagation into their interior milieu. In contrast to the cunner, heterospecifics occupying the same habitat have greater freeze protection because they produce AFP in the liver for export to the plasma as well as in external epithelia. Since the external epithelia would be the first tissue to come into contact with ice it is possible that one of the earliest steps involved in the evolution of freeze resistant fish could have been the expression of AFP in tissues such as the skin. We suggest that this epithelial-dominant AFP expression represents a primitive stage in AFP evolution and propose that cunner began to inhabit "freeze-risk ecozones" more recently than heterospecifics. PMID:23085291

  9. Saccharide antifreeze compositions

    DOEpatents

    Walters, Kent; Duman, John G; Serianni, Anthony S

    2013-12-10

    The invention provides an antifreeze glycolipid compounds and composition comprising a polysaccharide moiety of Formula I; ##STR00001## wherein D-Manp represents a D-mannopyranose moiety, D-Xylp represents a D-xylopyranose moiety, and n is about 5 to about 70; and one or more lipid moieties covalently linked to the polysaccharide moiety of Formula I or electrostatically associated with the polysaccaride moiety for Formula I. The antifreeze glycolipid compounds and compositions can be used for a variety of industrial, agricultural, medical, and cosmetic applications where recrystallization-inhibition, cyroprotection, or cryopreservation is desired. The antifreeze glycolipid compounds or compositions can be used as, for example, as cryoprotectants for tissue preservation and transplantation, improving the texture of processed frozen food and frozen meats, frostbit protection, crop protection, and green alternatives for land vehicle antifreeze and aircraft de-icing.

  10. Rational, yet simple, design and synthesis of an antifreeze-protein inspired polymer for cellular cryopreservation† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5cc04647e Click here for additional data file.

    PubMed Central

    Mitchell, Daniel E.; Cameron, Neil R.

    2015-01-01

    Antifreeze (glyco) proteins AF(G)Ps are potent ice recrystallization inhibitors, which is a desirable property to enhance cryopreservation of donor tissue/cells. Here we present the rational synthesis of a new, biomimetic, ice-recrystallization inhibiting polymer derived from a cheap commodity polymer, based on an ampholyte structure. The polymer is used to enhance the cryopreservation of red blood cells, demonstrating a macromolecular solution to tissue storage. PMID:26176027

  11. Validation of antifreeze properties of glutathione based on its thermodynamic characteristics and protection of baker's yeast during cryopreservation.

    PubMed

    Zhang, Chao; Zhang, Hui; Wang, Li; Yao, Huiyuan

    2007-06-13

    The antifreeze ability of glutathione was evaluated on the basis of its thermodynamic characteristics and protection of baker's yeast during cryopreservation at -30 degrees C. The thermodynamic characteristics and protection of baker's yeast of glutathione were similar to those of known antifreeze proteins, such as carrot antifreeze protein and holly antifreeze protein. These properties included lowering the freezing point at about 0.20 degrees C non-colligatively, decreasing freezable water content, controlling the movement of free water for its strong hydrophilicity, and improving baker's yeast survival during the simulated processing of frozen dough. Therefore, glutathione was viewed to be an antifreeze protein like substance on the basis of its unique thermodynamic characteristics and protection of baker's yeast. The method combining thermodynamic characteristic analysis and protection evaluation is a new and simple way to screen new antifreeze proteins. PMID:17508758

  12. Antifreeze glycopeptide adsorption on single crystal ice surfaces using ellipsometry

    PubMed Central

    Wilson, P. W.; Beaglehole, D.; DeVries, A. L.

    1993-01-01

    Antarctic fishes synthesise antifreeze proteins which can effectively inhibit the growth of ice crystals. The mechanism relies on adsorption of these proteins to the ice surface. Ellipsometry has been used to quantify glycopeptide antifreeze adsorption to the basal and prism faces of single ice crystals. The rate of accumulation was determined as a function of time and at concentrations between 0.0005 and 1.2 mg/ml. Estimates of packing density at saturation coverage have been made for the basal and prism faces. PMID:19431902

  13. Bacterial Ice Crystal Controlling Proteins

    PubMed Central

    Lorv, Janet S. H.; Rose, David R.; Glick, Bernard R.

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  14. Evaluation of propanediol, ethylene glycol, sucrose and antifreeze proteins on the survival of slow-cooled mouse pronuclear and 4-cell embryos.

    PubMed

    Shaw, J M; Ward, C; Trounson, A O

    1995-02-01

    Mouse pronuclear and 4-cell embryos were cryopreserved by slow cooling to -33 degrees C in 1.5 M 1,2-propanediol or 1.5 M ethylene glycol, with or without 0.1 M sucrose. Straws were thawed by immersion into a 37 degrees C water bath, immediately after their removal from liquid nitrogen (protocol 1), or after being held in air for 15 (protocol 2) or 30 s (protocol 3). Others were held in air until the ice melted (protocol 4). Embryos which formed blastocysts that hatched and attached to the Petri dish in vitro (plated) were considered viable. The thawing protocol did not significantly influence the viability of embryos frozen in propanediol with 0.1 M sucrose (52-72% of pronuclear and 69-97% of 4-cell embryos plated). In the other solutions tested, propanediol without sucrose and ethylene glycol with/without sucrose, only protocol 2 resulted in uniformly high development of both pronuclear (45-65% plating) and 4-cell embryos (70-97% plating). Thawing protocol 4 significantly reduced development, in particular for embryos frozen in ethylene glycol (0% 1-cell; 0-25% 4-cell plating). The difference between thawing protocols 2 and 4 was reduced by continuing slow cooling of ethylene glycol solutions to lower temperatures (-41 degrees C). Adding antifreeze proteins type I or III did not improve survival or development. Thus, although mouse pronuclear and 4-cell embryos can be frozen-thawed in either ethylene glycol or propanediol without significant loss of viability, an appropriate thawing protocol is essential for embryos frozen in ethylene glycol or propanediol-sucrose. PMID:7769070

  15. Conformational and dynamic properties of a 14 residue antifreeze glycopeptide from Antarctic cod.

    PubMed Central

    Lane, A. N.; Hays, L. M.; Feeney, R. E.; Crowe, L. M.; Crowe, J. H.

    1998-01-01

    The 1H and 13C NMR spectra of a 14-residue antifreeze glycopeptide from Antarctic cod (Tetramatomnus borchgrevinki) containing two proline residues have been assigned. 13C NMR relaxation experiments indicate motional anisotropy of the peptide, with a tumbling time in water at 5 degrees C of 3-4 ns. The relaxation data and lack of long-range NOEs are consistent with a linear peptide undergoing significant segmental motion. However, extreme values of some coupling constants and strong sequential NOEs indicate regions of local order, which are most evident at the two ATPA subsequences. Similar spectroscopic properties were observed in the 16-residue analogue containing an Arg-Ala dipeptide added to the C-terminus. Molecular modeling also showed no evidence of long-range order, but the two ATPA subsequences were relatively well determined by the experimental data. These motifs were quite distinct from helical structures or beta turns commonly found in proteins, but rather resemble sections of an extended polyproline helix. Thus, the NMR data provide a description of the local order, which is of relevance to the mechanism of action of the antifreeze activity of the antifreeze glycopeptides as well as their ability to protect cells during hypothermic storage. PMID:9684888

  16. Nontoxic Antifreeze for Insect Traps

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Propylene glycol in water is a safe and effective alternative to ethylene glycol as a capture liquid in insect traps (pitfalls, flight intercepts, pan traps). Propylene glycol formulations are readily available because it is the primary (95%) ingredient in certain automotive antifreeze formulations...

  17. NOVEL ICE-BINDING PROTEINS FROM A PSYCHROPHILIC ANTARCTIC ALGA (CHLAMYDOMONADACEAE, CHLOROPHYCEAE)(1).

    PubMed

    Raymond, James A; Janech, Michael G; Fritsen, Christian H

    2009-02-01

    Many cold-adapted unicellular plants express ice-active proteins, but at present, only one type of such proteins has been described, and it shows no resemblance to higher plant antifreezes. Here, we describe four isoforms of a second and very active type of extracellular ice-binding protein (IBP) from a unicellular chlamydomonad alga collected from an Antarctic intertidal location. The alga is a euryhaline psychrophile that, based on sequences of the alpha tubulin gene and an IBP gene, appears to be the same as a snow alga collected on Petrel Island, Antarctica. The IBPs, which do not resemble any known antifreezes, have strong recrystallization inhibition activity and have an ability to slow the drainage of brine from sea ice. These properties, by maintaining liquid environments, may increase survival of the cells in freezing environments. The IBPs have a repeating TXT motif, which has previously been implicated in ice binding in insect antifreezes and a ryegrass antifreeze. PMID:27033652

  18. Latent Ice Recrystallization Inhibition Activity in Nonantifreeze Proteins: Ca2+-Activated Plant Lectins and Cation-Activated Antimicrobial Peptides

    PubMed Central

    2015-01-01

    Organisms living in polar regions have evolved a series of antifreeze (glyco) proteins (AFGPs) to enable them to survive by modulating the structure of ice. These proteins have huge potential for use in cellular cryopreservation, ice-resistant surfaces, frozen food, and cryosurgery, but they are limited by their relatively low availability and questions regarding their mode of action. This has triggered the search for biomimetic materials capable of reproducing this function. The identification of new structures and sequences capable of inhibiting ice growth is crucial to aid our understanding of these proteins. Here, we show that plant c-type lectins, which have similar biological function to human c-type lectins (glycan recognition) but no sequence homology to AFPs, display calcium-dependent ice recrystallization inhibition (IRI) activity. This IRI activity can be switched on/off by changing the Ca2+ concentration. To show that more (nonantifreeze) proteins may exist with the potential to display IRI, a second motif was considered, amphipathicity. All known AFPs have defined hydrophobic/hydrophilic domains, rationalizing this choice. The cheap, and widely used, antimicrobial Nisin was found to have cation-dependent IRI activity, controlled by either acid or addition of histidine-binding ions such as zinc or nickel, which promote its amphipathic structure. These results demonstrate a new approach in the identification of antifreeze protein mimetic macromolecules and may help in the development of synthetic mimics of AFPs. PMID:26407233

  19. Latent Ice Recrystallization Inhibition Activity in Nonantifreeze Proteins: Ca2+-Activated Plant Lectins and Cation-Activated Antimicrobial Peptides.

    PubMed

    Mitchell, Daniel E; Gibson, Matthew I

    2015-10-12

    Organisms living in polar regions have evolved a series of antifreeze (glyco) proteins (AFGPs) to enable them to survive by modulating the structure of ice. These proteins have huge potential for use in cellular cryopreservation, ice-resistant surfaces, frozen food, and cryosurgery, but they are limited by their relatively low availability and questions regarding their mode of action. This has triggered the search for biomimetic materials capable of reproducing this function. The identification of new structures and sequences capable of inhibiting ice growth is crucial to aid our understanding of these proteins. Here, we show that plant c-type lectins, which have similar biological function to human c-type lectins (glycan recognition) but no sequence homology to AFPs, display calcium-dependent ice recrystallization inhibition (IRI) activity. This IRI activity can be switched on/off by changing the Ca2+ concentration. To show that more (nonantifreeze) proteins may exist with the potential to display IRI, a second motif was considered, amphipathicity. All known AFPs have defined hydrophobic/hydrophilic domains, rationalizing this choice. The cheap, and widely used, antimicrobial Nisin was found to have cation-dependent IRI activity, controlled by either acid or addition of histidine-binding ions such as zinc or nickel, which promote its amphipathic structure. These results demonstrate a new approach in the identification of antifreeze protein mimetic macromolecules and may help in the development of synthetic mimics of AFPs. PMID:26407233

  20. Inhibition of bacterial ice nucleators by fish antifreeze glycoproteins.

    PubMed

    Parody-Morreale, A; Murphy, K P; Di Cera, E; Fall, R; DeVries, A L; Gill, S J

    1988-06-23

    Certain bacteria promote the formation of ice in super-cooled water by means of ice nucleators which contain a unique protein associated with the cell membrane. Ice nucleators in general are believed to act by mimicking the structure of an ice crystal surface, thus imposing an ice-like arrangement on the water molecules in contact with the nucleating surface and lowering the energy necessary for the initiation of ice formation. Quantitative investigation of the bacterial ice-nucleating process has recently been made possible by the discovery of certain bacteria that shed stable membrane vesicles with ice nucleating activity. The opposite effect, inhibition of ice formation, has been described for a group of glycoproteins found in different fish and insect species. This group of substances, termed antifreeze glycoproteins (AFGPs), promotes the supercooling of water with no appreciable effect on the equilibrium freezing point or melting temperature. Substantial evidence now indicates that AFGPs act by binding to a growing ice crystal and slowing crystal growth. As the ice-nucleating protein surface is believed to have a structure similar to an embryonic ice crystal, AFGPs might be predicted to interact directly with a bacterial ice-nucleating site. We report here that AFGPs from the antarctic fish Dissostichus mawsoni inhibit the ice-nucleating activity of membrane vesicles from the bacterium Erwinia herbicola. The inhibition effect shows saturation at high concentration of AFGP and conforms to a simple binding reaction between the AFGP and the nucleation centre. PMID:3386720

  1. Structure and collective dynamics of hydrated anti-freeze protein type III from 180 K to 298 K by X-ray diffraction and inelastic X-ray scattering

    NASA Astrophysics Data System (ADS)

    Yoshida, Koji; Baron, Alfred Q. R.; Uchiyama, Hiroshi; Tsutsui, Satoshi; Yamaguchi, Toshio

    2016-04-01

    We investigated hydrated antifreeze protein type III (AFP III) powder with a hydration level h (=mass of water/mass of protein) of 0.4 in the temperature range between 180 K and 298 K using X-ray diffraction and inelastic X-ray scattering (IXS). The X-ray diffraction data showed smooth, largely monotonic changes between 180 K and 298 K without freezing water. Meanwhile, the collective dynamics observed by IXS showed a strong change in the sound velocity at 180 K, after being largely temperature independent at higher temperatures (298-220 K). We interpret this change in terms of the dynamic transition previously discussed using other probes including THz IR absorption spectroscopy and incoherent elastic and quasi-elastic neutron scattering. This finding suggests that the dynamic transition of hydrated proteins is observable on the subpicosecond time scale as well as nano- and pico-second scales, both in collective dynamics from IXS and single particle dynamics from neutron scattering. Moreover, it is most likely that the dynamic transition of hydrated AFP III is not directly correlated with its hydration structure.

  2. Structure and collective dynamics of hydrated anti-freeze protein type III from 180 K to 298 K by X-ray diffraction and inelastic X-ray scattering.

    PubMed

    Yoshida, Koji; Baron, Alfred Q R; Uchiyama, Hiroshi; Tsutsui, Satoshi; Yamaguchi, Toshio

    2016-04-01

    We investigated hydrated antifreeze protein type III (AFP III) powder with a hydration level h (=mass of water/mass of protein) of 0.4 in the temperature range between 180 K and 298 K using X-ray diffraction and inelastic X-ray scattering (IXS). The X-ray diffraction data showed smooth, largely monotonic changes between 180 K and 298 K without freezing water. Meanwhile, the collective dynamics observed by IXS showed a strong change in the sound velocity at 180 K, after being largely temperature independent at higher temperatures (298-220 K). We interpret this change in terms of the dynamic transition previously discussed using other probes including THz IR absorption spectroscopy and incoherent elastic and quasi-elastic neutron scattering. This finding suggests that the dynamic transition of hydrated proteins is observable on the subpicosecond time scale as well as nano- and pico-second scales, both in collective dynamics from IXS and single particle dynamics from neutron scattering. Moreover, it is most likely that the dynamic transition of hydrated AFP III is not directly correlated with its hydration structure. PMID:27059578

  3. Effect of Antifreeze Peptide Pretreatment on Ice Crystal Size, Drip Loss, Texture, and Volatile Compounds of Frozen Carrots.

    PubMed

    Kong, Charles H Z; Hamid, Nazimah; Liu, Tingting; Sarojini, Vijayalekshmi

    2016-06-01

    Ice crystal formation is of primary concern to the frozen food industry. In this study, the effects of antifreeze peptides (AFPs) on ice crystal formation were assessed in carrot during freezing and thawing. Three synthetic analogues based on naturally occurring antifreeze peptides were used in this study. The AFPs exhibited modification of ice crystal morphology, confirming their antifreeze activity in vitro. The ability of the synthetic AFPs to minimize drip loss and preserve color, structure, texture, and volatiles of frozen carrot was evaluated using the techniques of SEM, GC-MS, and texture analysis. The results prove the potential of these AFPs to preserve the above characteristics in frozen carrot samples. PMID:27138051

  4. Threonine side chain conformational population distribution of a type I antifreeze protein on interacting with ice surface studied via ¹³C-¹⁵N dynamic REDOR NMR.

