Protective action of trehalose and glucose on protein hydration shell clarified by using X-ray and neutron scattering

Ajito, Satoshi; Hirai, Mitsuhiro; Iwase, Hiroki; Igarashi, Noriyuki; Ohta, Nobuhiro

doi:10.1016/j.physb.2018.03.040

Cited by 28 publications

(37 citation statements)

References 31 publications

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“…The functional role of trehalose as an intracellular osmolyte is to manage the cell volume during exposure to intra-or extracellular osmotic, thermal, and oxidative stresses. Trehalose is a nonionic kosmotrope which preserves the protein hydration shell by reducing the water activity, a w (6). Moreover, during anhydrobiosis trehalose protects the cell membranes by direct binding to phospholipids, preventing water leakage during rehydration (7).…”

mentioning

confidence: 99%

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

Mestrom

Marsden

Dieters

et al. 2019

Appl Environ Microbiol

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LeLoir glycosyltransferases are important biocatalysts for the production of glycosidic bonds in natural products, chiral building blocks, and pharmaceuticals. Trehalose transferase (TreT) is of particular interest since it catalyzes the stereo-and enantioselective ␣,␣-(1¡1) coupling of a nucleotide sugar donor and monosaccharide acceptor for the synthesis of disaccharide derivatives. Heterologously expressed thermophilic trehalose transferases were found to be intrinsically aggregation prone and are mainly expressed as catalytically active inclusion bodies in Escherichia coli. To disfavor protein aggregation, the thermostable protein mCherry was explored as a fluorescent protein tag. The fusion of mCherry to trehalose transferase from Pyrobaculum yellowstonensis (PyTreT) demonstrated increased protein solubility. Chaotropic agents like guanidine or the divalent cations Mn(II), Ca(II), and Mg(II) enhanced the enzyme activity of the fusion protein. The thermodynamic equilibrium constant, K eq , for the reversible synthesis of trehalose from glucose and a nucleotide sugar was determined in both the synthesis and hydrolysis directions utilizing UDPglucose and ADP-glucose, respectively. UDP-glucose was shown to achieve higher conversions than ADP-glucose, highlighting the importance of the choice of nucleotide sugars for LeLoir glycosyltransferases under thermodynamic control. IMPORTANCE The heterologous expression of proteins in Escherichia coli is of great relevance for their functional and structural characterization and applications. However, the formation of insoluble inclusion bodies is observed in approximately 70% of all cases, and the subsequent effects can range from reduced soluble protein yields to a complete failure of the expression system. Here, we present an efficient methodology for the production and analysis of a thermostable, aggregation-prone trehalose transferase (TreT) from Pyrobaculum yellowstonensis via its fusion with mCherry as a thermostable fluorescent protein tag. This fusion strategy allowed for increased enzyme stability and solubility and could be applied to other (thermostable) proteins, allowing rapid visualization and quantification of the mCherry-fused protein of interest. Finally, we have demonstrated that the enzymatic synthesis of trehalose from glucose and a nucleotide sugar is reversible by approaching the thermodynamic equilibrium in both the synthesis and hydrolysis directions. Our results show that uridine establishes an equilibrium constant which is more in favor of the product trehalose than when adenosine is employed as the nucleotide under identical conditions. The influence of different nucleotides on the reaction can be generalized for all LeLoir glycosyltransferases under thermodynamic control as the position of the equilibrium depends solely on the reaction conditions and is not affected by the nature of the catalyst.

