The mechanism of folding of globular proteins. Suitability of a penicillinase from Staphylococcus Aureus as a model for refolding studies

The results of polyacrylamide disc electrophoretic, gel chromatographic and agar gel immunoelectrophoretic analyses of rabbit seminal plasma were compared to those for uterine secretions and serum. Two of the seminal plasma proteins seemed to be identical with the uterine proteins uteroglobin and prealbumin.     

Characterization of isoleucyl-tRNA synthetase from Staphylococcus aureus. II. Mechanism of inhibition by reaction intermediate and pseudomonic acid analogues studied using transient and steady-state kinetics

The interactions of isoleucyl-tRNA synthetase (IleRS, E) from Staphylococcus aureus with both intermediate analogues and pseudomonic acid (PS-A) have been investigated using transient and steady-state techniques. Non-hydrolyzable analogues of isoleucyl-AMP (I) were simple competitive inhibitors (Ile-ol-AMP, Ki = 50 nM and Ile-NHSO2-AMP, Ki = 1 nM;). PS-A (J) inhibits IleRS via a slow-tight binding competitive mechanism where E.J (Kj = approximately 2 nM), undergoes an isomerization to form a stabilized E*.J complex (K*j = 50 pM). To overcome tight-binding artifacts when K*j < [E], K*j values were estimated from PPi/ATP exchange where [S] > Km, thus raising K*j,app well above [E]. Using [3H]PS-A, it was confirmed that binding occurs with 1:1 stoichiometry and is reversible. Formation of inhibitor complexes was monitored directly through changes in enzyme tryptophan fluorescence. For Ile-ol-AMP and Ile-NHSO2-AMP, the fluorescence intensity of E.I was identical to that when E.Ile-AMP forms catalytically. Binding of PS-A induced only a small change in IleRS fluorescence that was characterized using transient kinetic competition. SB-205952, a PS-A analogue, produced a 37% quenching of IleRS fluorescence upon binding as a result of radiationless energy transfer. Inhibitor reversal rates were obtained by measuring relaxation between spectroscopically different complexes. Together, these data represent a comprehensive solution to the kinetics of inhibition by these compounds.     

Cooperative thermal transitions of bovine and human apo-alpha-lactalbumins: evidence for a new intermediate state

The thermal denaturation of bovine and human apo-alpha-lactalbumins at neutral pH has been studied by intrinsic protein fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC) methods. Apo-alpha-lactalbumin possesses a thermal transition with a midpoint about 25-30 degrees C under these conditions (pH 8.1, 10 mM borate, 1 mM EGTA), which is reflected in changes in both fluorescence emission maximum and quantum yield. However, the CD showed a decrease in ellipticity at 270 nm with a midpoint at about 10-15 degrees C, while DSC shows the transition within the region of 15-20 degrees C. The non-coincidence of transition monitored by different methods suggests the existence of an intermediate state in the course of the thermal denaturation process. This intermediate state is not the classical molten globule state which occurs at higher temperature (i.e. denatured state at these conditions) [D.A. Dolgikh, R.I. Gilmanshin, E.V. Brazhnikov, V.E. Bychkova, G.V. Semisotnov, S.Y. Venyaminov and O.B. Ptitsyn, FEBS Letters, 136 (1981) 311-315] and has physical properties intermediate between the native and molten globule states.     

Permethylation alters the conformational transitions and the complexing ability of melittin: a model for methylated proteins

A number of researches have used factor analyses and principal components analyses on handedness questionnaire data in order to learn something about handedness. However, these researchers have analyzed pooled data from righthanders and lefthanders. The practice of pooling groups that are known in advance to be heterogeneous is highly questionable because it is not possible to disentangle the sources of variance that are contributed by within group differences from those that arise from between group differences. The factor structure and item loadings that result from pooled data are misleading and cannot inform meaningfully about the relation of hand preference to handedness. Similar problems can be anticipated in other neuropsychological applications of factor analysis, where data from heterogeneous groups is pooled.     