    PubMed

    Mao, Yougang; Jeong, Myongho; Wang, Tieli; Ba, Yong

    2011-01-01

    Antifreeze proteins (AFPs) provide survival mechanism for species living in subzero environments by lowering the freezing points of their body fluids effectively. The mechanism is attributed to AFPs' ability to inhibit the growth of seed ice crystals through adsorption on specific ice surfaces. We have applied dynamic REDOR (Rotational Echo Double Resonance) solid state NMR to study the threonine (Thr) side chain conformational population distribution of a site-specific Thr ¹³C(γ) and ¹⁵N doubly labeled type I AFP in frozen aqueous solution. It is known that the Thr side chains together with those of the 4th and 8th Alanine (Ala) residues commencing from the Thrs (the 1st) in the four 11-residue repeat units form the peptide ice-binding surface. The conformational information can provide structural insight with regard to how the AFP side chains structurally interact with the ice surface. χ-squared statistical analysis of the experimental REDOR data in fitting the theoretically calculated dynamic REDOR fraction curves indicates that when the AFP interacted with the ice surface in the frozen AFP solution, the conformations of the Thr side chains changed from the anti-conformations, as in the AFP crystal structure, to partial population in the anti-conformation and partial population in the two gauche conformations. This change together with the structural analysis indicates that the simultaneous interactions of the methyl groups and the hydroxyl groups of the Thr side chains with the ice surface could be the reason for the conformational population change. The analysis of the theoretical dynamic REDOR fraction curves shows that the set of experimental REDOR data may fit a number of theoretical curves with different population distributions. Thus, other structural information is needed to assist in determining the conformational population distribution of the Thr side chains. PMID:21470833

  5. Modified Antifreeze Liquids for Use on Surfaces

    NASA Technical Reports Server (NTRS)

    Lynn, R. O.

    1983-01-01

    Report presents results of evaluation of two antifreeze liquids, dimethyl sulfoxide and ethylene glycol and five viscosity modifiers: gelatin, gum tragacanth, starch, agarose powder and citrus pectin. Purpose of evaluation to find best way of dealing with frost formation on Space Shuttle.

  6. Antifreeze glycoproteins from an Antarctic fish. Quasi-elastic light scattering studies of the hydrodynamic conformations of antifreeze glycoproteins.

    PubMed

    Ahmed, A I; Feeney, R E; Osuga, D T; Yeh, Y

    1975-05-10

    A quasi-elastic light-scattering technique was used to study the hydrodynamic conformations of antifreeze glycoproteins from an Antarctic fish. Antifreeze glycoprotein is composed of repeating units of Ala-Ala-Thr, with each threonine O-linked to a disaccharide, and it exists as several polymers of different numbers of this repeating unit. Molecular weights of the two major active polymers are 10,500 and 17,500 by such methods as centrifugation and osmotic pressure, but smaller than 20 by freezing-point depression. Translational diffusion coefficients at 20 degrees were 8.35 times 10-7 cm2 s-1 and 6.15 times 10-7 cm2 s-1 for the M-r-10,500 and 17,500 polymers, respectively. Measurements at -0.2 degrees in the presence of ice crystals did not indicate any conformational changes that might be related to the lowering of the freezing temperature. Lowering the temperature of these glycoprotein solutions close to temperatures of freezing caused a decrease in the effective hydrodynamic radius of both active and inactive glycoprotein components. PMID:1168194

  7. Antifreeze glycoproteins inhibit leakage from liposomes during thermotropic phase transitions.

    PubMed Central

    Hays, L M; Feeney, R E; Crowe, L M; Crowe, J H; Oliver, A E

    1996-01-01

    Antifreeze glycoproteins (AFGPs), found in the blood of polar fish at concentrations as high as 35 g/liter, are known to prevent ice crystal growth and depress the freezing temperature of the blood. Previously, Rubinsky et al. [Rubinsky, B., Mattioli, M., Arav, A., Barboni, B. & Fletcher, G. L. (1992) Am. J. Physiol. 262, R542-R545] provided evidence that AFGPs block ion fluxes across membranes during cooling, an effect that they ascribed to interactions with ion channels. We investigated the effects of AFGPs on the leakage of a trapped marker from liposomes during chilling. As these liposomes are cooled through the transition temperature, they leak approximately 50% of their contents. Addition of less than 1 mg/ml of AFGP prevents up to 100% of this leakage, both during chilling and warming through the phase transition. This is a general effect that we show here applies to liposomes composed of phospholipids with transition temperatures ranging from 12 degrees C to 41 degrees C. Because these results were obtained with liposomes composed of phospholipids alone, we conclude that the stabilizing effects of AFGPs on intact cells during chilling reported by Rubinsky et al. may be due to a nonspecific effect on the lipid components of native membranes. There are other proteins that prevent leakage, but only under specialized conditions. For instance, antifreeze proteins, bovine serum albumin, and ovomucoid all either have no effect or actually induce leakage. Following precipitation with acetone, all three proteins inhibited leakage, although not to the extent seen with AFGPs. Alternatively, there are proteins such as ovotransferrin that have no effect on leakage, either before or after acetone precipitation. PMID:8692905

  8. Antifreeze effect of carboxylated ε-poly-L-lysine on the growth kinetics of ice crystals.

    PubMed

    Vorontsov, Dmitry A; Sazaki, Gen; Hyon, Suong-Hyu; Matsumura, Kazuaki; Furukawa, Yoshinori

    2014-08-28

    Some biological substances control the nucleation and growth of inorganic crystals. Antifreeze proteins, which prohibit ice crystal growth in living organisms, promise are also important as biological antifreezes for medical applications and in the frozen food industries. In this work, we investigated the crystallization of ice in the presence of a new cryoprotector, carboxylated ε-poly-L-lysine (COOH-PLL). In order to reveal the characteristics and the mechanism of its antifreeze effect, free-growth experiments of ice crystals were carried out in solutions with various COOH-PLL concentrations and degrees of supercooling, and the depression of the freezing point and growth rates of the tips of ice dendrites were obtained using optical microscopy. Hysteresis of growth rates and depression of the freezing point was revealed in the presence of COOH-PLL. The growth-inhibition effect of COOH-PLL molecules could be explained on the basis of the Gibbs-Thomson law and the use of Langmuir's adsorption isotherm. Theoretical kinetic curves for hysteresis calculated on the basis of Punin-Artamonova's model were in good agreement with experimental data. We conclude that adsorption of large biological molecules in the case of ice crystallization has a non-steady-state character and occurs more slowly than the process of embedding of crystal growth units. PMID:25113284

  9. Translational dynamics of antifreeze glycoprotein in supercooled water.

    PubMed

    Krishnan, V V; Fink, William H; Feeney, Robert E; Yeh, Yin

    2004-08-01

    Structure and dynamics of biomolecules in supercooled water assume a particular and distinct importance in the case of antifreeze glycoproteins (AFGPs), which function at sub-zero temperatures. To investigate whether any large-scale structural digressions in the supercooled state are correlated to the function of AFGPs, self-diffusion behavior of the AFGP8, the smallest AFGP is monitored as a function of temperature from 243 to 303 K using nuclear magnetic resonance (NMR) spectroscopy. The experimental results are compared with the hydrodynamic calculations using the viscosity of water at the same temperature range. In order to evaluate results on AFGP8, the smallest AFGP, constituting approximately two-thirds of the total AFGP fraction in fish blood serum, similar experimental and computational calculations were also performed on a set of globular proteins. These results show that even though the general trend of translational dynamics of AFGP is similar to that of the other globular proteins, AFGP8 appears to be more hydrated (approximately 30% increase in the bead radius) than the others over the temperature range studied. These results also suggest that local conformational changes such as segmental librations or hydrogen bond dynamics that are closer to the protein surface are more likely the determining dynamic factors for the function of AFGPs rather than any large-scale structural rearrangements. PMID:15228958

  10. Dynamics of antifreeze glycoproteins in the presence of ice.

    PubMed Central

    Tsvetkova, Nelly M; Phillips, Brian L; Krishnan, Viswanathan V; Feeney, Robert E; Fink, William H; Crowe, John H; Risbud, Subhash H; Tablin, Fern; Yeh, Yin

    2002-01-01

    Antifreeze glycoproteins from the Greenland cod Boreogadus saida were dimethylated at the N-terminus (m*AFGP) and their dynamics and conformational properties were studied in the presence of ice using (13)C-NMR and FTIR spectroscopy. (13)C-NMR experiments of m*AFGP in D(2)O, in H(2)O, and of freeze-dried m*AFGP were performed as a function of temperature. Dynamic parameters ((1)H T(1 rho) and T(CH)) obtained by varying the contact time revealed notable differences in the motional properties of AFGP between the different states. AFGP/ice dynamics was dominated by fast-scale motions (nanosecond to picosecond time scale), suggesting that the relaxation is markedly affected by the protein hydration. The data suggest that AFGP adopts a similar type of three-dimensional fold both in the presence of ice and in the freeze-dried state. FTIR studies of the amide I band did not show a single prevailing secondary structure in the frozen state. The high number of conformers suggests a high flexibility, and possibly reflects the necessity to expose more ice-binding groups. The data suggest that the effect of hydration on the local mobility of AFGP and the lack of significant change in the backbone conformation in the frozen state may play a role in inhibiting the ice crystal growth. PMID:11751333

  11. Ice Growth Inhibition in Antifreeze Polypeptide Solution by Short-Time Solution Preheating.

    PubMed

    Nishi, Naoto; Miyamoto, Takuya; Waku, Tomonori; Tanaka, Naoki; Hagiwara, Yoshimichi

    2016-01-01

    The objective of this study is to enhance the inhibition of ice growth in the aqueous solution of a polypeptide, which is inspired by winter flounder antifreeze protein. We carried out measurements on unidirectional freezing of the polypeptide solution. The thickness of the solution was 0.02 mm, and the concentration of polypeptide was varied from 0 to 2 mg/mL. We captured successive microscopic images of ice/solution interfaces, and measured the interface velocity from the locations of tips of the pectinate interface in the images. We also simultaneously measured the temperature by using a small thermocouple. The ice/solution interface temperature was defined by the temperature at the tips. It was found that the interface temperature was decreased with an increasing concentration of polypeptide. To try varying the activity of the polypeptide, we preheated the polypeptide solution and cooled it before carrying out the measurements. Preheating for 1-5 hours was found to cause a further decrease in the interface temperature. Furthermore, wider regions of solution and ice with inclined interfaces in the pectinate interface structure were observed, compared with the case where the solution was not preheated. Thus, the ice growth inhibition was enhanced by this preheating. To investigate the reason for this enhancement, we measured the conformation and aggregates of polypeptide in the solution. We also measured the local concentration of polypeptide. It was found that the polypeptide aggregates became larger as a result of preheating, although the polypeptide conformation was unchanged. These large aggregates caused both adsorption to the interface and the wide regions of supercooled solution in the pectinate interface structure. PMID:27152720

  12. Ice Growth Inhibition in Antifreeze Polypeptide Solution by Short-Time Solution Preheating

    PubMed Central

    Nishi, Naoto; Miyamoto, Takuya; Waku, Tomonori; Tanaka, Naoki; Hagiwara, Yoshimichi

    2016-01-01

    The objective of this study is to enhance the inhibition of ice growth in the aqueous solution of a polypeptide, which is inspired by winter flounder antifreeze protein. We carried out measurements on unidirectional freezing of the polypeptide solution. The thickness of the solution was 0.02 mm, and the concentration of polypeptide was varied from 0 to 2 mg/mL. We captured successive microscopic images of ice/solution interfaces, and measured the interface velocity from the locations of tips of the pectinate interface in the images. We also simultaneously measured the temperature by using a small thermocouple. The ice/solution interface temperature was defined by the temperature at the tips. It was found that the interface temperature was decreased with an increasing concentration of polypeptide. To try varying the activity of the polypeptide, we preheated the polypeptide solution and cooled it before carrying out the measurements. Preheating for 1–5 hours was found to cause a further decrease in the interface temperature. Furthermore, wider regions of solution and ice with inclined interfaces in the pectinate interface structure were observed, compared with the case where the solution was not preheated. Thus, the ice growth inhibition was enhanced by this preheating. To investigate the reason for this enhancement, we measured the conformation and aggregates of polypeptide in the solution. We also measured the local concentration of polypeptide. It was found that the polypeptide aggregates became larger as a result of preheating, although the polypeptide conformation was unchanged. These large aggregates caused both adsorption to the interface and the wide regions of supercooled solution in the pectinate interface structure. PMID:27152720

  13. Bioinspired Antifreeze Secreting Frost-Responsive Pagophobic Coatings

    NASA Astrophysics Data System (ADS)

    Sun, Xiaoda; Damle, Viraj; Rykaczewski, Konrad

    2014-11-01

    Prevention of ice and frost accumulation is of interest to transportation, power generation, and agriculture industries. Superhydrophobic and lubricant impregnated pagophobic coatings have been proposed, however, they both fail in frosting conditions. Inspired by functional liquid secretion in natural systems, such as toxin secretion by poison dart frost in response to predator presence, we developed frost-responsive antifreeze secreting pagophobic coatings. These are bi-layered coatings with an inner superhydrophilic ``dermis'' infused with antifreeze and an outer permeable superhydrophobic ``epidermis.'' The superhydrophobic epidermis separates the antifreeze from the environment and prevents ice accumulation by repelling impinging water droplets. In frosting conditions, the antifreeze is secreted from the dermis through pores in the epidermis either due to contact with condensed droplets or temporary switch of the epidermis wettability from hydrophobic to hydrophilic caused by surface icing. Here we demonstrate superior performance of this multifunctional coating in simulated frosting, freezing mist/fog, and freezing spray/rain conditions. KR acknowledges startup funding from ASU.

  14. Observation of ice-like water layers at an aqueous protein surface.

    PubMed

    Meister, Konrad; Strazdaite, Simona; DeVries, Arthur L; Lotze, Stephan; Olijve, Luuk L C; Voets, Ilja K; Bakker, Huib J

    2014-12-16

    We study the properties of water at the surface of an antifreeze protein with femtosecond surface sum frequency generation spectroscopy. We find clear evidence for the presence of ice-like water layers at the ice-binding site of the protein in aqueous solution at temperatures above the freezing point. Decreasing the temperature to the biological working temperature of the protein (0 °C to -2 °C) increases the amount of ice-like water, while a single point mutation in the ice-binding site is observed to completely disrupt the ice-like character and to eliminate antifreeze activity. Our observations indicate that not the protein itself but ordered ice-like water layers are responsible for the recognition and binding to ice. PMID:25468976

  15. Observation of ice-like water layers at an aqueous protein surface

    PubMed Central

    Meister, Konrad; Strazdaite, Simona; DeVries, Arthur L.; Lotze, Stephan; Olijve, Luuk L. C.; Voets, Ilja K.; Bakker, Huib J.

    2014-01-01

    We study the properties of water at the surface of an antifreeze protein with femtosecond surface sum frequency generation spectroscopy. We find clear evidence for the presence of ice-like water layers at the ice-binding site of the protein in aqueous solution at temperatures above the freezing point. Decreasing the temperature to the biological working temperature of the protein (0 °C to −2 °C) increases the amount of ice-like water, while a single point mutation in the ice-binding site is observed to completely disrupt the ice-like character and to eliminate antifreeze activity. Our observations indicate that not the protein itself but ordered ice-like water layers are responsible for the recognition and binding to ice. PMID:25468976

  16. Separating proteins with activated carbon.

    PubMed

    Stone, Matthew T; Kozlov, Mikhail

    2014-07-15

    Activated carbon is applied to separate proteins based on differences in their size and effective charge. Three guidelines are suggested for the efficient separation of proteins with activated carbon. (1) Activated carbon can be used to efficiently remove smaller proteinaceous impurities from larger proteins. (2) Smaller proteinaceous impurities are most efficiently removed at a solution pH close to the impurity's isoelectric point, where they have a minimal effective charge. (3) The most efficient recovery of a small protein from activated carbon occurs at a solution pH further away from the protein's isoelectric point, where it is strongly charged. Studies measuring the binding capacities of individual polymers and proteins were used to develop these three guidelines, and they were then applied to the separation of several different protein mixtures. The ability of activated carbon to separate proteins was demonstrated to be broadly applicable with three different types of activated carbon by both static treatment and by flowing through a packed column of activated carbon. PMID:24898563

  17. Protein extraction from activated sludge.

    PubMed

    Denecke, M

    2006-01-01

    Two methods for the separation of protein originating from activated sludge were compared. In one method, the total protein was isolated out of the activated sludge (crude extract). These samples included all dissolved proteins originating from the bacterial cells and biofilm made up of extracellular polymeric substances (EPS). Every time polyacrylamide gel electrophoresis (PAGE) was done, the protein bands from samples of crude extract were covered by polymeric substances including carbohydrates, uronic acids or humic compounds. Using the immunoblot technique it was possible to demonstrate the presence of the heat shock protein HSP70 in crude extracts of activated sludge. The comparison of protein fingerprints required that clear and distinct bands appear on the PAGE analysis. To this end, a procedure to separates bacterial cells from the EPS was developed. Bacterial cells were separated by incubation with EDTA and subsequent filtration. The isolated cells were directly incubated in a sample buffer. PMID:16898150

  18. Ice growth in supercooled solutions of antifreeze glycoproteins

    NASA Technical Reports Server (NTRS)

    Harrison, K.; Hallett, J.; Burcham, T. S.; Feeney, R. E.; Kerr, W. L.

    1987-01-01

    The effects of different degrees of supercooling on the habit and rates of growth of ice crystals from solutions of antifreeze glycoproteins are reported. To isolate the influence of different solutions and supercooling alone, a system was devised that nucleated crystals in the middle of a uniformly supercooled sample. Alternatively, single crystals of selected orientation were inserted into free liquid surface. A crystallization rate up to five times greater than that in pure water was found. A mechanism explaining these results is suggested.

