Maintenance Of Protein Stability Research Articles

Tandem Affinity Purification of FITC-Labelled Target Protein for Biomedical Applications

Fluorescence-based assays are dynamic, sensitive and robust quantitative methods for studying the conformational plasticity, enzyme kinetics, dynamics, and macromolecular interactions. However, the caveat for most of these experimental approaches is to label the target protein with one or more extrinsic fluorophores having required photophysical characteristics. Fluorescein isothiocyanate (FITC), due to its high quantum efficiency and conjugate stability, is most widely used fluorescence labelling reagent for such experimental approaches. However, the limitations in this method are requirement of high protein concentration, maintenance of protein stability during the entire labelling process as well as presence of significant background fluorescence due to ineffective removal of unreacted FITC. Therefore, to circumvent these setbacks, we have devised a novel strategy through introduction of tandem affinity purification tags at both the N- and C-termini of the protein of interest. Using this method, we have efficiently labelled target protein with significant decrease in precipitation, degradation and background fluorescence due to residual FITC. This facile and rapid technique may also be used as a basis for labelling procedures with other fluorophores and hence has a broad application in the field of biomedical sciences. Figure 1

Effect of microwave heating on physicochemical properties, protein composition and structure, and micromorphology of camel and bovine milk samples

This study aimed to investigate the effect of microwave heating (0, 100, 300, 500, and 700 W; 2 min) on the physicochemical properties, protein composition, and structure and micromorphology of camel and bovine milk samples. Results showed that the physicochemical properties, including pH, protein content, ash content, and soluble solids of both milk samples, decreased after 300 W microwave processing (P < 0.05). The serum albumin and immunoglobulin levels of bovine milk decreased after 300 W processing (P < 0.05), and camel milk whey proteins were more heat resistant than bovine milk proteins. The decreased relative content of κ-casein and the absence of β-lactoglobulin were also found in camel milk during 300 to 700 W processing (P < 0.05). Besides, microwave heating after 300 W induced a conformational transformation and reduced the protein stability of both milk samples. The particle size and scanning electron microscopy images confirmed that the protein structure was changed after 300 W processing. These results indicated that microwave heating at 300 W for 2 min was beneficial to the retention of nutrients (proteins and soluble solids) and the maintenance of protein stability in both milk samples.

Ectoine and Hydroxyectoine Stabilize Antibodies in Spray-Dried Formulations at Elevated Temperature and during a Freeze/Thaw Process.

Maintenance of protein stability during manufacture, storage, and delivery is necessary for the successful development of a drug product. Herein, the utility of two compatible solutes-ectoine and hydroxyectoine-in stabilizing a model protein labeled Fab2 has been investigated. Specifically, the performance of ectoine and hydroxyectoine in stabilizing Fab2 in a spray-dried formulation at elevated temperature and after multiple freeze/thaw cycles has been compared with the performance of a formulation containing trehalose and a formulation containing no excipient as controls. In the solid state at 90 and 37 °C and in freeze concentrate systems, ectoine and hydroxyectoine suppress protein aggregation. Like trehalose, hydroxyectoine also limits N-terminal pyroglutamate formation in Fab2 in the solid state. The extent of protein stabilization is dependent on the excipient concentration in the formulation, but at a 1:1 excipient to protein mass ratio, hydroxyectoine is better than trehalose in stabilizing Fab2. The results presented here suggest that ectoine and hydroxyectoine are effective excipients for stabilizing therapeutic antibodies.

An efficient method for FITC labelling of proteins using tandem affinity purification.

