Contribution of amino acid substitutions at two different interior positions to the conformational stability of human lysozyme

To elucidate correlative relationships between structural changeand thermodynamic stability in proteins, a series of mutanthuman lysozymes modified at two buried positions (Ile56 andIle59) were examined. Their thermodynamic parameters of denaturationand crystal structures were studied by calorimetry and X-raycrystallography. The mutants at positions 56 and 59 exhibiteddifferent responses to a series of amino acid substitutions.The changes in stability due to substitutions showed a linearcorrelation with changes in hydrophobicity of substituted residues,having different slopes at each mutation site. However, thestability of each mutant was found to be represented by a uniqueequation involving physical properties calculated from mutantstructures. By fitting present and previous stability data formutant human lysozymes substituted at various positions to theequation, the magnitudes of the hydrophobicity of a carbon atomand the hydrophobicity of nitrogen and neutral oxygen atomswere found to be 0.178 and –0.013 kJ/mol.Ų, respectively.It was also found that the contribution of a hydrogen bond witha length of 3.0 Å to protein stability was 5.1 kJ/moland the entropy loss of newly introduction of a water moleculeswas 7.8 kJ/mol.     

Contribution of a proline residue and a salt bridge to the stability of a type I reverse turn in chymotrypsin inhibitor-2

The contributions of the components of a type I reverse turnto the stability of chymotrypsin inhibitor-2 (Lys43-Pro44-Gly45)have been determined by protein engineering methods. A double-mutantcycle was used to determine the interaction between Lys43 andGlu45 by replacing them with alanine. We also mutated Pro44,which gives the geometry of the turn, to alanine and analysedthe stability of the resulting mutants compared with wild-typechymotrypsin inhibitor-2, using equilibrium denaturation inducedby guanldinium chloride. There are decreases in stability (inkcal/mol) of 0.64 = 0.06 for Lys43 - Ala, 0.57 ± 0.15for Glu45 - Ala, 0.95 ± 0.06 for Lys43 - Ala/Glu45 -Ala and 1.93 ± 0.09 for Pro44 - Ala. The free energyof interaction between Lys43 and Glu45 is calculated to be only0.25 ± 0.09 kcal/mol. From the changes in denaturationmidpoint, T_m measured by circular dkhroism, we estimate theenergy of interaction between Lys43 and Glu45 to be 0.36 ±0.07 kcal/mol whereas the contribution of Pro44 is -2.0 kcal/mol.The contribution of the salt bridge to the stability of theprotein is very small and the residue Pro44 plays the key rolein stabilizing the turn     

The Trp-cage: optimizing the stability of a globular miniprotein

The Trp-cage, as the smallest miniprotein, remains the subject of numerous computational and experimental studies of protein folding dynamics and pathways. The original Trp-cage (NLYIQWLKDGGPSSGRPPPS, Tm = 42 degrees C) can be significantly stabilized by mutations; melting points as high as 64 degrees C are reported. In helical portions of the structure, each allowed replacement of Leu, Ile, Lys or Ser residues by Ala results in a 1.5 (+/-0.35) kJ/mol fold stabilization. No changes in structure or fluxionality of the core results upon stabilization. Contrary to the initial hypothesis, specific Pro/Trp interactions are not essential for core formation. The entropic advantage of Pro versus Ala (DeltaDeltaS(U) = 11 +/- 2 J/mol K) was measured at the solvent-exposed P17 site. Pro-Ala mutations at two of the three prolines (P12 and P18) that encage the indole ring result in less fold destabilization (2.3-3.4 kJ/mol). However, a P19A mutation reduces fold stability by 16 kJ/mol reflecting a favorable Y3/P19 interaction as well as Trp burial. The Y3/P19 hydrophobic staple interaction defines the folding motif as an 18-residue unit. Other stabilizing features that have been identified include a solvent-exposed Arg/Asp salt bridge (3.4-6 kJ/mol) and a buried H-bonded Ser side chain ( approximately 10 kJ/mol).     