  19. Adsorption to ice of fish antifreeze glycopeptides 7 and 8.

    PubMed Central

    Knight, C A; Driggers, E; DeVries, A L

    1993-01-01

    Experimental results show that fish antifreeze glycopeptides (AFGPs) 8 and 7 (with 4 and 5 repeats respectively of the Ala-Ala-Thr backbone sequence) bond onto ice prism planes aligned along a-axes, and inhibit crystal growth on prism planes and on surfaces close to that orientation. The 9.31-A repeat spacing of the AFGP in the polyproline II helix configuration, deduced from NMR studies, matches twice the repeat spacing of ice in the deduced alignment direction, 9.038 A, within 3%. A specific binding model is proposed for the AFGP and for the alpha-helical antifreeze peptide of winter flounder. For AFGP 7-8, two hydroxyl groups of each disaccharide (one disaccharide is attached to each threonine) reside within the ice surface, so that they are shared between the ice crystal and the disaccharide. This provides 24 hydrogen bonds between AFGP 8 and the ice and 30 for AFGP 7, explaining why the chemical adsorption is virtually irreversible and the crystal growth can be stopped virtually completely. The same scheme of sharing polar groups with the ice works well with the alpha-helical antifreeze of winter flounder, for which an amide as well as several hydroxyls are shared. The sharing of polar groups with the ice crystal, rather than hydrogen-bonding to the ice surface, may be a general requirement for adsoprtion-inhibition of freezing. Images FIGURE 2 FIGURE 5 PMID:8431545

  20. Isolation of an antifreeze peptide from the Antarctic sponge Homaxinella balfourensis

    PubMed Central

    Wilkins, S. P.; Blum, A. J.; Burkepile, D. E.; Rutland, T. J.; Wierzbicki, A.; Kelly, M.; Hamann, M.T.

    2016-01-01

    Polar plants and animals survive in subzero waters (−2°C) and many of these marine organisms produce antifreeze proteins (AFPs) to better adapt themselves to these conditions. AFPs prevent the growth of ice crystals which disrupt cellular membranes and destroy cells by inhibiting crystallization of water within the organism. The hydrophilic extract of an Antarctic sponge Homaxinella balfourensis exhibited a non-colligative freezing point depression effect on the crystal morphology of water. The extract was purified by repeated reverse phase high-pressure liquid chromatography, then assayed and shown to contain several AFPs. The major peptide was isolated, analyzed using matrix-assisted laser desorption ionization mass spectrometry and the partial structure of the peptide identified through amino acid sequencing. AFPs have potential applications in agriculture, medicine and the food industry. PMID:12568347

  1. Bacterial Heat Shock Protein Activity

    PubMed Central

    Maleki, Farajollah; Khosravi, Afra; Nasser, Ahmad; Taghinejad, Hamid

    2016-01-01

    Bacteria are exposed to different types of stress in their growth conditions. They have developed appropriate responses, modulated by the re-modeling of protein complexes and by phosphorylation dependent signal transduction systems, to adapt and to survive in a variety range of nature. Proteins are essential components for biologic activity in the eukaryotic and prokaryotic cell. Heat Shock Proteins (HSP) have been identified from various organisms and have critical role in cell hemostasis. Chaperone can sense environment and have different potential role in the organism evolution. PMID:27134861

  2. Directly probing the antifreeze glycoprotein kinetics at the ice/solution interface

    NASA Astrophysics Data System (ADS)

    Zepeda, Salvador; Yokoyama, Etsuro; Furukawa, Yoshinor

    2009-03-01

    Antifreeze proteins (AFP) and glycoproteins (AFGP) help fish, plants, insects and bacteria survive sub-freezing environments. It is well known that these proteins function via some surface interaction, but the exact mechanism has eluded scientists. Aside from mutagenesis experiments directed towards examining the functional importance of specific residues, conclusions about the mechanism have been drawn from indirect studies or more precisely from studies that describe the proteins effects on the ice interface. Our work is aimed at directly studying the protein kinetics at the ice/solution interface. Fluorescent microscopy is used to determine interaction planes, surface concentrations as well as adsorption characteristics and the segregation constants, while fourier transform infra-red attenuated total reßectance (FTIR-ATR) is used to determine the protein structure vs. temperature in the liquid and solid states as well as the ice interface characteristics. All data show that AFGP do not function by the characteristic Gibbs-Thomson mechanism. While the surface coverage is similar for the AFPIII, segregation (amount in ice/amount in solution) is non-zero.

  3. Antiviral activities of whey proteins.

    PubMed

    Ng, Tzi Bun; Cheung, Randy Chi Fai; Wong, Jack Ho; Wang, Yan; Ip, Denis Tsz Ming; Wan, David Chi Cheong; Xia, Jiang

    2015-09-01

    Milk contains an array of proteins with useful bioactivities. Many milk proteins encompassing native or chemically modified casein, lactoferrin, alpha-lactalbumin, and beta-lactoglobulin demonstrated antiviral activities. Casein and alpha-lactalbumin gained anti-HIV activity after modification with 3-hydroxyphthalic anhydride. Many milk proteins inhibited HIV reverse transcriptase. Bovine glycolactin, angiogenin-1, lactogenin, casein, alpha-lactalbumin, beta-lactoglobulin, bovine lactoferrampin, and human lactoferrampin inhibited HIV-1 protease and integrase. Several mammalian lactoferrins prevented hepatitis C infection. Lactoferrin, methylated alpha-lactalbumin and methylated beta-lactoglobulin inhibited human cytomegalovirus. Chemically modified alpha-lactalbumin, beta-lactoglobulin and lysozyme, lactoferrin and lactoferricin, methylated alpha-lactalbumin, methylated and ethylated beta-lactoglobulins inhibited HSV. Chemically modified bovine beta-lactoglobulin had antihuman papillomavirus activity. Beta-lactoglobulin, lactoferrin, esterified beta-lactoglobulin, and esterified lactoferrindisplayed anti-avian influenza A (H5N1) activity. Lactoferrin inhibited respiratory syncytial virus, hepatitis B virus, adenovirus, poliovirus, hantavirus, sindbis virus, semliki forest virus, echovirus, and enterovirus. Milk mucin, apolactoferrin, Fe(3+)-lactoferrin, beta-lactoglobulin, human lactadherin, bovine IgG, and bovine kappa-casein demonstrated antihuman rotavirus activity. PMID:26198883

  4. Why are hyperactive ice-binding-proteins so active?

    NASA Astrophysics Data System (ADS)

    Braslavsky, Ido; Celik, Yeliz; Pertaya, Natalya; Eun Choi, Young; Bar, Maya; Davies, Peter L.

    2008-03-01

    Ice binding proteins (IBPs), also called `antifreeze proteins' or `ice structuring proteins', are a class of proteins that protect organisms from freezing injury. These proteins have many applications in medicine and agriculture, and as a platform for future biotechnology applications. One of the interesting questions in this field focuses on the hyperactivity of some IBPs. Ice binding proteins can be classified in two groups: moderate ones that can depress the freezing point up to ˜1.0 ^oC and hyperactive ones that can depress the freezing point several-fold further even at lower concentrations. It has been suggested that the hyperactivity of IBPs stem from the fact that they block growth out of specific ice surfaces, more specifically the basal planes of ice. Here we show experimental results based on fluorescence microscopy, highlighting the differences between moderate IBPs and hyperactive IBPs. These include direct evidence for basal plane affinity of hyperactive IBPs, the effects of IBPs on growth-melt behavior of ice and the dynamics of their interaction with ice.

  5. Cold-Active Winter Rye Glucanases with Ice-Binding Capacity12

    PubMed Central

    Yaish, Mahmoud W.F.; Doxey, Andrew C.; McConkey, Brendan J.; Moffatt, Barbara A.; Griffith, Marilyn

    2006-01-01

    Extracellular pathogenesis-related proteins, including glucanases, are expressed at cold temperatures in winter rye (Secale cereale) and display antifreeze activity. We have characterized recombinant cold-induced glucanases from winter rye to further examine their roles and contributions to cold tolerance. Both basic β-1,3-glucanases and an acidic β-1,3;1,4-glucanase were expressed in Escherichia coli, purified, and assayed for their hydrolytic and antifreeze activities in vitro. All were found to be cold active and to retain partial hydrolytic activity at subzero temperatures (e.g. 14%–35% at −4°C). The two types of glucanases had antifreeze activity as measured by their ability to modify the growth of ice crystals. Structural models for the winter rye β-1,3-glucanases were developed on which putative ice-binding surfaces (IBSs) were identified. Residues on the putative IBSs were charge conserved for each of the expressed glucanases, with the exception of one β-1,3-glucanase recovered from nonacclimated winter rye in which a charged amino acid was present on the putative IBS. This protein also had a reduced antifreeze activity relative to the other expressed glucanases. These results support the hypothesis that winter rye glucanases have evolved to inhibit the formation of large, potentially fatal ice crystals, in addition to having enzymatic activity with a potential role in resisting infection by psychrophilic pathogens. Glucanases of winter rye provide an interesting example of protein evolution and adaptation aimed to combat cold and freezing conditions. PMID:16815958

  6. Mechanism of action of antifreeze polypeptide HPLC6 in solution: analysis of solvent behaviour by molecular dynamics

    NASA Astrophysics Data System (ADS)

    Brooke-Taylor, Charles A.; Grant, Guy H.; Elcock, Adrian H.; Graham Richards, W.

    1996-04-01

    The behaviour of the water surrounding the winter flounder antifreeze protein HPLC6 has been investigated using molecular dynamics with subsequent analysis of the trajectories. Two simulations were carried out: one at room temperature for comparison with pure (TIP3P) water and another at the temperature taken to represent the freezing point of TIP3P water. The peptide was found to remain in a helical conformation with the sidechains of the residues Thr2, Thr13, Thr24, and Thr35 roughly collinear and equally spaced. Furthermore, the structure of the water around these residues appeared to be much better defined than on the opposite face of the helix: this is predominatly hydrophobic. Comparison of the structure and dynamics of the water in the two environments serves to corroborate previously proposed ideas about the mode of antifreeze action. It would appear that the peptide inhibits ice-crystal growth by binding into the surface of trhe crystal via the polar residues, while the apolar residues restrict the binding of further water molecules and keep them in a flux, thus preventing further growth.

  7. Abrogation of fibroblast activation protein enzymatic activity attenuates tumor growth.

    PubMed

    Cheng, Jonathan D; Valianou, Matthildi; Canutescu, Adrian A; Jaffe, Eileen K; Lee, Hyung-Ok; Wang, Hao; Lai, Jack H; Bachovchin, William W; Weiner, Louis M

    2005-03-01

    Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting

  8. Antifreeze Polysaccharide Coating Study for De-icing Aircraft

    NASA Astrophysics Data System (ADS)

    Morita, Katsuaki; Sakaue, Hirotaka; Ando, Azuma; Matsuda, Yoshiyuki; Kawahara, Hidehisa

    2015-11-01

    Anti-icing or deicing of an aircraft is necessary for a safe flight operation. Mechanical processes, such as heating and deicer boot, are widely used. Deicing fluids, such as propyrene glycol and ethylene glycol, are used to coat the aircraft. However, these should be coated every time before the take-off, since the fluids come off from the aircraft while cruising. We study an antifreeze polysaccharide (AFPS) coating as a deicer for an aircraft. It is designed to coat on the aircraft without removal. Since an AFPS coating removes ice by reducing the interfacial energy, it would be an alternative way to prevent ice on the aircraft. We provide a temperature-controlled room, which can control its temperature under icing conditions (-8 and -4 °C). Ice adhesion tests are performed for AFPS coating and compared with a fundamental specimen without the coating.

  9. Antifreeze peptides and glycopeptides, and their derivatives: potential uses in biotechnology.

    PubMed

    Bang, Jeong Kyu; Lee, Jun Hyuck; Murugan, Ravichandran N; Lee, Sung Gu; Do, Hackwon; Koh, Hye Yeon; Shim, Hye-Eun; Kim, Hyun-Cheol; Kim, Hak Jun

    2013-06-01

    Antifreeze proteins (AFPs) and glycoproteins (AFGPs), collectively called AF(G)Ps, constitute a diverse class of proteins found in various Arctic and Antarctic fish, as well as in amphibians, plants, and insects. These compounds possess the ability to inhibit the formation of ice and are therefore essential to the survival of many marine teleost fishes that routinely encounter sub-zero temperatures. Owing to this property, AF(G)Ps have potential applications in many areas such as storage of cells or tissues at low temperature, ice slurries for refrigeration systems, and food storage. In contrast to AFGPs, which are composed of repeated tripeptide units (Ala-Ala-Thr)n with minor sequence variations, AFPs possess very different primary, secondary, and tertiary structures. The isolation and purification of AFGPs is laborious, costly, and often results in mixtures, making characterization difficult. Recent structural investigations into the mechanism by which linear and cyclic AFGPs inhibit ice crystallization have led to significant progress toward the synthesis and assessment of several synthetic mimics of AFGPs. This review article will summarize synthetic AFGP mimics as well as current challenges in designing compounds capable of mimicking AFGPs. It will also cover our recent efforts in exploring whether peptoid mimics can serve as structural and functional mimics of native AFGPs. PMID:23752356

  10. Antifreeze Peptides and Glycopeptides, and Their Derivatives: Potential Uses in Biotechnology

    PubMed Central

    Bang, Jeong Kyu; Lee, Jun Hyuck; Murugan, Ravichandran N.; Lee, Sung Gu; Do, Hackwon; Koh, Hye Yeon; Shim, Hye-Eun; Kim, Hyun-Cheol; Kim, Hak Jun

    2013-01-01

    Antifreeze proteins (AFPs) and glycoproteins (AFGPs), collectively called AF(G)Ps, constitute a diverse class of proteins found in various Arctic and Antarctic fish, as well as in amphibians, plants, and insects. These compounds possess the ability to inhibit the formation of ice and are therefore essential to the survival of many marine teleost fishes that routinely encounter sub-zero temperatures. Owing to this property, AF(G)Ps have potential applications in many areas such as storage of cells or tissues at low temperature, ice slurries for refrigeration systems, and food storage. In contrast to AFGPs, which are composed of repeated tripeptide units (Ala-Ala-Thr)n with minor sequence variations, AFPs possess very different primary, secondary, and tertiary structures. The isolation and purification of AFGPs is laborious, costly, and often results in mixtures, making characterization difficult. Recent structural investigations into the mechanism by which linear and cyclic AFGPs inhibit ice crystallization have led to significant progress toward the synthesis and assessment of several synthetic mimics of AFGPs. This review article will summarize synthetic AFGP mimics as well as current challenges in designing compounds capable of mimicking AFGPs. It will also cover our recent efforts in exploring whether peptoid mimics can serve as structural and functional mimics of native AFGPs. PMID:23752356

  11. Inhibition of Condensation Frosting by Arrays of Hygroscopic Antifreeze Drops.

    PubMed

    Sun, Xiaoda; Damle, Viraj G; Uppal, Aastha; Linder, Rubin; Chandrashekar, Sriram; Mohan, Ajay R; Rykaczewski, Konrad

    2015-12-29

    The formation of frost and ice can have negative impacts on travel and a variety of industrial processes and is typically addressed by dispensing antifreeze substances such as salts and glycols. Despite the popularity of this anti-icing approach, some of the intricate underlying physical mechanisms are just being unraveled. For example, recent studies have shown that in addition to suppressing ice formation within its own volume, an individual salt saturated water microdroplet forms a region of inhibited condensation and condensation frosting (RIC) in its surrounding area. This occurs because salt saturated water, like most antifreeze substances, is hygroscopic and has water vapor pressure at its surface lower than water saturation pressure at the substrate. Here, we demonstrate that for macroscopic drops of propylene glycol and salt saturated water, the absolute RIC size can remain essentially unchanged for several hours. Utilizing this observation, we demonstrate that frost formation can be completely inhibited in-between microscopic and macroscopic arrays of propylene glycol and salt saturated water drops with spacing (S) smaller than twice the radius of the RIC (δ). Furthermore, by characterizing condensation frosting dynamics around various hygroscopic drop arrays, we demonstrate that they can delay complete frosting over of the samples 1.6 to 10 times longer than films of the liquids with equivalent volume. The significant delay in onset of ice nucleation achieved by dispensing propylene glycol in drops rather than in films is likely due to uniform dilution of the drops driven by thermocapillary flow. This transport mode is absent in the films, leading to faster dilution, and with that facilitated homogeneous nucleation, near the liquid-air interface. PMID:26651017

  12. Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics.

