1. The antifreeze protein gene (Cn-AFP) from the Antarctic marine diatom Chaetoceros neogracile was cloned and characterized. The full-length Cn-AFP cDNA has an 849-bp open reading frame encoding a 29.2-kDa protein with a 30-amino-acid signal peptide at the N-terminus.
2. Both the coding regions with and without the signal sequence were cloned and expressed in Escherichia coli. Recombinant Cn-AFPs showed antifreeze activities. The mature recombinant Cn-AFP had 16-fold higher thermal hysteresis activity than the pre-mature form at the same concentration.
3. The presence of the mature recombinant Cn-AFP changed the ice crystal shape to an elongated hexagonal form.
4. Northern analysis indicated that Cn-AFP transcripts accumulated significantly under freezing stress. These results suggest that Cn-AFP plays an important role in the low-temperature adaptation of C. neogracile.
AFP050000
General Information
| Protein Name | Antifreeze protein |
| UniProt ID | D2DLE1 |
| Nucleotide Sequence ID in NCBI | FJ505233 |
| Protein Sequence ID in NCBI | ACU09498.1 |
| Species | Chaetoceros neogracile |
| Sequence Length | 252 |
| Sequence | |
| Structure | AF2 predicted Structure: AFP050000 |
| Solvent Accessible Surface Area | Total SASA | 9910.36 Ų | Polar SASA | 6801.98 Ų | Apolar SASA | 16712.34 Ų |
AFP050000000
| Mutation | Wild Type |
| Sequence |
Ice crystal morphology
| PMID | 20024694 |
| DOI | 10.1007/s10126-009-9250-x |
| Protein Name | Recombinant Mature Cn-AFP |
| Tag | Hexahistidine |
| Ice crystal morphology | Fig.7 Morphological changes of ice crystals in presence or absence of recombinant Cn-AFPs. a BSA(0.5 mg/ml), b recombinant mature Cn-AFP (0.5 mg/ml), c recombinant pre-mature Cn-AFP (0.5 mg/ml), scale bar 100 μm. |
Thermal Hysteresis
| PMID | 20024694 |
| DOI | 10.1007/s10126-009-9250-x |
| Protein Name | Recombinant Mature Cn-AFP |
| Tag | Hexahistidine |
| Thermal Hysteresis | Tab.1 Thermal hysteresis activity of recombinant Cn-AFP, with and without signal peptide. |
Brief description
|
PMID: 20024694
DOI: 10.1007/s10126-009-9250-x |
AFP050000001
| Mutation | 0_1insMSLITINHTLVVTALLFAAVALLGVPMAEG |
| Sequence |
Ice crystal morphology
| PMID | 20024694 |
| DOI | 10.1007/s10126-009-9250-x |
| Protein Name | Recombinant Pre-mature Cn-AFP |
| Tag | Hexahistidine |
| Ice crystal morphology | Fig.7 Morphological changes of ice crystals in presence or absence of recombinant Cn-AFPs. a BSA(0.5 mg/ml), b recombinant mature Cn-AFP (0.5 mg/ml), c recombinant pre-mature Cn-AFP (0.5 mg/ml), scale bar 100 μm. |
Thermal Hysteresis
| PMID | 20024694 |
| DOI | 10.1007/s10126-009-9250-x |
| Protein Name | Recombinant Pre-mature Cn-AFP |
| Tag | Hexahistidine |
| Thermal Hysteresis | Tab.1 Thermal hysteresis activity of recombinant Cn-AFP, with and without signal peptide. |
Brief description
|
PMID: 20024694
DOI: 10.1007/s10126-009-9250-x |
1. The antifreeze protein gene (Cn-AFP) from the Antarctic marine diatom Chaetoceros neogracile was cloned and characterized. The full-length Cn-AFP cDNA has an 849-bp open reading frame encoding a 29.2-kDa protein with a 30-amino-acid signal peptide at the N-terminus.