Evidence for an initiation site for hen lysozyme folding from the reduced form using its dissected peptide fragments

We prepared two dissected fragments of hen lysozyme and examinedwhether or not these two fragments associated to form a native-likestructure. One (Fragment I) is the peptide fragment Asn59–homoserine-105containing Cys64–Cys80 and Cys76–Cys94. The other(Fragment II) is the peptide fragment Lys1–homoserine-58connected by two disulfide bridges, Cys6–Cys127 and Cys30–Cys115,to the peptide fragment Asn106–Leu129. It was found thatthe Fragment I immobilized in the cuvette formed an equimolarcomplex with Fragment II (K_d = 3.3x10^–4 M at pH 8 and25°C) by means of surface plasmon resonance. Moreover, fromanalyses by circular dichroism spectroscopy and ion-exchangechromatography of the mixture of Fragments I and II at pH 8under non-reducing conditions, it was suggested that these fragmentsassociated to give the native-like structure. However, the mutantFragment I in which Cys64–Cys80 and Cys76–Cys94are lacking owing to the mutation of Cys to Ala, or the mutantfragment in which Trp62 is mutated to Gly, did not form thenative-like species with Fragment II, because the mutant FragmentI derived from mutant lysozymes had no local conformation dueto mutations. Considering our previous results where the preferentialoxidation of two inside disulfide bonds, Cys64–Cys80 andCys76–Cys94, occurred in the refolding of the fully reducedFragment I, we suggest that the peptide region correspondingto Fragment I is an initiation site for hen lysozyme folding.     

Free energy calculations of the mutation of Ile96 -> Ala in barnase: contributions to the difference in stability

Free energy calculations were carried out to determine the relativeunfolding free energy of the Ile96 wild type and Ala96 mutantbarnases. The total calculated free energies suggest that substitutionof Ile96 with Ala destabilizes barnase by 3.9 kcal/mol, whichis in good agreement with the independently determined experimentalvalues of 4.0 and 3.3 kcal/mol and a previous simulation. However,a decomposition of the free energy finds the dominant contributionsto this free energy arising from the noncovalent Interactionsbetween the perturbed group and distant residues of barnasein the sequence and water molecules and only a very small contributionfrom covalent interactions. This is in contrast to the previoussimulation, using the dual topology methodology, which produceda decomposition with an {small tilde}60% free energy contributionfrom changes in covalent interactions. The use of the singletopology employed in the present calculations and the dual topologyemployed in the previous study are analyzed in order to understandthe contrast between the present results and the results ofthe previous study.     

Construction of novel subtilisin E with high specificity, activity and productivity through multiple amino acid substitutions

Through three cumulative amino acid substitutions, we constructed novel mutant subtilisins E of Bacillus subtilis, all with high specificity, activity and productivity. The substitution of conserved Gly127, constituting P1 substrate-binding pocket, with Ala and Val showed a marked preference for the small P1 substrate. Leu was then substituted for Ile31 next to the catalytic Asp32 to enhance the catalytic activity. Both double mutants (I31L/G127A and I31L/G127V) showed a 3-5- fold increase in catalytic efficiency due to a large kcat, without any change in the specificity of the mutants at position 127. Molecular modeling suggests that large P1 residues were unable to access the pocket because of steric hindrance. A third mutation was introduced by replacing Tyr(-1) with Ala in the propeptide essential for autoprocessing to active mature subtilisin in vivo. A prominent 7-20- fold increase in active enzyme production occurred in the triple mutants (Y-1A/I31L/G127A and Y-1A/I31L/G127V).      

