Multidomain structure of a recombinant streptokinase. A differential scanning calorimetry study

The temperature dependence of the heat capacity function of a recombinant streptokinase (rSK) has been studied by high-sensitivity differential scanning microcalorimetry and circular dichroism as a function of pH in low- and high-ionic strength buffers. At low ionic strength it is found that this protein, between pH 7 and 10, undergoes four reversible and independent two-state transitions during its unfolding, suggesting the existence of four domains in the native structure of the protein. This result reconciles previous conflicting reports about the number of domains of this protein obtained by differential scanning calorimetry and small-angle X-ray scattering. The number of two-state transitions decreases when the pH of the medium is decreased, without noticeable changes in its circular dichroism spectrum. A plausible localization of the four domains in the streptokinase sequences is proposed and their thermodynamic parameters are given. Increase of ionic strength to 200 mM NaCl affects positively the protein stability and confirms the existence of four reversible two-state transitions. Above 200 mM NaCl the protein stability decreases, resulting in low percentage of reversibility, and even irreversible transitions.     

Use of high culture pH for the decreased proteolysis of human parathyroid hormone secreted by Saccharomyces cerevisiae

Human parathyroid hormone (hPTH) was expressed and secreted in Saccharomyces cerevisiae. In batch fermentations performed at pH = 5.6, 6.5, 7.2 and 7.5, optimal production of hPTH (12.1 mg/l) was obtained at pH 7.2 after 24 h of culture. At pH 5.6, most of secreted hPTH was degraded. Proteolysis of hPTH was significantly decreased by increasing the culture pH.     

南极细菌胞外多糖溶液冻结特性的差示扫描量热研究

采用差示扫描量热法,测定几种南极细菌胞外多糖(简称,EPSs)溶液的结晶、熔融、焓转变以及水合性质等冻结特性,分析了EPSs的浓度和分子量与其抗冻活性的关系.结果表明,在溶液冻结过程中,仅0.25%的Pseudoalteromonas sp.S-15-13 EPSs(分子量,6.2×104Da)可抑制冰核形成,溶液冻结温度较纯水的降低(1.07±0.62)℃;溶液的冻结焓降低说明冰核生长变缓,冰晶形成细小,0.125%的Shewanella sp.5-1-11-4 EPSs(分子量,1.2×103Da)和Moritella sp.2-5-10-1 EPSs(分子量,3.0×103Da)冻结焓分别较纯水的降低17.15%和29.13%,S-15-13 EPSs在0.125%～0.5%的范围内可降低冻结焓,0.125%时冻结焓较纯水的低30%,其不冻水含量为(0.292 ±0.05) g/g.在冰晶熔化过程中,几种EPSs均可降低溶液熔融温度和熔融焓,促进冰晶熔化,使冰晶细小;4.0%的5-1-11-4 EPSs、2-5-10-1 EPSs和0.5% S-15-13 EPSs的熔融温度较纯水的分别降低(2.70±0.15)℃、(2.30±0.39)℃和(4.66±0.42)℃.研究结果阐明EPSs可以通过改变菌体周围水的冻结特性,以抵御冰晶对微生物的损伤,大分子量EPSs对冰晶的抑制作用强于低分子量的.     

Thermal unfolding of staphylococcal nuclease and several mutant forms thereof studied by differential scanning calorimetry.

The effects of eight mutations on the thermodynamics of the reversible thermal unfolding of staphylococcal nuclease have been determined over a range of pH and protein concentration by means of differential scanning calorimetry. Variation of the protein concentration was included in our study because we found a significant dependence of the thermodynamics of protein unfolding on concentration. Values for the change in the standard free energy of unfolding, delta delta G0d, produced by the mutations in the pH range 5.0-7.0 varied from 1.9 kcal mol-1 (apparent stabilization) for H124L to -2.8 kcal mol-1 (apparent destabilization) for L25A. As has been observed in numerous other cases, there is no correlation in magnitude or sign between delta delta G0d and the corresponding values for delta delta Hd and T delta delta S0d, the latter quantities being in most cases much larger in magnitude than delta delta G0d. This fact emphasizes the difficulty in attempting to correlate the thermodynamic changes with structural changes observed by X-ray crystallography.     

