Free Energy Of Denaturation Research Articles

Determinants for 1-alkyl-3-methylimidazolium chloride-induced modulation of thermodynamic stability and CO-association dynamics of horse ferrocytochrome c

This manuscript determines the factors by which the water-miscible ionic liquid (IL), 1-alkyl-3-methylimidazolium chloride [Rmim]Cl affects the thermodynamic stability and CO association dynamics of horse ferrocytochrome c (Cyt cII) at pH 7. Investigation of rate constant and activation thermodynamic parameters for CO-association reaction of Cyt cII measured at different concentrations of [Rmim]Cl (1-Ethyl-3-methylimidazolium chloride [Emim]Cl, 1-butyl-3-methylimidazolium chloride [Bmim]Cl, 1-hexyl-3-methylimidazolium chloride [Hmim]Cl) revealed that the hydrophobicity of alkyl group of [Rmim]Cl modulates the CO-association dynamics of Cyt cII. The CO-association association kinetic results were further supported by intermolecular docking studies between the Cyt cII and [Rmim]+ ([Hmim]Cl, [Bmim]Cl, [Emim]Cl). Analysis of thermodynamic parameters calculated from thermal denaturation transitions of Cyt cII at different concentrations of [Rmim]Cl ([Hmim]Cl, [Bmim]Cl, [Emim]Cl) revealed that the [Rmim]Cl decreases the denaturation free energy in the order: [Hmim]Cl > [Bmim]Cl > [Emim]Cl, which suggests that the hydrophobicity of alkyl group of [Rmim]Cl modulates the [Rmim]Cl-induced decrease in thermodynamic stability of Cyt cII. Analysis of entropy − enthalpy plots showed that in addition to the hydrophobic effect, the enthalpic effect of [Rmim]Cl also decreases the thermodynamic stability of the protein. The dependence of unfolding free energy on [Rmim]Clconcentration was also translated into preferential interaction coefficients, Γ23. Experimental and theoretical estimations of Γ23 for Cyt cII provided the positive values of Γ23 at all across the [Rmim]Cl concentrations, indicating a robust and favorable interaction between the [Rmim]Cl and Cyt cII. Furthermore, the [Rmim]Cl-mediated increase in Γ23 typically follows the order: [Hmim]Cl > [Bmim]Cl > [Emim]Cl. Molecular dynamics (MD) simulation studies of Cyt cII performed at different concentrations of [Rmim]Cl ([Emim]Cl, [Bmim]Cl, and [Hmim]Cl) further showed that the hydrophobicity of the alkyl group modulates the structural stability and conformational-fluctuations of the Cyt cII.

Production, optimization, and purification of alkaline thermotolerant protease from newly isolated Phalaris minor seeds

The present work aims to purify and perform a preliminary analysis on a thermostable serine alkaline protease from a recently identified P. minor. The enzyme was purified 2.7-fold with a 12.4 % recovery using Sephadex G-100 chromatography, DEAE-cellulose, and ammonium sulphate precipitation. The isolated enzyme has a specific activity of 473 U/mg. The purified protease had a molecular mass of 29 kDa, and just one band was seen, which matched the band obtained using SDS-PAGE. High thermostability was demonstrated by the enzymes, which had half-lives of 31.79 and 6.0 min (a 5.3-fold improvement), enthalpies of denaturation (ΔH°) of 119.53 and 119.35 KJ mol−1, entropies of denaturation (ΔS°) of 32.96 and 41.11 J/mol·K, and free energies of denaturation (ΔG°) of 108.87 and 105.58 KJ mol−1 for the protease enzyme. Studies on the folding and stability of alkaline proteases are important since their use in biotechnology requires that they operate in settings of extreme pH and temperature. According to the kinetic and thermodynamic properties, the protease produced by P. minor is superior to that produced by other sources and previously described plants, and it might find utility in a variety of industrial fields.

Temperature-Dependent Conformational Evolution of SARS CoV-2 RNA Genome Using Network Analysis.

Understanding the dynamics of the SARS CoV-2 RNA genome and its dependence on temperature is necessary to fight the current COVID-19 crisis. Computationally, the handling of large data is a major challenge in the elucidation of the structures of RNA. This work presents network analysis as an important tool to see the conformational evolution and the most dominant structures of the RNA genome at six different temperatures. It effectively distinguished different communities of RNA having structural variation. It is found that at higher temperatures (348 K and above), 80% of the RNA structure is destroyed in both the SPC/E and mTIP3P water models. The thermal denaturation free energy change ΔΔG value calculated for the long-lived structure at higher temperatures of 348 and 363 K ranges from 2.58 to 2.78 kcal/mol for the SPC/E water model, which agrees well with the experimentally reported thermal denaturation free energy range of 2.874 kcal/mol of SARS CoV-NP at normal pH. At higher temperatures, the stability of RNA conformation is found to be due to the existence of non-native base pairs in the SPC/E water model.