    PubMed

    Drori, Ran; Celik, Yeliz; Davies, Peter L; Braslavsky, Ido

    2014-09-01

    Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH. PMID:25008081

  13. Protein Needs of Physically Active Children.

    PubMed

    Volterman, Kimberly A; Atkinson, Stephanie A

    2016-05-01

    Current Dietary Reference Intakes (DRI) for protein for children and youth require revision as they were derived primarily on nitrogen balance data in young children or extrapolated from adult values; did not account for the possible influence of above average physical activity; and did not set an upper tolerable level of intake. Revision of the protein DRIs requires new research that investigates: 1) long-term dose-response to identify protein and essential amino acid requirements of both sexes at various pubertal stages and under differing conditions of physical activity; 2) the acute protein needs (quantity and timing) following a single bout of exercise; 3) the potential adverse effects of chronic high intakes of protein; and 4) new measurement techniques (i.e., IAAO or stable isotope methodologies) to improve accuracy of protein needs. While active individuals may require protein in excess of current DRIs, most active Canadian children and youth have habitual protein intakes that exceed current recommendations. PMID:27137165

  14. Ancient climate change, antifreeze, and the evolutionary diversification of Antarctic fishes.

    PubMed

    Near, Thomas J; Dornburg, Alex; Kuhn, Kristen L; Eastman, Joseph T; Pennington, Jillian N; Patarnello, Tomaso; Zane, Lorenzo; Fernández, Daniel A; Jones, Christopher D

    2012-02-28

    The Southern Ocean around Antarctica is among the most rapidly warming regions on Earth, but has experienced episodic climate change during the past 40 million years. It remains unclear how ancient periods of climate change have shaped Antarctic biodiversity. The origin of antifreeze glycoproteins (AFGPs) in Antarctic notothenioid fishes has become a classic example of how the evolution of a key innovation in response to climate change can drive adaptive radiation. By using a time-calibrated molecular phylogeny of notothenioids and reconstructed paleoclimate, we demonstrate that the origin of AFGP occurred between 42 and 22 Ma, which includes a period of global cooling approximately 35 Ma. However, the most species-rich lineages diversified and evolved significant ecological differences at least 10 million years after the origin of AFGPs, during a second cooling event in the Late Miocene (11.6-5.3 Ma). This pattern indicates that AFGP was not the sole trigger of the notothenioid adaptive radiation. Instead, the bulk of the species richness and ecological diversity originated during the Late Miocene and into the Early Pliocene, a time coincident with the origin of polar conditions and increased ice activity in the Southern Ocean. Our results challenge the current understanding of the evolution of Antarctic notothenioids suggesting that the ecological opportunity that underlies this adaptive radiation is not linked to a single trait, but rather to a combination of freeze avoidance offered by AFGPs and subsequent exploitation of new habitats and open niches created by increased glacial and ice sheet activity. PMID:22331888

  15. Effect of antifreeze glycoprotein 8 supplementation during vitrification on the developmental competence of bovine oocytes.

    PubMed

    Liang, Shuang; Yuan, Bao; Kwon, Jeong-Woo; Ahn, Mija; Cui, Xiang-Shun; Bang, Jeong Kyu; Kim, Nam-Hyung

    2016-07-15

    The purpose of this study was to investigate the effect of antifreeze glycoprotein 8 (AFGP8) supplementation during vitrification on the survival, fertilization, and embryonic development of bovine oocytes and the underlying molecular mechanism(s). Survival, fertilization, early embryonic development, apoptosis, DNA double-strand breaks, reactive oxygen species levels, meiotic cytoskeleton assembly, chromosome alignment, and energy status of mitochondria were measured in the present experiments. Compared with that in the nonsupplemented group; survival, monospermy, blastocyst formation rates, and blastomere counts were significantly higher in the AFGP8-supplemented animals. Oocytes of the latter group also presented fewer double-strand breaks and lower cathepsin B and caspase activities. Rates of normal spindle organization and chromosome alignment, actin filament impairment, and mitochondrial distribution were significantly higher in the AFGP8-supplemented group. In addition, intracellular reactive oxygen species levels significantly decreased in the AFGP8-supplemented groups, maintaining a higher ΔΨm than that in the nonsupplemented group. Taken together, these results indicated that supplementation with AFGP8 during vitrification has a protective effect on bovine oocytes against chilling injury. PMID:26948296

  16. Antifreeze acceptability for ground-coupled heat pump ground loops in the United States

    SciTech Connect

    Den Braven, K.R.

    1998-10-01

    When designing and installing closed-loop ground-coupled heat pumps systems, it is necessary to be aware of applicable environmental regulations. Within the United States, nearly half of the states have regulations specifying or restricting the use of particular antifreezes or other fluids within the ground loop of a ground-coupled heat pump system. A number of other states have regulations pending. While all of these regulations are based on the need to preserve groundwater and/or aquifer quality, the list of acceptable antifreezes varies among those states with specified fluids. Typical antifreezes in use include ethylene glycol, propylene glycol, brines, alcohols, and potassium acetate. Each of these has its benefits and drawbacks. The status of the regulations has been determined for all of the states. An overview of the regulations is presented in this paper, along with a summary of the states` concerns.

  17. Long Wavelength Monitoring of Protein Kinase Activity

    PubMed Central

    Oien, Nathan P.; Nguyen, Luong T.; Jernigan, Finith E.; Priestman, Melanie A.

    2014-01-01

    A family of long wavelength protein kinase fluorescent reporters is described in which the probing wavelength is pre-programmed using readily available fluorophores. These agents can assess protein kinase activity within the optical window of tissue, as exemplified by monitoring endogenous cAMP-dependent protein kinase activity (1) in erythrocyte lysates and (2) in intact erythrocytes using a light-activatable reporter. PMID:24604833

  18. Safe antifreeze: The real difference between ethylene glycol and propylene glycol

    SciTech Connect

    Wray, T.K.

    1995-04-01

    Antifreeze-coolants are added to the radiators of internal combustion engines to prevent freezing during the winter and boil-over during the summer. Although ethylene glycol is the most commonly used coolant, products containing propylene glycol have been used--at least, experimentally--for years. Both substances have similar characteristics; however, some manufacturers claim that antifreeze-coolants containing propylene glycol are more environmentally friendly and safer to humans and animals than ethylene glycol products. This article examines these two substances, and addresses the similarities and differences of their physical and chemical compounds, thereby enabling users to determine whether such claims are valid or merely advertising hyperbole.

  19. Activity-Based Protein Profiling of Microbes

    SciTech Connect

    Sadler, Natalie C.; Wright, Aaron T.

    2015-02-01

    Activity-Based Protein Profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems include: enhancing protein annotation, characterizing protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

  20. Dietary protein considerations to support active aging.

    PubMed

    Wall, Benjamin T; Cermak, Naomi M; van Loon, Luc J C

    2014-11-01

    Given our rapidly aging world-wide population, the loss of skeletal muscle mass with healthy aging (sarcopenia) represents an important societal and public health concern. Maintaining or adopting an active lifestyle alleviates age-related muscle loss to a certain extent. Over time, even small losses of muscle tissue can hinder the ability to maintain an active lifestyle and, as such, contribute to the development of frailty and metabolic disease. Considerable research focus has addressed the application of dietary protein supplementation to support exercise-induced gains in muscle mass in younger individuals. In contrast, the role of dietary protein in supporting the maintenance (or gain) of skeletal muscle mass in active older persons has received less attention. Older individuals display a blunted muscle protein synthetic response to dietary protein ingestion. However, this reduced anabolic response can largely be overcome when physical activity is performed in close temporal proximity to protein consumption. Moreover, recent evidence has helped elucidate the optimal type and amount of dietary protein that should be ingested by the older adult throughout the day in order to maximize the skeletal muscle adaptive response to physical activity. Evidence demonstrates that when these principles are adhered to, muscle maintenance or hypertrophy over prolonged periods can be further augmented in active older persons. The present review outlines the current understanding of the role that dietary protein occupies in the lifestyle of active older adults as a means to increase skeletal muscle mass, strength and function, and thus support healthier aging. PMID:25355192

  1. Conformation of the antifreeze glycoprotein of polar fish.

    PubMed

    Bush, C A; Ralapati, S; Matson, G M; Yamasaki, R B; Osuga, D T; Yeh, Y; Feeney, R E

    1984-08-01

    High-field proton and 13C NMR spectroscopy has been used to test and refine the recent proposal, based on vacuum uv circular dichroism results, of a threefold left-handed helical conformation for antifreeze glycoprotein (AFGP). Partial assignment of the protons of the glycotripeptide repeating unit has been made by comparison with spectra of model compounds, by selective decoupling, and by measurements of nuclear Overhauser effect (nOe). At 40 degrees C, AFGP fraction 8 (Mr 2600) shows 2-Hz linewidths which broaden at lower temperature. Neither 1H nor 13C chemical shifts depend strongly on temperature, suggesting no abrupt conformational transition. The nOe between alanine alpha and beta protons vary with temperature and with field strength, from small positive enhancements at 50 degrees C and 80 MHz to large negative effects at 3 degrees C and 300 MHz, indicating a substantial change of rotational correlation time with temperature. The higher-molecular-weight fraction 1-4 shows negative nOe at all temperatures. The CD spectra of fraction 1-4 show bands characteristic of the polyproline II structure at both 3 and 50 degrees C, while those bands in fraction 8 are weaker at 50 than 3 degrees C. The 1H nOe, the 13C T1, and CD data are interpreted as indicating that AFGP fraction 8 is an extended "rod-like" conformation at low temperature which becomes a flexible coil at high temperature, while fraction 1-4 is a flexible rod with sufficient segmental mobility to eliminate any long-range order. PMID:6087734

  2. Ancient climate change, antifreeze, and the evolutionary diversification of Antarctic fishes

    PubMed Central

    Near, Thomas J.; Dornburg, Alex; Kuhn, Kristen L.; Eastman, Joseph T.; Pennington, Jillian N.; Patarnello, Tomaso; Zane, Lorenzo; Fernández, Daniel A.; Jones, Christopher D.

    2012-01-01

    The Southern Ocean around Antarctica is among the most rapidly warming regions on Earth, but has experienced episodic climate change during the past 40 million years. It remains unclear how ancient periods of climate change have shaped Antarctic biodiversity. The origin of antifreeze glycoproteins (AFGPs) in Antarctic notothenioid fishes has become a classic example of how the evolution of a key innovation in response to climate change can drive adaptive radiation. By using a time-calibrated molecular phylogeny of notothenioids and reconstructed paleoclimate, we demonstrate that the origin of AFGP occurred between 42 and 22 Ma, which includes a period of global cooling approximately 35 Ma. However, the most species-rich lineages diversified and evolved significant ecological differences at least 10 million years after the origin of AFGPs, during a second cooling event in the Late Miocene (11.6–5.3 Ma). This pattern indicates that AFGP was not the sole trigger of the notothenioid adaptive radiation. Instead, the bulk of the species richness and ecological diversity originated during the Late Miocene and into the Early Pliocene, a time coincident with the origin of polar conditions and increased ice activity in the Southern Ocean. Our results challenge the current understanding of the evolution of Antarctic notothenioids suggesting that the ecological opportunity that underlies this adaptive radiation is not linked to a single trait, but rather to a combination of freeze avoidance offered by AFGPs and subsequent exploitation of new habitats and open niches created by increased glacial and ice sheet activity. PMID:22331888

  3. DNA-based control of protein activity.

    PubMed

    Engelen, W; Janssen, B M G; Merkx, M

    2016-03-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  4. DNA-based control of protein activity

    PubMed Central

    Engelen, W.; Janssen, B. M. G.

    2016-01-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  5. Active Nuclear Import of Membrane Proteins Revisited

    PubMed Central

    Laba, Justyna K.; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M.

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast. PMID:26473931

  6. Active Nuclear Import of Membrane Proteins Revisited.

    PubMed

    Laba, Justyna K; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker's yeast. PMID:26473931

  7. Protein kinase activators alter glial cholesterol esterification

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-05-01

    Similar to nonneural tissues, the activity of glial acyl-CoA cholesterol acyltransferase is controlled by a phosphorylation and dephosphorylation mechanism. Manipulation of cyclic AMP content did not alter the cellular cholesterol esterification, suggesting that cyclic AMP is not a bioregulator in this case. Therefore, the authors tested the effect of phorbol-12-myristate 13-acetate (PMA) on cellular cholesterol esterification to determine the involvement of protein kinase C. PMA has a potent effect on cellular cholesterol esterification. PMA depresses cholesterol esterification initially, but cells recover from inhibition and the result was higher cholesterol esterification, suggesting dual effects of protein kinase C. Studies of other phorbol analogues and other protein kinase C activators such as merezein indicate the involvement of protein kinase C. Oleoyl-acetyl glycerol duplicates the effect of PMA. This observation is consistent with a diacyl-glycerol-protein kinase-dependent reaction. Calcium ionophore A23187 was ineffective in promoting the effect of PMA. They concluded that a calcium-independent and protein C-dependent pathway regulated glial cholesterol esterification.

  8. Petunia nectar proteins have ribonuclease activity.

    PubMed

    Hillwig, Melissa S; Liu, Xiaoteng; Liu, Guangyu; Thornburg, Robert W; Macintosh, Gustavo C

    2010-06-01

    Plants requiring an insect pollinator often produce nectar as a reward for the pollinator's visitations. This rich secretion needs mechanisms to inhibit microbial growth. In Nicotiana spp. nectar, anti-microbial activity is due to the production of hydrogen peroxide. In a close relative, Petunia hybrida, limited production of hydrogen peroxide was found; yet petunia nectar still has anti-bacterial properties, suggesting that a different mechanism may exist for this inhibition. The nectar proteins of petunia plants were compared with those of ornamental tobacco and significant differences were found in protein profiles and function between these two closely related species. Among those proteins, RNase activities unique to petunia nectar were identified. The genes corresponding to four RNase T2 proteins from Petunia hybrida that show unique expression patterns in different plant tissues were cloned. Two of these enzymes, RNase Phy3 and RNase Phy4 are unique among the T2 family and contain characteristics similar to both S- and S-like RNases. Analysis of amino acid patterns suggest that these proteins are an intermediate between S- and S-like RNases, and support the hypothesis that S-RNases evolved from defence RNases expressed in floral parts. This is the first report of RNase activities in nectar. PMID:20460362

  9. Petunia nectar proteins have ribonuclease activity

    PubMed Central

    Hillwig, Melissa S.; Liu, Xiaoteng; Liu, Guangyu; Thornburg, Robert W.; MacIntosh, Gustavo C.

    2010-01-01

    Plants requiring an insect pollinator often produce nectar as a reward for the pollinator's visitations. This rich secretion needs mechanisms to inhibit microbial growth. In Nicotiana spp. nectar, anti-microbial activity is due to the production of hydrogen peroxide. In a close relative, Petunia hybrida, limited production of hydrogen peroxide was found; yet petunia nectar still has anti-bacterial properties, suggesting that a different mechanism may exist for this inhibition. The nectar proteins of petunia plants were compared with those of ornamental tobacco and significant differences were found in protein profiles and function between these two closely related species. Among those proteins, RNase activities unique to petunia nectar were identified. The genes corresponding to four RNase T2 proteins from Petunia hybrida that show unique expression patterns in different plant tissues were cloned. Two of these enzymes, RNase Phy3 and RNase Phy4 are unique among the T2 family and contain characteristics similar to both S- and S-like RNases. Analysis of amino acid patterns suggest that these proteins are an intermediate between S- and S-like RNases, and support the hypothesis that S-RNases evolved from defence RNases expressed in floral parts. This is the first report of RNase activities in nectar. PMID:20460362

  10. Isolation and characterisation of sericin antifreeze peptides and molecular dynamics modelling of their ice-binding interaction.

    PubMed

    Wu, Jinhong; Rong, Yuzhi; Wang, Zhengwu; Zhou, Yanfu; Wang, Shaoyun; Zhao, Bo

    2015-05-01

    This study aimed to isolate and characterise a novel sericin antifreeze peptide and investigate its ice-binding molecular mechanism. The thermal hysteresis activity of ice-binding sericin peptides (I-SP) was measured and their activity reached as high as 0.94 °C. A P4 fraction, with high hypothermia protective activity and inhibition activity of ice recrystallisation, was obtained from I-SP, and a purified sericin peptide, named SM-AFP, with the sequence of TTSPTNVSTT and a molecular weight of 1009.50 Da was then isolated from the P4 fraction. Treatment of Lactobacillus delbrueckii Subsp. bulgaricus LB340 LYO with 100 μg/ml synthetic SM-AFP led to 1.4-fold increased survival (p < 0.05). Finally, an SM-AFP/ice binding model was constructed and results of molecular dynamics simulation suggested that the binding of SM-AFP with ice and prevention of ice crystal growth could be attributed to hydrogen bond formation, hydrophobic interaction and non-bond interactions. Sericin peptides could be developed into beneficial cryoprotectants and used in frozen food processing. PMID:25529728

  11. Evaluation of anti-freeze viscosity modifier for potential external tank applications

    NASA Technical Reports Server (NTRS)

    Lynn, R. O. L.

    1981-01-01

    Viscosity modifiers and gelling agents were evaluated in combination with ethylene glycol and dimethyl sulfoxide water eutectics. Pectin and agarose are found to gel these eutectics effectively in low concentration, but the anti-freeze protection afforded by these compositions is found to be marginal in simulations of the intended applications. Oxygen vent shutters and vertical metallic surfaces were simulated, with water supplied as a spray, dropwise, and by condensation from the air.

  12. Boiling Performance of Antifreeze Solutions in a Saturate Pool Boiling System

    NASA Astrophysics Data System (ADS)

    Matsumura, Kunihito; Kaminaga, Fumito

    Nucleate boiling of binary mixtures is of particular importance in a various industries. The purpose of the present study is to provide experimental data and prediction method for nucleate boiling heat transfer of anti-freeze solutions, Propylene-glycol (PG)/water and Ethylene-glycol (EG)/water. The pool nucleate boiling experiments were carried out under a saturated and atmospheric condition. The platinum wire of 0.3 mm diameter was used as the heating surface. The mole fractions of solutions are varied from 0.85 to 1. It was found that the heat transfer coefficient gradually decreases with increasing fraction of anti-freeze to water. It was also shown that a small addition of propylene-glycol and ethylene-glycol also decreases the CHF value far below that of pure water. It is concluded that the correlation proposed by Fujita for several binary mixtures can well predict the heat transfer coefficients within almost ±5% accuracy for every concentration of present anti-freeze solutions.