Fluorescence-based assays are extremely diverse, sensitive and robust experimental methods for investigating the conformational changes, enzyme kinetics, dynamics and molecular interactions. A prerequisite for most of these experimental approaches is to label the protein of interest with one or more extrinsic fluorophores with desired photophysical properties. Fluorescein isothiocyanate (FITC), due to its high quantum efficiency and conjugate stability, is most widely used fluorescence labelling reagent for such experimental approaches. However, the bottlenecks in this labelling reaction is requirement of high protein concentration, maintenance of protein stability during the labelling process as well as high background fluorescence due to ineffective removal of unreacted FITC, prior to fluorescence studies. Therefore, to overcome these inadequacies or limitations, we have modified the existing protocol by introducing tandem affinity purification tags at the N- and C-terminus of target protein. Using this modified method, we have efficiently labelled target protein with significant decrease in precipitation, degradation and background fluorescence of unreacted FITC. This facile and rapid technique may also be used as a basis for labelling procedures with other fluorophores and hence has a broad application in spectroscopic studies.

Changes in protein profile of Platycladus orientalis (L.) roots and leaves in response to drought stress

Platycladus orientalis (L.) Franco is widely used for afforestation in arid and semi-arid areas due to its high drought tolerance. To better understand the mechanism involved in drought tolerance in this important tree, responses to drought stress have been studied in 1-year-old P. orientalis via water withholding. Several physiological parameters were evaluated in four drought-treated groups. The root and leaf proteomes of two-dimensional electrophoresis (2-DE) gels were obtained, and a total of 162 proteins with significant quantitative variations and 1.5-fold differences in proteins were selected and identified by matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry (MS/MS). The majority of identified proteins were classified into functional categories, including stress response/defense, carbohydrate metabolic process, nitrogen metabolism, proteolysis, and photosynthesis. The proteome results revealed that a series of strategies were employed to survive in the drought environment, such as the maintenance of protein stability, activation, and folding; reactive oxygen species (ROS) detoxification; the regulation of cell osmotic conditions and cell wall integrity; energy metabolism; and stabilization of the cell skeleton. One of the most prominent findings in this study was the number of bark protein-like proteins and heat shock proteins detected in both root and leaf tissues in drought stress conditions. In addition, 12 differentially expressed proteins were selected for quantitative reverse transcription PCR analysis; not all of the protein expression levels were consistent with the mRNA expression levels. Our data provide a comprehensive picture of the root and leaf responses under varying watering regimes, which could be beneficial for further research and for understanding highly complex drought stress responses.

Optimization of an in vitro assay methodology for competitive binding of thyroidogenic xenobiotics with thyroxine on human transthyretin and albumin

Thyroid hormones (THs) are involved in the regulation of many physiological processes in vertebrates. Competition for TH binding sites on serum transport proteins can interfere with delivery of THs to target tissues, and this is a potential mechanism of action of exogenous thyroidogenic substances. To date, detailed accounts of in vitro methods for competitive binding with THs on TH transport proteins (human or wildlife) are sparse. In the limited number of published studies on in vitro radio-labelled TH-TH transport protein interactions, method descriptions were brief and with insufficient details for successful replication. Furthermore, upon review of these methodologies, we identified several opportunities for optimization. The present study addresses the methodological deficiencies and describes, in detail, a fully optimized and validated competitive T4 radio-ligand binding assay with human transthyretin (TTR) and albumin (ALB).•Significant improvements were made over previous methods, including better maintenance of protein stability and enhanced measurement of competition between different ligands.•Sample size was reduced to allow use of small pre-packed size exclusion chromatography columns, which eliminates the rinsing step during the separation procedure.•The assay was parameterized for use with T4 and human TTR and ALB.

