Significance of conservative asparagine residues in the thermal hysteresis activity of carrot antifreeze protein
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Abstract:
The ~24-amino-acid leucine-rich tandem repeat motif (PXXXXXLXXLXXLXLSXNXLXGXI) of carrot antifreeze protein comprises most of the processed protein and should contribute at least partly to the ice-binding site. Structural predictions using publicly available online sources indicated that the theoretical three-dimensional model of this plant protein includes a 10-loop β-helix containing the ~24-amino-acid tandem repeat. This theoretical model indicated that conservative asparagine residues create putative ice-binding sites with surface complementarity to the 1010 prism plane of ice. We used site-specific mutagenesis to test the importance of these residues, and observed a distinct loss of thermal hysteresis activity when conservative asparagines were replaced with valine or glutamine, whereas a large increase in thermal hysteresis was observed when phenylalanine or threonine residues were replaced with asparagine, putatively resulting in the formation of an ice-binding site. These results confirmed that the ice-binding site of carrot antifreeze protein consists of conservative asparagine residues in each β-loop. We also found that its thermal hysteresis activity is directly correlated with the length of its asparagine-rich binding site, and hence with the size of its ice-binding face.Keywords:
Antifreeze protein
Antifreeze protein
Psychrophile
Antifreeze
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Antifreeze proteins(AFPs),with the thermal hysteresis activity,are wildly distributed proteins which can change the growth and inhibit the recrystallization of the ice.The characteristics and structure as well as antifreeze molecular mechanisms of AFPs are reviewed in the article.Furthermore,the antifreeze gene and the application of AFPs are discussed.
Antifreeze protein
Antifreeze
Thermal hysteresis
Recrystallization (geology)
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Antifreeze protein
Psychrophile
Antifreeze
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Isoleucine
Essential amino acid
Alanine
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Antifreeze proteins (AFPs) are found in fish, insects, plants, and a variety of other organisms where they serve to prevent the growth of ice at subzero temperatures. Type III AFPs cloned from polar fishes have been studied extensively with X-ray crystallography, liquid-state NMR, and site directed mutagenesis and are, therefore, among the best characterized AFPs. A flat surface on the protein has previously been proposed to be the ice-binding site of type III AFP. The detailed nature of the ice binding remains controversial since it is not clear whether only polar or also hydrophobic residues are involved in ice binding and there is no structural information available of a type III AFP bound to ice. Here we present a high-resolution solid-state NMR study of a type III AFP (HPLC-12 isoform) in the presence of ice. The chemical-shift differences we detected between the frozen and the nonfrozen state agree well with the proposed ice-binding site. Furthermore, we found that the (1)H T(1) of HPLC-12 in frozen solution is very long compared to typical (1)H of proteins in the solid state as for example of ubiquitin in frozen solution.
Antifreeze protein
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Norleucine
Norvaline
Alanine
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Antifreeze proteins(AFPs) are the thermal hysteresis proteins that have the ability to modify the growth and inhibit the recrystallization of the ice.Antifreeze proteins aroused great interests of many researchers due to its special structure and functions.In this article,the recent advance in antifreeze protein was reviewed,and the types,properties,measurements,gene structures of antifreeze protein,and its applications in food industry were introduced.The application trials indicated that antifreeze protein could significantly improve the qualities of frozen foods,which suggested the potential food additives of antifreeze protein in future frozen food industry.
Antifreeze protein
Antifreeze
Thermal hysteresis
Recrystallization (geology)
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The branched-chain amino acids L-leucine, L-valine and L-isoleucine can, when supplied to the medium, inhibit the growth of the duckweed Spirodela polyrhiza. The responses to L-leucine and to L-valine could be described as all-or-none responses, that to L-isoleucine as a graded response. This information permitted unambiguous measurement of the growth responses. The relation between amino acid dose (initial concentration in the medium) and growth response was evaluated by means of probit analysis. Dose response curves were determined for each branched-chain amino acid when supplied singly to the medium. They were also determined when a second amino acid was added to the medium at a fixed initial concentration. It appeared that L-glutamic acid, glycine and L-alanine, in increasing order (1) antagonized, each to a distinct degree, the growth inhibitory action of irrespective which branched-chain amino acid and (2) inhibited the uptake rate of the branched-chain amino acids. D-valine, L-ornithine and L-lysine did not antagonize, nor did they inhibit uptake. Evidence is provided that the observed antagonisms result from inhibition of the uptake of the growth inhibitory amino acids.
Isoleucine
Alanine
Aspartic acid
Amino acid synthesis
Branched-chain amino acid
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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.
Antifreeze protein
Antifreeze
Recrystallization (geology)
Supercooling
Protein Adsorption
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Antifreeze protein
Antifreeze
Ice formation
Freezing tolerance
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