Hydrogen Bonding between Sugar and Protein Is Responsible for Inhibition of Dehydration-Induced Protein Unfolding (original) (raw)
Related papers
This review aims to provide an overview of current knowledge on stabilization of proteins by sugars in the solid state in relation to stress conditions commonly encountered during drying and storage. First protein degradation mechanisms in the solid state (i.e. physical and chemical degradation routes) and traditional theories regarding protein stabilization (vitrification and water replacement hypotheses) will be briefly discussed. Secondly, refinements to these theories, such as theories focusing on local mobility and protein-sugar packing density, are reviewed in relationship to the traditional theories and their analogies are discussed. The last section relates these mechanistic insights to the stress conditions against which these sugars are used to provide protection (i.e. drying, temperature, and moisture). In summary sugars should be able to adequately form interactions with the protein during drying, thereby maintaining it in its native conformation and reducing both local and global mobility during storage. Generally smaller sugars (disaccharides) are better at forming these interactions and reducing local mobility as they are less inhibited by steric hindrance, whilst larger sugars can reduce global mobility more efficiently. The principles outlined here can aid in choosing a suitable sugar as stabilizer depending on the protein, formulation and storage condition-specific dominant route of degradation.
International Journal of Pharmaceutics, 2015
In-line near infrared spectroscopy during freeze-drying as a tool to measure efficiency of hydrogen bond formulation between protein and sugar, predictive of protein storage stability. International Journal of Pharmaceutics 496 792-800 Abstract 18 Sugars are often used as stabilizers of protein formulations during freeze-drying. However, 19 not all sugars are equally suitable for this purpose. Using in-line near-infrared spectroscopy 20 during freeze-drying, it is here shown here that hydrogen bond formation during freeze-21 drying, under secondary drying conditions in particular, can be related to the preservation of 22 the functionality and structure of proteins during storage. The disaccharide trehalose was best 23 capable of forming hydrogen bonds with the model protein, lactate dehydrogenase, thereby 24 stabilizing it, followed by the molecularly flexible oligosaccharide inulin 4 kDa. The 25 molecularly rigid oligo-and polysaccharides dextran 5 kDa and 70 kDa, respectively, formed 26 the least amount of hydrogen bonds and provided least stabilization of the protein. It is 27 concluded that smaller and molecularly more flexible sugars are less affected by steric 28 hindrance, allowing them to form more hydrogen bonds with the protein, thereby stabilizing it 29 better. 30 Keywords 31 Near-infrared (NIR) spectroscopy, water-replacement, vitrification, molecular flexibility, 32 solid-state stability, Fourier transform infrared (FTIR) spectroscopy 33 34
FTIR Study of Hydration Phenomena in Protein–Sugar Systems
Journal of Colloid and Interface Science, 1998
The hydration of gelatin-sugar systems was studied by Fourier transform infrared spectroscopy in the transmission mode using direct difference methodology. Information on the hydration kinetics of gelatin was obtained from the changes in intensities of the amide bands. Evidence of molecular interactions between the sugar and the carbonyl group of the protein was obtained from the results describing the hydration of the protein. Such interactions depended on both the concentration and the type of sugar present in the system. The effects of the presence of sugars on the kinetics of hydration of gelatin were interpreted in terms of selective hydration of the protein at low RH and specific molecular interactions between the gelatin and the sugar. The partitioning of water between the two components in the system was found to be an important feature in this study.
