Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist: Role in restricted conformational mobility and compaction of native state

Abstract

Understanding the mechanism for sucroseinduced protein stabilization is important in many diverse fields, ranging from biochemistry and environmental physiology to pharmaceutical science. The current study measures the preferential exclusion of 1 M sucrose from a protein drug, recombinant interleukin 1 receptor antagonist (rhIL-1ra). It is proposed that the degree of preferential exclusion and increase in chemical potential are directly proportional to the protein surface area and that, hence, the system will favor the protein state with the smallest surface area. This mechanism explains the observed sucroseinduced restriction of rhIL-1ra conformational f luctuations, which were studied by hydrogen-deuterium exchange and cysteine reactivity measurements. Furthermore, infrared spectroscopy of rhlL-1ra suggested that a more ordered native conformation is induced by sucrose. Electron paramagnetic resonance spectroscopy demonstrated that in the presence of sucrose, spin-labeled cysteine 116 becomes more buried in the protein's interior and that the hydrodynamic diameter of the protein is reduced. The preferential exclusion of sucrose from the protein and the resulting shift in the equilibrium between protein states toward the most compact conformation account for sucrose-induced effects on rhIL-1ra.

Effect of viscogens on the kinetic response of a photoperturbed allosteric protein

“…Based on thermodynamic arguments, it has been argued that certain viscogens, such as in particular sucrose and glycerol, may be entirely excluded from the protein hydration shell. 63,64 This conclusion has found support from experimental techniques such as high precision densimetry, 46 neutron scattering experiments, 47 or the vibrational lifetime of a surface exposed protein labels. 48 While the question whether or not specific interactions of the viscogen molecule with the protein play a role might depend on the particular process studied, in our case they clearly do.…”

“…For example, it has been argued that both glycerol and sucrose are highly preferably excluded from the protein surface, [46][47][48] that gylcerol preferentially hydrates proteins, 60 and that these viscogens affects ligand dynamics in myoglobin only via the solvent viscosity, but not in a viscogen-specific way. 3 On the other hand, these viscogens also act as osmolytes and/or crowders, which often work through direct interactions with the protein, e.g., by attenuating the H-bond strength between polar protein residues and water, 61 or by interaction to the protein backbone with different strength.…”

“…To reach that value, a concentration of 40% w/w was needed for sucrose and 54% w/w for glycerol (see glycerol: MW 92), but both have been argued to be highly preferably excluded from the protein surface. [46][47][48] Nevertheless, Fig. 7 shows that glycerol significantly slows the relaxation kinetics in comparison with sucrose.…”

Applications of the second virial coefficient: protein crystallization and solubility

“…trehalose, sorbitol and mannitol) via their influence on the organization of and properties of solvent water. Other variables that can affect solubility of aqueous or membrane proteins include salt type/concentration, pH, temperature, reducing agents, type/concentration of detergent and other surfactants Arakawa & Timasheff, 1985;Bhat & Timasheff, 1992;Kendrick et al, 1997;Chi et al, 2003;Kaushik & Bhat, 2003;Lins et al, 2004;Timasheff, 1998;Valente et al, 2005). As noted previously, measured B values provide a qualitative assessment of protein-protein interactions, with more positive or more negative B values indicative of increased repulsion or increased attraction, respectively.…”

“…those conditions with B values within the crystallization slot). The approach of using an incomplete factorial (Kendrick et al, 1997) of crystallization conditions and outcomes combined with ANN analysis has previously been reported by DeLucas and coworkers (DeLucas et al, 2003(DeLucas et al, , 2005. The ability to improve or maximize the solubility of purified proteins is beneficial to many different protein research disciplines.…”