Molecular cloning and differential expression of sHSP gene family members from the resurrection plant Boea hygrometrica in response to abiotic stresses

AbstractSmall heat shock proteins (sHSPs) are a class of molecular chaperones that bind to and prevent aggregation of proteins. To assess the potential role of sHSPs in protection against abiotic stresses, we conducted a screening of sHSP genes from the desiccation-tolerant resurrection plant Boea hygrometrica, which is widespread in East Asia in alkaline, calcium-rich limestone crevices. In total, 25 sHSP genes, belonging to six subgroups, were identified from the desiccated leaves of B. hygrometrica. Ten of these genes were cloned and named according to the nomenclature proposed for sHSPs. Transcripts of all these BhsHSPs were detectable in fresh leaves, but only 6 genes were induced after desiccation, and remained high during rehydration. Four of the cytosol-targeted BhsHSP genes were up-regulated under treatments, such as heat, cold, alkaline conditions, high calcium, oxidation, or application of the phytohormone abscisic acid. Together, these results demonstrate that CI and CII sHSPs, especially Bh17.9CI and Bh17.4BCII, are associated with abiotic stresses, and may function in the maintenance of protein stability, aiding in the adaptations to extreme environmental conditions in which B. hygrometrica can survive.

The Relationship Between Conservation, Thermodynamic Stability, and Function in the SH3 Domain Hydrophobic Core

To investigate the relationships between sequence conservation, protein stability, and protein function, we have measured the thermodynamic stability, folding kinetics, and in vitro peptide-binding activity of a large number of single-site substitutions in the hydrophobic core of the Fyn SH3 domain. Comparison of these data to that derived from an analysis of a large alignment of SH3 domain sequences revealed a very good correlation between the distinct pattern of conservation observed at each core position and the thermodynamic stability of mutants. Conservation was also found to correlate well with the unfolding rates of mutants, but not to the folding rates, suggesting that evolution selects more strongly for optimal native state packing interactions than for maximal folding rates. Structural analysis suggests that residue–residue core packing interactions are very similar in all SH3 domains, which provides an explanation for the correlation between conservation and mutant stability effects studied in a single SH3 domain. We also demonstrate a correlation between stability and the in vivo activity of mutants, and between conservation and activity. However, the relationship between conservation and activity was very strong only for the three most conserved hydrophobic core positions. The weaker correlation between activity and conservation seen at the other seven core positions indicates that maintenance of protein stability is the dominant selective pressure at these positions. In general, the pattern of conservation at hydrophobic core positions appears to arise from conserved packing constraints, and can be effectively utilized to predict the destabilizing effects of amino acid substitutions.

The Role of N-Glycosylation in Transport to the Plasma Membrane and Sorting of the Neuronal Glycine Transporter GLYT2

Glycine transporter GLYT2 is an axonal glycoprotein involved in the removal of glycine from the synaptic cleft. To elucidate the role of the carbohydrate moiety on GLYT2 function, we analyzed the effect of the disruption of the putative N-glycosylation sites on the transport activity, intracellular traffic in COS cells, and asymmetrical distribution of this protein in polarized Madin-Darby canine kidney (MDCK) cells. Transport activity was reduced by 35-40% after enzymatic deglycosylation of the transporter reconstituted into liposomes. Site-directed mutagenesis of the four glycosylation sites (Asn-345, Asn-355, Asn-360, and Asn-366), located in the large extracellular loop of GLYT2, produced an inactive protein that was retained in intracellular compartments when transiently transfected in COS cells or in nonpolarized MDCK cells. When expressed in polarized MDCK cells, wild type GLYT2 localizes in the apical surface as assessed by transport and biotinylation assays. However, a partially unglycosylated mutant (triple mutant) was distributed in a nonpolarized manner in MDCK cells. The apical localization of GLYT2 occurred by a glycolipid rafts independent pathway.