Binding study of riboflavin-binding protein with riboflavin and its analogues by differential scanning calorimetry

Thermal unfolding parameters of hens' egg-white riboflavin-binding-protein (RBP) were measured by differential scanning calorimetry. Thermal denaturation scans of apoRBP and RBP complexes with riboflavin and its analogues (FMN, N10 DL-glyceryl isoalloxazine, and N10 -hydroxypentyl isoalloxazine) have been measured. It was found that ligand binding causes increase of RBP thermal stability, as manifested by a change of denaturation temperature from 60.8°C for apoRBP to 72.8°C for RBP—Rf complex. For RBP—FMN complex, the denaturation temperature of 73.0°C was even higher than for the RBP—Rf complex. The other two flavin analogues showed transition temperatures in between 66.9°C and 68.8°C, respectively. Analysis of excess heat capacity data showed that the best fit was the sum of two independent thermal transitions. One of the transitions, which contributed 70% to the total heat effect, has transition temperature in the broad range of 60.5–73.2°C; the other transition temperature is in the narrower range of 65.4–71.1°C. The observed transitions can be related to RBP domains.     

酿酒酵母ADH3基因的敲除

设计含有与酿酒酵母(Saccharomyces cerevisiae)编码乙醇脱氢酶Ⅲ的ADH3基因ORF两侧序列同源的长引物,以质粒pUG6为模板进行PCR构建带有Cre/loxP系统的敲除组件。转化酿酒酵母YS3(Saccharomyces cerevisiae),并将质粒pSH65转入阳性克隆子。半乳糖诱导表达Cre酶切除Kanr基因,在YPD培养基中连续传代培养丢失pSH65质粒,在原ORF处留下一个loxP位点,获得ADH3单倍体缺陷型菌株。利用同样的方法再次敲除双倍体的另一个等位基因。最终获得ADH3双倍体基因缺陷型突变株YS3-ADH3。     

Thermal porosity analysis of croscarmellose sodium and sodium starch glycolate by differential scanning calorimetry

The aim of the study was to demonstrate the applicability of differential scanning calorimetry (DSC) on porosity analysis for cellulose and starch. Croscarmellose sodium (CCS) and sodium starch glycolate (SSG) were allowed to sorb moisture in 85%, 90%, 95%, and 100% relative humidity (RH) at 40°C for 24 hours. The pretreated samples were then subjected to DSC running temperature ranging from 25°C to −50°C at a cooling rate of 10°C/min. The cooling traces of water crystallization, if present, were transformed to porosity distribution via capillary condensation using Kelvin's equation. The porosity analysis of CCS and SSG was also done using nitrogen adsorption as a reference method. It was found that sorbed water could not be frozen (in cases of 85% and 90% RH) until the moisture content exceeded a cutoff value (in cases of 95% and 100% RH). The nonfreezable moisture content was referred to tightly bound, plasticizing water, whereas the frozen one may be attributed to loosely bound water condensation in pore structure of CCS and SSG surfaces. Not only capillary condensation but also the tightly bound, nonfreezable monolayer water lying along the inner pores of the surface contributed to porosity determination. Good agreement with less than 5% deviation of mean pore size was observed when the results were compared with nitrogen adsorption. The narrower pore size distributions, however, were obtained because of the limitations of the technique. It was concluded that pore analysis by DSC could be successful. Further research needs to be done to account for limitations and to extend the applicability of the technique.     