  13. Total Cellular RNA Modulates Protein Activity.

    PubMed

    Majumder, Subhabrata; DeMott, Christopher M; Reverdatto, Sergey; Burz, David S; Shekhtman, Alexander

    2016-08-16

    RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein-RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states. PMID:27456029

  14. [Mitogen-activated protein kinases in atherosclerosis].

    PubMed

    Bryk, Dorota; Olejarz, Wioletta; Zapolska-Downar, Danuta

    2014-01-01

    Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases) intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase), JNK (c-Jun N-terminal kinase) and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis. PMID:24491891

  15. De Novo Construction of Redox Active Proteins.

    PubMed

    Moser, C C; Sheehan, M M; Ennist, N M; Kodali, G; Bialas, C; Englander, M T; Discher, B M; Dutton, P L

    2016-01-01

    Relatively simple principles can be used to plan and construct de novo proteins that bind redox cofactors and participate in a range of electron-transfer reactions analogous to those seen in natural oxidoreductase proteins. These designed redox proteins are called maquettes. Hydrophobic/hydrophilic binary patterning of heptad repeats of amino acids linked together in a single-chain self-assemble into 4-alpha-helix bundles. These bundles form a robust and adaptable frame for uncovering the default properties of protein embedded cofactors independent of the complexities introduced by generations of natural selection and allow us to better understand what factors can be exploited by man or nature to manipulate the physical chemical properties of these cofactors. Anchoring of redox cofactors such as hemes, light active tetrapyrroles, FeS clusters, and flavins by His and Cys residues allow cofactors to be placed at positions in which electron-tunneling rates between cofactors within or between proteins can be predicted in advance. The modularity of heptad repeat designs facilitates the construction of electron-transfer chains and novel combinations of redox cofactors and new redox cofactor assisted functions. Developing de novo designs that can support cofactor incorporation upon expression in a cell is needed to support a synthetic biology advance that integrates with natural bioenergetic pathways. PMID:27586341

  16. Synaptic Vesicle Proteins and Active Zone Plasticity

    PubMed Central

    Kittel, Robert J.; Heckmann, Manfred

    2016-01-01

    Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone (AZ). The complex molecular architecture of AZs mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of AZs vary significantly, even for a given connection. Thus, there appear to be distinct AZ states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the AZ. The protein-rich cytomatrix at the active zone (CAZ) provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1) and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and AZ states, which has heretofore received little attention. PMID:27148040

  17. Detection of protein C activation in humans.

    PubMed Central

    Bauer, K A; Kass, B L; Beeler, D L; Rosenberg, R D

    1984-01-01

    We have developed a radioimmunoassay (RIA) for the dodecapeptide that is liberated from protein C when this zymogen is activated by thrombin bound to thrombomodulin present on the vascular endothelium. The protein C activation peptide (PCP) was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used together with a 125I-labeled tyrosinated ligand and various concentrations of unlabeled PCP to construct a double antibody RIA capable of measuring as little as 10 pM of this component. We have established that the synthetic dodecapeptide has the same immunoreactivity as the native peptide and that the reactivity of protein C is less than 1/2,000 that of PCP on a molar basis. The extremely low levels of peptide in normal individuals as well as the nonspecific contributions of plasma constituents to the immunoreactive signal, necessitated the development of a procedure by which the PCP could be reproducibly extracted from plasma and concentrated approximately 20-fold. This methodology permitted us to demonstrate that the plasma PCP levels in 17 normal donors averaged 6.47 pM, and that elevations up to 180 pM were observed in individuals with evidence of disseminated intravascular coagulation. The validity of these measurements of protein C activation is supported by the fact that, in both of these situations, the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native dodecapeptide. We have also noted that the mean PCP concentration in seven patients fully anticoagulated with warfarin averaged 2.61 pM. Our studies also show that PCP is cleared from the plasma of primates with a t1/2 of approximately 5 min. Given that the t1/2 of activated protein C is estimated to be 10-15 min, the latter enzyme appears to exert its effects on the activated cofactors of the

  18. Crowding Activates Heat Shock Protein 90.

    PubMed

    Halpin, Jackson C; Huang, Bin; Sun, Ming; Street, Timothy O

    2016-03-18

    Hsp90 is a dimeric ATP-dependent chaperone involved in the folding, maturation, and activation of diverse target proteins. Extensive in vitro structural analysis has led to a working model of Hsp90's ATP-driven conformational cycle. An implicit assumption is that dilute experimental conditions do not significantly perturb Hsp90 structure and function. However, Hsp90 undergoes a dramatic open/closed conformational change, which raises the possibility that this assumption may not be valid for this chaperone. Indeed, here we show that the ATPase activity of Hsp90 is highly sensitive to molecular crowding, whereas the ATPase activities of Hsp60 and Hsp70 chaperones are insensitive to crowding conditions. Polymer crowders activate Hsp90 in a non-saturable manner, with increasing efficacy at increasing concentration. Crowders exhibit a non-linear relationship between their radius of gyration and the extent to which they activate Hsp90. This experimental relationship can be qualitatively recapitulated with simple structure-based volume calculations comparing open/closed configurations of Hsp90. Thermodynamic analysis indicates that crowding activation of Hsp90 is entropically driven, which is consistent with a model in which excluded volume provides a driving force that favors the closed active state of Hsp90. Multiple Hsp90 homologs are activated by crowders, with the endoplasmic reticulum-specific Hsp90, Grp94, exhibiting the highest sensitivity. Finally, we find that crowding activation works by a different mechanism than co-chaperone activation and that these mechanisms are independent. We hypothesize that Hsp90 has a higher intrinsic activity in the cell than in vitro. PMID:26797120

  19. Heat dissipation guides activation in signaling proteins.

    PubMed

    Weber, Jeffrey K; Shukla, Diwakar; Pande, Vijay S

    2015-08-18

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein-coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  20. Activation and activities of the p53 tumour suppressor protein

    PubMed Central

    Bálint, É; Vousden, K H

    2001-01-01

    The p53 tumour suppressor protein inhibits malignant progression by mediating cell cycle arrest, apoptosis or repair following cellular stress. One of the major regulators of p53 function is the MDM2 protein, and multiple forms of cellular stress activate p53 by inhibiting the MDM2-mediated degradation of p53. Mutations in p53, or disruption of the pathways that allow activation of p53, seem to be a general feature of all cancers. Here we review recent advances in our understanding of the pathways that regulate p53 and the pathways that are induced by p53, as well as their implications for cancer therapy. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11747320

  1. The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinases (MAPKAPKs) in Inflammation

    PubMed Central

    Moens, Ugo; Kostenko, Sergiy; Sveinbjørnsson, Baldur

    2013-01-01

    Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed. PMID:24705157

  2. STUDY ON PROPERTIES OF SKID RESISTANCE ON FREEZING PAVEMENTS AND QUANTITATIVE EVALUATION METHOD OF ANTIFREEZING EFFECTS

    NASA Astrophysics Data System (ADS)

    Tanaka, Shunsuke; Takeichi, Kiyoshi; Masuyama, Yukiei; Takahashi, Naoto

    Snow and ice control in winter roads trends to be controlled by the skid friction coefficients in North America and North European countries at present, but the measurements are not necessarily easy. We studied on a simplified measurement method based on the relationship between skid friction coefficients and the bare pavement ratio (BPR) in the laboratory tests and field tests. The factors of BPR, surface textures and antifreezing materials which affect the skid friction coefficient are reviewed by a multiple linear regression analysis and a spectrum analysis, considering different freezing surfaces. These studies indicate that conclusions induced by laboratory tests could be applied to roads in service.

  3. Phospholipases as GTPase activity accelerating proteins (GAPs) in plants.

    PubMed

    Pandey, Sona

    2016-05-01

    GTPase activity accelerating proteins (GAPs) are key regulators of the G-protein signaling cycle. By facilitating effective hydrolysis of the GTP bound on Gα proteins, GAPs control the timing and amplitude of the signaling cycle and ascertain the availability of the inactive heterotrimer for the next round of activation. Until very recently, the studies of GAPs in plants were focused exclusively on the regulator of G-protein signaling (RGS) protein. We now show that phospholipase Dα1 (PLDα1) is also a bona fide GAP in plants and together with the RGS protein controls the level of activeprotein. PMID:27124090

  4. Monooxygenase activity of type 3 copper proteins.

    PubMed

    Itoh, Shinobu; Fukuzumi, Shunichi

    2007-07-01

    The molecular mechanism of the monooxygenase (phenolase) activity of type 3 copper proteins has been examined in detail both in the model systems and in the enzymatic systems. The reaction of a side-on peroxo dicopper(II) model compound ( A) and neutral phenols proceeds via a proton-coupled electron-transfer (PCET) mechanism to generate phenoxyl radical species, which collapse each other to give the corresponding C-C coupling dimer products. In this reaction, a bis(mu-oxo)dicopper(III) complex ( B) generated by O-O bond homolysis of A is suggested to be a real active species. On the other hand, the reaction of lithium phenolates (deprotonated form of phenols) with the same side-on peroxo dicopper(II) complex proceeds via an electrophilic aromatic substitution mechanism to give the oxygenated products (catechols). The mechanistic difference between these two systems has been discussed on the basis of the Marcus theory of electron transfer and Hammett analysis. Mechanistic details of the monooxygenase activity of tyrosinase have also been examined using a simplified enzymatic reaction system to demonstrate that the enzymatic reaction mechanism is virtually the same as that of the model reaction, that is, an electrophilic aromatic substitution mechanism. In addition, the monooxygenase activity of the oxygen carrier protein hemocyanin has been explored for the first time by employing urea as an additive in the reaction system. In this case as well, the ortho-hydroxylation of phenols to catechols has been demonstrated to involve the same ionic mechanism. PMID:17461541

  5. Heat dissipation guides activation in signaling proteins

    PubMed Central

    Weber, Jeffrey K.; Shukla, Diwakar; Pande, Vijay S.

    2015-01-01

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein–coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  6. Protein-protein interactions in plant mitogen-activated protein kinase cascades.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2016-02-01

    Mitogen-activated protein kinases (MAPKs) form tightly controlled signaling cascades that play essential roles in plant growth, development, and defense. However, the molecular mechanisms underlying MAPK cascades are still elusive, due largely to our poor understanding of how they relay the signals. Extensive effort has been devoted to characterization of MAPK-substrate interactions to illustrate phosphorylation-based signaling. The diverse MAPK substrates identified also shed light on how spatiotemporal-specific protein-protein interactions function in distinct MAPK cascade-mediated biological processes. This review surveys various technologies used for characterizing MAPK-substrate interactions and presents case studies of MPK4 and MPK6, highlighting the multiple functions of MAPKs. Mass spectrometry-based approaches in identifying MAPK-interacting proteins are emphasized due to their increasing utility and effectiveness. The potential for using MAPKs and their substrates in enhancing plant stress tolerance is also discussed. PMID:26646897

  7. Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins.

    PubMed

    Hay, Debbie L; Walker, Christopher S; Gingell, Joseph J; Ladds, Graham; Reynolds, Christopher A; Poyner, David R

    2016-04-15

    Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine. PMID:27068971

  8. Just cool it! Cryoprotectant anti-freeze in immunocytochemistry and in situ hybridization.

    PubMed

    Hoffman, Gloria E; Le, Wei Wei

    2004-03-01

    Immunohistochemical techniques offer specificity as well as flexibility for visualizing antigens. Their use with freely floating sections provides a high signal-to-noise ratio and has become a gold standard for brain and a number of other tissues. Yet this approach initially suffered from inability to keep the antigenicity in tissue sections and required immediate processing of all cut sections. Use of sucrose solutions enabled storage at refrigerator temperatures for a few days but longer-term storage was risky and either bacterial/fungal growth or evaporation of the storage solution compromised the integrity of the tissue. Our discovery 25 years ago that tissue sections can be stored for many years at -20 degrees C in an anti-freeze cryoprotectant solution with no loss of antigenicity solved this problem and has become widely used. More recently the utility of tissue stored for many years in anti-freeze cryoprotectant was pushed to new levels by testing new non-radioactive in situ hybridization (ISH) techniques that are based on modern immunocytochemistry. This review touches upon these advances in immunocytochemical technology using examples from neuroscience applications. PMID:15134865

  9. Assessment of antifreeze solutions for ground-source heat pump systems

    SciTech Connect

    Heinonen, E.W.; Tapscott, R.E.; Wildin, M.W.; Beall, A.N.

    1997-12-31

    This paper assesses the risks of using six different fluids (methanol, ethanol, aqueous potassium acetate, propylene glycol, aqueous calcium magnesium acetate, and aqueous urea) as antifreezes in ground-source heat pump (GSHP) systems. Areas assessed included fire hazard; corrosion and leakage; health hazard; environmental; detailed heat pump system analysis, resulting in predictions of annual energy use, life-cycle cost, and power plant emissions; and regulatory risk to future use. For each area, each antifreeze was rated as having either significant potential for problems, minor potential for problems, or little or no potential for problems. Propylene glycol had low risk in all areas, despite having higher energy use; potassium acetate, calcium magnesium acetate, and urea had low to medium risk in all areas except leakage, which was high for all three fluids, and corrosion, which was high for urea; ethanol and methanol had high fire risk in their pure forms (but low risk in diluted form) and corrosion problems with iron compounds. In addition, ethanol had high environmental and health risks.

  10. Nucleolar protein B23 has molecular chaperone activities.

    PubMed Central

    Szebeni, A.; Olson, M. O.

    1999-01-01

    Protein B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly. Szebeni et al. (1997) showed stimulation of nuclear import in vitro by protein B23 and suggested that this effect was due to a molecular chaperone-like activity. Protein B23 was tested for chaperone activities using several protein substrates. The temperature-dependent and -independent aggregation of the HIV-1 Rev protein was measured using a zero angle light scattering (turbidity) assay. Protein B23 inhibited the aggregation of the Rev protein, with the amount of inhibition proportional to the concentration of B23 added. This activity was saturable with nearly complete inhibition when the molar ratio of B23:Rev was slightly above one. Protein B23 also protected liver alcohol dehydrogenase (LADH), carboxypeptidase A, citrate synthase, and rhodanese from aggregation during thermal denaturation and preserved the enzyme activity of LADH under these conditions. In addition, protein B23 was able to promote the restoration of activity of LADH previously denatured with guanidine-HCl. Protein B23 preferentially bound denatured substrates and exposed hydrophobic regions when complexed with denatured proteins. Thus, by several criteria, protein B23 behaves like a molecular chaperone; these activities may be related to its role in ribosome biogenesis. PMID:10211837

  11. Reconstitution of Membrane Proteins into Model Membranes: Seeking Better Ways to Retain Protein Activities

    PubMed Central

    Shen, Hsin-Hui; Lithgow, Trevor; Martin, Lisandra L.

    2013-01-01

    The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. The activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. The reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. However, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. This review will discuss the reconstitution of membrane protein activities in four different types of model membrane—monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. Variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity. We also discuss examples where the same membrane proteins have been successfully reconstituted into two or more model membrane systems with comparison of the observed activity in each system. Understanding of the behavioral changes for proteins in model membrane systems after membrane reconstitution is often a prerequisite to protein research. It is essential to find better solutions for retaining membrane protein activities for measurement and characterization in vitro. PMID:23344058

  12. Designing Mimics of Membrane Active Proteins

    PubMed Central

    Sgolastra, Federica; deRonde, Brittany M.; Sarapas, Joel M.; Som, Abhigyan; Tew, Gregory N.

