Structure-Function Relationships in a Type I Antifreeze Polypeptide

Several analogs of an alanine-rich, α-helical type I antifreeze polypeptide from the winter flounder were synthesized and studied to evaluate the role of threonine residues on antifreeze activity. In this series, the four Thr residues in the wild type polypeptide were substituted with from one to four Ser, allo-Thr, or Val residues. Circular dichroism studies determined that these substitutions did not significantly diminish α-helicity. Thermal hysteresis data showed that substitution of Thr by Ser resulted in moderate to complete loss of antifreeze activity, depending on the number and position of the substituted Thr residue(s). Replacement by Val, in confirmation of other recent reports, or by allo-Thr had a much less detrimental effect on activity though there were qualitative differences in activity between the mutants and the wild type AFP. Based on these results, we propose that both the methyl and hydroxyl groups of Thr, particularly of the central two Thr residues, Thr13 and Thr24, play key roles in the ice-binding properties of the antifreeze peptide. Specifically, the methyls participate in hydrophobic interactions with ice, which provide the driving force for binding and stability, whereas the hydroxyls and other polar residues control binding specificity and impart additional stability through hydrogen bonding. Several analogs of an alanine-rich, α-helical type I antifreeze polypeptide from the winter flounder were synthesized and studied to evaluate the role of threonine residues on antifreeze activity. In this series, the four Thr residues in the wild type polypeptide were substituted with from one to four Ser, allo-Thr, or Val residues. Circular dichroism studies determined that these substitutions did not significantly diminish α-helicity. Thermal hysteresis data showed that substitution of Thr by Ser resulted in moderate to complete loss of antifreeze activity, depending on the number and position of the substituted Thr residue(s). Replacement by Val, in confirmation of other recent reports, or by allo-Thr had a much less detrimental effect on activity though there were qualitative differences in activity between the mutants and the wild type AFP. Based on these results, we propose that both the methyl and hydroxyl groups of Thr, particularly of the central two Thr residues, Thr13 and Thr24, play key roles in the ice-binding properties of the antifreeze peptide. Specifically, the methyls participate in hydrophobic interactions with ice, which provide the driving force for binding and stability, whereas the hydroxyls and other polar residues control binding specificity and impart additional stability through hydrogen bonding. The type I antifreeze polypeptides (AFPs) 1The abbreviations used are: AFP, antifreeze polypeptide; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; TBTU, O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluroborate. 1The abbreviations used are: AFP, antifreeze polypeptide; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; TBTU, O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluroborate.are alanine-rich amphiphilic α-helices found in righteye flounders and sculpins (for reviews, see Refs. 1Yeh Y. Feeney R.E. Chem. Rev. 1996; 96: 601-617Crossref PubMed Scopus (457) Google Scholar, 2Ananthanarayanan V.S. Life Chem. Rep. 1989; 7: 1-32Google Scholar, 3Davies P.L. Hew C.L. FASEB J. 1990; 4: 2460-2468Crossref PubMed Scopus (370) Google Scholar). Type I AFPs present in flounder serum have a on an The studied of these the winter flounder V.S. Hew C.L. J. Chem. 1989; PubMed Google to see which four threonine residues. The studies of J. PubMed Scopus (457) Google the that a type I AFP, to the of the of the of polar groups on one of the hydrogen of polar of the to the have J. PubMed Scopus (457) Google Scholar, Hew C.L. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, Chem. Google Scholar, J. Chem. Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). In the binding and J. PubMed Scopus Google that both threonine and and on the and of the Several studies have this Chem. Google Scholar, J. Chem. Scopus Google Scholar, J. PubMed Scopus Google Scholar). Based on of and PubMed Scopus Google an binding the residues in the ice-binding and an ice-binding that binding to less by and J. PubMed Scopus Google Scholar). to this a recent that threonine and to and that a ice-binding in not P.L. 1996; PubMed Scopus Google role of threonine for antifreeze activity from the in the ice-binding of type I AFPs and from a hydrogen two recent studies P.L. PubMed Scopus Google Scholar, PubMed Scopus Google have that the threonine residues by not diminish antifreeze activity on the role of hydrogen in antifreeze activity. In PubMed Scopus Google on the that a in which of the Thr residues have by Val that hydrophobic interactions the antifreeze activity. that threonine a role in type I antifreeze activity J. PubMed Scopus (457) Google Scholar, Hew C.L. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, Chem. Google Scholar, J. Chem. Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google of the properties of the and the hydrophobic of the methyl in to the properties of the the role of the we studied analogs of the winter flounder AFP, in which the four Thr residues were by Ser, allo-Thr, or these we that both the methyl and hydroxyl groups of particularly of the central two have roles in the antifreeze activity of type I AFPs that an of polar one of the The winter flounder in of Thr and to J. PubMed Scopus (457) Google Scholar, Hew C.L. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, Scopus Google Scholar, Google that AFPs by hydrogen of the polar to on the The studies of J. PubMed Scopus (457) Google which showed that to with a on the and that the the between Thr residues, to the that hydrogen the force in binding of the winter flounder to that other of In that to a binding that hydrogen and J. PubMed Scopus Google that the of the two residues in had a to the of the with the showed that of these residues by to a loss of antifreeze activity J. Chem. PubMed Google Scholar). Based on Chem. Google that the of to the and of the for in a and J. PubMed Scopus Google that the of the or hydrophobic not with the two residues, with the Thr methyl groups and two residues, and that to the by a of hydrogen and of of a in of a that methyl groups have Scopus Google Scholar, J. Chem. Scopus Google and PubMed Google for with methyl groups to liquid not to present in ice, the found in the of ice, in which by hydrogen provide a The of and the between the of the and the of an or that the of the polar the of the The of and J. PubMed Scopus Google showed that of the methyl groups of of four two on the and two on the of the that hydrophobic interactions between the methyl groups of Thr were from studies with mutants of by P.L. PubMed Scopus Google showed that of Thr13 and by Ser a loss in activity in the present that by Val had effect on activity PubMed Scopus Google showed that four Thr residues by Val a loss of activity and that hydroxyl groups not play a role in the of with that of of the methyl groups of Thr and not and residues that with the the present we of the of P.L. PubMed Scopus Google and of PubMed Scopus Google and provide on the role of Thr residues in of Thr residues by Ser that Ser not a for substitutions Thr24, and showed moderate loss of activity. not Hew C.L. J. Chem. 1989; PubMed Google had that of the four residues from effect on antifreeze Ser studies showed that of the central two Thr residues a loss of activity, whereas of the two resulted in a these results, we that the central two of for antifreeze activity. not of the two complete binding the P.L. PubMed Scopus Google Scholar). Replacement of or four of the Thr residues by Ser complete loss of activity that of the methyl of threonine detrimental to antifreeze PubMed Scopus Google that of four Thr residues by Val a that In to the Thr residues, these two to that substitution by Val a were to with the that we activity. of the of of the we did PubMed Scopus Google that there to the that the both the and found not did P.L. PubMed Scopus Google for that did not the in the hysteresis high Val a for Thr in not and that there differences in the of and with mutants were to with of the high of we two Replacement of Thr13 by allo-Thr had effect on activity that of substitution of four Thr residues a that had the activity and properties that to high and have antifreeze that the hydroxyl though present in not to participate in the of for to for and on the determined by J. PubMed Scopus (457) Google Scholar). the by J. Chem. Scopus Google Scholar, PubMed Scopus Google showed that the of synthesized with the between the and the or interactions threonine for by P.L. PubMed Scopus Google and PubMed Scopus Google and the of the two residues in in a of antifreeze activity J. Chem. PubMed Google Scholar). number of other particularly of Thr and a loss of activity not of loss of J. Chem. PubMed Google Scholar, J. Chem. PubMed Google from the and P.L. PubMed Scopus Google