The effect of temperature and solution pH on the nucleation of tetragonal lysozyme crystals.

Part of the challenge of macromolecular crystal growth for structure determination is obtaining crystals with a volume suitable for x-ray analysis. In this respect an understanding of the effect of solution conditions on macromolecule nucleation rates is advantageous. This study investigated the effects of supersaturation, temperature, and pH on the nucleation rate of tetragonal lysozyme crystals. Batch crystallization plates were prepared at given solution concentrations and incubated at set temperatures over 1 week. The number of crystals per well with their size and axial ratios were recorded and correlated with solution conditions. Crystal numbers were found to increase with increasing supersaturation and temperature. The most significant variable, however, was pH; crystal numbers changed by two orders of magnitude over the pH range 4.0-5.2. Crystal size also varied with solution conditions, with the largest crystals obtained at pH 5.2. Having optimized the crystallization conditions, we prepared a batch of crystals under the same initial conditions, and 50 of these crystals were analyzed by x-ray diffraction techniques. The results indicate that even under the same crystallization conditions, a marked variation in crystal properties exists.     

Structural and conformational stability of horseradish peroxidase: effect of temperature and pH

Detailed circular dichroism and fluorescence studies at different pHs have been carried out to monitor thermal unfolding of horseradish peroxidase isoenzyme c (HRPc). The change in CD in the 222 nm region corresponds to changes in the overall secondary structure of the enzyme, while that in the 400 nm region (Soret region) corresponds to changes in the tertiary structure around the heme in the enzyme. The temperature dependence of the tertiary structure around the heme also affected the intrinsic tryptophan fluorescence emission spectrum of the enzyme. The results suggested that melting of the tertiary structure to a pre-molten globule form takes place at 45 degrees C, which is much lower than the temperature (T(m) = 74 degrees C) at which depletion of heme from the heme cavity takes place. The melting of the tertiary structure was found to be associated with a pK(a) of approximately 5, indicating that this phase possibly involves breaking of the hydrogen-bonding network of the heme pocket, keeping the heme moiety still inside it. The stability of the secondary structure of the enzyme was also found to decrease at pH below 4.5. A 'high temperature' unfolding phase was observed which was, however, independent of pH. The stability of the secondary structure was found to drastically decrease in the presence of DTT (dithiothreitol), indicating that the 'high temperature' form is possibly stabilized due to interhelical disulfide bonds. Depletion of Ca(2+) ions resulted in a marked decrease in the stability of the secondary structure of the enzyme.     

Linear correlation between thermal stability and folding kinetics of lysozyme

We have studied the refolding and thermal denaturation of hen egg white lysozyme in a wide range of pH values (from 1.5 to 9.4) using stopped-flow circular dichroism (CD) and differential scanning calorimetry (DSC). A linear correlation was found between the thermal denaturation temperature (T(m)) and the logarithm of the refolding rate of the slow folding phase of hen egg white lysozyme (lnk(2)).     

Remarkable thermal stability of doubly intramolecularly cross-linked hen lysozyme

In order to examine how a protein can be effectively stabilized, two intramolecular cross-links, Glu35-Trp108 and Lys1-His15, which have few unfavorable interactions in the folded state, were simultaneously introduced into hen lysozyme. Both of the intramolecularly cross-linked lysozymes, 35-108 CL and 1-15 CL, containing cross-links Glu35-Trp108 and Lys1-His15, respectively, showed increases in thermal stability of 13.9 and 5.2 degrees C, respectively, over that of wild type, at pH 2.7. On the other hand, a doubly cross-linked lysozyme showed an increase in thermal stability of 20.8 degrees C over that of wild type, under identical conditions. Since the sum of the differences in denaturation temperature between wild type and each of the cross-linked lysozymes was nearly equal to that between wild type and the doubly cross-linked lysozyme, we suggest that the efficient stabilization of the lysozyme molecule was the direct result of the double intramolecular cross-links.     