Borate Volatility from SOFC Sealing Glasses

The volatility of borate species from glasses developed for solid oxide fuel cell seals was studied using thermodynamic calculations and compared with experimental results. Vapor pressure diagrams were used to identify the most volatile compounds under a range of expected operational conditions, e.g. oxidizing and reducing atmospheres with water vapor, at temperatures in the range of 700°–1000°C. The species with the highest vapor pressures were BO₂( g ) under dry conditions and B₃H₃O₆( g ) under wet, reducing conditions. The depletion of boron from glass surfaces to depths beyond 100 nm was characterized using Auger electron spectroscopy depth profile analysis. Weight loss experiments were conducted on several different glass compositions. The cumulative weight loss from a glass with 20 mol% B₂O₃ ("glass #59") was about 10 times greater than from a glass with 2 mol% B₂O₃ ("glass #27"), under the same conditions. The activation energy for volatilization from glass #59 was 371±86 kJ/mol and was 272±65 kJ/mol for "glass #27." The cumulative weight loss of each composition in forming gas with 30% water vapor was greater than in dry air at 800°C. Volatile species were collected in a water trap, and these results confirmed predictions about the effect of atmosphere and B₂O₃ content on volatilization behavior.     

六水氯化钐与甘氨酸丙氨酸三元固态配合物的热化学

合成了 Sm3 +与甘氨酸丙氨酸混配体配合物 ,通过红外光谱分析、热重分析和化学分析 ,确定了配合物的组成为 :Sm( Gly) 2 ( Ala) 3 Cl3 · 2 H2 O,并用溶解量热法分别测定了 Sm Cl3 · 6H2 O( s) ,2 Gly( s) + 3Ala( s)和 Sm( Gly) 2 ( Ala) 3 Cl3 · 2 H2 O( s)在 2 mol/L HCl中的溶解焓 ;再根据盖斯定律设计了一个热化学循环 ,计算得到了六水氯化钐与甘氨酸丙氨酸反应的反应焓△ r Hθm( 2 98.1 5 K) =2 .1 60 k J/mol,并求出了 Sm( Gly) 2 ( Ala) 3 Cl3 · 2 H2 O( s)标准生成焓△f Hθm[Sm( Gly) 2 ( Ala) 3 Cl3 ·2 H2 O,s,2 98.1 5 K]=- 44 81 .5 k J/mol     

Formation and Dehydration Enthalpy of Potassium Hexaniobate

The formation energetics of hydrous and dehydrated potassium hexaniobates are investigated using high‐temperature oxide melt solution calorimetry. The enthalpies of formation of K₄Nb₆O₁₇ and K₄Nb₆O₁₇·3H₂O from oxides are (?864.42 ± 10.63) and (?899.32 ± 11.48) kJ/mol, respectively. The formation enthalpy of K₄Nb₆O₁₇ from elements is (?7289.64 ± 12.50) kJ/mol, and of K₄Nb₆O₁₇·3H₂O is (?8181.94 ± 13.24) kJ/mol. The enthalpy of dehydration (ΔH_dehy) for the reaction K₄Nb₆O₁₇·3H₂O (xl, 25°C) = K₄Nb₆O₁₇ (xl, 25°C) + 3H₂O (l, 25°C) is endothermic and is 34.60 ± 7.56 kJ/mol. The ΔH_dehy per mole of water, 11.53 ± 2.52 kJ/mol, indicates the water molecules in K₄Nb₆O₁₇·3H₂O are not just physically adsorbed, but loosely bonded in the K₄Nb₆O₁₇ phase, presumably in specific interlayer sites. The loss of this water near 100°C on heating is consistent with the weak bonding of water.     

Hyperthermostable mutants of Bacillus licheniformis {alpha}-amylase: multiple amino acid replacements and molecular modelling

We have identified previously two critical positions for thethermostability of the highly thermostable -amylase from Bacilluslicheniformis. We have now introduced all 19 possible aminoacid residues to these two positions, His 133 and Ala209. Themost favourable substitutions were to Ile and Val, respectively,which both increased the half-life of the enzyme at 80°Cby a factor of 3. At both positions a stabilizing effect ofhydrophobic residues was observed, although only in the caseof position 133 could a clear correlation be drawn between thehydrophobicity of the inserted amino acid and the gain in proteinstability. The construction of double mutants showed a cumulativeeffect of the most favourable and/or deleterious substitutions.Computer modelling was used to generate a 3-D structure of thewild-type protein and to model substitutions at position 209,which lies in the conserved (/ß)₈ barrel domain of-amylase; Ala209 would be located at the beginning of the thirdhelix of the barrel, in the bottom of a small cavity facingthe fourth helix. The model suggests that replacement by, forexample, a valine could fill this cavity and therefore increaseintra- and interhelical compactness and hydrophobic interactions.     