Scholar, PubMed Scopus Google that Ser not a for Thr in the that Thr analogs and which the methyl the of J. PubMed Scopus Google Scholar, P.L. PubMed Scopus Google Scholar, PubMed Scopus Google that the methyl in of hydrophobic or with the the of and from that of by antifreeze activity and the to of the or of the hydroxyl which hydrogen to J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). in that there two on the for binding the of hydrophobic for binding the methyl and a hydrogen for the hydroxyl Thr of both Thr substituted by Val or allo-Thr, one the of allo-Thr, Val, the hydrophobic which activity, to the ice-binding of we the of and J. PubMed Scopus Google by and J. PubMed Scopus Google additional hydrogen the for interactions the methyl particularly of Thr, and residues, with the of the of residues two on the of of the and that the force driving from the from hydrophobic interactions in and and J. PubMed Scopus Google Scholar, P.L. 1996; 4: PubMed Scopus Google that the hydrophobic effect in by of hydrophobic groups with on the The hydrophobic of residues which to particularly for activity, the for with the role of hydrogen that to specificity and stability to the The of a or with the or or less The between the to the between the hydrophobic on hydrophobic interactions were in binding of to this one the binding to the hydrophobic in the In this not a the had In from a the In the with the the the there the other the to hydrogen with the ice, in with a of this by and PubMed Scopus Google for in which two in a by interactions of hydrophobic residues, with the of a of residues in the to the hydrogen the to an one that and both and of the hydrophobic interactions provide stability, the hydrogen between the residues in of the that for the in the PubMed Scopus Google a that hydrogen not provide a for binding of AFPs to there not much in between a hydrogen to and one to there driving force for of an to hydrophobic interactions in and the on the to hydrogen with polar groups by In the the stability of the the of interactions and hydrogen additional that hydrophobic or interactions of threonine methyl groups for antifreeze activity in a type I antifreeze from winter flounder the hydroxyl play an Specifically, we propose that hydrophobic interactions between hydrophobic of methyl groups in the and other with the of the provide the driving force for binding and the hydrogen between the and the hydroxyl and groups of Thr and or less J. PubMed Scopus Google Scholar, J. PubMed Scopus Google additional stability and the mutants of the hydrophobic methyl groups for and mutants not hydroxyl groups for hydrogen in on the and that binding to this the that to to the and by of of binding groups and which on the other of the the of binding The type I antifreeze polypeptides (AFPs) 1The abbreviations used are: AFP, antifreeze polypeptide; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; TBTU, O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluroborate. 1The abbreviations used are: AFP, antifreeze polypeptide; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; TBTU, O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluroborate.are alanine-rich amphiphilic α-helices found in righteye flounders and sculpins (for reviews, see Refs. 1Yeh Y. Feeney R.E. Chem. Rev. 1996; 96: 601-617Crossref PubMed Scopus (457) Google Scholar, 2Ananthanarayanan V.S. Life Chem. Rep. 1989; 7: 1-32Google Scholar, 3Davies P.L. Hew C.L. FASEB J. 1990; 4: 2460-2468Crossref PubMed Scopus (370) Google Scholar). Type I AFPs present in flounder serum have a on an The studied of these the winter flounder V.S. Hew C.L. J. Chem. 1989; PubMed Google to see which four threonine residues. The studies of J. PubMed Scopus (457) Google the that a type I AFP, to the of the of the of polar groups on one of the hydrogen of polar of the to the have J. PubMed Scopus (457) Google Scholar, Hew C.L. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, Chem. Google Scholar, J. Chem. Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). In the binding and J. PubMed Scopus Google that both threonine and and on the and of the Several studies have this Chem. Google Scholar, J. Chem. Scopus Google Scholar, J. PubMed Scopus Google Scholar). Based on of and PubMed Scopus Google an binding the residues in the ice-binding and an ice-binding that binding to less by and J. PubMed Scopus Google Scholar). to this a recent that threonine and to and that a ice-binding in not P.L. 1996; PubMed Scopus Google Scholar). The role of threonine for antifreeze activity from the in the ice-binding of type I AFPs and from a hydrogen two recent studies P.L. PubMed Scopus Google Scholar, PubMed Scopus Google have that the threonine residues by not diminish antifreeze activity on the role of hydrogen in antifreeze activity. In PubMed Scopus Google on the that a in which of the Thr residues have by Val that hydrophobic interactions the antifreeze activity. that threonine a role in type I antifreeze activity J. PubMed Scopus (457) Google Scholar, Hew C.L. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, Chem. Google Scholar, J. Chem. Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google of the properties of the and the hydrophobic of the methyl in to the properties of the the role of the we studied analogs of the winter flounder AFP, in which the four Thr residues were by Ser, allo-Thr, or these we that both the methyl and hydroxyl groups of particularly of the central two have roles in the antifreeze activity of type I AFPs that an of polar one of the The winter flounder in of Thr and to J. PubMed Scopus (457) Google Scholar, Hew C.L. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, Scopus Google Scholar, Google that AFPs by hydrogen of the polar to on the The studies of J. PubMed Scopus (457) Google which showed that to with a on the and that the the between Thr residues, to the that hydrogen the force in binding of the winter flounder to that other of In that to a binding that hydrogen and J. PubMed Scopus Google that the of the two residues in had a to the of the with the showed that of these residues by to a loss of antifreeze activity J. Chem. PubMed Google Scholar). Based on Chem. Google that the of to the and of the for in a and J. PubMed Scopus Google that the of the or hydrophobic not with the two residues, with the Thr methyl groups and two residues, and that to the by a of hydrogen and of of a in of a that methyl groups have Scopus Google Scholar, J. Chem. Scopus Google and PubMed Google for with methyl groups to liquid not to present in ice, the found in the of ice, in which by hydrogen provide a The of and the between the of the and the of an or that the of the polar the of the The of and J. PubMed Scopus Google showed that of the methyl groups of of four two on the and two on the of the that hydrophobic interactions between the methyl groups of Thr were from studies with mutants of by P.L. PubMed Scopus Google showed that of Thr13 and by Ser a loss in activity in the present that by Val had effect on activity PubMed Scopus Google showed that four Thr residues by Val a loss of activity and that hydroxyl groups not play a role in the of with that of of the methyl groups of Thr and not and residues that with the the present we of the of P.L. PubMed Scopus Google and of PubMed Scopus Google and provide on the role of Thr residues in of Thr residues by Ser that Ser not a for substitutions Thr24, and showed moderate loss of activity. not Hew C.L. J. Chem. 1989; PubMed Google had that of the four residues from effect on antifreeze Ser studies showed that of the central two Thr residues a loss of activity, whereas of the two resulted in a these results, we that the central two of for antifreeze activity. not of the two complete binding the P.L. PubMed Scopus Google Scholar). Replacement of or four of the Thr residues by Ser complete loss of activity that of the methyl of threonine detrimental to antifreeze PubMed Scopus Google that of four Thr residues by Val a that In to the Thr residues, these two to that substitution by Val a were to with the that we activity. of the of of the we did PubMed Scopus Google that there to the that the both the and found not did P.L. PubMed Scopus Google for that did not the in the hysteresis high Val a for Thr in not and that there differences in the of and with mutants were to with of the high of we two Replacement of Thr13 by allo-Thr had effect on activity that of substitution of four Thr residues a that had the activity and properties that to high and have antifreeze that the hydroxyl though present in not to participate in the of for to for and on the determined by J. PubMed Scopus (457) Google Scholar). the by J. Chem. Scopus Google Scholar, PubMed Scopus Google showed that the of synthesized with the between the and the or interactions threonine for by P.L. PubMed Scopus Google and PubMed Scopus Google and the of the two residues in in a of antifreeze activity J. Chem. PubMed Google Scholar). number of other particularly of Thr and a loss of activity not of loss of J. Chem. PubMed Google Scholar, J. Chem. PubMed Google from