Las teorías modernas de DEL sustentadas en diversos enfoques multidisciplinarios.

on Ice

Over the last 50 years various mechanisms have been proposed for the effect of AF(G)Ps on ice, but no single mechanism has been agreed upon to date. Of course, it is possible that there might be a variety of mechanisms at work for the different proteins. A selection of the more popular mechanisms are reviewed below.

The Adsorption-Inhibition Mechanism:

For a long time the adsorption-inhibition mechanism, published by Raymond and De- Vries in 1977,287was generally accepted,278, 299–302but more recently this mechanism has been criticised due to new evidence of irreversible ice binding. This model states that after the antifreeze has bound to the ice surface, the ice continues to grow between the protein molecules with a high surface curvature. There is now a high energetic cost of adding a water molecule to this convex ice surface (due to the Gibbs-Thomson or Kelvin effect), causing non-equilibrium freezing point depression (Figure 6.12).230, 287, 303, 304 Ice growth will only resume if the temperature decreases further.

Early static models for ice growth in the presence of AF(G)Ps based on the this mechanism are the “mattress model”230, 266and the “step pinning model”230, 266, 303, 306, 307

Figure 6.11: Evidence of reversible ice binding adapted from Zepeda et al.297 showing confocal images of an ice crystal with AFGP adsorbed onto particular planes. Images b and c were taken 4 and 9.6 seconds after image a. The arrows in image a indicate an ice plane where a new surface of AFGP has just formed (01-10). Image b and c show ice growth in a new direction (1-100) after desorption of the protein. The plot shows the AFGP intensity in the rectangles highlighted in the images. The black line indicates the significantly higher concentration of protein on the ice plane before it desorbs (b, yellow line) and the ice growth continues (c, red line).

Figure 6.12: The adsorption-inhibition mechanism from Knight.305 After AF molecules have bound to the ice surface, ice growth continues in the gaps between the protein molecules for a short while until the curvature of the ice surface makes it energetically unfavourable for water molecules to join the ice lattice.

(see Figure 6.13). The mattress model is a 2D model whereby the AF molecules inhibit ice growth perpendicular to the surface, whereas the step pinning model is a 3D model where the AF molecules are adsorbed in a mono-step layer blocking the growth of that step. Kuiper and co-workers produced dynamics simulations of an insect AFP solution in the presence of ice that clearly showed the adsorption-inhibition mechanism in ac- tion, confirming that this species of AFP binds irreversibly to ice.293 However, these models are by no means perfect, mainly due to the growing evidence of reversible ice binding.230, 252, 297–301, 308

The Two-Step Reversible Adsorption Mechanism:

Figure 6.11 shows that AFGP adsorption is not instant, in fact it takes several seconds to reach maximum coverage. Interestingly, after just a small portion of the protein molecules have adsorbed onto ice the surface there is no further ice growth, showing that maximum coverage is not required to halt the growth. Zepeda and co-workers proposed, based on their observations of the interactions between AFGPs (types 4-6) and ice, that initially a number of protein molecules strongly bind to the ice surface, halting the ice growth, after which further proteins continue to bind weakly.297

The reversible binding reported here shows that some of the proteins must be adsorbed weakly enough that the addition of water molecules to the ice plane can eject the protein molecules, allowing ice growth to continue where it was previously halted. This indicates that the AFGP-ice interactions are much weaker and more dynamic than originally predicted.297, 309

The two-step reversible adsorption mechanism also considers the role of protein solvation and the QLL; AFGPs are highly flexible310and ice surfaces are highly dynamic, so a rigid match between these, such as the adsorption-inhibition mechanism, is a poor description of the antifreeze mechanism. A much more fitting mechanism is that the protein molecules disrupt the quasi-ordering of the surface liquid at the interface by purely kinetic effects.297 Measurements show that the solvation entropy can dominate

the whole process311 and that both AGFP and AFP III reduce the surface free energy

of the water which can be attributed to a significant increase in the thickness of the

Figure 6.13: The (a) “mattress model” and (b) “step pinning model”, examples of adsorption-inhibition models for ice growth in the presence of AF(G)P.

QLL.297, 312–314This research acts as a reminder that it is critical to consider the protein solvation and QLL dynamics in an antifreeze mechanism, in addition to the structure of the protein and ice lattice.254, 297, 304, 315

The Anchored Clathrate Water Mechanism:

In 2008, the anchored clathrate water mechanism was proposed by Nutt and Smith to explain the interaction between ice, AF(G)Ps and their neighbouring water molecules.316 In an AF(G)P solution, when the temperature becomes low enough, the water molecules in the solvation shell become more structured and less dynamic at the ice-binding site (Figure 6.14a). This ordered, ice-like region of water at the IBS now has a lower energy barrier for ice formation. When the protein approaches the ice and comes into con- tact with the QLL, the ordered waters at the IBS merge with the QLL (Figure 6.14b) facilitating local ice growth. If the growing ice surface is the correct plane and the protein is orientated favourably, then the ice growth will incorporate the IBS of the protein into the ice lattice (Figure 6.14c).258, 316 The hydrophilic surface of the non-IBS disrupts the solvent shells in order to prevent the protein being fully surrounded by ice,316, 317 highlighting that the non-IBS could play a crucial role in the antifreeze effect of AF(G)Ps.258, 261

Figure 6.14: The anchored clathrate water mechanism from Nutt and Smith.316 _Ordering

at the ice-water and protein-water interfaces is indicated by shading (black represents more structured water and white less structured water). The figure shows, a) the protein with ordered ice-like water molecules at the ice-binding site, b) the protein approaching the ice surface causing the ordered regions of water to overlap and c) the local ice growth incorporating the IBS of the protein into the ice plane.

Several studies, using a combination of x-ray diffraction (XRD) and computa- tional simulations, have revealed clathrate waters at the ice-binding sites of AFPs.268, 318, 319

These studies also revealed that the “maxi” AFP has at least 400 intramolecular ice-like water molecules that are critical for stabilisation and has ice-binding residues within the protein core, strongly supporting the anchored clathrate water mechanism.268