Comparative kinetic analysis of alginate lyase and mannanase co-produced via solid-state fermentation of cow dung supplemented with seaweed wastes by a novel Streptomyces sp. Alg-S23

The global industrial interest in the application of biocatalysts to manufacture green and sustainable chemicals necessitates cost-effective production of microbial enzymes via screening out potent microbial isolates from different environmental niches and identifying cheap feedstocks for their propagation. In this study, the biocatalytic efficacy of a novel Streptomyces sp. Alg-S23 (GenBank accession no. MK098188) isolated from putrefying Sargassum seaweed waste was assessed for the first time for co-production of alginate lyase and mannanase via solid-state fermentation of cow dung supplemented with decomposed Sargassum as the feedstocks. The optimization of the SSF feedstock components by using a 22-factorial central composite statistical design resulted in the enhanced co-production of alginate lyase and mannanase at 15 g of cow dung and 4.5 g of decomposed Sargassum biomass. Maximal activities of extracted alginate lyase and mannanase were recorded at pH 7.5 and at temperature 50 °C and 55 °C, respectively. The co-produced alginate lyase and mannanase had strong affinity toward the substrate by displaying Km and Vmax values as 0.72 mg/mL and 10.2 U/mg-protein, and 1.83 mg/mL and 78.7 U/mg-protein, respectively. Thermal denaturation kinetic analysis of the co-produced alginate lyase and mannanase at 40-60 °C suggested that both the enzymes were thermodynamically efficient by exhibiting higher values of activation, enthalpy, and free energies of denaturation. These promising enzymatic traits highlight the novel potential of cow dung supplemented with decomposed Sargassum seaweed waste as the inexpensive feedstocks for sustainable production of alginate lyase and mannanase.

Conformational Entropy as a Determinant of the Thermodynamic Stability of the p53 Core Domain.

Mutations in the core domain of tumor suppressor protein p53 have been associated with ∼50% of the occurrences of human cancers. A majority of these mutations inactivate p53 function by destabilizing its native structure. Although studies have shown p53's function can be restored by stabilizing the mutants to their wild-type conformation with immense therapeutic potential, its applicability has been restricted because of our limited understanding of the precise nature of destabilization arising from changes in the mutant p53's structure and dynamics. Here, using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations, we have probed the conformational flexibility in three of the most widespread and clinically important "hot spot" mutants of the p53 core domain. Our results show that NMR order parameter-derived conformational entropy is linearly correlated with the change in free energy of urea-mediated denaturation, the latter being a well-established reporter of stability in p53 core domain mutants. Using a linear regression function, we show that the three parameters of equilibrium denaturation experiments, i.e., the free energy of denaturation (Δ GD-NH2O), the slope of the transition ( m), and the urea concentration at 50% denaturation ([urea]50%), can be used to predict the conformational entropy in p53 core domain mutants, thereby demonstrating a method for using these parameters as predictors of a protein's conformational entropy, which has been known to shape the functional properties of proteins.

Quinary interactions with an unfolded state ensemble.

Anfinsen's thermodynamic hypothesis states that the native three-dimensional fold of a protein represents the structure with the lowest Gibbs free energy. Changes in the free energy of denaturation can arise from changes to the folded state, the unfolded state, or both. It has been recently recognized that quinary interactions, transient contacts that take place only in cells, can modulate protein stability through interactions involving the folded state. Here we show that the cellular environment can also remodel the unfolded state ensemble.

Thermodynamic and saccharification analysis of cloned GH12 endo-1,4-β-glucanase from Thermotoga petrophila in a mesophilic host.