Lattice deformation and thermal stability of crystals in spider silk

The X-ray diffraction of dragline silks, produced by Nephila and Cyrtophora spiders, were measured by synchrotron radiation in their original states or in situ during stretching and heating. Nephila pilipes spiders construct a two-dimensional orb web that must be rebuilt in one or 2 days, but Cyrtophora spiders form a three-dimensional tent web that can exist for several weeks in a tropical forest. Diffraction patterns of N. pilipes and Cyrtophora draglines resemble each other. Crystals of two kinds are identified in these draglines; one is aligned parallel to the silk direction and another is less oriented. The less oriented crystal in Cyrtophora dragline is aligned better than that in N. pilipes dragline, which generates about three times stronger diffract intensity. Crystals in N. pilipes and C. moluccensis dragline silks have remarkable thermal stability. Equatorial reflections remain undiminished until 350 and 450 °C for N. pilipes and C. moluccensis, respectively. In contrast, the meridional reflections S and (0 0 2), which are parallel to the silk thread, disappear at a temperature less than 100 °C for C. moluccensis but remain for Nephila up to 100 °C. Meridional reflections S and (0 0 2) shift to a smaller angle during stretching, whereas equatorial reflections remain constant in a range 1.0–1.3 times the original length. The position of the S reflection shifts rapidly in the first 10% of elongation from the original length but remains constant during subsequent stretching, whereas the (0 0 2) reflection shifts rapidly during the first 5% elongation from the original length and continues to shift subsequently. In contrast, the features of N. pilipes dragline alter insignificantly during stretching. Examination of the composition of amino acids of the draglines of N. pilipes and C. moluccensis indicates that a dragline of N. pilipes contains more glycine, but much less alanine, than that of C. moluccensis.     

Reversible and non-reversible thermal denaturation of lysozyme with varying pH at low ionic strength

DSC analysis has been used to quantify the reversibility of unfolding following thermal denaturation of lysozyme. Since the temperature at which protein unfolding occurs, T_m, varies with different solution conditions, the effect on the melting temperature and the degree of refolding after thermal denaturation in low ionic strength sodium phosphate buffers (5–1000 mM) over a range of pH (5–9) in the presence/absence of disaccharides is examined. This study compares the enthalpies of unfolding during successive heating cycles to quantify reversibility following thermal denaturation. The disaccharides, trehalose and maltose were used to assess if the disaccharide induced increase in T_m is reflected in the reversibility of thermally induced denaturation. There was extensive overlap between the T_m values where non-reversible and reversible thermal denaturation occurred. Indeed, for pH 6, at the highest and lowest T_m, no refolding was observed whereas refolding was observed for intermediate values, but with similar T_m values having different proportions of refolded protein. We established a method to measure the degree of reversible unfolding following thermal denaturation and hence indirectly, the degree to which protein is lost to irreversible aggregation, and show that solution conditions which increase melt transition temperatures do not automatically confer an increase in reversibility. This type of analysis may prove useful in assessing the stability of proteins in both the biopharmaceutical and food industries.     

Stereoselectivity and conformational stability of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110: the effect of pH and temperature

The effect of pH and temperature on structure, stability, activity and enantioselectivity of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110 was investigated in this study. Conformational changes have been assessed by circular dichroism spectroscopy, functional changes by kinetic analysis, while quaternary structure was studied by gel filtration chromatography. Our study shows that the DbjA enzyme is highly tolerant to pH changes. Its secondary and tertiary structure was not affected by pH in the ranges 5.3-10.3 and 6.2-10.1, respectively. Oligomerization of DbjA was strongly pH-dependent: monomer, dimer, tetramer and a high molecular weight cluster of the enzyme were distinguished in solution at different pH conditions. Moreover, different oligomeric states of DbjA possessed different thermal stabilities. The highest melting temperature (T(m) = 49.1 ± 0.2 °C) was observed at pH 6.5, at which the enzyme occurs in dimeric form. Maximal activity was detected at 50 °C and in the pH interval 7.7-10.4. While pH did not have any effect on enantiodiscriminination of DbjA, temperature significantly altered DbjA enantioselectivity. A decrease in temperature results in significantly enhanced enantioselectivity. The temperature dependence of DbjA enantioselectivity was analysed with 2-bromobutane, 2-bromopentane, methyl 2-bromopropionate and ethyl 2-bromobutyrate, and differential activation parameters Δ(R-S)ΔH and Δ(R-S)ΔS were determined. The thermodynamic analysis revealed that the resolution of β-bromoalkanes was driven by both enthalpic and entropic terms, while the resolution of α-bromoesters was driven mainly by an enthalpic term. Unique catalytic activity and structural stability of DbjA in a broad pH range, combined with high enantioselectivity with particular substrates, make this enzyme a very versatile biocatalyst. Enzyme EC3.8.1.5 haloalkane dehalogenase.     

pH and thermal stability studies of chitinase from Trichoderma harzianum: A thermodynamic consideration

The combined effect of pH and temperature on chitinase was investigated using response surface methodology. A central composite design for two variables was employed. The optimal pH and temperature for the least degree of deactivation were found out to be 5.4 and 24°C respectively. The deactivation rate constants and the half life of chitinase were estimated at different pH and temperature combinations. At the optimal pH of 5.4, the rate of the deactivation was found to be the least. Thermodynamic parameters, viz., ΔH^*, ΔS^*, ΔG^* and activation energy of thermal deactivation of chitinase were calculated in the temperature range from 50°C to 60°C.     

