Co‐operative effect of the isoforms of type III antifreeze protein expressed in Notched‐fin eelpout, <i>Zoarces elongatus Kner</i>

Nishimiya, Yoshiyuki; Sato, Ryoko; Takamichi, Manabu; Miura, Ai; Tsuda, Sakae

doi:10.1111/j.1742-4658.2004.04490.x

Cited by 62 publications

(95 citation statements)

References 36 publications

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“…Wild-type nfeAFP11 was ligated into the pET20b expression vector as previously described [4]. All mutants of nfeAFP11 were created using standard overlap extension PCR methodologies [13] and were verified by DNA sequencing.…”

Section: Site-directed Mutagenesis and Cloningmentioning

confidence: 99%

“…All proteins were tested in 100 mM sodium hydrogen carbonate buffer (pH 8.5) as previously described [4,5].…”

Section: Ice Crystal Morphology and Thermal Hysteresis Measurementsmentioning

confidence: 99%

“…Like nfeAFP6, nfeAFP11 (a QAE2 isoform) is also unable to prevent ice crystal growth at undercooling temperatures, despite its ability to shape ice into hexagonal trapezohedrons [4,5]. NfeAFP11 differs in its ice-binding residues in having Val, Val, Gly, and Ile at positions 9, 19, 20, and 41, respectively (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…QAE1-isoforms are fully active as antifreezes and completely arrest ice crystal growth at temperatures below the equilibrium freezing point of a solution. QAE2-and SP-isoforms have the ability to shape ice crystals into hexagonal trapezohedrons, but are incapable of preventing their growth unless supplemented with some QAE1-isoform [4,5].…”

Section: Introductionmentioning

confidence: 99%

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Engineering a naturally inactive isoform of type III antifreeze protein into one that can stop the growth of ice

et al. 2012

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a b s t r a c tType III antifreeze proteins (AFPs) can be sub-divided into three classes of isoforms. SP and QAE2 isoforms can slow, but not stop, the growth of ice crystals by binding to pyramidal ice planes. The other class (QAE1) binds both pyramidal and primary prism planes and is able to halt the growth of ice. Here we describe the conversion of a QAE2 isoform into a fully-active QAE1-like isoform by changing four surface-exposed residues to develop a primary prism plane binding site. Molecular dynamics analyses suggest that the basis for gain in antifreeze activity is the formation of ice-like waters on the mutated protein surface.

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Section: Site-directed Mutagenesis and Cloningmentioning

confidence: 99%

“…All proteins were tested in 100 mM sodium hydrogen carbonate buffer (pH 8.5) as previously described [4,5].…”

Section: Ice Crystal Morphology and Thermal Hysteresis Measurementsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Engineering a naturally inactive isoform of type III antifreeze protein into one that can stop the growth of ice

et al. 2012

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“…These two isoforms possess different pIs and share approximately 50% sequence identity. The Japanese notched-fin eelpout, Zoarces elongates Kner, produces 13 different AFPIII isoforms (denoted nfeAFP), which have been divided into six SP (nfeAFP1-6) and seven QAE (nfeAFP7-13) isoforms, and the latter was further divided into QAE1 (nfeAFP7-10) and QAE2 (nfeAFP11-13) isoforms (Nishimiya et al 2005). Among them, only the QAE1 isoforms are fully active variants exhibiting both TH and ice-shaping activities, while the others only shape the ice morphology.…”