Domain structure and molecular flexibility of streptococcal M proteinIn Situ probed by limited proteolysis

Serologically distinct group A streptococcal M proteins, the antiphagocytic determinants of the bacteria, have a highly repetitive sequence and exhibit a heptad periodicity characteristic of alpha-helical coiled-coil proteins. Based on the differences in the pattern of heptad periodicity, the coiled-coil region of the complete M molecule has been divided into three distinct domains: I, II, and III. Domains I and II together constitute the variable part of M protein, whereas domain III is conserved among serotypes. Pepsin treatment of the M5, M6, and M24 streptococci results in a preferential cleavage of their M molecules between the predicted domains II and III, releasing biologically active fragments of the respective M proteins. Thus, a pepsin cleavage site at the junction of their variable and conserved regions is conserved in the M5, M6, and M24 proteins. In contrast, in the case of the M49 streptococci, the primary site of pepsin cleavage was observed to be within the conserved region of the M49 molecule, rather than at the junction of its variable and conserved regions. Despite containing part of the conserved region, the PepM49 protein is significantly smaller than the pepsin fragments of the M5, M6, and M24 proteins, which contain only the variable regions. However, in addition to the major PepM49 species, the pepsin digest of the type-49 streptococci also contained a smaller fragment, PepM49/a, as a minor component. Its formation was extremely sensitive to thepH of pepsin digestion. PepM49/a, which retains both the propensity to attain an alpha-helical conformation and the opsonic antibody epitope of the M49 molecule, contains only domains I and II like the other PepM proteins. Thus, as in the M5, M6, and M24 proteins, a pepsin cleavage site at the junction of the variable and conserved regions is indeed present in the M49 molecule, but is much less accessible relative to the other serotypes. Thus, the pepsin cleavage sites in the M protein correlate quite well with the boundaries of structurally distinct domains reflected by the predictive analysis. These sites apparently represent the flexible/hinge regions of the molecule. PepM49/a is the least repetitive and the shortest of the M protein pepsin fragments isolated so far. These results suggest that the flexibility of the interdomain regions in M protein may be dependent on the molecular size of their variable domains. The placement of a more accessible hinge within the conserved part of the M49 molecule, rather than at the junction of the variable and conserved domains, suggests that a critical molecular size may be essential for the efficient functioning of the M molecule.     

Interactions of tryptophan-rich cathelicidin antimicrobial peptides with model membranes studied by differential scanning calorimetry

The 13-residue cathelicidins indolicidin and tritrpticin are part of a group of relatively short tryptophan-rich antimicrobial peptides that hold potential as future substitutes for antibiotics. Differential scanning calorimetry (DSC) has been applied here to study the effect of indolicidin and tritrpticin as well as five tritrpticin analogs on the phase transition behaviour of model membranes made up of zwitterionic dimyristoylphosphatidylcholine (DMPC, DMPC/cholesterol) and anionic dimyristoylphosphatidyl glycerol (DMPG) phospholipids. Most of the peptides studied significantly modified the phase transition profile, suggesting the importance of hydrophobic forces for the peptide interactions with the lipid bilayers and their insertion into the bilayer. Indolicidin and tritrpticin are both known to be flexible in aqueous solution, but they adopt turn-turn structures when they bind to and insert in a membrane surface. Pro-to-Ala substitutions in tritrpticin, which result in the formation of a stable α-helix in this peptide, lead to a substantial increase in the peptide interactions with both zwitterionic and anionic phospholipid vesicles. In contrast, the substitution of the three Trp residues by Tyr or Phe resulted in a significant decrease of the peptide's interaction with anionic vesicles and virtually eliminated binding of these peptides to the zwitterionic vesicles. An increase of the cationic charge of the peptide induced much smaller changes to the peptide interaction with all lipid systems than substitution of particular amino acids or modification of the peptide conformation. The presence of multiple lipid domains with a non-uniform peptide distribution was noticed. Slow equilibration of the lipid-peptide systems due to peptide redistribution was observed in some cases. Generally good agreement between the present DSC data and peptide antimicrobial activity data was obtained.     

Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis

The SH3 domains are small protein modules of 60-85 amino acid residues that are found in many proteins involved in intracellular signal transduction. The SH3 domain of the p85alpha subunit of bovine phosphatidylinositol 3'-kinase (PI3-SH3) under acidic solution adopts a compact denatured state from which amyloid fibrils are readily formed. This aggregation process has been found to be modulated substantially by solution conditions. Here, we have analyzed the conformational features of the native and acid denatured states of PI3-SH3 by limited proteolysis experiments using proteinase K and pepsin, respectively. Moreover, we have analyzed the propensity of PI3-SH3 to be hydrolyzed by pepsin at different stages in the process of aggregation and amyloid formation at pH 1.2 and 2.0 and compared the sites of proteolysis under these conditions with the conformational features of both native and aggregated PI3-SH3. The results demonstrate that the denatured state of PI3-SH3 formed at low pH is relatively resistant to proteolysis, indicating that it is partially folded. The long loop connecting beta-strands b and c in the native protein is the region in this structure most susceptible to proteolysis. Remarkably, aggregates of PI3-SH3 that are formed initially from this denatured state in acid solution display enhanced susceptibility to proteolysis of the long loop, suggesting that the protein becomes more unfolded in the early stages of aggregation. By contrast, the more defined amyloid fibrils that are formed over longer periods of time are completely resistant to proteolysis. We suggest that the protein aggregates formed initially are relatively dynamic species that are able readily to reorganize their interactions to enable formation of very well ordered fibrillar structures. In addition, the disordered and non-native character of the polypeptide chains in the early aggregates could be important in determining the high cytotoxicity that has been revealed in previous studies of these species.     

Interaction of cAMP receptor protein from Escherichia coli with cAMP and DNA studied by differential scanning calorimetry

The cyclic AMP receptor protein (CRP) regulates the expression of many genes in Escherichia coli. The protein is a homodimer, and each monomer is folded into two distinct structural domains. In this study, we have used differential scanning calorimetry (DSC) and circular dichroism (CD) to measure the enthalpy change and melting temperature of the apo-CRP and CRP complexes with cAMP or DNA sequences lac, gal, and palindromic ICAP. DSC and CD measurements showed irreversible thermal denaturation process of CRP. Enthalpy of dissociation of the protein–DNA complex, as measured by DSC, depends on the DNA sequence. The thermal transition of the protein in CRP-DNA complexes, measured by CD, indicates that the protein stability in the complex is also DNA sequence-dependent.     

Perturbations of membrane structure by optical probes: II. Differential scanning calorimetry of dipalmitoyllecithin and its analogs interacting with merocyanine 540

Summary Differential scanning calorimetry of multilamellar liposomes, interacting with the optical probe Merocyanine 540, yields quantitative information about perturbances of the bilayer structure induced by this dye. At low dye: lipid ratios, the dye perturbs primarily its own microenvironment, which is laterally separated from the unmodified lipid domain and exhibits modified thermotropic properties. A further increase in the dye concentration results in a perturbance of the whole lipid bilayer. The degree of perturbance is sensitive to structural modifications in the head-group region of the lipids. It is concluded that Merocyanine 540 reports in every case, even at infinite dilution, on localized events originating from a perturbed microenvironment.An abstract of this paper was presented at the XI Congress of Biochemistry, Toronto, in July 1979.     

Analysis of IgG kinetic stability by differential scanning calorimetry,probe fluorescence and light scattering

Monoclonal antibodies of the immunoglobulin G (IgG) type have become mainstream therapeutics for the treatment of many life‐threatening diseases. For their successful application in the clinic and a favorable cost‐benefit ratio, the design and formulation of these therapeutic molecules must guarantee long‐term stability for an extended period of time. Accelerated stability studies, e.g., by employing thermal denaturation, have the great potential for enabling high‐throughput screening campaigns to find optimal molecular variants and formulations in a short time. Surprisingly, no validated quantitative analysis of these accelerated studies has been performed yet, which clearly limits their application for predicting IgG stability. Therefore, we have established a quantitative approach for the assessment of the kinetic stability over a broad range of temperatures. To this end, differential scanning calorimetry (DSC) experiments were performed with a model IgG, testing chaotropic formulations and an extended temperature range, and they were subsequently analyzed by our recently developed three‐step sequential model of IgG denaturation, consisting of one reversible and two irreversible steps. A critical comparison of the predictions from this model with data obtained by an orthogonal fluorescence probe method, based on 8‐anilinonaphthalene‐1‐sulfonate binding to partially unfolded states, resulted in very good agreement. In summary, our study highlights the validity of this easy‐to‐perform analysis for reliably assessing the kinetic stability of IgGs, which can support accelerated formulation development of monoclonal antibodies by ranking different formulations as well as by improving colloidal stability models.     

