Preferential Ordering and Organization of Hydration Water Favor Nucleation of Ice by Ice-Nucleating Proteins over Antifreeze Proteins.

Bacteria containing ice-nucleating proteins (INPs) evolved in nature to nucleate ice at the high sub-zero ambiance. The ability of the INPs to induce order in the hydration layer and their aggregation propensity appear to be key factors of their ice nucleation abilities. However, the mechanism of the process of ice nucleation by INPs is yet to be understood clearly. Here, we have performed all-atom molecular dynamics simulations and analyzed the structure and dynamics of the hydration layer around the proposed ice-nucleating surface of a model INP. Results are compared with the hydration of a topologically similar non-ice-binding protein (non-IBP) and another ice-growth inhibitory antifreeze protein (sbwAFP). We observed that the hydration structure around the ice-nucleating surface of INP is highly ordered and the dynamics of the hydration water are slower, compared to the non-IBP. Even the ordering of the hydration layer is more evident around the ice-binding surface of INP, compared to the antifreeze protein sbwAFP. Particularly with increasing repeat units of INP, we observe an increased population of ice-like water. Interestingly, the distances between the hydroxyl groups of the threonine ladder and its associated channel water of the ice-binding surface (IBS) of INP in the X and Y direction mimic the oxygen atom distances of the basal plane of hexagonal ice. However, the structural synergies between the hydroxyl group distances of the threonine ladder and its associated channel water of the IBS of sbwAFP and oxygen atom distances of the basal plane are less evident. This difference makes the IBS of the INP a better template for ice nucleation than AFP, although both of them bind to the ice surface efficiently.

Number of Hydrogen Bonds per Unit Solvent Accessible Surface Area: A Descriptor of Functional States of Proteins.

Proteins function close to native and near-native conformations. These states are evolutionarily selected to ensure the effect of mutations is minimized. The structural organization of a protein is hierarchical and modular, which reduces the dimensionality of the configurational space of the native states. Thus, finding appropriate descriptors that define the native state among all possible states of a protein is a problem of immense interest. The present study explores the correlation between solvent accessible surface areas (SASAs) and different intraprotein as well as protein-water hydrogen bonds of 55 single-chain globular proteins from four different structural classes (all α, all ß, α+ß, and α/ß), 16 multichain proteins, and 4 macromolecular complexes. A systematic analysis of the solvent accessible surface area and intraprotein and protein-water hydrogen bonds suggests a linear relationship between SASAs and hydrogen bonds. The number of protein-water hydrogen bonds per unit SASA ranges from 3 to 4 for all the different structural protein classes. In contrast, the number of intramolecular hydrogen bonds per unit SASA, including the mainchain-mainchain, mainchain-sidechain, and sidechain-sidechain, varies between 0.75 to 2. The solvation free energy of a protein linearly decreases with SASA. Our study also shows that the solvation free energy/SASA varies from -75 to -105 kJ mol-1 nm-2 across all the native states studied here. The number conservancy of intraprotein hydrogen bonds per unit SASA possibly imparts structural stability to the native structure. On the other hand, 3-4 protein-water hydrogen bonds per unit SASA are possibly required to maintain a balance between the solubility and functionality of the native states. This study provides a basis for synthetic biologists to design new folds with improved functionality.

Molecular Factors of Ice Growth Inhibition for Hyperactive and Globular Antifreeze Proteins: Insights from Molecular Dynamics Simulation.