    2014-01-01

    CONSPECTUS As a semi-permeable barrier that controls the flux of biomolecules in and out the cell, the plasma membrane is critical in cell function and survival. Many proteins interact with the plasma membrane and modulate its physiology. Within this large landscape of membrane-active molecules, researchers have focused significant attention on two specific classes of peptides, antimicrobial peptides (AMPs) and cell penetrating peptides (CPPs) because of their unique properties. In this account, we describe our efforts over the last decade to build and understand synthetic mimics of antimicrobial peptides (SMAMPs). These endeavors represent one specific example of a much larger effort to understand how synthetic molecules interact with and manipulate the plasma membrane. Using both defined molecular weight oligomers and easier to produce, but heterogeneous, polymers, it has been possible to generate scaffolds with biological potency superior to the natural analogs. In one case, a compound has progressed through a phase II clinical trial for pan)staph infections. Modern biophysical assays highlighted the interplay between the synthetic scaffold and lipid composition leading to negative Gaussian curvature, a requirement for both pore formation and endosomal escape. The complexity of this interplay between lipids, bilayer components, and the scaffolds remains to be better resolved, but significant new insight has been provided. It is worthwhile to consider the various aspects of permeation and how these are related to ‘pore formation.’ More recently, our efforts have expanded toward protein transduction domains, or cell penetrating peptide, mimics. The combination of unique molecular scaffolds and guanidinium) rich side chains has produced an array of polymers with robust transduction (and delivery) activity. Being a new area, the fundamental interactions between these new scaffolds and the plasma membrane are just beginning to be understood. Negative Gaussian

  13. Activating AMP-activated protein kinase (AMPK) slows renal cystogenesis.

    PubMed

    Takiar, Vinita; Nishio, Saori; Seo-Mayer, Patricia; King, J Darwin; Li, Hui; Zhang, Li; Karihaloo, Anil; Hallows, Kenneth R; Somlo, Stefan; Caplan, Michael J

    2011-02-01

    Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) involves both fluid secretion and abnormal proliferation of cyst-lining epithelial cells. The chloride channel of the cystic fibrosis transmembrane conductance regulator (CFTR) participates in secretion of cyst fluid, and the mammalian target of rapamycin (mTOR) pathway may drive proliferation of cyst epithelial cells. CFTR and mTOR are both negatively regulated by AMP-activated protein kinase (AMPK). Metformin, a drug in wide clinical use, is a pharmacological activator of AMPK. We find that metformin stimulates AMPK, resulting in inhibition of both CFTR and the mTOR pathways. Metformin induces significant arrest of cystic growth in both in vitro and ex vivo models of renal cystogenesis. In addition, metformin administration produces a significant decrease in the cystic index in two mouse models of ADPKD. Our results suggest a possible role for AMPK activation in slowing renal cystogenesis as well as the potential for therapeutic application of metformin in the context of ADPKD. PMID:21262823

  14. Endoplasmic reticulum stress activates transglutaminase 2 leading to protein aggregation

    PubMed Central

    LEE, JIN-HAENG; JEONG, JAEHO; JEONG, EUI MAN; CHO, SUNG-YUP; KANG, JEONG WOOK; LIM, JISUN; HEO, JINBEOM; KANG, HYUNSOOK; KIM, IN-GYU; SHIN, DONG-MYUNG

    2014-01-01

    Aberrant activation of transglutaminase 2 (TGase2) contributes to a variety of protein conformational disorders such as neurodegenerative diseases and age-related cataracts. The accumulation of improperly folded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), which promotes either repair or degradation of the damaged proteins. Inadequate UPR results in protein aggregation that may contribute to the development of age-related degenerative diseases. TGase2 is a calcium-dependent enzyme that irreversibly modifies proteins by forming cross-linked protein aggregates. Intracellular TGase2 is activated by oxidative stress which generates large quantities of unfolded proteins. However, the relationship between TGase2 activity and UPR has not yet been established. In the present study, we demonstrated that ER stress activated TGase2 in various cell types. TGase2 activation was dependent on the ER stress-induced increase in the intracellular calcium ion concentration but not on the TGase2 protein expression level. Enzyme substrate analysis revealed that TGase2-mediated protein modification promoted protein aggregation concurrently with decreasing water solubility. Moreover, treatment with KCC009, a TGase2 inhibitor, abrogated ER stress-induced TGase2 activation and subsequent protein aggregation. However, TGase2 activation had no effect on ER stress-induced cell death. These results demonstrate that the accumulation of misfolded proteins activates TGase2, which further accelerates the formation of protein aggregates. Therefore, we suggest that inhibition of TGase2 may be a novel strategy by which to prevent the protein aggregation in age-related degenerative diseases. PMID:24481335

  15. Modeling Protein Folding and Applying It to a Relevant Activity

    ERIC Educational Resources Information Center

    Nelson, Allan; Goetze, Jim

    2004-01-01

    The different levels of protein structure that can be easily understood by creating a model that simulates protein folding, which can then be evaluated by applying it to a relevant activity, is presented. The materials required and the procedure for constructing a protein folding model are mentioned.

  16. Cross-flow heat exchangers for anti-freezing of liquid nitrogen

    NASA Astrophysics Data System (ADS)

    Chang, Ho-Myung; Gwak, Kyung Hyun; Yang, Hyung Suk; Hwang, Si-Dole

    2013-10-01

    Cross-flow heat exchangers are proposed and experimentally investigated as an anti-freezing scheme of liquid nitrogen. The possibility of freeze-out of liquid nitrogen is an important design issue in developing long superconducting cables, as the supply temperature of liquid nitrogen is close to its freezing temperature (63.3 K). Plate-fin heat exchangers are fabricated as typical counter-flow and newly proposed two-pass cross-flow in laboratory scale, and tested with cold helium gas at temperatures below 60 K. The experimental results show that the cross-flow heat exchanger is less vulnerable to the freeze-out condition, since the temperature distribution is basically two-dimensional. The cross-flow heat exchangers are effective in avoiding a complete clog-up of all passages and reducing the risk of freeze-out of liquid nitrogen.

  17. Nanomaterials for efficiently lowering the freezing point of anti-freeze coolants.

    PubMed

    Hong, Haiping; Zheng, Yingsong; Roy, Walter

    2007-09-01

    In this paper, we report, for the first time, the effect of the lowered freezing point in a 50% water/50% anti-freeze coolant (PAC) or 50% water/50% ethylene glycol (EG) solution by the addition of carbon nanotubes and other particles. The experimental results indicated that the nano materials are much more efficient (hundreds fold) in lowering the freezing point than the regular ionic materials (e.g., NaCl). The possible explanation for this interesting phenomenon is the colligative property of fluid and relative small size of nano material. It is quite certain that the carbon nanotubes and metal oxide nano particles could be a wonderful candidate for the nano coolant application because they could not only increase the thermal conductivity, but also efficiently lower the freezing point of traditional coolants. PMID:18019146

  18. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  19. Global Analysis of Protein Activities Using Proteome Chips

    NASA Astrophysics Data System (ADS)

    Zhu, Heng; Bilgin, Metin; Bangham, Rhonda; Hall, David; Casamayor, Antonio; Bertone, Paul; Lan, Ning; Jansen, Ronald; Bidlingmaier, Scott; Houfek, Thomas; Mitchell, Tom; Miller, Perry; Dean, Ralph A.; Gerstein, Mark; Snyder, Michael

    2001-09-01

    To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.

  20. DIETARY PROTEIN AND LACTOSE INCREASE TRANSLATION INITIATION FACTOR ACTIVATION AND TISSUE PROTEIN SYNTHESIS IN NEONATAL PIGS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in muscle and liver of pigs parenterally infused with amino acids and insulin. To examine the effects of enteral protein and carbohydrate on protein synthesis, pigs (n = 42, 1.7 kg body wt) were fed isocaloric milk die...

  1. Poliovirus protein 2C has ATPase and GTPase activities.

    PubMed

    Rodríguez, P L; Carrasco, L

    1993-04-15

    Poliovirus protein 2C belongs to an expanding group of proteins containing a nucleotide binding motif in their sequence. We present evidence that poliovirus 2C has nucleoside triphosphatase (NTPase) activity and binds to RNA. Poliovirus 2C was expressed in Escherichia coli cells as a fusion protein with the maltose binding protein (MBP). The fusion protein MBP-2C is efficiently cut by protease Xa within the 2C region. Thus, the fusion protein as such was used to assay for the putative activities of poliovirus 2C. Deletion mutants were constructed which lacked different portions of the 2C carboxyl terminus: mutant 2C delta 1 lacked the last 169 amino acids, whereas mutant 2C delta 2 had the last 74 amino acids deleted. The fusion proteins MBP-2C, MBP-2BC, and the mutant MBP-2C delta 2 that contained the first 255 amino acids of 2C had NTPase activity. Both ATPase and GTPase activities are inhibited by antibodies directed against the MBP-2C protein. Analysis of the ability of the different proteins to bind to labeled RNA indicates that MBP-2C and MBP-2BC form a complex, whereas none of the mutants interacted with RNA, indicating that the RNA binding domain lies beyond amino acid 255. None of the fusion proteins had detectable helicase activity. We suggest that poliovirus protein 2C shows similarities to the GTPases group involved in vesicular traffic and transports the viral RNA replication complexes. These results provide the first experimental evidence that poliovirus protein 2C is an NTPase and that this protein has affinity for nucleic acids. PMID:8385138

  2. Protein determination in seeds by proton activation

    NASA Astrophysics Data System (ADS)

    Morales, J. R.; Dinator, M. I.; Cerda, P.

    1989-04-01

    A proton beam of 6.6 MeV has been used to produce 11C and 13N in Araucaria Araucana seeds. Their positron decay allows determination of the N/C ratio. In seeds the nitrogen content is associated to proteins while carbon is spread in the organic material. Samples were irradiated for about 10 min with a beam intensity of 5 nA on areas of 1 mm 2. Slices of the seed were radially explored, showing a larger concentration of protein in the center.

  3. Monitoring G protein activation in cells with BRET

    PubMed Central

    Masuho, Ikuo; Martemyanov, Kirill A.; Lambert, Nevin A.

    2016-01-01

    Summary Live-cell assays based on fluorescence and luminescence are now indispensable tools for the study of G protein signaling. Assays based on fluorescence and bioluminescence resonance energy transfer (FRET and BRET) have been particularly valuable for monitoring changes in second messengers, protein-protein interactions, and protein conformation. Here we describe a BRET assay that monitors the release of free Gβγ dimers after activation of heterotrimers containing Gα subunits from all four G protein subfamilies. This assay provides useful kinetic and pharmacological information with reasonably high throughput using standard laboratory equipment. PMID:26260597

  4. New constitutive latex osmotin-like proteins lacking antifungal activity.

    PubMed

    Freitas, Cleverson D T; Silva, Maria Z R; Bruno-Moreno, Frederico; Monteiro-Moreira, Ana C O; Moreira, Renato A; Ramos, Márcio V

    2015-11-01

    Proteins that share similar primary sequences to the protein originally described in salt-stressed tobacco cells have been named osmotins. So far, only two osmotin-like proteins were purified and characterized of latex fluids. Osmotin from Carica papaya latex is an inducible protein lacking antifungal activity, whereas the Calotropis procera latex osmotin is a constitutive antifungal protein. To get additional insights into this subject, we investigated osmotins in latex fluids of five species. Two potential osmotin-like proteins in Cryptostegia grandiflora and Plumeria rubra latex were detected by immunological cross-reactivity with polyclonal antibodies produced against the C. procera latex osmotin (CpOsm) by ELISA, Dot Blot and Western Blot assays. Osmotin-like proteins were not detected in the latex of Thevetia peruviana, Himatanthus drasticus and healthy Carica papaya fruits. Later, the two new osmotin-like proteins were purified through immunoaffinity chromatography with anti-CpOsm immobilized antibodies. Worth noting the chromatographic efficiency allowed for the purification of the osmotin-like protein belonging to H. drasticus latex, which was not detectable by immunoassays. The identification of the purified proteins was confirmed after MS/MS analyses of their tryptic digests. It is concluded that the constitutive osmotin-like proteins reported here share structural similarities to CpOsm. However, unlike CpOsm, they did not exhibit antifungal activity against Fusarium solani and Colletotrichum gloeosporioides. These results suggest that osmotins of different latex sources may be involved in distinct physiological or defensive events. PMID:26231325

  5. Protein kinase activity associated with pancreatic zymogen granules.

    PubMed

    Burnham, D B; Munowitz, P; Thorn, N; Williams, J A

    1985-05-01

    Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues. PMID:4004796

  6. Protein kinase activity associated with pancreatic zymogen granules.

    PubMed Central

    Burnham, D B; Munowitz, P; Thorn, N; Williams, J A

    1985-01-01

    Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues. Images Fig. 1. Fig. 2. Fig. 7. Fig. 8. PMID:4004796

  7. The kinetics of ER fusion protein activation in vivo

    PubMed Central

    Wilson, Catherine H.; Gamper, Ivonne; Perfetto, Alessandra; Auw, Jeremy; Littlewood, Trevor D.; Evan, Gerard I.

    2014-01-01

    Reversibly switchable proteins are powerful tools with which to explore protein function in vitro and in vivo. For example, the activity of many proteins fused to the hormone-binding domain of the modified estrogen receptor (ERTAM) can be regulated by provision or removal of 4-hydroxytamoxifen (4-OHT). Despite the widespread use of ERTAM fusions in vivo, inadequate data are available as to the most efficacious routes for systemic tamoxifen delivery. In this study, we have used two well-characterised ERTAM fusion proteins, both reversibly activated by 4-OHT, to compare the effectiveness and kinetics of 4-OHT delivery in mice in vivo by either tamoxifen in food or by intraperitoneal injection. Our data indicate that dietary tamoxifen offers an effective, facile and ethically preferable means for long term activation of ERTAM fusion proteins in vivo. PMID:24662815

  8. Hemagglutinating activity of proteins from Parkia speciosa seeds.

    PubMed

    Chankhamjon, Kanokwan; Petsom, Amorn; Sawasdipuksa, Narumon; Sangvanich, Polkit

    2010-01-01

    Proteins from Parkia speciosa Hassk. (Fabaceae) seeds were extracted and stepwise precipitated using ammonium sulfate. Proteins precipitated with 25% ammonium sulfate were separated by affinity chromatography on Affi-Gel Blue gel followed by protein liquid chromatography on Superdex 200. The protein Gj, which was identified as a protein similar to putative aristolochene synthase, 3'-partial from Oryza sativa L. (Poaceae), had hemagglutinating activity of 0.39 mug/muL. Moreover, fraction C2 from the proteins precipitated with 60% ammonium sulfate, separated by lectin-specific adsorption chromatography using Con A Sepharose, had hemagglutinating activity of 1.17 mug/muL. Using gel electrophoresis, two proteins C2a and C2b were separated, having molecular weights of 45 kDa and 23 kDa, respectively. From protein identification, C2a was found to be similar to the hypothetical protein B1342F01.11 from Oryza sativa, and C2b was similar to the hypothetical protein At1g51560 from Arabidopsis thaliana (L.) Heynh. (Brassicaceae). PMID:20645760

  9. Modeling the SHG activities of diverse protein crystals

    SciTech Connect

    Haupert, Levi M.; DeWalt, Emma L.; Simpson, Garth J.

    2012-11-01

    The origins of the diversity in the SHG signal from protein crystals are investigated and potential protein-crystal coverage by SHG microscopy is assessed. A symmetry-additive ab initio model for second-harmonic generation (SHG) activity of protein crystals was applied to assess the likely protein-crystal coverage of SHG microscopy. Calculations were performed for 250 proteins in nine point-group symmetries: a total of 2250 crystals. The model suggests that the crystal symmetry and the limit of detection of the instrument are expected to be the strongest predictors of coverage of the factors considered, which also included secondary-structural content and protein size. Much of the diversity in SHG activity is expected to arise primarily from the variability in the intrinsic protein response as well as the orientation within the crystal lattice. Two or more orders-of-magnitude variation in intensity are expected even within protein crystals of the same symmetry. SHG measurements of tetragonal lysozyme crystals confirmed detection, from which a protein coverage of ∼84% was estimated based on the proportion of proteins calculated to produce SHG responses greater than that of tetragonal lysozyme. Good agreement was observed between the measured and calculated ratios of the SHG intensity from lysozyme in tetragonal and monoclinic lattices.

  10. Evidence for a receptor protein of activated carcinogen

    PubMed Central

    Mainigi, Kumar D.; Sorof, Sam

    1977-01-01

    During carcinogenesis in rat liver by 3′-methyl-4-dimethylaminoazobenzene, the two moieties of the principal liver carcinogen-protein complex have considerably different turnover rates. With continued ingestion of the azocarcinogen by rats, the bound azo dye in the complex has a half-life of 2.5 ± 0.25 (SD) days, while the protein moiety has a half-life of 8.7 ± 1.6 days (probability of identity <0.001). In addition, the interaction of the azocarcinogen with the principal target protein in vivo appears to extend the half-life of the protein itself from 3.3 ± 0.2 days in normal liver to 8.7 ± 1.6 days (P < 0.001). The slowing of the turnover of the protein by the carcinogen appears to be readily reversible, since soon after the cessation of azocarcinogen feeding, the half-life of the protein returns to that of the target protein in normal liver. The considerable difference in turnover rates of the two moieties of the complex and the reversible effects of the carcinogen in slowing the turnover of the protein moiety suggest that the two moieties of the native azoprotein are noncovalently linked and that they have different biological activities. The native complex appears to contain azo dye in an activated state that is capable of yielding a reactive electrophile, because after protein denaturation the bound azo dye was previously found to have properties that are indicative of covalent linkage to the protein. The retardation in the biological turnover rate of the protein moiety, apparently resulting from interaction with azocarcinogen, is in agreement with the known ligand-induced stabilization in vitro and reduced rate of proteolytic degradation in vivo of other proteins that result from conformational change to a more compact configuration. Our evidence is consistent with the hypothesis that the principal liver carcinogen-protein complex contains hydrophobically bound activated azocarcinogen, whose specificity of reaction with critical macromolecule(s) in

  11. Cloning of three novel neuronal Cdk5 activator binding proteins.

    PubMed

    Ching, Y P; Qi, Z; Wang, J H

    2000-01-25

    Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics. The active kinase consists of a catalytic subunit, Cdk5, and a 25 kDa activator protein (p25nck5a) derived from a 35 kDa neuronal-specific protein (p35nck5a). As an extension of our previous study (Qi, Z., Tang, D., Zhu, X., Fujita, D.J., Wang, J.H., 1998. Association of neurofilament proteins with neuronal Cdk5 activator. J. Biol. Chem. 270, 2329-2335), which showed that neurofilament is one of the p35nck5a-associated proteins, we now report the isolation of three other novel p35nck5a-associated proteins using the yeast two-hybrid screen. The full-length forms of these three novel proteins, designated C42, C48 and C53, have a molecular mass of 66, 24, and 57 kDa, respectively. Northern analysis indicates that these novel proteins are widely expressed in human tissues, including the heart, brain, skeletal muscle, placenta, lung, liver, kidney and pancreas. The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35nck5a complexed with Cdk5 from insect cell lysate. Among these three proteins, only C48 and C53 can be phosphorylated by Nclk, suggesting that they may be the substrates of Nclk. Sequence homology searches have suggested that the C48 protein is marginally related to restin protein, whereas the C42 protein has homologues of unknown function in Caenorhabditis elegans and Arabidopsis thaliana. PMID:10721722