Conformational changes in the NS3 protease from hepatitis C virus strain Bk monitored by limited proteolysis and mass spectrometry.

Conformational changes occurring within the NS3 protease domain from the hepatitis C virus Bk strain (NS3(1-180)) under different physico-chemical conditions either in the absence or in the presence of its cofactor Pep4A were investigated by limited proteolysis experiments. Because the surface accessibility of the protein is affected by conformational changes, when comparative experiments were carried out on NS3(1-180) either at different glycerol concentrations or in the presence of Pep4A, differential peptide maps were obtained from which protein regions involved in the structural changes could be inferred. The surface topology of isolated NS3(1-180) in solution was essentially consistent with the crystal structure of the protein with the N-terminal segment showing a high conformational flexibility. At higher glycerol concentration, the protease assumed a more compact structure showing a decrease in the accessibility of the N-terminal segment that either was forced to interact with the protein or originate intermolecular interactions with neighboring molecules. Binding of the cofactor Pep4A caused the displacement of the N-terminal arm from the protein moiety, leading this segment to again adopt an open and flexible conformation, thus suggesting that the N-terminus of the protease contributes only marginally to the stability of the complex. The observed conformational changes might be directly correlated with the activation mechanism of the protease by either the cosolvent or the cofactor peptide because they lead to tighter packing of the substrate binding site.     

利用SPT3的定向进化提高工业酿酒酵母乙醇耐受性

利用对转录因子的定向进化可对多基因控制的性状进行有效的代谢工程改造。本研究对酿酒酵母负责胁迫相关基因转录的SAGA复合体成分SPT3编码基因进行易错PCR随机突变,并研究了SPT3的定向进化对酿酒酵母乙醇耐性的影响。将SPT3的易错PCR产物连接改造的pYES2.0表达载体并转化酿酒酵母Saccharomyces cerevisiae4126,构建了突变体文库。通过筛选在高浓度乙醇中耐受性提高的突变株,获得了一株在10%(V/V)乙醇中生长较好的突变株M25。该突变株利用125g/L的葡萄糖进行乙醇发酵时,终点乙醇产量比对照菌株提高了11.7%。由此表明,SPT3是对酿酒酵母乙醇耐性进行代谢工程改造的一个重要的转录因子。     

Thermotropic phase behaviour of lipid bilayers containing carotenoid pigment canthaxanthin: a differential scanning calorimetry study

In this study we address the problem of the effect of canthaxanthin on the thermotropic properties of lipid membranes formed with lipids which differ in the thickness of their hydrophobic core, size of polar heads or presence of the ester carbonyl group. For all the lipids a decrease in main transition enthalpy has been observed, indicating that canthaxanthin alters the membrane properties in its gel phase. The strongest influence of canthaxanthin on main phase transition and pretransition has been observed for the lipid having the thinnest hydrophobic region. Component analysis indicates a distinct cooperativity change, which most probably colligates with the formation of new thermotropic phases. The effect of canthaxanthin has been almost negligible in the case of phosphatidylethanolamines. The absence of the ester carbonyl group results in different thermotropic behavior, especially for low canthaxanthin concentrations. The effect of canthaxanthin is explained in terms of its organization within the membrane.     

Three-dimensional reconstruction of the valyl-tRNA synthetase/elongation factor-1H complex and localization of the delta subunit

Eukaryotic valyl-tRNA synthetase (ValRS) and the heavy form of elongation factor 1 (EF-1H) are isolated as a stable high molecular mass complex that catalyzes consecutive steps in protein biosynthesis--aminoacylation of tRNA and its transfer to elongation factor. Herein is the first three-dimensional structure of the particle as calculated from electron microscopic images of negatively stained samples of the human ValRS/EF-1H complex. The ca. 12 x 8 nm particle has two distinct domains and each appears to have twofold symmetry. Bound antibodies place two delta subunits near the particle's center. These data support a dimeric head-to-head arrangement of particle components.