Effect of foreign N-terminal residues on the conformational stability of human lysozyme.

To minutely understand the effect of foreign N-terminal residues on the conformational stability of human lysozyme, five mutant proteins were constructed: two had Met or Ala in place of the N-terminal Lys residue (K1M and K1A, respectively), and others had one additional residue, Met, Gly or Pro, to the N-terminal Lys residue (Met(-1), Gly(-1) and Pro(-1), respectively). The thermodynamic parameters for denaturation of these mutant proteins were examined by differential scanning calorimetry and were compared with that of the wild-type protein. Three mutants with the extra residue were significantly destabilized: the changes in unfolding Gibbs energy (DeltaDeltaG) were -9.1 to -12.2 kJ.mol-1. However, the stability of two single substitutions at the N-terminal slightly decreased; the DeltaDeltaG values were only -0.5 to -2.5 kJ.mol-1. The results indicate that human lysozyme is destabilized by an expanded N-terminal residue. The crystal structural analyses of K1M, K1A and Gly(-1) revealed that the introduction of a residue at the N-terminal of human lysozyme caused the destruction of hydrogen bond networks with ordered water molecules, resulting in the destabilization of the protein.     

Effect of alkylation with different sized substituents on thermal stability of lysozyme.

The amino groups of hen egg white lysozyme were reductively alkylated by the reaction with aliphatic aldehydes of various chain lengths and with two aldehydes of different steric hindrance at pH 7.5 and 4 degrees for 3 h. About four of the original six lysine residues were modified by the reaction with acetaldehyde, n-butylaldehyde or n-hexylaldehyde. About three lysine residues were 2,2-dimethylpropylated with trimethylacetaldehyde while a single residue was modified with benzaldehyde. The thermal stabilities of these alkylated lysozymes were investigated by differential scanning calorimetry (DSC) at different acidic pH values. Alkylation thermally destabilized the proteins, depending not only on the extent of modification but also on the size of the substituent. The alkylated derivatives were 8-19 kJ/mol less stable than native lysozyme at 25 degrees and pH 3.0. The temperature dependences of the activities of the alkylated lysozymes against ethylene glycol chitin indicated that the orders of the optimum temperatures and the maximum activities were exactly the same as the order of the thermal stabilities.     

Structure and stability of complex coacervate core micelles with lysozyme

Encapsulation of enzymes by polymers is a promising method to influence their activity and stability. Here, we explore the use of complex coacervate core micelles for encapsulation of enzymes. The core of the micelles consists of negatively charged blocks of the diblock copolymer PAA42PAAm417 and the positively charged homopolymer PDMAEMA150. For encapsulation, part of the positively charged homopolymer was replaced by the positively charged globular protein lysozyme. We have studied the formation, structure, and stability of the resulting micelles for three different mixing ratios of homopolymer and lysozyme: a system predominantly consisting of homopolymer, a system predominantly consisting of lysozyme, and a system where the molar ratio between the two positively charged molecules was almost one. We also studied complexes made of only lysozyme and PAA42PAAm417. Complex formation and the salt-induced disintegration of the complexes were studied using dynamic light-scattering titrations. Small-angle neutron scattering was used to investigate the structures of the cores. We found that micelles predominantly consisting of homopolymer are spherical but that complex coacervate core micelles predominantly consisting of lysozyme are nonspherical. The stability of the micelles containing a larger fraction of lysozyme is lower.     

Shape evolution and thermal stability of lysozyme crystals: effect of pH and temperature

The properties of crystalline protein materials are closely linked to crystal shape. However, the effective strategies for the shape control of protein crystals are lacking. The conventional sitting-drop vapor-diffusion method was employed to investigate the influence of pH and temperature on the crystal nucleation behavior of hen egg white lysozyme. Moreover, the size distributions of protein crystals grown at different conditions were analyzed. Differential scanning calorimetry was employed to evaluate the thermal stability of lysozyme crystals. The results indicated that pH and temperature will affect the supersaturation and electrostatic interactions among protein molecules in the nucleation process. In particular, the crystals with different aspect ratios can be selectively nucleated, depending upon the choice of pH and temperature. Therefore, this study provided a simple method for obtaining shape-controlled lysozyme crystals and supplied some information on thermal behaviors of lysozyme crystals grown at different pH values.     

Effect of pH on the stability of type-C toxin ofClostridium botulinum

Stability of type-C botulinum toxin at pH 1.8–12.0 and during exposure to 5 and 28°C for 20 and 16 h, respectively, was tested by titration on adult mice. The toxin was found in the samples kept at pH of 2.7–10.2, whereas, at the pH extremes of 1.8 and 12.0, it was inactivated.     