2. Both the coding regions with and without the signal sequence were cloned and expressed in Escherichia coli. Recombinant Cn-AFPs showed antifreeze activities. The mature recombinant Cn-AFP had 16-fold higher thermal hysteresis activity than the pre-mature form at the same concentration. 3. The presence of the mature recombinant Cn-AFP changed the ice crystal shape to an elongated hexagonal form. 4. Northern analysis indicated that Cn-AFP transcripts accumulated significantly under freezing stress. These results suggest that Cn-AFP plays an important role in the low-temperature adaptation of C. neogracile. |
AFP050000002
| Mutation | 220_252del,1_19del,N/A |
| Sequence |
Ice crystal morphology
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP |
| Tag | Hexahistidine |
| Ice crystal morphology | Fig.7.B Morphological changes in ice crystals of wild-type Cn-AFP and mutant proteins at various protein concentrations. All ice crystals formed in the presence of the wild-type protein; G124Y and G124T proteins showed a burst form at protein concentrations 1.25 mg/ml. Ice crystals formed by other mutant proteins showed hexagonal or asterisk crystal shapes, indicating weak ice-binding activity regardless of protein concentration. All experiments were performed in triplicate. Scale bars 100 μm. |
Thermal Hysteresis
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP |
| Tag | Hexahistidine |
| Thermal Hysteresis | Fig.7.A TH activity of wild-type Cn-AFP and mutant proteins at various protein concentrations.(A) The maximum TH activity of Cn-AFP was 1.28℃ at 10 mg/ml protein. TH activities of T19Y, T41Y, D175Y, T193Y, and T211Y mutants were 10% of the activity of the wild-type protein. G124Y and G124T showed 1.9℃ TH values. |
Ice Binding Sites
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP |
| Tag | Hexahistidine |
| IBS | The b face of Cn-AFP is the ice-binding face. |
Brief description
|
PMID: 25114178
DOI: 10.1096/fj.14-256388 |
1. The study investigates the structure and function of the antifreeze protein (Cn-AFP) from the Antarctic marine diatom Chaetoceros neogracile, as well as its expression levels and ice-binding site characteristics.
2. The Cn-AFP promoter contains elements responsive to light and temperature. Both Cn-AFP transcript and protein expression are rapidly stimulated by freezing, temperature, and high light stress. 3. Recombinant Cn-AFP exhibits clear antifreeze activity. Protein-folding simulation and site-directed mutagenesis confirm the identification of the ice-binding sites in Cn-AFP. |
AFP050000003
| Mutation | 220_252del,1_19del,T19Y |
| Sequence |
Ice crystal morphology
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (T19Y) |
| Tag | Hexahistidine |
| Ice crystal morphology | Fig.7.B Morphological changes in ice crystals of wild-type Cn-AFP and mutant proteins at various protein concentrations. All ice crystals formed in the presence of the wild-type protein; G124Y and G124T proteins showed a burst form at protein concentrations 1.25 mg/ml. Ice crystals formed by other mutant proteins showed hexagonal or asterisk crystal shapes, indicating weak ice-binding activity regardless of protein concentration. All experiments were performed in triplicate. Scale bars 100 μm. |
Thermal Hysteresis
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (T19Y) |
| Tag | Hexahistidine |
| Thermal Hysteresis | Fig.7.A TH activity of wild-type Cn-AFP and mutant proteins at various protein concentrations.(A) The maximum TH activity of Cn-AFP was 1.28℃ at 10 mg/ml protein. TH activities of T19Y, T41Y, D175Y, T193Y, and T211Y mutants were 10% of the activity of the wild-type protein. G124Y and G124T showed 1.9℃ TH values. |
Brief description
|
PMID: 25114178
DOI: 10.1096/fj.14-256388 |
1. T19Y, T41Y, D175Y, T193Y, T211Y:TH activity was <10% of that of the wild-type protein.
2. Ice crystals showed hexagonal or asterisk shapes, indicating weak ice-binding activity, regardless of concentration. 3. The reduction in activity was attributed to steric hindrance from tyrosine residues on the β-face. |
AFP050000004
| Mutation | 220_252del,1_19del,T41Y |
| Sequence |
Ice crystal morphology
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (T41Y) |
| Tag | Hexahistidine |
| Ice crystal morphology | Fig.7.B Morphological changes in ice crystals of wild-type Cn-AFP and mutant proteins at various protein concentrations. All ice crystals formed in the presence of the wild-type protein; G124Y and G124T proteins showed a burst form at protein concentrations 1.25 mg/ml. Ice crystals formed by other mutant proteins showed hexagonal or asterisk crystal shapes, indicating weak ice-binding activity regardless of protein concentration. All experiments were performed in triplicate. Scale bars 100 μm. |
Thermal Hysteresis
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (T41Y) |
| Tag | Hexahistidine |
| Thermal Hysteresis | Fig.7.A TH activity of wild-type Cn-AFP and mutant proteins at various protein concentrations.(A) The maximum TH activity of Cn-AFP was 1.28℃ at 10 mg/ml protein. TH activities of T19Y, T41Y, D175Y, T193Y, and T211Y mutants were 10% of the activity of the wild-type protein. G124Y and G124T showed 1.9℃ TH values. |