Section: Biological Contextmentioning

confidence: 99%

NMR structure note: a defective isoform and its activity-improved variant of a type III antifreeze protein from Zoarces elongates Kner

et al. 2013

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Biological contextAntifreeze proteins (AFPs) produced in various cold-adapted animals and plants can specifically bind to ice crystals and inhibit their growth (Fletcher et al. 2001;Graether and Sykes, 2004). The ice-binding ability of AFPs depresses the freezing temperature (T f ) of water non-colligatively, which leads to the protection of cells and tissues from freezing. The level of T f depression has been evaluated by measuring the T f and melting temperature (T m ) for an ice crystal created in an aqueous solution of an AFP. The difference between these two temperatures is defined as the thermal hysteresis (TH), which is a measure of the AFP's ice-growth inhibition. AFPs also modify the shape of an ice crystal uniquely, hexagonal bipyramid for example, in the temperature range of TH. These two activities have been assumed to be common for all AFPs; however, it has recently been found that they are not always the rule for every species, such as the AFP type III (denoted AFPIII) found in fish (Takamichi et al. 2008).Fish AFPIII is a globular protein made up of many short β-strands and one helical turn. AFPIII is generally produced in vivo as a mixture of quaternary-amino-ethyl (QAE)-Sephadex-and sulfopropyl (SP)-Sephadex-binding isoforms. These two isoforms possess different pIs and share approximately 50% sequence identity. The Japanese notched-fin eelpout, Zoarces elongates Kner, produces 13 different AFPIII isoforms (denoted nfeAFP), which have been divided into six SP (nfeAFP1-6) and seven QAE (nfeAFP7-13) isoforms, and the latter was further divided into QAE1 (nfeAFP7-10) and QAE2 (nfeAFP11-13) isoforms (Nishimiya et al. 2005). Among them, only the QAE1 isoforms are fully active variants exhibiting both TH and ice-shaping activities, while the others only shape the ice morphology. The reason for such defective activity for the SP-and QAE2-isoforms is not well understood.Recently, we found that alterations of the 9 th , 19 th , and 20 th residues of a QAE2 isoform make them the same as their counterparts in the QAE1 isoform, leading to the successful conversion of the former into a QAE1-like fully active isoform . That is, the QAE-2 isoform nfeAFP11 can only shape ice crystals, but has no TH activity, while its triple mutant nfeAFP11-V9Q/V19L/G20V (denoted nfeAFP11-tri) perfectly exhibits both activities. These residues were thought to construct a "compound" ice-binding site (IBS) that consists of two adjacent flat surfaces inclined at an angle of approximately 150° to each other (Garnham et al. 2010). To determine if the triple mutations in nfeAFP11 led to changes in the structure of the IBS that could affect its icebinding activity, the multidimensional NMR spectra of both nfeAFP11 and nfeAFP11-tri were examined. Special attention was paid to the surface-bound water molecules, for which the unique role of anchoring the IBS to ice lattice has been postulated (Garnham et 3 al. 2011;Kondo et al. 2012).The tertiary structures of AFPIII have been determined mostly for the QAE1-isoform HPLC12, which was ...

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NMR study of the antifreeze activities of active and inactive isoforms of a type III antifreeze protein

et al. 2016

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The quaternary-amino-ethyl 1 (QAE1) isoforms of type III antifreeze proteins (AFPs) prevent the growth of ice crystals within organisms living in polar regions. We determined the antifreeze activity of wild-type and mutant constructs of the Japanese notched-fin eelpout (Zoarces elongates Kner) AFP8 (nfeAFP8) and characterized the structural and dynamics properties of their ice-binding surface using NMR. We found that the three constructs containing the V20G mutation were incapable of stopping the growth of ice crystals and exhibited structural changes, as well as increased conformational flexibility, in the first 3 helix (residues 18-22) of the sequence. Our results suggest that the inactive nfeAFP8s are incapable of anchoring water molecules due to the unusual and flexible backbone conformation of their primary prism plane-binding surface.

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Co‐operative effect of the isoforms of type III antifreeze protein expressed in Notched‐fin eelpout, Zoarces elongatus Kner

Cited by 62 publications

References 36 publications

Engineering a naturally inactive isoform of type III antifreeze protein into one that can stop the growth of ice

Engineering a naturally inactive isoform of type III antifreeze protein into one that can stop the growth of ice

NMR structure note: a defective isoform and its activity-improved variant of a type III antifreeze protein from Zoarces elongates Kner

NMR study of the antifreeze activities of active and inactive isoforms of a type III antifreeze protein

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