Effect of pH and denaturants on the folding and stability of murine interleukin-6.

The conformation and stability of a recombinant mouse interleukin-6 (mIL-6) has been investigated by analytical ultracentrifugation, fluorescence spectroscopy, urea-gradient gel electrophoresis, and near- and far-ultraviolet circular dichroism. On decreasing the pH from 8.0 to 4.0, the tryptophan fluorescence of mIL-6 was quenched 40%, the midpoint of the transition occurring at pH 6.9. The change in fluorescence quantum yield was not due to unfolding of the molecule because the conformation of mIL-6, as judged by both urea-gradient gel electrophoresis and CD spectroscopy, was stable over the pH range 2.0-10.0. Sedimentation equilibrium experiments indicated that mIL-6 was monomeric, with a molecular mass of 22,500 Da over the pH range used in these physicochemical studies. Quenching of tryptophan fluorescence (20%) also occurred in the presence of 6 M guanidine hydrochloride upon going from pH 7.4 to 4.0 suggesting that an amino acid residue vicinal in the primary structure to one or both of the two tryptophan residues, Trp-36 and Trp-160, may be partially involved in the quenching of endogenous fluorescence. In this regard, similar results were obtained for a 17-residue synthetic peptide, peptide H1, which corresponds to an N-terminal region of mIL-6 (residues Val-27-Lys-43). The pH-dependent acid quenching of endogenous tryptophan fluorescence of peptide H1 was 30% in the random coil conformation and 60% in the presence of alpha-helix-promoting solvents. Replacement of His-33 with Ala-33 in peptide H1 alleviated a significant portion of the pH-dependent quenching of fluorescence suggesting that the interaction of the imidazole ring of His-33 with the indole ring of Trp-36 is a major determinant responsible for the quenching of the endogenous protein fluorescence of mIL-6.     

Effect of polyols on the conformational stability and biological activity of a model protein lysozyme

The purpose of this study was to investigate the stabilizing action of polyols against various protein degradation mechanisms (eg, aggregation, deamidation, oxidation), using a model protein lysozyme. Differential scanning calorimeter (DSC) was used to measure the thermodynamic parameters, mid point transition temperature and calorimetric enthalpy, in order to evaluate conformational stability. Enzyme activity assay was used to corroborate the DSC results. Mannitol, sucrose, lactose, glycerol, and propylene glycol were used as polyols to stabilize lysozyme against aggregation, deamidation, and oxidation. Mannitol was found to stabilize lysozyme against aggregation, sucrose against deamidation both at neutral pH and at acidic pH, and lactose against oxidation. Stabilizers that provided greater conformational stability of lysozyme against various degradation mechanisms also protected specific enzyme activity to a greater extent. It was concluded that DSC and bioassay could be valuable tools for screening stabilizers in protein formulations.     

Effect of the pH of the medium and of temperature on tylosin stability

The stability of tylosin, a macrolide antibiotic, in solutions with varying pH and temperature was determined quantitatively. It was shown that tylosin was the most stable at pH about 3.5 and 9.0, which corresponded to the salt and nondissociated forms of the substance. Outside these stability ranges significant inactivation of the antibiotic was observed. The inactivation markedly increased with an increase in the temperature level and the exposure period. Satisfactory correlation between the data on microbiological and spectrophotometric determinations of tylosin in solutions is indicative of the advisability of the use of spectrophotometry in production of tylosin.     

Structure and conformation of linear peptides. XII. Structure of tryptophanyl-glycyl-glycine dihydrate

The crystal structure of a tripeptide, tryptophanyl-glycyl-glycine dihydrate (C15H18N4O4.2H2O, molecular weight = 354) has been determined. The crystals are orthorhombic, space group P2(1)2(1)2(1), with a = 7.875 (1) A, b = 9.009(1), c = 24.307(1) and Z = 4. The final R-index is 0.058 for 1488 reflections [sin theta)/lambda less than or equal to 0.6 A-1) with I greater than 2 sigma (I). The molecule exists as a zwitterion, with terminal NH3+ and COO- groups. The peptide units are trans and nearly perpendicular to the plane of the carboxyl group. The backbone torsion angles are: psi 1 = 132.7 degrees, omega 1 = 174.2 degrees, phi 2 = 88.2 degrees, psi 2 = 8.6 degrees, omega 2 = -179.8 degrees, phi 3 = -85.2 degrees, psi 31 = -178.1 degrees, psi 32 = 5.0 degrees. For the sidechain of tryptophan, chi 1 = -171.6 degrees, chi 2 = 101.0 degrees.