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mentioning

confidence: 99%

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

Mestrom

Marsden

Dieters

et al. 2019

Appl Environ Microbiol

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“…maximally separated), they would be separated at a distance of 43 Å rather than 36 Å as shown here. This short distance indicates that, in the two-component sample, where the size of protein is estimated to be about 17 Å [45], barely, a single water layer is permitted in between the protein molecules. It can also partly explain why the peak for the Mb-D 2 O sample is much weaker, since the scattering contrast reduces with almost no solvent between the protein molecules.…”

Section: Resultsmentioning

confidence: 98%

“…If the radius of myoglobin in a trehalose solution is 16 Å (based on the measurements of Ajito et al [45]), the reported inter-protein distance in the three-component system suggests that there would exist regions of water/sugar-solution in between the proteins which are in the order of 8-10 Å. This kind of separation permits both water and trehalose molecules to enter in between protein molecules (at least in regions where the space between the protein molecules is larger than at the closest distance).…”

Section: Resultsmentioning

confidence: 99%

The role of disaccharides for protein–protein interactions – a SANS study

Olsson

Swenson

2019

Molecular Physics

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The disaccharide trehalose has shown outstanding anti-aggregation properties for proteins, which are highly important for the possibility to treat neurodegenerative diseases, such as Alzheimer's and Huntington's disease. However, the role and mechanism of trehalose for such stabilising effects are still largely unknown, partly because a direct structural picture of how trehalose organises around proteins in an aqueous system is missing. Here we compare small-angle neutron scattering (SANS) data on myoglobin in aqueous solutions of either sucrose or trehalose, in order to investigate their effect on protein-protein interactions. We find that both trehalose and sucrose induces a well-defined protein-protein distance, which could explain why these inhibit protein-protein interactions and associated protein aggregation. It does not however explain the superior antiaggregation effect of trehalose and suggests that the local solvent structures are highly important for explaining the protein stabilisation mechanism. In a broader perspective, these findings are important for understanding the role of sugars in biological stabilisation, and could provide a structural explanation for why trehalose is a promising candidate for the treatment of neurodegenerative and other protein aggregation related diseases.

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“…In the light of renewed studies on trehalose behavior in water [35,36], or of trehalose preferential exclusion from protein hydration shell [37], we report on trehalose effect on amyloid fibrillation of two model proteins explored as a function of ionic strength. For this purpose, we have performed spectrophotometric experiments with Congo Red as specific amyloid dye, Circular Dichroism (CD), and Syncrothron Small Angle X-ray Scattering (SAXS) to monitor the temporal evolution of protein fibrillation, either in the absence or in the presence of NaCl.…”

Section: Introductionmentioning

confidence: 99%

Trehalose Effect on The Aggregation of Model Proteins into Amyloid Fibrils

Mari

Ricci

Pieraccini

et al. 2020

Life

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Protein aggregation into amyloid fibrils is a phenomenon that attracts attention from a wide and composite part of the scientific community. Indeed, the presence of mature fibrils is associated with several neurodegenerative diseases, and in addition these supramolecular aggregates are considered promising self-assembling nanomaterials. In this framework, investigation on the effect of cosolutes on protein propensity to aggregate into fibrils is receiving growing interest, and new insights on this aspect might represent valuable steps towards comprehension of highly complex biological processes. In this work we studied the influence exerted by the osmolyte trehalose on fibrillation of two model proteins, that is, lysozyme and insulin, investigated during concomitant variation of the solution ionic strength due to NaCl. In order to monitor both secondary structures and the overall tridimensional conformations, we have performed UV spectroscopy measurements with Congo Red, Circular Dichroism, and synchrotron Small Angle X-ray Scattering. For both proteins we describe the effect of trehalose in changing the fibrillation pattern and, as main result, we observe that ionic strength in solution is a key factor in determining trehalose efficiency in slowing down or blocking protein fibrillation. Ionic strength reveals to be a competitive element with respect to trehalose, being able to counteract its inhibiting effects toward amyloidogenesis. Reported data highlight the importance of combining studies carried out on cosolutes with valuation of other physiological parameters that may affect the aggregation process. Also, the obtained experimental results allow to hypothesize a plausible mechanism adopted by the osmolyte to preserve protein surface and prevent protein fibrillation.

show abstract

Protective action of trehalose and glucose on protein hydration shell clarified by using X-ray and neutron scattering

Cited by 28 publications

References 31 publications

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

The role of disaccharides for protein–protein interactions – a SANS study

Trehalose Effect on The Aggregation of Model Proteins into Amyloid Fibrils

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