The Role of N and C Termini in the Antifreeze Activity of Winter Flounder (Pleuronectes americanus) Antifreeze Proteins

Abstract

Antifreeze proteins (AFPs) are found in many marine fish and have been classified into five biochemical classes: AFP types I-IV and the antifreeze glycoproteins. Type I AFPs are ␣-helical, partially amphipathic, Alarich polypeptides. The winter flounder (Pleuronectes americanus) produces two type I AFP subclasses, the liver-type AFPs (wflAFPs) and the skin-type AFPs (wfsAFPs), that are encoded by distinct gene families with different tissue-specific expression. wfsAFPs and wflAFPs share a high level of identity even though the wfsAFPs have approximately half the activity of the wflAFPs. Synthetic polypeptides based on two representative wflAFPs and wfsAFPs were generated to examine the role of the termini in antifreeze activity. Through systematic exchange of N and C termini between wflAFP-6 and wfsAFP-2, the termini were determined to be the major causative agents for the variation in activity levels between the two AFPs. Furthermore, the termini of wflAFP-6 possessed greater helix-stabilizing ability compared with their wfsAFP-2 counterparts. The observed 50% difference in activity between wflAFP-6 and wfsAFP-2 can be divided into ϳ20% for differences at each termini and ϳ10% for differences in the core. Furthermore, the N terminus was determined to be the most critical component for antifreeze activity.Fish antifreeze proteins are diverse in structure and have been grouped into five biochemical classes based on their structural characteristics: antifreeze proteins (AFPs) 1 types I-IV and antifreeze glycoproteins (AFGPs) (1-3). Although diverse in structure, all AF(G)Ps act by what is known as the adsorption inhibition mechanism (2, 4 -6), where the AF(G)Ps lower the observed freezing point in a non-colligative manner creating a hysteresis between the equilibrium melting point and the observed freezing point. The degree of thermal hysteresis is used as a measure of AF(G)P activity.Type I AFPs are Ala-rich, partially amphipathic single ␣-helical polypeptides found in several sculpins and righteye flounders (2,7,8). The winter flounder (Pleuronectes americanus) produces two subclasses of type I AFPs, the liver-type (wflAFPs) and the skin-type AFPs (wfsAFPs), which are encoded by distinct gene families (9). wflAFP-6 (formerly known as HPLC-6) is the major winter flounder plasma AFP and is produced in the liver as a prepro-precursor, and then secreted into circulation, where it is then processed into the mature polypeptide of 37 residues (10, 11). Conversely, the wfsAFPs have a wider tissue distribution and are produced as mature intracellular polypeptides (9).The wflAFPs and wfsAFPs share a high level of structural identity even though the wfsAFPs have approximately half the activity of wflAFP-6 (9). Both wflAFPs and wfsAFPs possess two 11-residue motifs of the structure TaaXAXXAAXX (where the first Thr is always conserved, uppercase A is a conserved Ala, lowercase a is almost always Ala, and X can be one of several amino acids), with wflAFP-6 having a third full motif at the N terminus (see Fig. 1). Furthermore,...

Solution Structure of a Recombinant Type I Sculpin Antifreeze Protein,

“…While activity levels have been reported to increase with helical content for two homologues of the flounder AFPs (59), there is no clear correlation between helicity and activity. For example, highly helical mutants of type I AFPs can show marked decreases in activity (58) and specific mutations in skin-type AFP have been shown to increase activity by 15%, while reducing the helical content from 80 to 60% (59). The data presented for rSS3 confirm that there is not a simple correlation of this type, and that to some extent the determinants of hysteresis are somewhat more subtle.…”

“…Several AFPs have been shown to maintain their fold at very low temperatures and an increase in R-helical content is observed with a decrease in temperature in other type I AFPs (58). While activity levels have been reported to increase with helical content for two homologues of the flounder AFPs (59), there is no clear correlation between helicity and activity. For example, highly helical mutants of type I AFPs can show marked decreases in activity (58) and specific mutations in skin-type AFP have been shown to increase activity by 15%, while reducing the helical content from 80 to 60% (59).…”

Hydrogen Exchange Study of Winter Flounder Type I Antifreeze Protein

“…10 It was reported that termini of the HPLC6 isoform possess greater helix-stabilizing ability compared to the HPLC2 isoform, leading to a 50% difference in the thermal hysteresis activity between these two AFPs. 11 These results suggested that helical stabilization of the N and C termini is the critical component for antifreeze activity of the type I AFP. 11 It was recently reported that flexibility of the C-terminal region causes a loss of thermal hysteresis activity because its dynamic nature strongly prevents binding to the ice surface.…”

“…11 These results suggested that helical stabilization of the N and C termini is the critical component for antifreeze activity of the type I AFP. 11 It was recently reported that flexibility of the C-terminal region causes a loss of thermal hysteresis activity because its dynamic nature strongly prevents binding to the ice surface. 12 Here, to further understand the correlation between the dynamic properties and function of type I AFPs, we performed NMR hydrogen exchange experiments on the winter flounder type I AFP (HPLC6 isoform) (Fig.…”

Diffusion NMR studies on fish antifreeze proteins and synthetic analogues

“…1B), identified the requirement for a hydrophobic face on the peptide to maintain thermal hysteresis. While molecular simulations of the interaction of TTTT with the fluid ice–water interface has assisted in understanding the balance between hydrophobic and hydrophilic residues in ice growth inhibition [11], the subtle role(s) of the helix‐dipole and positioning of individual residues within the chain remain poorly understood [12]. Thus, attempts to design synthetic AFPs related to TTTT have been of limited success to date [13,14].…”