the and P.L. PubMed Scopus Google Scholar, PubMed Scopus Google that Ser not a for Thr in the that Thr analogs and which the methyl the of J. PubMed Scopus Google Scholar, P.L. PubMed Scopus Google Scholar, PubMed Scopus Google that the methyl in of hydrophobic or with the the of and from that of by antifreeze activity and the to of the or of the hydroxyl which hydrogen to J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). in that there two on the for binding the of hydrophobic for binding the methyl and a hydrogen for the hydroxyl Thr of both Thr substituted by Val or allo-Thr, one the of allo-Thr, Val, the hydrophobic which activity, to the ice-binding of we the of and J. PubMed Scopus Google by and J. PubMed Scopus Google additional hydrogen the for interactions the methyl particularly of Thr, and residues, with the of the of residues two on the of of the and that the force driving from the from hydrophobic interactions in and and J. PubMed Scopus Google Scholar, P.L. 1996; 4: PubMed Scopus Google that the hydrophobic effect in by of hydrophobic groups with on the The hydrophobic of residues which to particularly for activity, the for with the role of hydrogen that to specificity and stability to the The of a or with the or or less The between the to the between the hydrophobic on hydrophobic interactions were in binding of to this one the binding to the hydrophobic in the In this not a the had In from a the In the with the the the there the other the to hydrogen with the ice, in with a of this by and PubMed Scopus Google for in which two in a by interactions of hydrophobic residues, with the of a of residues in the to the hydrogen the to an one that and both and of the hydrophobic interactions provide stability, the hydrogen between the residues in of the that for the in the PubMed Scopus Google a that hydrogen not provide a for binding of AFPs to there not much in between a hydrogen to and one to there driving force for of an to hydrophobic interactions in and the on the to hydrogen with polar groups by In the the stability of the the of interactions and hydrogen The type I AFPs that an of polar one of the The winter flounder in of Thr and to J. PubMed Scopus (457) Google Scholar, Hew C.L. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, Scopus Google Scholar, Google that AFPs by hydrogen of the polar to on the The studies of J. PubMed Scopus (457) Google which showed that to with a on the and that the the between Thr residues, to the that hydrogen the force in binding of the winter flounder to that other of In that to a binding that hydrogen and J. PubMed Scopus Google that the of the two residues in had a to the of the with the showed that of these residues by to a loss of antifreeze activity J. Chem. PubMed Google Scholar). Based on Chem. Google that the of to the and of the for in a and J. PubMed Scopus Google that the of the or hydrophobic not with the two residues, with the Thr methyl groups and two residues, and that to the by a of hydrogen and of of a in of a that methyl groups have Scopus Google Scholar, J. Chem. Scopus Google and PubMed Google for with methyl groups to liquid not to present in ice, the found in the of ice, in which by hydrogen provide a The of and the between the of the and the of an or that the of the polar the of the The of and J. PubMed Scopus Google showed that of the methyl groups of of four two on the and two on the of the that hydrophobic interactions between the methyl groups of Thr were from studies with mutants of by P.L. PubMed Scopus Google showed that of Thr13 and by Ser a loss in activity in the present that by Val had effect on activity PubMed Scopus Google showed that four Thr residues by Val a loss of activity and that hydroxyl groups not play a role in the of with that of of the methyl groups of Thr and not and residues that with the In the present we of the of P.L. PubMed Scopus Google and of PubMed Scopus Google and provide on the role of Thr residues in of Thr residues by Ser that Ser not a for substitutions Thr24, and showed moderate loss of activity. not Hew C.L. J. Chem. 1989; PubMed Google had that of the four residues from effect on antifreeze Ser studies showed that of the central two Thr residues a loss of activity, whereas of the two resulted in a these results, we that the central two of for antifreeze activity. not of the two complete binding the P.L. PubMed Scopus Google Scholar). Replacement of or four of the Thr residues by Ser complete loss of activity that of the methyl of threonine detrimental to antifreeze activity. PubMed