Dendrimer-Linked Antifreeze Proteins Have Superior Activity and Thermal Recovery

Abstract

By binding to ice, antifreeze proteins (AFPs) depress the freezing point of a solution and inhibit ice recrystallization if freezing does occur. Previous work showed that the activity of an AFP was incrementally increased by fusing it to another protein. Even larger increases in activity were achieved by doubling the number of ice-binding sites by dimerization. Here, we have combined the two strategies by linking multiple outward-facing AFPs to a dendrimer to significantly increase both the size of the molecule and the number of ice-binding sites. Using a heterobifunctional cross-linker, we attached between 6 and 11 type III AFPs to a second-generation polyamidoamine (G2-PAMAM) dendrimer with 16 reactive termini. This heterogeneous sample of dendrimer-linked type III constructs showed a greater than 4-fold increase in freezing point depression over that of monomeric type III AFP. This multimerized AFP was particularly effective at ice recrystallization inhibition activity, likely because it can simultaneously bind multiple ice surfaces. Additionally, attachment to the dendrimer has afforded the AFP superior recovery from heat denaturation. Linking AFPs together via polymers can generate novel reagents for controlling ice growth and recrystallization.

Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant

“…Studies from the laboratories of Tsuda, Davies, and Holland have demonstrated that the multimerization of native type III AFP can increase TH activity [210,211,212]. Recently, Steven et al claimed the dendrimer-like AFPs showed higher TH values [213], and Phippen et al demonstrated 12 AFP-fused protein cage nanoparticles that increased the TH value to more than 50-fold that of monomeric AFP [214]. A few groups have attempted to synthesize AFP or AFGP derivatives to elucidate the underlying mechanism of action and to develop practical applications [215,216,217,218,219,220,221].…”

Influence of Block Copolymerization on the Antifreeze Protein Mimetic Ice Recrystallization Inhibition Activity of Poly(vinyl alcohol)

“… 24 To address this, we have investigated (supramolecular) branched PVAs and found that addition of a third arm to a PVA has no effect on IRI activity, 25 as has been seen for branched AFPs. 26 This is significant, as although the molecular weight was increased, there was no enhancement compared to increasing the molecular weight of a linear polymer. Brush-like PVA also shows identical activity to linear equivalents 12 despite having far higher molecular weight.…”

A Supramolecular Ice Growth Inhibitor

“…This behavior is similar to AFPs that do not bind to the basal {0001} planes. 9 As illustrated in Figure 3A, the TH activity for S + Cl -(0.31 kDa) is less than that reported for TmAFP (9 kDa; 9,10 ) and AFPIII (7 kDa; 10,33 ), but similar to the glycoprotein AFGP8 (2.6 kDa). 34 The growth velocity along the a-axis was negligible until the sudden burst of growth as the temperature was lowered, precluding measurement of growth velocity along this direction.…”