Stabilization of lysozyme against irreversible inactivation by alterations of the Asp-Gly sequences

Site-directed mutagenesis was performed at Asp-Gly (48–49,66–67, 101–102) and Asn-Gly (103–104) sequencesof hen egg-white lysozyme to protect the enzyme against irreversiblethermoinactivation. Because the lysozyme inactivation was causedby the accumulation of multiple chemical reactions, includingthe isomerization of the Asp-Gly sequence and the deamidationof Asn [Tomizawa et al.(1994) Biochemistry, 33, 13032–13037],the suppression of these reactions by the substitution of Glyto Ala, or the introduction of a sequence of human-type lysozyme,was attempted and the mutants (where each or all labile sequenceswere replaced) were prepared. The substitution resulted in thereversible destabilization from 1 to 2 kcal/mol per substitution.The destabilization was caused by the introduction of ß-carbonto the constrained position that had conformational angles withinthe allowed range for the Gly residue. Despite the decreasein the reversible conformational stability, the mutants hadmore resistance to irreversible inactivation at pH 4 and 100°C.In particular, the rate of irreversible inactivation of themutant, which was replaced at four chemically labile sequences,was the latest and corresponded to 18 kcal/mol of the reversibleconformational stability. Therefore, replacement of the chemicallylabile sequence was found to be more effective at protectingenzymes against irreversible thermoinactivation than at strengtheningreversible conformational stability.     

Modeling protein stability: a theoretical analysis of the stability of T4 lysozyme mutants

Free energy calculations were conducted to determine the relative stability of the unnatural amino acid mutants of T4 lysozyme norvaline (Nvl) and O-methyl-serine (Mse) and of alanine at residue 133, which is leucine in the native sequence. These calculations were performed both to assess the validity of the methodology and to gain a better understanding of the forces which contribute to protein stability. Peptides of different length were used to model the denatured state. Restraints were employed to force sampling of the side chain chi1 dihedral of the perturbed side chain, and the effect of protein repacking in response to mutation was studied through the use of different constraint sets. In addition, the convergence behavior and hysteresis of the simulations in the folded and unfolded states were determined. The calculated results agree well with experiment, + 1.84 versus + 1.56 kcal/mol for Mse-->Nvl and -3.48 versus -2.2 to -3.6 kcal/mol for Nvl-->Ala. We find that free energy calculations can provide useful insights to protein stability when conducted carefully on a well chosen system. Our results suggest that loss of packing interactions in the native state is a major source of destabilization for mutants which decrease the amount of buried nonpolar surface area and that subtle responses of the backbone affect the magnitude of the loss of stability. We show that the conformational freedom of the chi1 dihedral has a noticeable effect on protein stability and that the solvation of amino acid side chains is strongly influenced by interactions with the peptide backbone.      

氯亚乙基肼基甲酸乙酯生成反应热动力学研究

利用微量热量计测定了氯乙醛和肼基甲酸乙酯在水溶液中(298.15K)生成氯亚乙基肼基甲酸乙酯的反应焓变为(-13.035±0.088)kJ/mol。计算得出该反应的热动力学参数:反应级数n=1;速率常数k=2.018×10-3s-1及反应活化自由能ΔG ≠=88.408kJ/mol。结果表明,该反应在室温下易于进行。     

Contribution of a disulfide bridge to the stability of 1,3-1,4-P-D-glucan 4-glucanohydrolase from Bacillus licheniformis