BOOK. Organic Solvents: Properties, Toxicity, and Industrial Effects, 2011
This review describes the basic principles of a novel method for studying the structure of the dehydrated proteins in the presence of organic solvents. This method, based on combined calorimetric and FTIR spectroscopic measurements, allows the simultaneous monitoring of the thermochemical parameters (interaction enthalpies, DSC thermograms) of the dried proteins and the corresponding changes in the protein structure in anhydrous organic solvents. This review aims to analyse the effect of organic solvents on dehydrated protein systems in order to understand what intra- and intermolecular processes produce the main effect on the structure and functioning of proteins in low water organic media. Two unrelated proteins with a high -helix content (human serum albumin, HSA) and with a high -sheet content (bovine pancreatic -chymotrypsin, CT) were used as models. Two groups of model organic solvents were used. The first group included hydrogen bond accepting solvents. The second group included hydrogen bond donating liquids. The results obtained showed that: 1) The enthalpy and integral structural changes accompanying the interaction of dried proteins with anhydrous organic solvents depend cooperatively on the solvent hydrophilicity. The solvent hydrophilicity was characterized by an excess molar Gibbs energy of water in organic solvent at infinite dilution and 25oC. Based on this solvent hydrophilicity parameter, the solvents were divided into two groups. The first group included hydrophilic solvents such as methanol, ethanol, and dimethylsulphoxide (DMSO). Considerable structural rearrangements were observed in this group of solvents. The interaction enthalpies of the dried proteins with hydrophilic liquids were strongly exothermic. The second group included the hydrophobic and medium hydrophilic liquids such as benzene, dioxane, butanol-1, and propanol-1. The enthalpy and structural changes in the second group of solvents were close to zero. 2) The FTIR spectroscopic results can be attributed to the formation of different unfolded states of CT and HSA obtained upon dehydration-, alcohol- and DMSO-induced denaturation. The denatured state obtained in DMSO has a maximal degree of unfolding compared with that observed in alcohols or in the presence of dry air. 3) The effect of the organic solvent on the protein structure is “protein selective”. On the other hand, the organic solvent-induced integral structural changes versus solvent hydrophilicity profiles do not depend on the predominant form of secondary structure in the protein. 4) Heat-induced exothermic peaks were observed on the DSC thermograms of the dried proteins in anhydrous organic solvents in the temperature range 60-105 oC. This means that dehydrated proteins in anhydrous solvents is the non-equilibrium state at room temperature. These results give strong support to the idea that the non-equilibrium status of the dehydrated proteins results from the protein–organic solvent interactions being “frozen” at near room temperature. The thermodynamic and structural data were analysed to give a unified picture of the state of the dried proteins in anhydrous organic solvents. According to this model, the dehydration-induced protein-protein contacts and the potential of the organic solvent to form the hydrogen bonds are key factors in determining the structure of the dehydrated proteins in the liquids under study.
Protein Modifications in High Protein-Oil and Protein-Oil-Sugar Systems at Low Water Activity
Food Biophysics, 2013
Physicochemical and thermal properties of high protein systems during storage at 20 and 40°C were investigated for 14 weeks. Component interactions of whey protein isolate (WPI)-olive oil (OO), WPI-sunflower oil (SO) (75:25), WPI-(glucose-fructose; G-F) (45:40), WPI-OO-(G-F), and WPI-SO-(G-F) (45:15:40) systems at low water contents during storage were derived from differential scanning calorimetry (DSC), colorimetric, water activity (a w ), reducing and nonreducing SDS-PAGE electrophoresis data. The degree of unsaturation of oil affected color (yellowness) and microstructure of the systems as well as variations in water migration and nonenzymatic browning kinetics (NEB) during storage. These effects were evident in the SO systems. All systems at 40°C showed changes in protein conformation to those favoring hydrophobic interactions with oil. These systems showed decreased a w , insolubilization, hardening as a result of carbonyl-amine polymerization and covalent cross-linking of proteins in the NEB. The DSC data showed a protein hydration transition for rehumidified-WPI, WPI-oil, WPI-sugar, and WPI-oil-sugar. The rehumidified-WPI and WPI-oil also showed a w -dependent denaturation endotherms (irreversible transition) for α-lactalbumin and β-lactoglobulin at higher temperatures (T). The WPI-sugar and WPI-oil-sugar showed an exotherm for the browning reaction (irreversible transition) at T onset~9 0°C. An exothermic protein hydration in the systems containing sugar was storage time-dependent, and indicated changes of protein conformation. The presence of oil in WPI-oil-sugar caused an increase in the glass transition of sugars during storage, especially for SO. The WPI-(G-F) and WPI-oil-(G-F) showed broadened glass transition during a reheating scan in DSC that was a result of polymerization in protein, oil, and sugar components mixture. Stability of high protein systems is dependent on hydration and reactions in both hydrophilic and hydrophobic phases.