  12. Pestivirus NS3 (p80) protein possesses RNA helicase activity.

    PubMed Central

    Warrener, P; Collett, M S

    1995-01-01

    The pestivirus bovine viral diarrhea virus (BVDV) p80 protein (referred to here as the NS3 protein) contains amino acid sequence motifs predictive of three enzymatic activities: serine proteinase, nucleoside triphosphatase, and RNA helicase. We have previously demonstrated that the former two enzymatic activities are associated with this protein. Here, we show that a purified recombinant BVDV NS3 protein derived from baculovirus-infected insect cells possesses RNA helicase activity. BVDV NS3 RNA helicase activity was specifically inhibited by monoclonal antibodies to the p80 protein. The activity was dependent on the presence of nucleoside triphosphate and divalent cation, with a preference for ATP and Mn2+. Hydrolysis of the nucleoside triphosphate was necessary for strand displacement. The helicase activity required substrates with an un-base-paired region on the template strand 3' of the duplex region. As few as three un-base-paired nucleotides were sufficient for efficient oligonucleotide displacement. However, the enzyme did not act on substrates having a single-stranded region only to the 5' end of the duplex or on substrates lacking single-stranded regions altogether (blunt-ended duplex substrates), suggesting that the directionality of the BVDV RNA helicase was 3' to 5' with respect to the template strand. The BVDV helicase activity was able to displace both RNA and DNA oligonucleotides from RNA template strands but was unable to release oligonucleotides from DNA templates. The possible role of this activity in pestivirus replication is discussed. PMID:7853509

  13. Activation of an Endoribonuclease by Non-intein Protein Splicing.

    PubMed

    Campbell, Stephen J; Stern, David B

    2016-07-29

    The Chlamydomonas reinhardtii chloroplast-localized poly(A)-binding protein RB47 is predicted to contain a non-conserved linker (NCL) sequence flanked by highly conserved N- and C-terminal sequences, based on the corresponding cDNA. RB47 was purified from chloroplasts in association with an endoribonuclease activity; however, protein sequencing failed to detect the NCL. Furthermore, while recombinant RB47 including the NCL did not display endoribonuclease activity in vitro, versions lacking the NCL displayed strong activity. Both full-length and shorter forms of RB47 could be detected in chloroplasts, with conversion to the shorter form occurring in chloroplasts isolated from cells grown in the light. This conversion could be replicated in vitro in chloroplast extracts in a light-dependent manner, where epitope tags and protein sequencing showed that the NCL was excised from a full-length recombinant substrate, together with splicing of the flanking sequences. The requirement for endogenous factors and light differentiates this protein splicing from autocatalytic inteins, and may allow the chloroplast to regulate the activation of RB47 endoribonuclease activity. We speculate that this protein splicing activity arose to post-translationally repair proteins that had been inactivated by deleterious insertions or extensions. PMID:27311716

  14. Iron-chelating activity of chickpea protein hydrolysate peptides.

    PubMed

    Torres-Fuentes, Cristina; Alaiz, Manuel; Vioque, Javier

    2012-10-01

    Chickpea-chelating peptides were purified and analysed for their iron-chelating activity. These peptides were purified after affinity and gel filtration chromatography from a chickpea protein hydrolysate produced with pepsin and pancreatin. Iron-chelating activity was higher in purified peptide fractions than in the original hydrolysate. Histidine contents were positively correlated with the iron-chelating activity. Hence fractions with histidine contents above 20% showed the highest chelating activity. These results show that iron-chelating peptides are generated after chickpea protein hydrolysis with pepsin plus pancreatin. These peptides, through metal chelation, may increase iron solubility and bioavailability and improve iron absorption. PMID:25005984

  15. Inclusion bodies and purification of proteins in biologically active forms.

    PubMed

    Mukhopadhyay, A

    1997-01-01

    Even though recombinant DNA technology has made possible the production of valuable therapeutic proteins, its accumulation in the host cell as inclusion body poses serious problems in the recovery of functionally active proteins. In the last twenty years, alternative techniques have been evolved to purify biologically active proteins from inclusion bodies. Most of these remain only as inventions and very few are commercially exploited. This review summarizes the developments in isolation, refolding and purification of proteins from inclusion bodies that could be used for vaccine and non-vaccine applications. The second section involves a discussion on inclusion bodies, how they are formed, and their physicochemical properties. In vivo protein folding in Escherichia coli and kinetics of in vitro protein folding are the subjects of the third and fourth sections respectively. The next section covers the recovery of bioactive protein from inclusion bodies: it includes isolation of inclusion body from host cell debris, purification in denatured state alternate refolding techniques, and final purification of active molecules. Since purity and safety are two important issues in therapeutic grade proteins, the following three sections are devoted to immunological and biological characterization of biomolecules, nature, and type of impurities normally encountered, and their detection. Lastly, two case studies are discussed to demonstrate the sequence of process steps involved. PMID:8939059

  16. Activators of G Protein Signaling in the Kidney

    PubMed Central

    2015-01-01

    Heterotrimeric G proteins play a crucial role in regulating signal processing to maintain normal cellular homeostasis, and subtle perturbations in its activity can potentially lead to the pathogenesis of renal disorders or diseases. Cell-surface receptors and accessory proteins, which normally modify and organize the coupling of individual G protein subunits, contribute to the regulation of heterotrimeric G protein activity and their convergence and/or divergence of downstream signaling initiated by effector systems. Activators of G protein signaling (AGS) are a family of accessory proteins that intervene at multiple distinct points during the activation–inactivation cycle of G proteins, even in the absence of receptor stimulation. Perturbations in the expression of individual AGS proteins have been reported to modulate signal transduction pathways in a wide array of diseases and disorders within the brain, heart, immune system, and more recently, the kidney. This review will provide an overview of the expression profile, localization, and putative biologic role of the AGS family in the context of normal and diseased states of the kidney. PMID:25628392

  17. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    SciTech Connect

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; Rathore, Rajendra; Ramchandran, Ramani

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.

  18. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    DOE PAGESBeta

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; et al

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function.more » We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.« less

  19. Notum deacylates Wnt proteins to suppress signalling activity.

    PubMed

    Kakugawa, Satoshi; Langton, Paul F; Zebisch, Matthias; Howell, Steven A; Chang, Tao-Hsin; Liu, Yan; Feizi, Ten; Bineva, Ganka; O'Reilly, Nicola; Snijders, Ambrosius P; Jones, E Yvonne; Vincent, Jean-Paul

    2015-03-12

    Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase. PMID:25731175

  20. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    NASA Astrophysics Data System (ADS)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  1. Regulatory Crosstalk by Protein Kinases on CFTR Trafficking and Activity

    PubMed Central

    Farinha, Carlos M.; Swiatecka-Urban, Agnieszka; Brautigan, David L.; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease. PMID:26835446

  2. Activation of a human chromosomal replication origin by protein tethering

    PubMed Central

    Chen, Xiaomi; Liu, Guoqi; Leffak, Michael

    2013-01-01

    The specification of mammalian chromosomal replication origins is incompletely understood. To analyze the assembly and activation of prereplicative complexes (pre-RCs), we tested the effects of tethered binding of chromatin acetyltransferases and replication proteins on chromosomal c-myc origin deletion mutants containing a GAL4-binding cassette. GAL4DBD (DNA binding domain) fusions with Orc2, Cdt1, E2F1 or HBO1 coordinated the recruitment of the Mcm7 helicase subunit, the DNA unwinding element (DUE)-binding protein DUE-B and the minichromosome maintenance (MCM) helicase activator Cdc45 to the replicator, and restored origin activity. In contrast, replication protein binding and origin activity were not stimulated by fusion protein binding in the absence of flanking c-myc DNA. Substitution of the GAL4-binding site for the c-myc replicator DUE allowed Orc2 and Mcm7 binding, but eliminated origin activity, indicating that the DUE is essential for pre-RC activation. Additionally, tethering of DUE-B was not sufficient to recruit Cdc45 or activate pre-RCs formed in the absence of a DUE. These results show directly in a chromosomal background that chromatin acetylation, Orc2 or Cdt1 suffice to recruit all downstream replication initiation activities to a prospective origin, and that chromosomal origin activity requires singular DNA sequences. PMID:23658226

  3. Computational study on the antifreeze glycoproteins as inhibitors of clathrate-hydrate formation.

    PubMed

    Cruz-Torres, Armando; Romero-Martínez, Ascención; Galano, Annia

    2008-08-01

    The ability of antifreeze glycoproteins to inhibit clathrate-hydrate formation is studied using DFT. A 5(12) cavity, dodecahedral (H(2)O)(20), and the AATA peptide are used to model the inhibitor-clathrate interaction. The presence of AATA in the vicinity of the water cavities not only leads to the formation of complexes, with different peptide/cavity ratios, but also to the deformation of the cavity and to the elongation of several of the hydrogen bonds responsible for keeping the dodecahedral (H(2)O)(20) together. The complexes are formed through hydrogen bonding between the peptides and the water cavities. The glycoproteins are expected to anchor onto the clathrate surface, blocking the access of new water molecules and preventing the incipient crystals from growing. They are also expected to weaken the clathrate structure. Amide IR bands are associated with the complexes' formation. They are significantly red-shifted in the hydrogen-bonded systems compared to isolated AATA. The amide A band is the most sensitive to hydrogen bonding. In addition a distinctive band around 3100 cm(-1) is proposed for the identification of clathrate-peptide hydrogen-bonded complexes. PMID:18618535

  4. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. PMID:26579181

  5. G Protein Activation without a GEF in the Plant Kingdom

    PubMed Central

    Wang, Hao; Matthews, Melissa; Bradford, William; Bennetzen, Jeffrey L.; Jones, Alan M.

    2012-01-01

    Animal heterotrimeric G proteins are activated by guanine nucleotide exchange factors (GEF), typically seven transmembrane receptors that trigger GDP release and subsequent GTP binding. In contrast, the Arabidopsis thaliana G protein (AtGPA1) rapidly activates itself without a GEF and is instead regulated by a seven transmembrane Regulator of G protein Signaling (7TM-RGS) protein that promotes GTP hydrolysis to reset the inactive (GDP-bound) state. It is not known if this unusual activation is a major and constraining part of the evolutionary history of G signaling in eukaryotes. In particular, it is not known if this is an ancestral form or if this mechanism is maintained, and therefore constrained, within the plant kingdom. To determine if this mode of signal regulation is conserved throughout the plant kingdom, we analyzed available plant genomes for G protein signaling components, and we purified individually the plant components encoded in an informative set of plant genomes in order to determine their activation properties in vitro. While the subunits of the heterotrimeric G protein complex are encoded in vascular plant genomes, the 7TM-RGS genes were lost in all investigated grasses. Despite the absence of a Gα-inactivating protein in grasses, all vascular plant Gα proteins examined rapidly released GDP without a receptor and slowly hydrolyzed GTP, indicating that these Gα are self-activating. We showed further that a single amino acid substitution found naturally in grass Gα proteins reduced the Gα-RGS interaction, and this amino acid substitution occurred before the loss of the RGS gene in the grass lineage. Like grasses, non-vascular plants also appear to lack RGS proteins. However, unlike grasses, one representative non-vascular plant Gα showed rapid GTP hydrolysis, likely compensating for the loss of the RGS gene. Our findings, the loss of a regulatory gene and the retention of the “self-activating” trait, indicate the existence of divergent

  6. Oxidative Stress Impairs the Stimulatory Effect of S100 Proteins on Protein Phosphatase 5 Activity.

    PubMed

    Yamaguchi, Fuminori; Tsuchiya, Mitsumasa; Shimamoto, Seiko; Fujimoto, Tomohito; Tokumitsu, Hiroshi; Tokuda, Masaaki; Kobayashi, Ryoji

    2016-01-01

    Oxidative stress is the consequence of an imbalance between the production of harmful reactive oxygen species and the cellular antioxidant system for neutralization, and it activates multiple intracellular signaling pathways, including apoptosis signal-regulating kinase 1 (ASK1). Protein phosphatase 5 (PP5) is a serine/threonine phosphatase involved in oxidative stress responses. Previously, we reported that S100 proteins activate PP5 in a calcium-dependent manner. S100 proteins belong to a family of small EF-hand calcium-binding proteins involved in many processes such as cell proliferation, differentiation, apoptosis, and inflammation. Therefore, we investigated the effects of oxidative stress on S100 proteins, their interaction with PP5, and PP5 enzyme activity. Recombinant S100A2 was easily air-oxidized or Cu-oxidized, and oxidized S100A2 formed cross-linked dimers and higher molecular-mass complexes. The binding of oxidized S100A2 to PP5 was reduced, resulting in decreased PP5 activation in vitro. Oxidation also impaired S100A1, S100A6, S100B, and S100P to activate PP5, although the low dose of oxidized S100 proteins still activated PP5. Hydrogen peroxide (H2O2) induced S100A2 oxidation in human keratinocytes (HaCaT) and human hepatocellular carcinoma (Huh-7) cells. Furthermore, H2O2 reduced the binding of S100A2 to PP5 and decreased PP5 activation in HaCaT and Huh-7 cells. Importantly, even the low dose of S100A2 achieved by knocking down increased dephosphorylation of ASK1 and reduced caspase 3/7 activity in Huh-7 cells treated with H2O2. These results indicate that oxidative stress impairs the ability of S100 proteins to bind and activate PP5, which in turn modulates the ASK1-mediated signaling cascades involved in apoptosis. PMID:27600583

  7. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins. PMID:27080133

  8. Anthelmintic activity of Leucaena leucocephala protein extracts on Haemonchus contortus.

    PubMed

    Soares, Alexandra Martins dos Santos; de Araújo, Sandra Alves; Lopes, Suzana Gomes; Costa Junior, Livio Martins

    2015-01-01

    The objective of this study was to evaluate the effects of protein extracts obtained from the plant Leucaena leucocephala on the nematode parasite Haemonchus contortus. The seeds, shell and cotyledon of L. leucocephala were separated and their proteins extracted using a sodium phosphate buffer, and named as TE (total seed extract), SE (shell extract) and CE (cotyledon extract). Soluble protein content, protease, protease inhibitory and chitinase activity assays were performed. Exsheathment inhibition of H. contortus larvae were performed at concentrations of 0.6 mg mL-1, and egg hatch assays were conducted at protein concentrations of 0.8, 0.4, 0.2, 0.1 and 0.05 mg mL-1. The effective concentration for 50% hatching inhibition (EC50) was estimated by probit. Different proportions of soluble proteins, protease and chitinase were found in TE and CE. Protease inhibitory activity was detected in all extracts. The EC50 of the CE and TE extracts were 0.48 and 0.33 mg mL-1, respectively. No ovicidal effects on H. contortus were detected in SE extracts, and none of the protein extracts demonstrated larvicidal effects on H. contortus. We therefore conclude that protein extracts of L. leucocephala had a detrimental effect on nematode eggs, which can be correlated with the high protease and chitinase activity of these extracts. PMID:26689178

  9. Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1998-01-01

    In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

  10. Liposomal packaging generates Wnt protein with in vivo biological activity.

    PubMed

    Morrell, Nathan T; Leucht, Philipp; Zhao, Ludan; Kim, Jae-Beom; ten Berge, Derk; Ponnusamy, Karthik; Carre, A Lyonel; Dudek, Henryk; Zachlederova, Marie; McElhaney, Michael; Brunton, Shirley; Gunzner, Janet; Callow, Marinella; Polakis, Paul; Costa, Mike; Zhang, Xiaoyan M; Helms, Jill A; Nusse, Roel

    2008-01-01

    Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context. PMID:18698373

  11. Activation of protein kinase C induces mitogen-activated protein kinase dephosphorylation and pronucleus formation in rat oocytes.