Bacillus 1,3-1,4-ß-glucanases possess a highly conserveddisulfide bridge connecting a ß-strand with a solventexposedloop lying on top of the extended binding site cleft The contributionof the disulfide bond and of both individual cysteines (Cys61and Cys90) in the Bacillus licheniformis enzyme to stabilityand activity has been evaluated by protein engineering methods.Reduction of the disulfide bond has no effect on kinetic parameters,has only a minor effect on the activity-temperature profileat high temperatures, and destabilizes the protein by less than0.7 kcal/mol as measured by equilibrium urea denatu ration at37°C. Replacing either of the Cys residues with Ala destabilizesthe protein and lowers the specific activity. C90A retains 70%of wild-type (wt) activity (in terms of Vmax), whereas C61Aand the double mutant C61A–C90A have 10% of wt V_max. Alarger change in free energy of unfolding is seen by equilibriumurea denaturation for the C61A mutation (loop residue, 3.2 kcal/molrelative to reduced wt) as compared with the C90A mutation (ß-strandresidue, 1.8 kcal/mol relative to reduced wt), while the doublemutant C61A–C90A is 0.8 kcal/mol less stable than thesingle C61A mutant. The effects on stability are interpretedas a result of the change in hydrophobic packing that occursupon removal of the sulfur atoms in the Cys to Ala mutations     

Thermodynamics of Nanoscale Calcium and Strontium Titanate Perovskites

The surface enthalpies of nanocrystalline CaTiO₃ and SrTiO₃ perovskites were determined using high‐temperature oxide melt solution calorimetry in conjunction with water adsorption calorimetry. The nanocrystalline samples were synthesized by a hydrothermal method and characterized using powder X‐ray diffraction, FTIR spectroscopy, and Brunauer–Emmett–Teller surface area measurements. The integral heats of water vapor adsorption on the surfaces of nanocrystalline CaTiO₃ and SrTiO₃ are ?78.63 ± 4.71 kJ/mol and ?69.97 ± 4.43 kJ/mol, respectively. The energies of the hydrous and anhydrous surfaces are 2.49 ± 0.12 J/m² and 2.79 ± 0.13 J/m² for CaTiO₃ and 2.55 ± 0.15 J/m² and 2.85 ± 0.15 J/m² for SrTiO₃, respectively. The stability of the perovskite compounds in this study is discussed according to the lattice energy and tolerance factor approach. The energetics of different perovskites suggest that the formation enthalpy becomes more exothermic and surface energy increases with an increase in ionic radius of the “A” site cation (Ca, Sr, and Ba), or with the tolerance factor. PbTiO₃ shows a lower surface energy, weaker water binding, and a less exothermic enthalpy of formation than the alkaline‐earth perovskites.     

Kinetics analysis of the curing of a diepoxy-diamine system

The reaction between the diglycidylether of bisphenol A (DGEBA) and 4,9-dioxa-1,12-dodecanediamine (DDDD) has been studied by means of isothermal and dynamic differential scanning calorimetry. The enthalpy of the reaction of an epoxy group with an amino-hydrogen has been determined to be 112 ± 5 kJ/mol. A kinetic model has been validated. It involves two competitive mechanisms: one is catalysed by the hydroxy groups initially present on the epoxy chain or generated during the reaction (activation energy 77 ± 5 kJ/mol), the other is not catalysed with a higher activation energy (103 ± 3 kJ/mol). For each isothermal curing, the kinetics are not modified by gelation. Evaluated from the gel times, the overall activation energy of the reaction is equal to 62 ± 2 kJ/mol.     

Directed evolution of Thermotoga neapolitana xylose isomerase: high activity on glucose at low temperature and low pH

The Thermotoga neapolitana xylose isomerase (TNXI) is extremelythermostable and optimally active at 95°C. Its derivative,TNXI Val185Thr (V185T), is the most active type II xylose isomerasereported, with a catalytic efficiency of 25.1 s^–1 mM^–1toward glucose at 80°C (pH 7.0). To further optimize TNXI’spotential industrial utility, two rounds of random mutagenesisand low temperature/low pH activity screening were performedusing the TNXI V185T-encoding gene as the template. Two highlyactive mutants were obtained, 3A2 (V185T/L282P) and 1F1 (V185T/L282P/F186S).1F1 was more active than 3A2, which in turn was more activethan TNXI V185T at all temperatures and pH values tested. 3A2and 1F1’s high activities at low temperatures were dueto significantly lower activation energies (57 and 44 kJ/mol,respectively) than that of TNXI and V185T (87 kJ/mol). MutationL282P introduced a kink in helix     