The thermotolerant endo-1,4-β-glucanase gene, of Thermotoga petrophila RKU-1, was cloned and over-expressed in E. coli strain BL21 CodonPlus. Enzyme was purified to homogeneity, producing a single band on SDS-PAGE corresponding to 38 kDa, by purification steps of heat treatment combined with ion-exchange column chromatography. The purified enzyme was optimally active, with specific activity of 530 Umg(-1) against carboxymethyl cellulose (CMC), at pH 6.0 and 95°C and was also stable upto 8 h at 80°C. The enzyme also showed activity against β-glucan barley: 303 %, laminarin: 13.7 %, Whatman filter paper: 0.017 % with no activity against starch and Avicel. The recombinant enzyme exhibited Km, Vmax and Kcat of 12.5 mM, 735 µmol mg-1min-1 and 2351.23 s-1, respectively against CMC as a substrate. The stable recombinant enzyme manifested half life (t1/2) of 6.6 min even at temperature as high as 97°C, with free energy of denaturation (ΔG*D), enthalpy of denaturation (ΔH*D), and entropy of denaturation (ΔS*D) of 98.2 kJ mol(-1), 528.9 kJ mol(-1), and 1.17 kJ mol(-1)K(-1), respectively at 97°C. In addition, the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for hydrolysis of CMC substrate by endo-1,4-β-glucanase were calculated at 95°C as 48.2 kJ mol(-1), 54.6 kJ mol(-1) and -17.4 J mol(-1) K(-1), respectively. The recombinant enzyme saccharified pre-treated wheat straw and bagasse to 3.32 % and 3.2 %, respectively after 6 h incubation at 85°C. Its thermostability, resistance to heavy metal ions and specific activity make this enzyme an interesting candidate for industrial applications.

Characterization of salt-tolerant β-glucosidase with increased thermostability under high salinity conditions from Bacillus sp. SJ-10 isolated from jeotgal, a traditional Korean fermented seafood.

The β-glucosidase gene, bglC, was cloned from Bacillus sp. SJ-10 isolated from the squid jeotgal. Recombinant BglC protein overexpression was induced in Escherichia coli. The optimal pH and temperature of the enzyme, using p-nitrophenyl-β-D-glucopyranoside (pNPβGlc) as a substrate, were pH 6 and 40 °C, respectively. Enzymatic activity increased by 3.3- and 3.5-fold in the presence of 15% NaCl and KCl, respectively. Furthermore, enzyme thermostability improved in the presence of NaCl or KCl. At 45 °C in the presence of salts, the enzyme was stable for 2 h and maintained 80% activity. In the absence of salts, BglC completely lost activity after 110 min at 45 °C. Comparison of the kinetic parameters at various salt concentrations revealed that BglC had approximately 1.5- and 1.2-fold higher affinity and hydrolyzed pNPβGlc 1.9- and 2.1-fold faster in the presence of 15% NaCl and KCl, respectively. Additionally, the Gibb's free energy for denaturation was higher in the presence of 15% salt than in the absence of salt at 45 and 50 °C. Since enzymatic activity and thermostability were enhanced under high salinity conditions, BglC is an ideal salt-tolerant enzyme for further research and industrial applications.

Design, expression and characterization of a highly stable tetratricopeptide-based protein scaffold for phage display application.

Tetratricopeptide repeat (TPR) is a structural motif mediating variety of protein-protein interactions. It has a high potential to serve as a small, stable and robust, non-immunoglobulin ligand binding scaffold. In this study, we showed the consensus approach to design the novel protein called designed tetratricopeptide repeat (dTPR), composed of three repeated 34 amino-acid tetratricopeptide motifs. The designed sequence was efficiently overexpressed in E. coli and purified to homogeneity. Recombinant dTPR is monomeric in solution and preserves its secondary structure within the pH range from 2.0 to 11.0. Its denaturation temperature at pH 7.5 is extremely high (104.5°C) as determined by differential scanning calorimetry. At extreme pH values the protein is still very stable: denaturation temperature is 90.1°C at pH 2.0 and 60.4°C at pH 11. Chemical unfolding of the dTPR is a cooperative, two-state process both at pH 7.5 and 2.0. The free energy of denaturation in the absence of denaturant equals to 15.0 kcal/mol and 13.5 kcal/mol at pH 7.5 and 2.0, respectively. Efficient expression and extraordinary biophysical properties make dTPR a promising framework for a biotechnological application, such as generation of specific ligand- binding molecules.

