205:) determined by Mass Spectroscopy, Edman degradation and by constructing a partial cDNA sequence and PCR have shown that a TxTxTxT internal repeat exists. Sequence logos constructed from the RiAFP internal repeats, have been particularly helpful in the determination of the consensus sequence of these repeats. The TxTxTxT domains are irregularly spaced within the protein and have been shown to be conserved from the TxT binding motif of other AFPs. The
191:
25:
216:
residues fits well, when spaced as they are in the internal repeats, with the hydroxyl moieties of externally facing water molecules in the forming ice lattice. This mimics the formation of the growth cone at a nucleation site in the absence of AFPs. Thus, the binding of RiAFP inhibits the growth of
142:
point. At a certain low temperature, the maximum convexity of the ice nucleation site is reached. Any further cooling will actually result in a "spreading" of the nucleation site away from this convex region, causing rapid, uncontrollable nucleation of the ice crystal. The temperature at which this
134:
onto the exposed ice crystal force the growth of the ice crystal in a convex fashion as the temperature drops, which elevates the ice vapour pressure at the nucleation sites. Ice vapour pressure continues to increase until it reaches equilibrium with the surrounding solution (water), at which point
253:). On the basis of these observations, it has been predicted that the need for insect AFPs came about after insect evolutionary divergence, much like the evolution of fish AFPs; thus, different AFPs most likely evolved in parallel from adaptations to cold (environmental) stress. As a result,
149:
is further supported by the observation that antifreeze activity increases with increasing AFP concentration – the more AFPs adsorb onto the forming ice crystal, the more 'crowded' these proteins become, making ice crystal nucleation less favourable.
103:, as well as varying numbers of 12- or 13-mer repeats of 8.3-12.5kDa, RiAFP is notable for containing only one disulfide bridge. This property of RiAFP makes it particularly attractive for recombinant expression and biotechnological applications.
169:
allows the tissues and fluids within the beetle to withstand freezing up to -30 °C (the thermal hysteresis point for this AFP). This strategy provides an obvious survival benefit to these beetles, who are endemic to cold climates, such as
94:
prevents its body fluids from freezing altogether. This contrasts with freeze-tolerant species, whose AFPs simply depress levels of ice crystal formation in low temperatures. Whereas most insect antifreeze proteins contain
443:
Kristiansen E, Ramløv H, Hagen L, Pedersen SA, Andersen RA, Zachariassen KE (September 2005). "Isolation and characterization of hemolymph antifreeze proteins from larvae of the longhorn beetle
566:
Graether SP, Kuiper MJ, Gagnè SM, Walker VK, Jia Z, Sykes BD, Davies PL (July 2000). "β-helix structure and ice-binding properties of a hyperactive antifreeze protein from an insect".
399:
Kristiansen E, Ramløv H, Højrup P, Pedersen SA, Hagen L, Zachariassen KE (February 2011). "Structural characteristics of a novel antifreeze protein from the longhorn beetle
130:
of the ice crystal lattice are blocked by the AFP, inhibiting the rapid growth of the crystal that could be fatal for the organism. In physical chemistry terms, the AFPs
126:, AFPs disrupt the thermodynamically favourable growth of an ice crystal via kinetic inhibition of contact between solid ice and liquid water. In this manner, the
229:
queries have returned no viable matches, has led some researchers to suggest that RiAFP represents a new type of AFP – one that differs from the heavily studied
197:
A sequence logo constructed from the 13-nucleotide repeat regions found by EBI-RADAR, showing a clear TxTxTxT binding motif embedded within these regions.
617:
Lin FH, Davies PL, Graham LA (May 2011). "The Thr- and Ala-Rich
Hyperactive Antifreeze Protein from Inchworm Folds as a Flat Silk-like-β-Helix".
86:
longhorned beetle. It is a type V antifreeze protein with a molecular weight of 12.8 kDa; this type of AFP is noted for its hyperactivity.
171:
111:
AFPs work through an interaction with small ice crystals that is similar to an enzyme-ligand binding mechanism which inhibits
272:
291:
protein, having six β-strand regions consisting of 13-amino acids (including one TxTxTxT binding motif) per strand.
225:
The fact that the binding motif appears as a "triplet" of the conserved TxT repeat, as well as the observation that
736:
Sicheri, F; Yang DS (1995). "Ice-binding structure and mechanism of an antifreeze protein from winter flounder".
115:
of ice. This explanation of the interruption of the ice crystal structure by the AFP has come to be known as the
299:
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Scott GK, Fletcher GL, Davies PL (1986). "Fish
Antifreeze Proteins: Recent Gene Evolution".