Journal of Pharmaceutical Sciences, 2009
The purpose of this study is to investigate protein-sugar interactions in dried protein solids as a function of sucrose level using water sorption isotherm data and secondary structure information from Fourier transform infrared (FTIR) spectroscopy. Three IgG1 fusion proteins and two cytokines were freeze-dried with sucrose at different sucrose/protein mass ratios. The water monolayer of the colyophilized sucrose/protein samples, as determined by BET analysis of water sorption data, was found to be lower than that expected based on additive contributions of pure protein and pure sucrose. This negative deviation suggests the presence of a solid-state interaction between protein and sucrose that reduces the availability of total water-binding sites. The difference in water monolayer between colyophilized and a physical mixture of protein and sucrose reached a maximum value at sucrose/protein mass ratio of 1/1 for these proteins, suggesting saturation of the protein-sugar interaction at this ratio. In addition, for four proteins studied, the normalized peak height of the major band in the FTIR spectra reached a plateau at about a 1/1 mass ratio. Therefore, it appears that there is a coupling between the preservation of protein secondary structure and the protein-sugar interaction as measured by water sorption isotherms. ß
BOOK. Analysis of the Organic Solvent Effect on the Hydration-Dehydration and Structure of Proteins by FTIR Spectroscopy., 2007
Sirotkin, V.A. Analysis of the Organic Solvent Effect on the Hydration-Dehydration and Structure of Proteins by FTIR Spectroscopy. In: Methods in Protein Structure and Stability Analysis, Uversky, V.N. and Permyakov, E.A. (Eds.). - Nova Science Publishers, Inc., Hauppauge, NY, - 2007, P. 195-229. ABSTRACT This work describes basic principles of a novel method for studying the hydration-dehydration and structure of proteins in the presence and absence of organic solvents. This method based on FTIR spectroscopic measurements provides the simultaneous monitoring of the adsorption-desorption of water and organic solvent on protein films and the corresponding changes in the protein secondary structure in the thermodynamic water activity range from 0 to 1.0 at 25oC. Two unrelated proteins with a high -helix content (human serum albumin) and with a high -sheet content (bovine pancreatic -chymotrypsin) were used as models. Dioxane was used as a model organic solvent. This organic solvent may be considered as an informative molecular probe for analyzing the effect of hydrogen bonding and hydrophobic interactions on the hydration-dehydration and structure of proteins. The obtained results show that (1) The hydration and structure of proteins depend markedly on how the proteins have been hydrated – whether in the presence or in the absence of organic solvent. (2) The sorption and structural isotherms for the first adsorption-desorption cycle obtained in the presence of organic solvent show pronounced hysteresis. This result is consistent with the idea that the state of dehydrated proteins is a non-equilibrium state relative to the sorption of organic solvent. (3) The isotherms for the second adsorption-desorption cycle exhibit no hysteresis within the limits of experimental error. This means that the sorption and structural isotherms started from the high water activity value (aw~0.98) represent true equilibrium conditions in the whole range of water activity. (4) The organic solvent effect on the protein secondary structure is “protein selective”. On the other hand, the organic solvent-induced structural changes – water activity profiles do not depend on the predominant form of secondary structure in the protein. The results from the thermodynamic and structural measurements were analysed to give a unified picture of the hydration-dehydration process of proteins in the presence of organic solvent. According to this model, the dehydration-induced protein-protein contacts are one of the important factors that determine the protein activity – water activity and organic solvent-induced conformational rearrangements – water activity profiles.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2001
Calorimetric heat effects and structural rearrangements assessed by means of Fourier transform infrared (FTIR) amide I spectra were followed by immersing dry human serum albumin and bovine pancreatic K-chymotrypsin in low water organic solvents and in pure water at 298 K. Enthalpy changes upon immersion of the proteins in different media are in a good linear correlation with the corresponding IR absorbance changes. Based on calorimetric and FTIR data the solvents were divided into two groups. The first group includes carbon tetrachloride, benzene, nitromethane, acetonitrile, 1,4-dioxane, n-butanol, n-propanol and pyridine where no significant heat evolution and structural changes were found during protein immersion. Due to kinetic reasons no significant protein^solvent interactions are expected in such systems. The second group of solvents includes dimethyl sulfoxide, methanol, ethanol, and water. Immersion of proteins in these media results in protein swelling and involves significant exothermic heat evolution and structural changes in the protein. Dividing of different media in the two groups is in a qualitative correlation with the solvent hydrophilicity defined as partial excess molar Gibbs free energy of water at infinite dilution in a given solvent. The first group includes the solvents with hydrophilicity exceeding 2.7 kJ/mol. More hydrophilic second group solvents have this energy values less than 2.3 kJ/mol. The hydrogen bond donating ability of the solvents also assists in protein swelling. Hydrogen bonding between protein and solvent is assumed to be a main factor controlling the swelling of dry solid proteins in the studied solvents. ß
Insufficiently dehydrated hydrogen bonds as determinants of protein interactions
Proceedings of the National Academy of Sciences, 2003
The prediction of binding sites and the understanding of interfaces associated with protein complexation remains an open problem in molecular biophysics. This work shows that a crucial factor in predicting and rationalizing protein-protein interfaces can be inferred by assessing the extent of intramolecular desolvation of backbone hydrogen bonds in monomeric structures. Our statistical analysis of native structures shows that, in the majority of soluble proteins, most backbone hydrogen bonds are thoroughly wrapped intramolecularly by nonpolar groups except for a few ones. These latter underwrapped hydrogen bonds may be dramatically stabilized by removal of water. This fact implies that packing defects are ''sticky'' in a way that decisively contributes to determining the binding sites for proteins, as an examination of numerous complexes demonstrates.