Scopus Google that of four Thr residues by Val a that In to the Thr residues, these two to that substitution by Val a were to with the that we activity. of the of of the we did PubMed Scopus Google that there to the that the both the and found not did P.L. PubMed Scopus Google for that did not the in the hysteresis high Val a for Thr in not and that there differences in the of and with mutants were to with of the high of we two Replacement of Thr13 by allo-Thr had effect on activity that of substitution of four Thr residues a that had the activity and properties that to high and have antifreeze that the hydroxyl though present in not to participate in the of for to for and on the determined by J. PubMed Scopus (457) Google Scholar). the by J. Chem. Scopus Google Scholar, PubMed Scopus Google showed that the of synthesized with the between the and the or interactions threonine for by P.L. PubMed Scopus Google and PubMed Scopus Google and the of the two residues in in a of antifreeze activity J. Chem. PubMed Google Scholar). number of other particularly of Thr and a loss of activity not of loss of J. Chem. PubMed Google Scholar, J. Chem. PubMed Google Scholar). from the and P.L. PubMed Scopus Google Scholar, PubMed Scopus Google that Ser not a for Thr in the that Thr analogs and which the methyl the of J. PubMed Scopus Google Scholar, P.L. PubMed Scopus Google Scholar, PubMed Scopus Google that the methyl in of hydrophobic or with the the of and from that of by antifreeze activity and the to of the or of the hydroxyl which hydrogen to J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). in that there two on the for binding the of hydrophobic for binding the methyl and a hydrogen for the hydroxyl Thr of both Thr substituted by Val or allo-Thr, one the of allo-Thr, Val, the hydrophobic which activity, to the ice-binding of we the of and J. PubMed Scopus Google by and J. PubMed Scopus Google additional hydrogen the for interactions the methyl particularly of Thr, and residues, with the of the of residues two on the of of the and that the force driving from the from hydrophobic interactions in and and J. PubMed Scopus Google Scholar, P.L. 1996; 4: PubMed Scopus Google that the hydrophobic effect in by of hydrophobic groups with on the The hydrophobic of residues which to particularly for activity, the for with the role of hydrogen that to specificity and stability to the The of a or with the or or less The between the to the between the hydrophobic on hydrophobic interactions were in binding of to this one the binding to the hydrophobic in the In this not a the had In from a the In the with the the the there the other the to hydrogen with the ice, in with a of this by and PubMed Scopus Google for in which two in a by interactions of hydrophobic residues, with the of a of residues in the to the hydrogen the to an one that and both and of the hydrophobic interactions provide stability, the hydrogen between the residues in of the that for the in the PubMed Scopus Google Scholar). a that hydrogen not provide a for binding of AFPs to there not much in between a hydrogen to and one to there driving force for of an to hydrophobic interactions in and the on the to hydrogen with polar groups by In the the stability of the the of interactions and hydrogen additional that hydrophobic or interactions of threonine methyl groups for antifreeze activity in a type I antifreeze from winter flounder the hydroxyl play an Specifically, we propose that hydrophobic interactions between hydrophobic of methyl groups in the and other with the of the provide the driving force for binding and the hydrogen between the and the hydroxyl and groups of Thr and or less J. PubMed Scopus Google Scholar, J. PubMed Scopus Google additional stability and the mutants of the hydrophobic methyl groups for and mutants not hydroxyl groups for hydrogen in on the and that binding to this the that to to the and by of of binding groups and which on the other of the the of binding additional that hydrophobic or interactions of threonine methyl groups for antifreeze activity in a type I antifreeze from winter flounder the hydroxyl play an Specifically, we propose that hydrophobic interactions between hydrophobic of methyl groups in the and other with the of the provide the driving force for binding and the hydrogen between the and the hydroxyl and groups of Thr and or less J. PubMed Scopus Google Scholar, J. PubMed Scopus Google additional stability and the mutants of the hydrophobic methyl groups for and mutants not hydroxyl groups for hydrogen in on the and