The Role of Protein Denaturation Energetics and Molecular Chaperones in the Aggregation and Mistargeting of Mutants Causing Primary Hyperoxaluria Type I

Primary hyperoxaluria type I (PH1) is a conformational disease which result in the loss of alanine:glyoxylate aminotransferase (AGT) function. The study of AGT has important implications for protein folding and trafficking because PH1 mutants may cause protein aggregation and mitochondrial mistargeting. We herein describe a multidisciplinary study aimed to understand the molecular basis of protein aggregation and mistargeting in PH1 by studying twelve AGT variants. Expression studies in cell cultures reveal strong protein folding defects in PH1 causing mutants leading to enhanced aggregation, and in two cases, mitochondrial mistargeting. Immunoprecipitation studies in a cell-free system reveal that most mutants enhance the interactions with Hsc70 chaperones along their folding process, while in vitro binding experiments show no changes in the interaction of folded AGT dimers with the peroxisomal receptor Pex5p. Thermal denaturation studies by calorimetry support that PH1 causing mutants often kinetically destabilize the folded apo-protein through significant changes in the denaturation free energy barrier, whereas coenzyme binding overcomes this destabilization. Modeling of the mutations on a 1.9 Å crystal structure suggests that PH1 causing mutants perturb locally the native structure. Our work support that a misbalance between denaturation energetics and interactions with chaperones underlie aggregation and mistargeting in PH1, suggesting that native state stabilizers and protein homeostasis modulators are potential drugs to restore the complex and delicate balance of AGT protein homeostasis in PH1.

Ternary Interpolyelectrolyte Complexes Insulin-Poly(methylaminophosphazene)-Dextran Sulfate for Oral Delivery of Insulin

Ternary interpolyelectrolyte complexes of insulin with biodegradable synthetic cationic polymer, poly(methylaminophosphazene) hydrochloride (PMAP), and dextran sulfate (DS) were investigated by means of turbidimetry, dynamic light scattering, phase analysis, and high-sensitivity differential scanning calorimetry. Formation of ternary insoluble stoichiometric Insulin-PMAP-DS complexes was detected under conditions imitating the human gastric environment (pH 2, 0.15 M NaCl). A complete immobilization of insulin in the complexes was observed in a wide range of the reaction mixture compositions. The ternary complexes were shown to dissolve and dissociate under conditions imitating the human intestinal environment (pH 8.3, 0.15 M NaCl). The products of the complex dissociation were free insulin and soluble binary Insulin-PMAP complexes. The conformational stability of insulin in the soluble complexes of various compositions was investigated by high-sensitivity differential scanning calorimetry. The dependence of the excess denaturation free energy of insulin in these complexes on the PMAP content was obtained. The binding constants of the folded and unfolded forms of insulin to the PMAP polycation were estimated. Proteolysis of insulin involved in the insoluble ternary complexes by pepsin was investigated under physiological conditions. It was found that the complexes ensure an almost 100% protection of insulin against proteolytic degradation. The obtained results provide a perspective basis for development of oral insulin preparations.

Structural and Energetic Basis of Protein Kinetic Destabilization in Human Phosphoglycerate Kinase 1 Deficiency

Protein kinetic destabilization is a common feature of many human genetic diseases. Human phosphoglycerate kinase 1 (PGK1) deficiency is a rare genetic disease caused by mutations in the PGK1 protein, which often shows reduced kinetic stability. In this work, we have performed an in-depth characterization of the thermal stability of the wild type and four disease-causing mutants (I47N, L89P, E252A, and T378P) of human PGK1. PGK1 thermal denaturation is a process under kinetic control, and it is described well by a two-state irreversible denaturation model. Kinetic analysis of differential scanning calorimetry profiles shows that the disease-causing mutations decrease PGK1 kinetic stability from ~5-fold (E252A) to ~100000-fold (L89P) compared to that of wild-type PGK1, and in some cases, mutant enzymes are denatured on a time scale of a few minutes at physiological temperature. We show that changes in protein kinetic stability are associated with large differences in enthalpic and entropic contributions to denaturation free energy barriers. It is also shown that the denaturation transition state becomes more nativelike in terms of solvent exposure as the protein is destabilized by mutations (Hammond effect). Unfolding experiments with urea further suggest a scenario in which the thermodynamic stability of PGK1 at least partly determines its kinetic stability. ATP and ADP kinetically stabilize PGK1 enzymes, and kinetic stabilization is nucleotide- and mutant-selective. Overall, our data provide insight into the structural and energetic basis underlying the low kinetic stability displayed by some mutants causing human PGK1 deficiency, which may have important implications for the development of native state kinetic stabilizers for the treatment of this disease.

Communication: Nanoscale electrostatic theory of epistructural fields at the protein-water interface

Nanoscale solvent confinement at the protein-water interface promotes dipole orientations that are not aligned with the internal electrostatic field of a protein, yielding what we term epistructural polarization. To quantify this effect, an equation is derived from first principles relating epistructural polarization with the magnitude of local distortions in water coordination causative of interfacial tension. The equation defines a nanoscale electrostatic model of water and enables an estimation of protein denaturation free energies and the inference of hot spots for protein associations. The theoretical results are validated vis-à-vis calorimetric data, revealing the destabilizing effect of epistructural polarization and its molecular origin.

