Type III AFPs

We used type-III AFP (RD3-NI retrieved from Antarctic eel pout) and trehalose (research-grade Hayashibara Biochemical Laboratories, Inc.) as the inhibitors. The characteristics of this AFP are described in detail in Miura et al. We added a small amount of AFP (from 0.01 to 1 mg mf water) to distilled and deionized water (approximately 18 MQ in resistivity) for the experiments. Trehalose was added at concentrations ranging from 1 to 50 wt%. CO2 was delivered from Hokkaido Air Water, Inc., and its purity was approximately 99%. 

Effects of type-III AFP on the formation process of the COi-hydrate film... 

The inhibition effects of type-III AFP and trehalose, two cryoprotecting materials produced in animals, on type-I CO2 clathrate-hydrates were examined. For comparison with the results of a previous study in which the lateral growth rates of COi-hydrate film were dependent on temperature, pressure and NaCl concentration, the solution droplet was observed in a high pressure vessel filled with CO2. Type-III AFP was found to increase the induction period and to reduce the lateral growth rate of C02-hydrate films. It worked well at low concentrations, indicating that AFP works as a kinetic inhibitor. It was also indicated that AFP would weaken the memory effect of C02-hydrate formation. Trehalose had similar inhibition effects on both the induction period and the lateral growth rate, but it had little apparent concentration-dependence on them. Since trehalose also causes the equilibrium conditions of the CO2 hydrate to shift to lower temperatures, it works not only as a thermodynamic inhibitor but also as a kinetic inhibitor, especially as an anti-agglomerant. 

Case 2 An experimental density map for recombinant type III antifreeze protein from eel pout (AFP), which contains 66 residues and in its crystalline form is a member of the P212121 space group [20]. 

The complete TCR vp, Dp and Jp-chain is synthesized using 9 fragments (Fig. 2), the AFP-Type III protein is synthesized using 8 fragments (Fig. 3). 

Fig. 4 5 The analytical RP-HPLC chromatogram and the CE chromatogram of the purified AFP-Type III protein are shown. HPLC conditions VYDAC RP-18 column gradient 20/20/60% (H20/ACN/MeOH)+0.1%TFA into 30 min. to 0/10/90% into 35 min. 0/100/0%. The CE conditions are as discribed into the material section.

The small chemical shift dispersion of the NH region in the NMR spectrum of the protein indicates a mostly unfolded structure in the presence of a small fraction of the folded protein (Fig. 7a c), as could be appreciated from the ID-spectrum of the native AFP-Type III sequence (Fig. 7b). Whether the N-terminal Gin is responsible for unfolding is currently under investigation. 

Fig, 7a-c The ID-NMR spectrum of the purified AFP-Type III protein (7a c) and the ID-NMR spectrum of the native AFP-Type III HPLC-6 (7b) isolated from Macrozoarces americanus [7] are given. 

Fragment condensation on the solid support has been shown to be a feasible approach towards chemical synthesis of proteins. A progress report on the example of the TCR p-chain and AFP type III has been given. In the latter case a pure AFP type III peptide was obtained that however did not fold properly. 

This work is supported by DFG under grant Gr 1211/4-1 and by the Fonds der Chemischen Industrie. We thank Dr. Zechel and Dr. Haupt, BASF, Ludwigshafen, Dr. Savelsberg, Merck, Darmstadt and Dr. Fehlhaber, Hoechst, Frankfurt for the recording of mass spectra. Prof Dr. Davies, Kingston, Canada for the measurement of the freezing activity and Dr. Sbnnichsen, Edmonton, Canada for the ID NMR spectrum of the native AFP-Type III. 

Whereas a wealth of structural information derived from x-ray and NMR spectroscopy[9, 11, 12], is available for Types 1 and III AFPs, very little is known about the three dimensional structure of the largest, Type II AFP and Type IV. A three dimensional structure of Type II antifreeze protein was recently proposed using comparative modeling[13]. This structure was derived using a sequence similarity to the carbohydrate recognition domain (CDR) of... 

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