Brief description
|
PMID: 25114178
DOI: 10.1096/fj.14-256388 |
1. T19Y, T41Y, D175Y, T193Y, T211Y:TH activity was <10% of that of the wild-type protein.
2. Ice crystals showed hexagonal or asterisk shapes, indicating weak ice-binding activity, regardless of concentration. 3. The reduction in activity was attributed to steric hindrance from tyrosine residues on the β-face. |
AFP050000005
| Mutation | 220_252del,1_19del,G124Y |
| Sequence |
Ice crystal morphology
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (G124Y) |
| Tag | Hexahistidine |
| Ice crystal morphology | Fig.7.B Morphological changes in ice crystals of wild-type Cn-AFP and mutant proteins at various protein concentrations. All ice crystals formed in the presence of the wild-type protein; G124Y and G124T proteins showed a burst form at protein concentrations 1.25 mg/ml. Ice crystals formed by other mutant proteins showed hexagonal or asterisk crystal shapes, indicating weak ice-binding activity regardless of protein concentration. All experiments were performed in triplicate. Scale bars 100 μm. |
Thermal Hysteresis
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (G124Y) |
| Tag | Hexahistidine |
| Thermal Hysteresis | Fig.7.A TH activity of wild-type Cn-AFP and mutant proteins at various protein concentrations.(A) The maximum TH activity of Cn-AFP was 1.28℃ at 10 mg/ml protein. TH activities of T19Y, T41Y, D175Y, T193Y, and T211Y mutants were 10% of the activity of the wild-type protein. G124Y and G124T showed 1.9℃ TH values. |
Brief description
|
PMID: 25114178
DOI: 10.1096/fj.14-256388 |
1. G124Y and G124T:Both exhibited enhanced TH activity (~1.9°C), higher than the wild-type.
2. At concentrations >1.25 mg/ml, ice crystals formed a burst form, indicating hyperactive antifreeze activity. |
AFP050000006
| Mutation | 220_252del,1_19del,T148Y |
| Sequence |
Ice crystal morphology
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (T148Y) |
| Tag | Hexahistidine |
| Ice crystal morphology | Fig.7.B Morphological changes in ice crystals of wild-type Cn-AFP and mutant proteins at various protein concentrations. All ice crystals formed in the presence of the wild-type protein; G124Y and G124T proteins showed a burst form at protein concentrations 1.25 mg/ml. Ice crystals formed by other mutant proteins showed hexagonal or asterisk crystal shapes, indicating weak ice-binding activity regardless of protein concentration. All experiments were performed in triplicate. Scale bars 100 μm. |
Thermal Hysteresis
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (T148Y) |
| Tag | Hexahistidine |
| Thermal Hysteresis | Fig.7.A TH activity of wild-type Cn-AFP and mutant proteins at various protein concentrations.(A) The maximum TH activity of Cn-AFP was 1.28℃ at 10 mg/ml protein. TH activities of T19Y, T41Y, D175Y, T193Y, and T211Y mutants were 10% of the activity of the wild-type protein. G124Y and G124T showed 1.9℃ TH values. |
Brief description
|
PMID: 25114178
DOI: 10.1096/fj.14-256388 |
1. T148Y:TH activity was slightly lower than that of the wild-type but higher than other mutants, suggesting that T148 is less critical for antifreeze activity.
|
AFP050000007
| Mutation | 220_252del,1_19del,D175Y |
| Sequence |
Ice crystal morphology
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (D175Y) |
| Tag | Hexahistidine |
| Ice crystal morphology | Fig.7.B Morphological changes in ice crystals of wild-type Cn-AFP and mutant proteins at various protein concentrations. All ice crystals formed in the presence of the wild-type protein; G124Y and G124T proteins showed a burst form at protein concentrations 1.25 mg/ml. Ice crystals formed by other mutant proteins showed hexagonal or asterisk crystal shapes, indicating weak ice-binding activity regardless of protein concentration. All experiments were performed in triplicate. Scale bars 100 μm. |
Thermal Hysteresis
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (D175Y) |
| Tag | Hexahistidine |
| Thermal Hysteresis | Fig.7.A TH activity of wild-type Cn-AFP and mutant proteins at various protein concentrations.(A) The maximum TH activity of Cn-AFP was 1.28℃ at 10 mg/ml protein. TH activities of T19Y, T41Y, D175Y, T193Y, and T211Y mutants were 10% of the activity of the wild-type protein. G124Y and G124T showed 1.9℃ TH values. |