Desiccation tolerance of prokaryotes

The removal of cell-bound water through air drying and the addition of water to air-dried cells are forces that have played a pivotal role in the evolution of the prokaryotes. In bacterial cells that have been subjected to air drying, the evaporation of free cytoplasmic water (Vf) can be instantaneous, and an equilibrium between cell-bound water (Vb) and the environmental water (vapor) potential (psi wv) may be achieved rapidly. In the air-dried state some bacteria survive only for seconds whereas others can tolerate desiccation for thousands, perhaps millions, of years. The desiccated (anhydrobiotic) cell is characterized by its singular lack of water--with contents as low as 0.02 g of H2O g (dry weight)-1. At these levels the monolayer coverage by water of macromolecules, including DNA and proteins, is disturbed. As a consequence the mechanisms that confer desiccation tolerance upon air-dried bacteria are markedly different from those, such as the mechanism of preferential exclusion of compatible solutes, that preserve the integrity of salt-, osmotically, and freeze-thaw-stressed cells. Desiccation tolerance reflects a complex array of interactions at the structural, physiological, and molecular levels. Many of the mechanisms remain cryptic, but it is clear that they involve interactions, such as those between proteins and co-solvents, that derive from the unique properties of the water molecule. A water replacement hypothesis accounts for how the nonreducing disaccharides trehalose and sucrose preserve the integrity of membranes and proteins. Nevertheless, we have virtually no insight into the state of the cytoplasm of an air-dried cell. There is no evidence for any obvious adaptations of proteins that can counter the effects of air drying or for the occurrence of any proteins that provide a direct and a tangible contribution to cell stability. Among the prokaryotes that can exist as anhydrobiotic cells, the cyanobacteria have a marked capacity to do so. One form, Nostoc commune, encompasses a number of the features that appear to be critical to the withstanding of a long-term water deficit, including the elaboration of a conspicuous extracellular glycan, synthesis of abundant UV-absorbing pigments, and maintenance of protein stability and structural integrity. There are indications of a growing technology for air-dried cells and enzymes. Paradoxically, desiccation tolerance of bacteria has virtually been ignored for the past quarter century. The present review considers what is known, and what is not known, about desiccation, a phenomenon that impinges upon every facet of the distributions and activities of prokaryotic cells.

Desiccation tolerance of prokaryotes.

The removal of cell-bound water through air drying and the addition of water to air-dried cells are forces that have played a pivotal role in the evolution of the prokaryotes. In bacterial cells that have been subjected to air drying, the evaporation of free cytoplasmic water (Vf) can be instantaneous, and an equilibrium between cell-bound water (Vb) and the environmental water (vapor) potential (psi wv) may be achieved rapidly. In the air-dried state some bacteria survive only for seconds whereas others can tolerate desiccation for thousands, perhaps millions, of years. The desiccated (anhydrobiotic) cell is characterized by its singular lack of water--with contents as low as 0.02 g of H2O g (dry weight)-1. At these levels the monolayer coverage by water of macromolecules, including DNA and proteins, is disturbed. As a consequence the mechanisms that confer desiccation tolerance upon air-dried bacteria are markedly different from those, such as the mechanism of preferential exclusion of compatible solutes, that preserve the integrity of salt-, osmotically, and freeze-thaw-stressed cells. Desiccation tolerance reflects a complex array of interactions at the structural, physiological, and molecular levels. Many of the mechanisms remain cryptic, but it is clear that they involve interactions, such as those between proteins and co-solvents, that derive from the unique properties of the water molecule. A water replacement hypothesis accounts for how the nonreducing disaccharides trehalose and sucrose preserve the integrity of membranes and proteins. Nevertheless, we have virtually no insight into the state of the cytoplasm of an air-dried cell. There is no evidence for any obvious adaptations of proteins that can counter the effects of air drying or for the occurrence of any proteins that provide a direct and a tangible contribution to cell stability. Among the prokaryotes that can exist as anhydrobiotic cells, the cyanobacteria have a marked capacity to do so. One form, Nostoc commune, encompasses a number of the features that appear to be critical to the withstanding of a long-term water deficit, including the elaboration of a conspicuous extracellular glycan, synthesis of abundant UV-absorbing pigments, and maintenance of protein stability and structural integrity. There are indications of a growing technology for air-dried cells and enzymes. Paradoxically, desiccation tolerance of bacteria has virtually been ignored for the past quarter century. The present review considers what is known, and what is not known, about desiccation, a phenomenon that impinges upon every facet of the distributions and activities of prokaryotic cells.