M

| Fig. 1.3.24. Antifreeze proteins (AFPs) of fish. A Various aianine-rich a-helical AFPs. / Winter flounder; 2 yellow tail flounder; 3 plaice; 4-8 different AFPs from sculpin. The N- and C-terminal caps, which stabilise the a-helical structures, are shown by structure symbols. Ice- (water-) binding motifs (i-iv) are shown by circles. B Side view of the AFP B of the winter flounder, in which the unilateral orientation of the hydrophilic groups can be clearly recognised. K22 and D26 form a salt bridge (amino acids are specified in single letter code). (After Sicheri and Yang 1995)

| Fig. 1.3.24. Antifreeze proteins (AFPs) of fish. A Various aianine-rich a-helical AFPs. / Winter flounder; 2 yellow tail flounder; 3 plaice; 4-8 different AFPs from sculpin. The N- and C-terminal caps, which stabilise the a-helical structures, are shown by structure symbols. Ice- (water-) binding motifs (i-iv) are shown by circles. B Side view of the AFP B of the winter flounder, in which the unilateral orientation of the hydrophilic groups can be clearly recognised. K22 and D26 form a salt bridge (amino acids are specified in single letter code). (After Sicheri and Yang 1995)

crystal. The matrix for this attachment is set by the ice crystal, while the manner in which the attachment takes place is determined by the protein. Since a-helical polypeptides are dipoles, the AFP preferentially attaches to the prism surfaces of the ice crystal in an antiparallel manner. Due to the blocking of its normal growth surfaces, the ice crystal now grows less rapidly and extends mainly in the direction of the c-axis. This leads to the formation of small, needle-like crystals (Antikainen et al. 1996; Fig. 1.3.25).

It is quite evident that the effect of AFPs in delaying freezing depends on the concentration of the proteins. Since they must be bound to the surface of the ice to achieve their effect, they can be said to be used up. This also explains why AFPs do not provide a general protection against freezing. AFPs reduce the formation of ice in the organism, but they cannot suppress it completely. The partially frozen state of the tis sue liquid arrived at in this manner is certainly more stable during supercooling than the completely liquid state, which tends to freeze abruptly. It is quite plausible that many small ice crystals which are inhibited in their growth and cloaked in proteins are less damaging to the organism than is a large crystal. The protein cloak of the ice crystal is possibly the deciding factor in frost protection by AFPs. A further effect of AFPs could be to prevent recrystallisation of ice concomitant with the formation of larger ice crystals during the thawing process (Carpenter and Hansen 1992). This is of particular importance with regard to ice crystals in the body fluids of cold-water fish and insects, where the formation of larger ice crystals would lead to circulatory damage. The combination of INPs and AFPs could constitute an effective method of regulating the formation of ice crystals in tissues (Griffith et al. 1993; Worland and Block

Fig, 1.3.25. The effect of antifreeze proteins (AFPs) on the formation of ice crystals. A-C Schematic drawings of crystals in the presence of increasing concentrations of winter flounder AFP. In the most dilute solution, there is not yet any interaction between the individual protein molecules, which orient themselves mainly according to the ice crystal vectors. In the more concentrated solutions, the protein molecules interact and bind in an antiparallel manner to the prism surfaces. E, F Micrographs of ice crystals which have formed in the presence of protein extracts from the apoplast of unhardened (E) and frost-hardened rye leaves (F). Length of the bar: 25 |.im. For comparison an ice crystal in pure water is shown in D. G Molecular model of the binding of the winter flounder protein (grey) to an ice crystal (circles denote water molecules). The four ice-binding motifs correspond to the distance between the water molecules in the ice crystal. (A-C after Yang et al. 1988; D-F after Griffith et al. 1992; G after Sicheri and Yang 1995)

Crystal vector 112

Basal plain c-axis

Antifreeze proteins AFP concentration A: 0.242 mM B: 0.363 mM

Fig, 1.3.25. The effect of antifreeze proteins (AFPs) on the formation of ice crystals. A-C Schematic drawings of crystals in the presence of increasing concentrations of winter flounder AFP. In the most dilute solution, there is not yet any interaction between the individual protein molecules, which orient themselves mainly according to the ice crystal vectors. In the more concentrated solutions, the protein molecules interact and bind in an antiparallel manner to the prism surfaces. E, F Micrographs of ice crystals which have formed in the presence of protein extracts from the apoplast of unhardened (E) and frost-hardened rye leaves (F). Length of the bar: 25 |.im. For comparison an ice crystal in pure water is shown in D. G Molecular model of the binding of the winter flounder protein (grey) to an ice crystal (circles denote water molecules). The four ice-binding motifs correspond to the distance between the water molecules in the ice crystal. (A-C after Yang et al. 1988; D-F after Griffith et al. 1992; G after Sicheri and Yang 1995)

Crystal vector 112

Basal plain c-axis

Antifreeze proteins AFP concentration A: 0.242 mM B: 0.363 mM

1999). Attempts to transform potato plants with the AFP of flounder genetically engineered to optimise codon usage yielded plants with significantly increased cold tolerance (Wallis et al. 1997).

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