The effect of surface charge on the thermal stability and ice recrystallization inhibition activity of antifreeze protein III (AFP III).

The aim of this study was to examine the effect of chemical cationization on the structure and function of antifreeze protein III (AFP III) over an extreme temperature range (-40°C to +90°C) using far-UV synchrotron radiation circular dichroism (SRCD) and ice recrystallization inhibition (IRI) assays. Chemical cationization was able to produce a modified AFP III with a net cationic charge at physiological pH that had enhanced resistance to denaturation at elevated temperatures, with no immediate negative impact on protein structure at subzero temperatures. Furthermore, cationized AFP III retained an IRI activity similar to that of native AFP III. Consequently, chemical cationization may provide a pathway to the development of more robust antifreeze proteins as supplementary cryoprotectants in the cryopreservation of clinically relevant cells.

Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase.

Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3(10)-helices, and two ß-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.

Purification, crystal structure determination and functional characterization of type III antifreeze proteins from the European eelpout Zoarces viviparus.

Antifreeze proteins (AFPs) are essential components of many organisms adaptation to cold temperatures. Fish type III AFPs are divided into two groups, SP isoforms being much less active than QAE1 isoforms. Two type III AFPs from Zoarces viviparus, a QAE1 (ZvAFP13) and an SP (ZvAFP6) isoform, are here characterized and their crystal structures determined. We conclude that the higher activity of the QAE1 isoforms cannot be attributed to single residues, but rather a combination of structural effects. Furthermore both ZvAFP6 and ZvAFP13 crystal structures have water molecules around T18 equivalent to the tetrahedral-like waters previously identified in a neutron crystal structure. Interestingly, ZvAFP6 forms dimers in the crystal, with a significant dimer interface. The presence of ZvAFP6 dimers was confirmed in solution by native electrophoresis and gel filtration. To our knowledge this is the first report of dimerization of AFP type III proteins.

Activity of a two-domain antifreeze protein is not dependent on linker sequence.

The reported NMR structure of RD3, a naturally occurring two-domain antifreeze protein, suggests that the two nearly identical domains are oriented to allow simultaneous binding of their active regions to the ice surface. It is implied that the nine residues linking the two domains play a role in this alignment, but this has not been established. We have designed and expressed a modified form of RD3 that replaces the nine-residue linker with a generic sequence of one serine and eight glycine residues to test the importance of the linker amino acid sequence. The modified linker is shown to have significantly different characteristics compared to the original linker. Heteronuclear nuclear Overhauser effect experiments show that the new linker residues have more mobility than the linker residues in the native protein. Further, NMR data show that the folding of the C-terminal domain is somewhat perturbed by the altered linker. Finally, distributions of residual dipolar couplings indicate that the two domains tumble and move independently of each other. Nevertheless, the thermal hysteresis activity of the modified protein is indistinguishable from that of native RD3, proving that increased activity of the two-domain antifreeze protein is not dependent on structure of the linker.