The molecular mechanism behind the ice growth inhibition by antifreeze proteins (AFPs) is yet to be understood completely. Also, what physical parameters differentiate between the AFP and non-AFP are largely unknown. Thus, to get an atomistic overview of the differential antifreeze activities of different classes of AFPs, we have studied ice growth from different ice surfaces in the presence of a moderately active globular type III AFP and a hyperactive spruce budworm (sbw) AFP. Results are compared with the observations of ice growth simulations in the presence of topologically similar non-AFPs using all-atom molecular dynamics simulations. Simulation data suggest that the ice surface coverage is a critical factor in ice growth inhibition. Due to the presence of an ice binding surface (IBS), AFPs form a high affinity complex with ice, accompanied by a transition of hydration water around the IBS from clathrate-like to ice-like. Several residues around the periphery of the IBS anchor the AFP to the curved ice surface mediated by multiple strong hydrogen bonds, stabilizing the complex immensely. In the high surface coverage regime, the slow unbinding kinetics dominates over the ice growth kinetics and thus facilitates the ice growth inhibition. Due to the non-availability of a proper IBS, non-AFPs form a low-affinity complex with the growing ice surface. As a result, the non-AFPs are continuously repelled by the surface. If the concentration of AFPs is low, then the effective surface coverage is reduced significantly. In this low surface coverage regime, AFPs can also behave like impurities and are engulfed by the growing ice crystal.

Direct Participation of Solvent Molecules in the Formation of Supramolecular Polymers.

This article reports supramolecular polymerization of two bis-amide functionalized naphthalene-diimide (NDI) building blocks (NDI-L and NDI-C) in two solvents, namely n-heptane (Hep) and methylcyclohexane (MCH). NDI-L and NDI-C differ only by the peripheral hydrocarbon wedges, consisting of linear C7 chains or cyclic methylcyclohexane rings, respectively. UV/Vis and FTIR spectroscopy studies reveal distinct internal order and H-bonding pattern for NDI-L and NDI-C aggregates irrespective of the solvent system, indicating the dominant role of the intrinsic packing parameters of the individual building block, possibly influenced by the peripheral steric crowding. However, NDI-L produces a significantly stronger gel in Hep compared to MCH as evident from the rheological and thermal properties. In contrast, NDI-C exhibits a clear preference for MCH, producing gel with moderate strength but in Hep it fails to produce 1D morphology or gelation. All-atom molecular dynamics (MD) simulation studies corroborate with the experimental observation and provide the rationale for the observed solvent-shape effect by revealing a quantitative estimate regarding the thermodynamics of self-assembly in these four combinations. Such clear-cut shape-matching effect (between the peripheral hydrocarbon wedge and the solvent system) unambiguously support a direct participation of the solvent molecules during supramolecular polymerization and presence of a closely-adhered solvent shell around the supramolecular polymers, similar to the first layer of water molecules around the protein surface. Solvent induced CD experiments support this hypothesis as induced CD band was observed only from a chiral co-solvent of matching shape. This is reconfirmed by the higher de-solvation temperature of the shape-matching NDI/solvent system combination compared to the shape mis-match combination in variable temperature UV/Vis experiments, revealing transformation to a different aggregate at higher temperatures rather than disassembly to the monomer for all four combinations.

Deciphering the Role of the Non-ice-binding Surface in the Antifreeze Activity of Hyperactive Antifreeze Proteins.

Antifreeze proteins (AFPs) show thermal hysteresis through specific interaction with the ice crystal. Hyperactive AFPs interact with the ice surface through a threonine-rich motif present at their ice-binding surface (IBS). Ordering of water around the IBS was extensively investigated. However, the role of non-IBS in ice growth inhibition is yet to be understood completely. The present study explores the nature of hydration and its length-scale evaluation around the non-IBS for hyperactive AFPs. We observed that the hydration layer of non-IBS is liquid-like, even in highly supercooled conditions, and the nature of hydration is drastically different from the hydration pattern of non-AFP surfaces. In similar conditions, the hydration layer around the IBS is ice-like ordered. Non-IBS of the hyperactive AFP exposes toward the bulk and is able to maintain the liquid-like character of its hydration water up to 15 Å. We also find that the amino acid compositions and their spatial distribution on the non-IBS are markedly different from those of the IBS and non-AFP surfaces. These results elucidate the combined role of IBS and non-IBS in ice-growth inhibition. While IBS is required to adsorb on ice efficiently, the exposed non-IBS may prevent ice nucleation/growth on top of the bound AFPs.