Diffusion NMR studies on fish antifreeze proteins and synthetic analogues.
(9/16)
Pulsed field gradient spin echo NMR spectroscopy was used to measure diffusion coefficients of the alpha-helical type I antifreeze protein from the winter flounder, two synthetic derivatives in which the four Thr residues were replaced with Val and Ala, respectively, and the low molecular weight fraction antifreeze glycoprotein. Under the conditions studied, the natural type I antifreeze protein and low molecular weight glycoprotein gave diffusion values that were consistent with the presence of monomeric protein in solution. While significant aggregation of the Ala analogue was observed (2-10 mM), there was no evidence for aggregation in the Val analogue (1-3 mM). These results are compared with previously reported solubility and thermal hysteresis data and the implications for the design of synthetic antifreeze proteins are discussed. (+info)
Cryoprotection and cryosterilization effects of type I antifreeze protein on E. coli cells.
(10/16)
To establish the effects of type I antifreeze protein (AFP) on E. coli cells, we have focused on the survival rate of the E. coli cells using type I AFP at various concentrations under rapid cooling conditions using liquid N2 at atmospheric or low pressure. The survival rate of E. coli was enhanced by the addition of type I AFP at a concentration of 10 microg/ml, and its value shifted from 0.73% to 2.96%. When the concentration of type I AFP was 100 microg/ml, the cell survival rate markedly decreased to 0.090%. This low survival rate was further decreased (0.022%) by the application of the same freeze-thaw treatment for four times. Also, the effect of type I AFP as a bactericidal agent did not vary according to the varying initial cell densities from 10(4) to 10(8) cells / ml. Furthermore, the effects of using type I AFP at 1.0 MPa with N2 gas under conditions of low pressure and low oxygen tension using a simple device were examined. When the actions of type I AFP as a cryoprotectant were stimulated, the survival rate of the E. coil cells increased to 57.8%. In addition, the bactericidal effect of type I AFP at 100 micro g/ml of protein concentration could also be enhanced. The survival rate using 100 g/ml of type I AFP under low pressure was 0.35% of that using 10 microg/ml under the same conditions. This is the first report on the cryoprotectant and cryosterilization effects of type I AFP of E. coli cells under various conditions. (+info)
Protein digestibility evaluations of meat and fish substrates using laboratory, avian, and ileally cannulated dog assays.
(11/16)
Threonine side chain conformational population distribution of a type I antifreeze protein on interacting with ice surface studied via (1)(3)C-(1)(5)N dynamic REDOR NMR.
(14/16)
Effects of a type I antifreeze protein (AFP) on the melting of frozen AFP and AFP+solute aqueous solutions studied by NMR microimaging experiment.
(15/16)
Skin-type antifreeze protein from the shorthorn sculpin, Myoxocephalus scorpius. Expression and characterization of a Mr 9, 700 recombinant protein.
(16/16)
A cDNA clone encoding a presumptive antifreeze protein was isolated from a skin library from shorthorn sculpin, Myoxocephalus scorpius. The clone encodes a 92-residue mature polypeptide (sssAFP-2) without any signal and prosequence, which suggests an intracellular localization. It is the largest alanine-rich, alpha-helical type I antifreeze protein known. A recombinant fusion protein containing an N-terminal-linked His-tag was produced and purified from Escherichia coli. This protein is alpha-helical at 0 degreesC and exhibits significant antifreeze activity. Northern blot and reverse transcription-polymerase chain reaction analyses indicate that sssAFP-2 mRNA has limited tissue distribution and is present in peripheral tissues such as skin and dorsal fin, but is notably absent in the liver. These studies reinforce recent evidence that indicate that the external tissues of cold water marine fishes are major organs for antifreeze protein synthesis and are likely the first line of defense against the threat of freezing. (+info)