    PubMed

    Lu, Qing; Smith, Gary D; Chen, Da-Yuan; Han, Zhi-Ming; Sun, Qing-Yuan

    2002-07-01

    Mammalian oocytes are arrested at metaphase of the second meiotic division (MII) before fertilization. When oocytes are stimulated by spermatozoa, they exit MII stage and complete meiosis. It has been suggested that an immediate increase in intracellular free calcium concentration and inactivation of maturation promoting factor (MPF) are required for oocyte activation. However, the underlying mechanism is still unclear. In the present study, we investigated the role of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase, and their interplay in rat oocyte activation. We found that MAP kinase became dephosphorylated in correlation with pronucleus formation after fertilization. Protein kinase C activators, phorbol 12-myriatate 13-acetate (PMA) and 1,2-dioctanoyl-rac-glycerol (diC8), triggered dephosphorylation of MAP kinase and pronucleus formation in a dose-dependent and time-dependent manner. Dephosphorylation of MAP kinase was also correlated with pronucleus formation when oocytes were treated with PKC activators. Effects of PKC activators were abolished by the PKC inhibitors, calphostin C and staurosporine, as well as a protein phosphatase blocker, okadaic acid (OA). These results suggest that PKC activation may cause rat oocyte pronucleus formation via MAP kinase dephosphorylation, which is probably mediated by OA-sensitive protein phosphatases. We also provide evidence supporting the involvement of such a process in fertilization. PMID:12080000

  12. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  13. Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

    PubMed

    Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

    2005-01-01

    The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research. PMID:16302727

  14. Counteracting Protein Kinase Activity in the Heart: The Multiple Roles of Protein Phosphatases

    PubMed Central

    Weber, Silvio; Meyer-Roxlau, Stefanie; Wagner, Michael; Dobrev, Dobromir; El-Armouche, Ali

    2015-01-01

    Decades of cardiovascular research have shown that variable and flexible levels of protein phosphorylation are necessary to maintain cardiac function. A delicate balance between phosphorylated and dephosphorylated states of proteins is guaranteed by a complex interplay of protein kinases (PKs) and phosphatases. Serine/threonine phosphatases, in particular members of the protein phosphatase (PP) family govern dephosphorylation of the majority of these cardiac proteins. Recent findings have however shown that PPs do not only dephosphorylate previously phosphorylated proteins as a passive control mechanism but are capable to actively control PK activity via different direct and indirect signaling pathways. These control mechanisms can take place on (epi-)genetic, (post-)transcriptional, and (post-)translational levels. In addition PPs themselves are targets of a plethora of proteinaceous interaction partner regulating their endogenous activity, thus adding another level of complexity and feedback control toward this system. Finally, novel approaches are underway to achieve spatiotemporal pharmacologic control of PPs which in turn can be used to fine-tune misleaded PK activity in heart disease. Taken together, this review comprehensively summarizes the major aspects of PP-mediated PK regulation and discusses the subsequent consequences of deregulated PP activity for cardiovascular diseases in depth. PMID:26617522

  15. Activities of the Sex-lethal protein in RNA binding and protein:protein interactions.

    PubMed Central

    Samuels, M; Deshpande, G; Schedl, P

    1998-01-01

    The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain. PMID:9592147

  16. Heated Proteins are Still Active in a Functionalized Nanoporous Support

    SciTech Connect

    Chen, Baowei; Qi, Wen N.; Li, Xiaolin; Lei, Chenghong; Liu, Jun

    2013-07-08

    We report that even under the heated condition, the conformation and activity of a protein can be hoarded in a functionalized nanoporous support via non-covalent interaction, although the hoarded protein was not exhibiting the full protein activity, the protein released subsequently still maintained its native conformation and activity. Glucose oxidase (GOX) was spontaneously and largely entrapped in aminopropyl-functionalized mesoporous silica (NH2-FMS) at 20 oC via a dominant electrostatic interaction. Although FMS-GOX displayed 45% activity of the free enzyme in solution, the GOX released from FMS exhibited its 100% activity prior to the entrapment. Surprisingly, the released GOX from FMS still maintained 89% of its initial activity prior to the entrapment after FMS-GOX was incubated at 60 oC for 1 h prior to release, while the free GOX in solution lost nearly all activity under the same incubation. Intrinsic fluorescence emission of GOX and native electrophoresis demonstrated that the heating resulted in significant conformational changes and oligomeric structures of the free GOX, but FMS efficiently maintained the thermal stability of GOX therein and resisted the thermal denaturation and oligomeric aggregation.

  17. Probing heterotrimeric G protein activation: applications to biased ligands

    PubMed Central

    Denis, Colette; Saulière, Aude; Galandrin, Ségolène; Sénard, Jean-Michel; Galés, Céline

    2012-01-01

    Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of “ligand-directed trafficking” has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting activation of specific signals and thus generating textured responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligand responses. However, considering that a single receptor has pleiotropic signalling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even if some G protein-independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discuss them in the context of G protein biased -ligands. PMID:22229559

  18. Probing heterotrimeric G protein activation: applications to biased ligands.

    PubMed

    Denis, Colette; Saulière, Aude; Galandrin, Segolene; Sénard, Jean-Michel; Galés, Céline

    2012-01-01

    Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of "ligand-directed trafficking" has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting the activation of specific signals and thus generating texture responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligands. However, considering that a single receptor has pleiotropic signaling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even, if some G protein independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discussed them in the context of G protein biased-ligands. PMID:22229559

  19. Bryostatins activate protein kinase C in intact human platelets

    SciTech Connect

    Smith, J.B.; Tallant, E.A.; Pettit, G.R.; Wallace, R.W.

    1986-05-01

    Bryostatins, macrocyclic lactones isolated from a marine bryozoan, have antineoplastic activity in the P388 lymphocytic leukemia system. These compounds also stimulate growth in Swiss 3T3 cells, induce secretion in leukocytes, inhibit phorbol dibutyrate binding to a high affinity receptor, and activate the C-kinase in vitro. In human platelets, phorbol esters induce aggregation and activate protein kinase C, resulting in phosphorylation of a 47K protein and the 20K myosin light chain. The authors now show that bryostatin 7 (B-7) triggers platelet aggregation to the same rate and extent as phorbol 12-myristate 13-acetate (PMA). B-7 also causes the in vivo activation of the C-kinase, resulting in phosphorylation of both the 47K and the 20K proteins; the time courses and dose-responses of these B-7-induced phosphorylations were similar to those found with PMA. In addition, B-7 increases the level of /sup 32/P-incorporation into the platelet polyphosphoinositides, which also occurs in response to PMA. Bryostatin 3 (B-3), which has been shown to be much less potent than B-7 in mimicking other PMA effects, was much less effective than PMA or B-7 in inducing platelet aggregation and in stimulating /sup 32/P-incorporation into both proteins and the phosphoinositides. These results demonstrate that, intact human platelets, bryostatins mimic the phorbol esters tumor promoters and directly activate protein kinase C.

  20. NALP3 inflammasome activation in protein misfolding diseases.

    PubMed

    Shi, Fushan; Kouadir, Mohammed; Yang, Yang

    2015-08-15

    Protein-misfolding diseases, such as Alzheimer's disease, type 2 diabetes, Prion diseases, and Parkinson's disease, are characterized by inflammatory reactions. In all these diseases, IL-1β (Interlukine-1β) has been shown to be an important regulator, and the misfolded proteins are proved to be triggers of the release of IL-1β. Recently, several reports demonstrated that the inflammasome activation is involved in the progress of the misfolded protein diseases, and that the inflammasome can recognize pathogenic proteins leading to the release of IL-1β. In this review, we discuss the role of inflammasome in the pathogenesis of misfolded protein diseases and the potential of inflammasome-targeting therapeutic interventions in the management of these diseases. PMID:26037399

  1. Enhancer-specific modulation of E protein activity.

    PubMed

    Markus, Maurice; Du, Zhimei; Benezra, Robert

    2002-02-22

    Homodimeric complexes of members of the E protein family of basic helix-loop-helix (bHLH) transcription factors are important for tissue-specific activation of genes in B lymphocytes (Bain, G., Gruenwald, S., and Murre, C. (1993) Mol. Cell Biol. 13, 3522-3529; Shen, C. P., and Kadesch, T. (1995) Mol. Cell Biol. 15, 4518-4524; Jacobs, Y., et al. (1994) Mol. Cell Biol. 14, 4087-4096; Wilson, R. B., et al. (1991) Mol. Cell Biol. 11, 6185-6191). These homodimers, however, have little activity on myogenic enhancers (Weintraub, H., Genetta, T., and Kadesch, T. (1994) Genes Dev. 8, 2203-2211). We report here the identification of a novel cis-acting transcriptional repression domain in the E protein family of bHLH transcription factors. This domain, the Rep domain, is present in each of the known vertebrate E proteins. Extensive mapping analysis demonstrates that this domain is an acidic region of 30 amino acids with a predicted loop structure. Fusion studies indicate that the Rep domain can repress both of the E protein transactivation domains (AD1 and AD2). Physiologically, the Rep domain plays a key role in maintaining E protein homodimers in an inactive state on myogenic enhancers. In addition, we demonstrate that Rep domain mediated repression of AD1 is a necessary for the function of MyoD-E protein heterodimeric complexes. These studies demonstrate that the Rep domain is important for modulating the transcriptional activity of E proteins and provide key insights into both the selectivity and mechanism of action of E protein containing bHLH protein complexes. PMID:11724804

  2. Peptides and proteins with antimicrobial activity

    PubMed Central

    Coutinho, Henrique Douglas Melo; Lôbo, Katiuscia Menezes; Bezerra, Denise Aline Casimiro; Lôbo, Inalzuir

    2008-01-01

    The increase of microbial resistance to antibiotics has led to a continuing search for newer and more effective drugs. Antimicrobial peptides are generally found in animals, plants, and microorganisms and are of great interest to medicine, pharmacology, and the food industry. These peptides are capable of inhibiting pathogenic microorganisms. They can attack parasites, while causing little or no harm to the host cells. The defensins are peptides found in granules in the polymorphonuclear neutrophils (PMNs) and are responsible for the defense of the organism. Several animal defensins, like dermaseptin, antileukoprotease, protegrin, and others, have had their activities and efficacy tested and been shown to be effective against bacteria, fungi, and protists; there are also specific defensins from invertebrates, e.g., drosomycin and heliomicin; from plants, e.g., the types A and B; and the bacteriocins, e.g., acrocin, marcescin, etc. The aim of the present work was to compile a comprehensive bibliographic review of the diverse potentially antimicrobial peptides in an effort to systematize the current knowledge on these substances as a contribution for further researches. The currently available bibliography does not give a holistic approach on this subject. The present work intends to show that the mechanism of defense represented by defensins is promising from the perspective of its application in the treatment of infectious diseases in human, animals and plants. PMID:21264153

  3. Chemical labelling of active serum thioester proteins for quantification.

    PubMed

    Holm, Lotta; Ackland, Gareth L; Edwards, Mark R; Breckenridge, Ross A; Sim, Robert B; Offer, John

    2012-02-01

    The complement serum proteins C3 and C4 and the protease inhibitor α-2 macroglobulin are all members of the C3/α-2M thioester protein family, an evolutionarily ancient and conserved family that contains an intrachain thioester bond. The chemistry of the thioester bond is a key to the function of the thioester proteins. All these proteins function by covalently linking to their target by acyl transfer of the protein via the thioester moiety. We show that the signature thioester bond can be targeted with nucleophiles linked to a bioreporter molecule, site-specifically modifying the whole, intact thioester protein. Conditions were optimised to label selectively and efficiently pull-down unprocessed thioester-containing proteins from serum. We demonstrated pull-down of full-length C3, α-2M and C4 from sera in high salt, using a biotinylated nucleophile and streptavidin-coated resin, confirmed by MALDI-TOF MS identification of the gel bands. The potential for the development of a quantitative method for measuring active C3 in serum was investigated in patient sera pre and post operation. Quantifying active C3 in clinical assays using current methods is difficult. Methods based on antibody detection (e.g. nephelometry) do not distinguish between active C3 and inactive breakdown products. C3-specific haemolytic assays can be used, but these require use of relatively unstable reagents. The current work represents a promising robust, enzyme- and antibody-free chemical method for detecting active thioester proteins in blood, plasma or serum. PMID:21852021

  4. Endocytosis of Seven-Transmembrane RGS Protein Activates G- protein Coupled Signaling in Arabidopsis

    PubMed Central

    Urano, Daisuke; Phan, Nguyen; Jones, Janice C.; Yang, Jing; Huang, Jirong; Grigston, Jeffrey; Taylor, J. Philip; Jones, Alan M.

    2012-01-01

    Signal transduction typically begins by ligand-dependent activation of a concomitant partner which is otherwise in its resting state. However, in cases where signal activation is constitutive by default, the mechanism of regulation is unknown. The Arabidopsis thaliana heterotrimeric Gα protein self-activates without accessory proteins, and is kept in its resting state by the negative regulator, AtRGS1 (Regulator of G protein Signaling 1), which is the prototype of a seven transmembrane receptor fused with an RGS domain. Endocytosis of AtRGS1 by ligand-dependent endocytosis physically uncouples the GTPase accelerating activity of AtRGS1 from the Gα protein, permitting sustained activation. Phosphorylation of AtRGS1 by AtWNK8 kinase causes AtRGS1 endocytosis, required both for G protein-mediated sugar signaling and cell proliferation. In animals, receptor endocytosis results in signal desensitization, whereas in plants, endocytosis results in signal activation. These findings reveal how different organisms rearrange a regulatory system to result in opposite outcomes using similar phosphorylation-dependent endocytosis. PMID:22940907

  5. Cyclic AMP-dependent protein kinase activity in Trypanosoma cruzi.

    PubMed Central

    Ulloa, R M; Mesri, E; Esteva, M; Torres, H N; Téllez-Iñón, M T

    1988-01-01

    A cyclic AMP-dependent protein kinase activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns, giving two peaks of kinase activity, which were eluted at 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band. Images Fig. 7. Fig. 8. PMID:2848508

  6. MAPK-Activated Protein Kinases (MKs): Novel Insights and Challenges

    PubMed Central

    Gaestel, Matthias

    2016-01-01

    Downstream of MAPKs, such as classical/atypical ERKs and p38 MAPKs, but not of JNKs, signaling is often mediated by protein kinases which are phosphorylated and activated by MAPKs and, therefore, designated MAPK-activated protein kinases (MAPKAPKs). Recently, novel insights into the specificity of the assembly of MAPK/MAPKAPK hetero-dimeric protein kinase signaling complexes have been gained. In addition, new functional aspects of MKs have been described and established functions have been challenged. This short review will summarize recent developments including the linear motif (LM) in MKs, the ERK-independent activation of RSK, the RSK-independent effects of some RSK-inhibitors and the challenged role of MK5/PRAK in tumor suppression. PMID:26779481

  7. MAPK-Activated Protein Kinases (MKs): Novel Insights and Challenges.

    PubMed

    Gaestel, Matthias

    2015-01-01

    Downstream of MAPKs, such as classical/atypical ERKs and p38 MAPKs, but not of JNKs, signaling is often mediated by protein kinases which are phosphorylated and activated by MAPKs and, therefore, designated MAPK-activated protein kinases (MAPKAPKs). Recently, novel insights into the specificity of the assembly of MAPK/MAPKAPK hetero-dimeric protein kinase signaling complexes have been gained. In addition, new functional aspects of MKs have been described and established functions have been challenged. This short review will summarize recent developments including the linear motif (LM) in MKs, the ERK-independent activation of RSK, the RSK-independent effects of some RSK-inhibitors and the challenged role of MK5/PRAK in tumor suppression. PMID:26779481

  8. Hydrodynamic collective effects of active proteins in biological membranes.

    PubMed

    Koyano, Yuki; Kitahata, Hiroyuki; Mikhailov, Alexander S

    2016-08-01

    Lipid bilayers forming biological membranes are known to behave as viscous two-dimensional fluids on submicrometer scales; usually they contain a large number of active protein inclusions. Recently, it was shown [A. S. Mikhailov and R. Kapral, Proc. Natl. Acad. Sci. USA 112, E3639 (2015)PNASA60027-842410.1073/pnas.1506825112] that such active proteins should induce nonthermal fluctuating lipid flows leading to diffusion enhancement and chemotaxislike drift for passive inclusions in biomembranes. Here, a detailed analytical and numerical investigation of such effects is performed. The attention is focused on the situations when proteins are concentrated within lipid rafts. We demonstrate that passive particles tend to become attracted by active rafts and are accumulated inside them. PMID:27627343

  9. Proteins of the ETS family with transcriptional repressor activity.

    PubMed

    Mavrothalassitis, G; Ghysdael, J

    2000-12-18

    ETS proteins form one of the largest families of signal-dependent transcriptional regulators, mediating cellular proliferation, differentiation and tumorigenesis. Most of the known ETS proteins have been shown to activate transcription. However, four ETS proteins (YAN, ERF, NET and TEL) can act as transcriptional repressors. In three cases (ERF, NET and TEL) distinct repression domains have been identified and there are indications that NET and TEL may mediate transcription via Histone Deacetylase recruitment. All four proteins appear to be regulated by MAPKs, though for YAN and ERF this regulation seems to be restricted to ERKs. YAN, ERF and TEL have been implicated in cellular proliferation although there are indications suggesting a possible involvement of YAN and TEL in differentiation as well. Other ETS-domain proteins have been shown to repress transcription in a context specific manner, and there are suggestions that the ETS DNA-binding domain may act as a transcriptional repressor. Transcriptional repression by ETS domain proteins adds an other level in the orchestrated regulation by this diverse family of transcription factors that often recognize similar if not identical binding sites on DNA and are believed to regulate critical genes in a variety of biological processes. Definitive assessment of the importance of this novel regulatory level will require the identification of ETS proteins target genes and the further analysis of transcriptional control and biological function of these proteins in defined pathways. PMID:11175368

  10. Signal peptides are allosteric activators of the protein translocase.

    PubMed

    Gouridis, Giorgos; Karamanou, Spyridoula; Gelis, Ioannis; Kalodimos, Charalampos G; Economou, Anastassios

    2009-11-19

    Extra-cytoplasmic polypeptides are usually synthesized as 'preproteins' carrying amino-terminal, cleavable signal peptides and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA. Most proteins destined for the periplasm and beyond are exported post-translationally by SecA. Preprotein targeting to SecA is thought to involve signal peptides and chaperones like SecB. Here we show that signal peptides have a new role beyond targeting: they are essential allosteric activators of the translocase. On docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, 'triggering' that drives the translocase to a lower activation energy state; second, 'trapping' that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus; and third, 'secretion' during which trapped mature domains undergo several turnovers of translocation in segments. A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases. PMID:19924216