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583:
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are located at the ends of β-strand regions. These data suggest that RiAFP is a well-folded
209:
127:
112:
749:
681:"Expression, purification, crystallization, and preliminary crystallographic studies of
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346:
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543:
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298:, have been published on a RiAFP crystal (which diffracted to 1.3Å resolution) in the
795:
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Duman JG (2001). "Antifreeze and ice nucleator proteins in terrestrial arthropods".
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783:
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have determined that the internal repeats are spaced sufficiently to tend towards
519:"Theoretical study of interaction of winter flounder antifreeze protein with ice"
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161:, a fluid that bathes all the cells of the beetle and fills a cavity called the
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The aforementioned effect of AFPs on ice crystal nucleation is lost at the
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is a freeze-avoidant species, meaning that, due to its AFP,
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21), with unit-cell parameters a = b = 46.46, c = 193.21Å.
201:
The primary structure of RiAFP (the sequence may be found
329:
Graham LA, Liou YC, Walker VK, Davies PL (August 1997).
49:
39:
217:
the crystal in the basal and prism planes of the ice.
679:Hakim A, Thakral D, Zhu DF, Nguyen JB (May 2012).
143:phenomenon occurs is the thermal hysteresis point.
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283:regions include the conserved repeats; and all
331:"Hyperactive antifreeze protein from beetles"
8:
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517:Jorov A, Zhorov BS, Yang DS (June 2004).
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273:Secondary structure modelling algorithms
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135:the growth of the ice crystal stops.
7:
14:
496:10.1146/annurev.physiol.63.1.327
147:adsorption-inhibition hypothesis
117:adsorption-inhibition hypothesis
23:
157:beetle, AFPs are found in the
1:
269:would prove to be fruitless.
823:
461:10.1016/j.cbpc.2005.06.004
417:10.1016/j.ibmb.2010.11.002
165:. The presence of AFPs in
701:10.1107/S1744309112010421
221:RiAFP Predicted Structure
296:crystallographic studies
38:, as no other articles
449:Comp Biochem Physiol B
198:
80:(AFP) produced by the
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99:at least every sixth
654:Can J Fish Aquat Sci
405:Insect Biochem Molec
300:trigonal space group
750:1995Natur.375..427S
685:antifreeze protein"
580:2000Natur.406..325G
535:10.1110/ps.04641104
347:1997Natur.388..727G
689:Acta Crystallogr F
683:Rhagium inquisitor
484:Annu. Rev. Physiol
445:Rhagium inquisitor
401:Rhagium inquisitor
279:configuration; no
255:homology modelling
199:
140:thermal hysteresis
122:According to this
83:Rhagium inquisitor
78:antifreeze protein
57:for suggestions.
47:to this page from
16:Antifreeze protein
744:(6530): 427–431.
631:10.1021/bi2003108
625:(21): 4467–4478.
574:(6793): 325–328.
186:RiAFP Ice Binding
113:recrystallization
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285:turn regions
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807:Cryobiology
523:Protein Sci
172:Scandinavia
796:Categories
767:11375/7005
490:: 327–57.
314:References
235:T. molitor
159:haemolymph
124:hypothesis
53:; try the
40:link to it
373:205029622
306:21 (or P3
289:β-helical
195:Figure 1:
163:haemocoel
97:cysteines
62:June 2016
43:. Please
802:Proteins
719:22691785
639:21486083
596:10917537
553:15152087
504:11181959
469:15993638
425:21078390
294:Primary
277:β-strand
207:hydroxyl
132:adsorbed
776:7760940
746:Bibcode
710:3374510
604:4345188
576:Bibcode
544:2279984
365:9285581
343:Bibcode
281:helical
245:), and
212:of the
176:Siberia
153:In the
101:residue
784:758990
782:
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738:Nature
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568:Nature
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335:Nature
249:(from
241:(from
233:(from
227:blastp
210:moiety
180:Alaska
178:, and
36:orphan
34:is an
780:S2CID
600:S2CID
369:S2CID
267:CfAFP
265:, or
263:DcAFP
259:TmAFP
257:with
247:CfAFP
239:DcAFP
231:TmAFP
74:RiAFP
772:PMID
715:PMID
635:PMID
592:PMID
549:PMID
500:PMID
465:PMID
421:PMID
361:PMID
203:here
145:The
107:AFPs
762:hdl
754:doi
742:375
705:PMC
697:doi
662:doi
627:doi
584:doi
572:406
539:PMC
531:doi
492:doi
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