Communication: Epistructural thermodynamics of soluble proteins

The epistructural tension of a soluble protein is defined as the reversible work per unit area required to span the interfacial solvent envelope of the protein structure. It includes an entropic penalty term to account for losses in hydrogen-bonding coordination of interfacial water and is determined by a scalar field that indicates the expected coordination of a test water molecule at any given spatial location. An exhaustive analysis of structure-reported monomeric proteins reveals that disulfide bridges required to maintain structural integrity provide the thermodynamic counterbalance to the epistructural tension, yielding a tight linear correlation. Accordingly, deviations from the balance law correlate with the thermal denaturation free energies of proteins under reducing conditions. The picomolar-affinity toxin HsTX1 has the highest epistructural tension, while the metastable cellular form of the human prion protein PrP(C) represents the least tension-balanced protein.

Kinetic and thermodynamic study of cloned thermostable endo-1,4-β-xylanase from Thermotoga petrophila in mesophilic host

The 1,044 bp endo-1,4-β-xylanase gene of a hyperthermophilic Eubacterium, "Thermotoga petrophila RKU 1" (T. petrophila) was amplified, from the genomic DNA of donor bacterium, cloned and expressed in mesophilic host E. coli strain BL21 Codon plus. The extracellular target protein was purified by heat treatment followed by anion and cation exchange column chromatography. The purified enzyme appeared as a single band, corresponding to molecular mass of 40 kDa, upon SDS-PAGE. The pH and temperature profile showed that enzyme was maximally active at 6.0 and 95 °C, respectively against birchwood xylan as a substrate (2,600 U/mg). The enzyme also exhibited marked activity towards beech wood xylan (1,655 U/mg). However minor activity against CMC (61 U/mg) and β-Glucan barley (21 U/mg) was observed. No activity against Avicel, Starch, Laminarin and Whatman filter paper 42 was observed. The K(m), V(max) and K (cat) of the recombinant enzyme were found to be 3.5 mg ml(-1), 2778 μmol mg(-1)min(-1) and 2,137,346.15 s(-1), respectively against birchwood xylan as a substrate. The recombinant enzyme was found very stable and exhibited half life (t(½)) of 54.5 min even at temperature as high as 96 °C, with enthalpy of denaturation (ΔH*(D)), free energy of denaturation (ΔG*(D)) and entropy of denaturation (ΔS*(D)) of 513.23 kJ mol(-1), 104.42 kJ mol(-1) and 1.10 kJ mol(-1)K(-1), respectively at 96 °C. Further the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for birchwood xylan hydrolysis by recombinant endo-1,4-β-xylanase were calculated at 95 °C as 62.45 kJ mol(-1), 46.18 kJ mol(-1) and 44.2 J mol(-1) K(-1), respectively.

Enhancing the activity and thermostability of thermostable β-glucosidase from a marine Aspergillus niger at high salinity

To evaluate the effect of salinity on the catalyzing ability of β-glucosidase in the marine fungus Aspergillus niger, the thermodynamic parameters of the β-glucosidase were investigated at different salinities. At the optimum salinity of 6% NaCl (w/v) solution, the optimum temperature and pH of the β-glucosidase activity was 66°C and 5.0, respectively. Under these conditions, the β-glucosidase activity increased 1.46 fold. The half-life of denaturation in 6% NaCl (w/v) solution was approximately twice as long as that in NaCl free solution. The Gibb's free energy for denaturation, ΔG, was 2kJ/mol higher in 6% NaCl (w/v) solution than in NaCl free solution. The melting point (68.51°C) in 6% NaCl (w/v) solution was 1.71°C higher than that (66.80°C) in NaCl free solution. Similarly, the activity and thermostability of the pure β-glucosidase increased remarkably at high salinity. The thermostable β-glucosidase, of which the activity and the thermostability are remarkably enhanced at high salinity, is valuable for industrial hydrolyzation of cellulose in high salinity environments.