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.     

The evolution of lysozyme and alpha-lactalbumin

From the analysis of phylogenetic trees constructed from the amino acid sequences and metal-binding properties of various lysozymes c and alpha-lactalbumins, it was found that before the divergence of the lineages of birds and mammals, calcium-binding lysozyme diverged from non-calcium-binding lysozyme. alpha-Lactalbumin evolved from the calcium-binding lysozyme along the mammalian lineage after the divergence of birds and mammals. Rapid evolution took place, not in the process of acquisition of the activity of alpha-lactalbumin, but after the loss of lysozyme activity, due to the change in the distribution of selective pressure on each amino acid site. A general process for the change in function of a protein during evolution is suggested to be as follows: after duplication of the gene, one of their protein products acquires a new function, besides that already present; the old function is eventually lost.     

Human Calprotectin： Effect of Calcium and Zinc on its Secondary and Tertiary Structures, and Role of pH in its Thermal Stability

Calprotectin, a heterodimeric complex belonging to the S 100 protein family, has been found predominantly in the cytosolic fraction of neutrophils. In the present study, human calprotectin was purified from neutrophils using two-step ion exchange chromatography. The purified protein was used for circular dichroism study and fluorescence analysis in the presence of calcium and zinc at physiological concentrations, as well as for assessment of its inhibitory activity on the K562 leukemia cell line. The thermal stability of the protein at pH 7.0 （physiological pH） and 8.0 （similar to intestinal pH） was also compared. The results of cell proliferation analysis revealed that human calprotectin initiated growth inhibition of the tumor cells in a dose- dependent manner. The intrinsic fluorescence emission spectra of human calprotectin （50 ktg/ml） in the presence of calcium and zinc ions show a reduction in fluorescence intensity, reflecting a conformational change within the protein with exposure of aromatic residues to the protein surface that is important for the biological function of calprotectin. The far ultraviolet-circular dichroism spectra of human calprotectin in the presence of calcium and zinc ions at physiological concentrations show a decrease in the m-helical content of the protein and an increase in [3- and other structures. Our results also show that increasing the pH level from 7.0 to 8.0 leads to a marked elevation in the thermal stability of human calprotectin, indicating a significant role for pH in the stability of calprotectin in the gut.     

Cloud-point temperatures for lysozyme in electrolyte solutions: effect of salt type, salt concentration and pH

Liquid-liquid phase-separation data were obtained for aqueous saline solutions of hen egg-white lysozyme at a fixed protein concentration (87 g/l). The cloud-point temperature (CPT) was measured as a function of salt type and salt concentration to 3 M, at pH 4.0 and 7.0. Salts used included those from mono and divalent cations and anions. For the monovalent cations studied, as salt concentration increases, the CPT increases. For divalent cations, as salt concentration rises, a maximum in the CPT is observed and attributed to ion binding to the protein surface and subsequent water structuring. Trends for sulfate salts were dramatically different from those for other salts because sulfate ion is strongly hydrated and excluded from the lysozyme surface. For anions at fixed salt concentration, the CPT decreases with rising anion kosmotropic character. Comparison of CPTs for pH 4.0 and 7.0 revealed two trends. At low ionic strength for a given salt, differences in CPT can be explained in terms of repulsive electrostatic interactions between protein molecules, while at higher ionic strength, differences can be attributed to hydration forces. A model is proposed for the correlation and prediction of the CPT as a function of salt type and salt concentration. NaCl was chosen as a reference salt, and CPT deviations from that of NaCl were attributed to hydration forces. The Random Phase Approximation, in conjunction with a square-well potential, was used to calculate the strength of protein-protein interactions as a function of solution conditions for all salts studied.     

Spectrum of Newcastle disease virus stability in gradients of temperature and pH

Newcastle disease (ND) is one of the highly pathogenic viral diseases of avian species. The disease is endemic in many developing countries where agriculture serves as the primary source of national income. Newcastle disease virus (NDV) belongs to the family Paramyxoviridae and is well characterized member among the avian paramyxovirus serotypes. The failure of vaccination is one of the major causes of NDV outbreaks in field condition. The present study gives a brief picture about the biology of NDV genome and its proteins under different conditions of temperature and pH. Our results indicate that the NDV is non-infective above 42 °C and unstable above 72 °C. The study will be useful in defining an optimum storage condition for NDV without causing any deterioration in its viability.