Brief description
|
PMID: 25114178
DOI: 10.1096/fj.14-256388 |
1. T19Y, T41Y, D175Y, T193Y, T211Y:TH activity was <10% of that of the wild-type protein.
2. Ice crystals showed hexagonal or asterisk shapes, indicating weak ice-binding activity, regardless of concentration. 3. The reduction in activity was attributed to steric hindrance from tyrosine residues on the β-face. |
AFP050000008
| Mutation | 220_252del,1_19del,T193Y |
| Sequence |
Ice crystal morphology
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (T193Y) |
| Tag | Hexahistidine |
| Ice crystal morphology | Fig.7.B Morphological changes in ice crystals of wild-type Cn-AFP and mutant proteins at various protein concentrations. All ice crystals formed in the presence of the wild-type protein; G124Y and G124T proteins showed a burst form at protein concentrations 1.25 mg/ml. Ice crystals formed by other mutant proteins showed hexagonal or asterisk crystal shapes, indicating weak ice-binding activity regardless of protein concentration. All experiments were performed in triplicate. Scale bars 100 μm. |
Thermal Hysteresis
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (T193Y) |
| Tag | Hexahistidine |
| Thermal Hysteresis | Fig.7.A TH activity of wild-type Cn-AFP and mutant proteins at various protein concentrations.(A) The maximum TH activity of Cn-AFP was 1.28℃ at 10 mg/ml protein. TH activities of T19Y, T41Y, D175Y, T193Y, and T211Y mutants were 10% of the activity of the wild-type protein. G124Y and G124T showed 1.9℃ TH values. |
Brief description
|
PMID: 25114178
DOI: 10.1096/fj.14-256388 |
1. T19Y, T41Y, D175Y, T193Y, T211Y:TH activity was <10% of that of the wild-type protein.
2. Ice crystals showed hexagonal or asterisk shapes, indicating weak ice-binding activity, regardless of concentration. 3. The reduction in activity was attributed to steric hindrance from tyrosine residues on the β-face. |
AFP050000009
| Mutation | 220_252del,1_19del,T211Y |
| Sequence |
Ice crystal morphology
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (T211Y) |
| Tag | Hexahistidine |
| Ice crystal morphology | Fig.7.B Morphological changes in ice crystals of wild-type Cn-AFP and mutant proteins at various protein concentrations. All ice crystals formed in the presence of the wild-type protein; G124Y and G124T proteins showed a burst form at protein concentrations 1.25 mg/ml. Ice crystals formed by other mutant proteins showed hexagonal or asterisk crystal shapes, indicating weak ice-binding activity regardless of protein concentration. All experiments were performed in triplicate. Scale bars 100 μm. |
Thermal Hysteresis
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (T211Y) |
| Tag | Hexahistidine |
| Thermal Hysteresis | Fig.7.A TH activity of wild-type Cn-AFP and mutant proteins at various protein concentrations.(A) The maximum TH activity of Cn-AFP was 1.28℃ at 10 mg/ml protein. TH activities of T19Y, T41Y, D175Y, T193Y, and T211Y mutants were 10% of the activity of the wild-type protein. G124Y and G124T showed 1.9℃ TH values. |
Brief description
|
PMID: 25114178
DOI: 10.1096/fj.14-256388 |
1. T19Y, T41Y, D175Y, T193Y, T211Y:TH activity was <10% of that of the wild-type protein.
2. Ice crystals showed hexagonal or asterisk shapes, indicating weak ice-binding activity, regardless of concentration. 3. The reduction in activity was attributed to steric hindrance from tyrosine residues on the β-face. |
AFP050000010
| Mutation | 220_252del,1_19del,G124T |
| Sequence |
Ice crystal morphology
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (G124T) |
| Tag | Hexahistidine |
| Ice crystal morphology | Fig.7.B Morphological changes in ice crystals of wild-type Cn-AFP and mutant proteins at various protein concentrations. All ice crystals formed in the presence of the wild-type protein; G124Y and G124T proteins showed a burst form at protein concentrations 1.25 mg/ml. Ice crystals formed by other mutant proteins showed hexagonal or asterisk crystal shapes, indicating weak ice-binding activity regardless of protein concentration. All experiments were performed in triplicate. Scale bars 100 μm. |
Thermal Hysteresis
| PMID | 25114178 |
| DOI | 10.1096/fj.14-256388 |
| Protein Name | Cn-AFP (G124T) |
| Tag | Hexahistidine |
| Thermal Hysteresis | Fig.7.A TH activity of wild-type Cn-AFP and mutant proteins at various protein concentrations.(A) The maximum TH activity of Cn-AFP was 1.28℃ at 10 mg/ml protein. TH activities of T19Y, T41Y, D175Y, T193Y, and T211Y mutants were 10% of the activity of the wild-type protein. G124Y and G124T showed 1.9℃ TH values. |
Brief description
|
PMID: 25114178
DOI: 10.1096/fj.14-256388 |
1. G124Y and G124T:Both exhibited enhanced TH activity (~1.9°C), higher than the wild-type.
2. At concentrations >1.25 mg/ml, ice crystals formed a burst form, indicating hyperactive antifreeze activity. |