Pressure- and Urea-Induced Denaturation of Bovine Serum Albumin: Considerations about Protein Heterogeneity

Urea denatures proteins at different concentrations, depending on the experimental conditions and the protein. We in-vestigated the pressure-induced denaturation of bovine serum albumin (BSA) in the presence of subdenaturing concen-trations of urea based on a two-state equilibrium. Pressure-induced denaturation was enhanced at urea concentrations ([U]) of 3.5 M to 8.0 M, with the free energy of denaturation at atmospheric pressure ranging from +5.0 to –2.5 kJ/mol of BSA. The m values appeared to be biphasic, with m1 and m2 of 0.92 and 2.35 kJ mol–1?M–1, respectively. Plots of versus ln[U] yielded values of u, the apparent stoichiometric coefficient, of 1.68 and 6.67 mol of urea/mol of BSA for m1 and m2, respectively. These values were compared with the m and u values of other monomeric proteins reported in or calculated from the literature. The very low values of u systematically observed for proteins were suggestive of heterogeneity in the free energy of denaturation. Thus, a u value of 140 mol of urea/mol of BSA may indicate the existence of a heterogeneous molecular population with respect to the free energy of dena-turation.

Superhelical Duplex Destabilization and the Recombination Position Effect

The susceptibility to recombination of a plasmid inserted into a chromosome varies with its genomic position. This recombination position effect is known to correlate with the average G+C content of the flanking sequences. Here we propose that this effect could be mediated by changes in the susceptibility to superhelical duplex destabilization that would occur. We use standard nonparametric statistical tests, regression analysis and principal component analysis to identify statistically significant differences in the destabilization profiles calculated for the plasmid in different contexts, and correlate the results with their measured recombination rates. We show that the flanking sequences significantly affect the free energy of denaturation at specific sites interior to the plasmid. These changes correlate well with experimentally measured variations of the recombination rates within the plasmid. This correlation of recombination rate with superhelical destabilization properties of the inserted plasmid DNA is stronger than that with average G+C content of the flanking sequences. This model suggests a possible mechanism by which flanking sequence base composition, which is not itself a context-dependent attribute, can affect recombination rates at positions within the plasmid.

Characterization of a β-xylosidase produced by a mutant derivative of Humicola lanuginosa in solid state fermentation

The production of extracellular β-xylosidase by a newly isolated mutant derivative of Humicola lanuginosa M7D on lignocellulosic substrates and xylan was maximized in solid state fermentation by adopting a search technique varying one parameter at a time. The H. lanuginosa mutant achieved maximum production of β-xylosidase (728 IU g−1 substrate, YP/S) when grown on Vogel’s medium containing xylan, followed by medium containing corncobs (669 IU g−1) supplemented with corn steep liquor (initial pH 6.5, moisture level 75%) at 45°C. Purified mutant- and parent-derived enzyme exhibited Km values of 1.8 and 2.0 mM, respectively. Both enzymes were optimally active at pH 8.5 and a temperature of 60°C. Both enzymes displayed high thermostability, with a half-life of 2.9 and 0.9 min, enthalpy of denaturation (ΔH*) of 102.1 and 110.10 kJ mol−1, entropy of denaturation (ΔS*) of −38.5 and −4.5 J/mol K, and free energy of denaturation (ΔG*) of 115.7 and 111.7 kJ/mol at 80°C for the mutant- and parent organism-derived enzymes, respectively. The kinetic and thermodynamic properties suggest that the β-xylosidases from both strains are superior to several previously reported enzymes from other thermophilic species, and may have potential applications in various industrial fields.

Structural and functional consequences of the Milano mutation (R173C) in human apolipoprotein A-I

Carriers of the apolipoprotein A-I(Milano) (apoA-I(M)) variant, R173C, have reduced levels of plasma HDL but no increase in cardiovascular disease. Despite intensive study, it is not clear whether the removal of the arginine or the introduction of the cysteine is responsible for this altered functionality. We investigated this question using two engineered variations of the apoA-I(M) mutation: R173S apoA-I, similar to apoA-I(M) but incapable of forming a disulfide bond, and R173K apoA-I, a conservative mutation. Characterization of the lipid-free proteins showed that the order of stability was wild type approximately R173K>R173S>R173C. Compared with wild-type apoA-I, apoA-I(M) had a lower affinity for lipids, while R173S apoA-I displayed intermediate affinity. The in vivo effects of the apoA-I variants were measured by injecting apoA-I-expressing adeno-associated virus into apoA-I-null mice. Mice that expressed the R173S variant again showed an intermediate phenotype. Thus, both the loss of the arginine and its replacement by a cysteine contribute to the altered properties of apoA-I(M). The arginine is potentially involved in an intrahelical salt bridge with E169 that is disrupted by the loss of the positively charged arginine and repelled by the cysteine, destabilizing the helix bundle domain in the apoA-I molecule and modifying its lipid binding characteristics.