Dissecting the Effects of Concentrated Carbohydrate Solutions on Protein Diffusion, Hydration, and Internal Dynamics

Abstract

We present herein a thorough description of the effects of high glucose concentrations on the diffusion, hydration and internal dynamics of ubiquitin, as predicted from extensive molecular dynamics simulations on several systems described at fully atomistic level. We observe that the protein acts as a seed that speeds up the natural propensity of glucose to cluster at high concentration; the sugar molecules thus aggregate around the protein trapping it inside a dynamic cage. This process extensively dehydrates the protein surface, restricts the motions of the remaining water molecules, and drags the large-scale, collective motions of protein atoms slowing down the rate of exploration of the conformational space despite only a slight dampening of fast, local dynamics. We discuss how these effects could be relevant to the function of sugars as preservation agents in biological materials, and how crowding by small sticky molecules could modulate proteins across different reaction coordinates inside the cellular cytosol. ■ INTRODUCTIONSugars play roles as agents for the preservation of biological material in nature and in biotechnological manufacture, 1−3 most importantly through their capacity to stabilize proteins against cold and hot denaturation, both in solution and in the solid state. 4−7 Other potential effects of sugars on protein properties have been less explored, but changes in activity, dynamics, and regulation can be expected by analogy to the effects known to be caused by high concentrations of other molecules. Whereas most hydrophilic molecules have the capacity to agglomerate and disrupt water structure, sugars and polyols generally have exceptionally large solubilities, which allows them to strongly dehydrate other molecules and to cluster at very high concentrations forming glassy states. 8−11 Because of these special properties, questions about the effects of sugars on protein properties are intimately related to those revolving around the effects of viscosity, molecular crowding, encapsulation and even vitrification on proteins, all meeting at the crossroads between chemistry, biology, medicine, and applications in the food and pharmacological industries. Within all these closely related fields, the effects of high concentrations of small hydrophilic molecules on protein stability and translational and rotational diffusion have been explored, but studies of their effects on protein hydration and on internal protein dynamics are scarce. 4−7,12−16 More specifically to sugars, reports on the structure and dynamics of sugar-only solutions abound, 8,17−23 but works dealing with proteins in sugar solutions are mostly focused on the stability of the proteins and do not pay much attention to other effects related to hydration, diffusion, and internal mobility. 4−7 We recently reported in a preliminary work that concentrated glucose solutions can perturb protein dynamics by restricting exploration of the conformational space, through a mechanism that presumably involves protein−sugar interac...

Relative Cosolute Size Influences the Kinetics of Protein-Protein Interactions

“…We also evaluated CaM in the presence of the osmolyte sucrose (340 Da), which minimizes the solvent-exposed surface area of proteins (27,28,48) through an osmophobicity effect, where burial of the backbone is favored (49,50). Osmolytes are also preferentially excluded from protein surfaces and have the potential to create a caging effect where water molecules trapped on protein surfaces have decreased ability to exchange with the bulk solvent (22). Sucrose has been shown to stabilize compact conformers of protein ensembles, and has been suggested to reduce structural fluctuations significantly when proteins are in loose extended conformations with high internal hydration (26,51,52).…”

“…These highly abundant osmolytes make up~25% of solutes inside cells (3,22), play vital roles in maintaining protein function through stabilizing protein structure (23)(24)(25), and may exert frictional effects at smaller spatial scales than macromolecules. Unlike macromolecules, osmolytes are not traditionally considered to contribute to the excluded volume effect, but rather minimize protein surface area (26) to favor more compact states (27,28).…”

An optimized strategy to measure protein stability highlights differences between cold and hot unfolded states

“…The range of crowder concentration existing in living cells is rather large and ill defined1; thus, it is not uncommon to find studies on crowding at concentrations lower than 10% or as large as 40%. Some studies have been performed using low molecular weight crowders whose effect can hardly be defined as crowding as it is better described as solvent perturbation56. Other authors have made use of artificial crowders such as dextran789, Ficoll101112, polyethylene glycol (PEG)913 or different proteins1415 that could potentially mimic the cellular environment.…”

Crowding in Cellular Environments at an Atomistic Level from Computer Simulations

“…The available simulations suggest that fluctuations around the native state, as measured by S 2 order parameters or Debye–Waller B -factors, are only moderately affected by crowding. 97,98,143,174,208 Other results point at increased fluctuations as a result of protein–protein contacts in CI2 when interacting with lysozyme, 170 whereas somewhat reduced backbone dynamics were observed in other studies as a result of crowding. 112,174 Experiments also indicate moderate effects of crowding on fast internal motions but a stronger influence on slower conformational dynamics.…”

“…29 The slowdown in conformational transitions has been correlated with crowders, such as glucose, or water molecules becoming trapped between solutes and exhibiting slow diffusion and long residence times. 29,208 It remains difficult, however, to clearly separate cause and effect with respect to retarded solvation causing slow conformational dynamics or vice versa . In any case, as conformational transitions are essential in many biological mechanisms, understanding how much such kinetic processes are slowed down in cellular environments is critical for fully understanding biological processes.…”