Folding and conformational studies on SCR1-3 domains of human complement receptor 1

Short consensus repeats SCR3 and SCR1-3 are soluble recombinantproteins, consisting of the third and first three N-terminaldomains of complement receptor 1, respectively, which retainsome anti-complement activity. The conformational stabilitiesand folding/unfolding of SCR3 and SCR1-3 have been studied usingcircular dichroism and equilibrium and pre-equilibrium fluorescencespectroscopy. Denaturation by guanidinium hydrochloride (GdnHCl)is rapid and completely reversible. Reduction of disulphidebridges in the folded proteins by ß-mercaptoethanolleads to an increase in fluorescence intensity. The fluorescenceintensity of the folded proteins is {small tilde}7.5% of thatof the respective unfolded proteins. The data can be approximatedto a two-state transition between native and denatured formsof the proteins. SCR3 has a conformational stability in waterof 12–13 kJ/mol whereas that of SCR1-3 is 19.5–19.9kJ/mol depending upon the technique utilized. The heat capacitychange associated with the unfolding of SCR1-3 was obtainedby a series of GdnHCl unfolding experiments over a range oftemperatures and was found to be 6.6 kJ/K.mol or 33.8 J/K.mol_residue.The refolding process of SCR3 was found to be simple, describedby a single exponential equation, whereas that of SCR1-3 wasfound to be complex and could be fitted to a double exponentialequation indicating the presence of folding intermediates.     

Access of the substrate to the active site of yeast oxidosqualene cyclase: an inhibition and site-directed mutagenesis approach

A structural model of Saccharomyces cerevisiae oxidosqualene cyclase (SceOSC) suggests that some residues of the conserved sequence Pro-Ala-Glu-Val-Phe-Gly (residues 524-529) belong to a channel constriction that gives access to the active-site cavity. Starting from the SceOSC C457D mutant, which lacks the cysteine residue next to the catalytic Asp456 residue Cys457 has been replaced but Asp456 is still there, we prepared two further mutants where the wild-type residues Ala525 and Glu526 were individually replaced by cysteine. These mutants, especially E526C, were very sensitive to the thiol-reacting agent dodecyl-maleimide. Moreover, both the specific activity and the thermal stability of E526C were severely reduced. A similar decrease of the enzyme functionality was obtained by replacing Glu526 with alanine, while substitution with the conservative residues aspartate or glutamine did not alter catalytic activity. Molecular modeling of the yeast wild-type OSC and mutants on the template structure of human OSC confirms that the channel constriction is an important aspect of the protein structure and suggests a critical structural role for Glu526.     

Immobilization influence on the water sorption and diffusion in poly(3-hydroxybutyrate)

The temperature dependency of water vapor sorption and diffusion in poly(3-hydroxybutyrate) (PHB) was studied for the first time. Equilibrium sorption and diffusion kinetics were determined by a quartz McBain's vacuum microbalance technique in the temperature range of 303–333 K. A probability of water molecule interaction with the polymer matrix was analyzed for wet PHB films by FTIR spectroscopy technique. Sorption isotherms are interpreted as the solution of free water molecules estimated by the Flory–Huggins equation and the sorption of water molecules immobilized on the carbonyl groups of PHB. The immobilization effect was described by a Langmuir-type equation. The dependency of diffusivity on water concentration was described in the frames of Fujita's immobilization model in which the growing function D_w versus C_w characterized the filling degree of carbonyl groups as sites of immobilization in the polymer. Enthalpy of free water sorption (12 kJ/mol) and water immobilization (42 kJ/mol), as well as the activation energy of water diffusion coefficients (71 kJ/mol), in noncrystalline areas of PHB were determined. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 73: 981–985, 1999