Antifreeze polypeptides from the Newfoundland ocean pout,Macrozoarces americanus: presence of multiple and compositionally diverse components

Hew, Choy L.; Slaughter, Donald; Joshi, Shashikant B.; Fletcher, George P.; Ananthanarayanan, V. S.

doi:10.1007/bf00688795

Cited by 59 publications

(37 citation statements)

References 28 publications

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“…Type III AFPs are very small (;65 amino acids long) proteins that are seasonally found at high concentrations in the blood of some fishes living in polar or highlatitude temperate seas (Hew et al 1984;Fletcher et al 2001). Type III AFPs show a globular fold that contains short b-strands and exhibit a flat ice-binding patch on their surface (Fig.…”

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Thermodynamic stability of a cold‐adapted protein, type III antifreeze protein, and energetic contribution of salt bridges

et al. 2007

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A thermodynamic analysis of a cold-adapted protein, type III anti-freeze protein (AFP), was carried out. The results indicate that the folding equilibrium of type III AFP is a reversible, unimolecular, two-state process with no populated intermediates. Compared to most mesophilic proteins whose folding is twostate, the psychrophilic type III AFP has a much lower thermodynamic stability at 25°C, ;3 kcal/mol, and presents a remarkably downshifted stability-temperature curve, reaching a maximum of 5 kcal/mol around 0°C. Type III AFPs contain few and non-optimally distributed surface charges relative to their mesophilic homologs, the C-terminal domains of sialic acid synthases. We used thermodynamic double mutant cycles to evaluate the energetic role of every surface salt bridge in type III AFP. Two isolated salt bridges provided no contribution to stability, while the Asp36-Arg39 salt bridge, involved in a salt bridge network with the C-terminal carboxylate, had a substantial contribution (;1 kcal/mol). However, this contribution was more than counteracted by the destabilizing effect of the Asp36 carboxylate itself, whose removal led to a net 30% increase in stability at 25°C. This study suggests that type III AFPs may have evolved for a minimally acceptable stability at the restricted, low temperature range (around 0°C) at which AFPs must function. In addition, it indicates that salt bridge networks are used in nature also for the stability of psychrophilic proteins, and has led to a type III AFP variant of increased stability that could be used for biotechnological purposes.Keywords: psychrophilic protein; cold adaptation; antifreeze protein; thermodynamic stability; salt bridge Psychrophilic, or cold-adapted, proteins are widespread in organisms living in cold environments. These proteins constitute excellent models to study molecular adaptations to extreme conditions (Jaenicke 1990; Gerday 1997, 2003;Jaenicke and Böhm 1998;Smalas et al. 2000;Arnold et al. 2001;D'Amico et al. 2002;Marx et al. 2004), and may have many biotechnological applications (Feller and Gerday 1997;Russell 1998;Cavicchioli et al. 2002;Marx et al. 2004). Most studies on the stability of cold-adapted proteins (Ushakov 1964;Chen and Berns 1978;Cheng and DeVries 1989; Reprint requests to: Mauricio G. Mateu, Centro de Biología Molecular ''Severo Ochoa'' (UAM-CSIC), Universidad Autó noma de Madrid, Cantoblanco, 28049 Madrid, Spain; e-mail: mgarcia@cbm. uam.es; fax: +34-914974799.Abbreviations: AFP, antifreeze protein; CD, circular dichroism; GdmHCl, guanidinium hydrochloride; T m , transition temperature; DH u Tm , enthalpy of unfolding at the transition temperature; DG u H2O , free energy of unfolding extrapolated to absence of denaturant; m, variation in the free energy of unfolding with the denaturant concentration; DDG u H2O , the change in DG u H2O upon mutation; DDG intXY , the coupling energy between groups X and Y in a protein.Article published online ahead of print. Article and publication date are at

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Thermodynamic stability of a cold‐adapted protein, type III antifreeze protein, and energetic contribution of salt bridges

et al. 2007

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“…These species include the winter flounder [4-61, shorthorn sculpin [7], sea raven [8] and ocean pout [9]. Isolation and characterization of these AFP have indicated three distinct types of polypeptides based on the differences in amino acid composition and secondary structure [4-91.…”

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Structures of shorthorn sculpin antifreeze polypeptides

Hew

Joshi

Wang

et al. 1985

European Journal of Biochemistry

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The amino acid sequences of the two major antifreeze polypeptides (AFP) from the shorthorn sculpin have been determined using an automatic protein sequencer and enzymic digestion. These two polypeptides, SS-3 and SS-8, consist of 33 and 45 amino acid residues respectively. The N-terminal methionyl residue is blocked in both the polypeptides. When aligned for maximum structural similarity these two AFP are 80% homologous, and there appears a deletion of 12 amino acid residues at the N-terminal portion of SS-3. Like the winter flounder AFP, both the sculpin AFP also contain the 11-amino-acid repeat sequences. The secondary structure of the sculpin AFP is mainly a-helical as deduced from circular dichroic spectral data. The helical content of SS-8 is high (73%), while that of SS-3 is moderate (about 45%). The latter exhibits a relatively weak antifreeze activity. Removal of the blocked N-terminal residue in SS-8 did not alter the helical content significantly but did reduce the antifreeze activity. Helical contents of proteolytically generated fragments of AFP are much lower, and they are devoid of activity. The a-helix in the SS-8 component is seen to be amphiphilic in character. The relevance of this feature to the mechanism of the antifreeze action is briefly discussed.To avoid freezing, many species ofmarine fishes inhabiting the Newfoundland and Labrador coastal waters produce antifreeze polypeptides (AFP) in the winter months [I -31. These species include the winter flounder [4-61, shorthorn sculpin [7], sea raven [8] and ocean pout [9]. Isolation and characterization of these AFP have indicated three distinct types of polypeptides based on the differences in amino acid composition and secondary structure [4-91. The AFP from flounder and sculpin comprise one such class.The AFP from the winter flounder, Pseudopleuronectus americunus can be fractionated into a t least seven active components by reverse-phase high-performance liquid chromatography (HPLC) [6]. The two major components, each with a relative molecular mass of about 3300, are closely related. Both consist of eight or nine different amino acids of which alanine accounts for 60% of the residues [4-61. The amino acid sequence of these two components have been elucidated [lo -121. The 37-residue-long polypeptide chain in each component has been found to contain three repeating sequences of 11 amino acid residues, Thr-(X)2-Y-(X)7-, where X represents a non-polar amino acid (mainly alanine) and Y is a polar amino acid [lo-121. The AFP from the shorthorn sculpin, Myoxocephulus scorpius, are similar to those of the winter flounder with respect to the abundance of alanine in their amino acid compositions and their high cr-helical contents [7, 131. However, they contain 12 different amino acids, and have slightly larger molecular masses withCorrespondence to

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“…Type III AFPs are found in eelpouts, suborder Zoarcoidei from Antarctic, Arctic, and temperate waters (Hew et al, 1984;Schrag et al, 1987;Sørensen and Ramløv, 2001;Nishimaya et al, 2005). All known type III AFPs have masses of about 6e7 kDa, and several isoforms have been reported in various species (Hew et al, 1988;Wang et al, 1995;Sørensen et al, 2006).…”

Section: Afp Type IIImentioning

confidence: 99%