Sec-independent insertion of thylakoid membrane proteins. Analysis of insertion forces and identification of a loop intermediate involving the signal peptide

A group of membrane proteins are synthesized with cleavable signal sequences but inserted into the thylakoid membrane by an unusual Sec/SRP-independent mechanism. In this report we describe a key intermediate in the insertion of one such protein, photosystem II subunit W (PSII-W). A single mutation in the terminal cleavage site partially blocks processing and leads to the formation of an intermediate-size protein in the thylakoid membrane during chloroplast import assays. This protein is in the form of a loop structure: the N and C termini are exposed on the stromal face, whereas the cleavage site has been translocated into the lumen. In this respect the insertion of this protein resembles that of M13 procoat, which also adopts a loop structure during insertion, and we present preliminary evidence that a similar mechanism is used by another thylakoid protein, PSII-X. However, whereas the negatively charged region of procoat is translocated by an apparently electrophoretic mechanism using the DeltamuH+, the corresponding region of PSII-W is equally acidic but insertion is DeltamuH+ independent. We furthermore show that neutralization of this region has no apparent effect on the insertion process. We propose that a central element in this insertion mechanism is a loop structure whose formation is driven by hydrophobic interactions.     

The lipase from Staphylococcus aureus. Expression in Escherichia coli, large-scale purification and comparison of substrate specificity to Staphylococcus hyicus lipase

The genes coding for the mature part of the lipases from Staphylococcus aureus NCTC8530 and Staphylococcus hyicus have been cloned and overexpressed in Escherichia coli as fusion proteins with an N-terminal hexa-histidine tag. The enzymes accumulated in the cytoplasm and were purified using sequential precipitation with protamine sulphate and ammonium sulphate, followed by metal-affinity and hydroxyapatite chromatography. The yield of pure lipase was 4.5 mg/g wet cells for S. aureus lipase and 13 mg/g for S. hyicus lipase. The purified enzymes need calcium for activity, albeit with different affinities, and a low residual activity was found in the absence of calcium. In contrast to S. hyicus lipase, not only strontium but also barium can replace calcium with full retention of activity of S. aureus lipase. Whereas S. hyicus lipase is optimally active at pH 8.5, the optimum pH for enzymatic activity for S. aureus lipase was found to be pH 6.5. The S. aureus lipase has a narrow substrate specificity: short-chain triacylglycerols and acyl esters of both p-nitrophenol and umbelliferone are readily degraded, whereas medium- and long-chain lipids, as well as phospholipids, are poor substrates. In contrast, S. hyicus lipase prefers phospholipids as substrate and hydrolyses neutral lipids irrespective of their chain length. The results are discussed in view of the large sequence similarity between both lipases.     

Structural and dynamical characterization of a biologically active unfolded fibronectin-binding protein from Staphylococcus aureus

A 130-residue fragment (D1-D4) taken from a fibronectin-binding protein of Staphylococcus aureus, which contains four fibronectin-binding repeats and is unfolded but biologically active at neutral pH, has been studied extensively by NMR spectroscopy. Using heteronuclear multidimensional techniques, the conformational properties of D1-D4 have been defined at both a global and a local level. Diffusion studies give an average effective radius of 26.2 +/- 0.1 A, approximately 75% larger than that expected for a globular protein of this size. Analysis of chemical shift, 3JHNalpha coupling constant, and NOE data show that the experimental parameters agree well overall with values measured in short model peptides and with predictions from a statistical model for a random coil. Sequences where specific features give deviations from these predictions for a random coil have however been identified. These arise from clustering of hydrophobic side chains and electrostatic interactions between charged groups. 15N relaxation studies demonstrate that local fluctuations of the chain are the dominant motional process that gives rise to relaxation of the 15N nuclei, with a persistence length of approximately 7-10 residues for the segmental motion. The consequences of the structural and dynamical properties of this unfolded protein for its biological role of binding to fibronectin have been considered. It is found that the regions of the sequence involved in binding have a high propensity for populating extended conformations, a feature that would allow a number of both charged and hydrophobic groups to be presented to fibronectin for highly specific binding.     

Consecutive isolation of homologous strains of methicillin-resistant and methicillin-susceptible Staphylococcus aureus from a hospitalized child

Considering that there's a lack of information concerning the risks of radiation exposure in pregnancy, the author identifies such risks and estimates its magnitude. He underlines the fact that radiological examinations deliver lower doses in relation to those that can be proved to be malefic to the embryo, both in the somatic and genetic plan.     

On the mechanism of human stefin B folding: II. Folding from GuHCl unfolded, TFE denatured, acid denatured, and acid intermediate states

In this report, we demonstrate the utility of interleukin-2 (IL-2) stimulation of spleen cell cultures and bivariate flow cytometry in the analysis and purification of the C57BL/6J mouse Y Chromosome. We determined that the DNA content of the C57BL/6J Y Chromosome is approximately 94.7 Mb, making it similar in size to human Chromosome 16 and significantly larger than previous estimates. In addition, we describe the bulk isolation of mouse Y Chromosomes and demonstrate enrichment of the isolated material using a fluorescence in situ hybridization strategy. We detail the construction of two small insert Y Chromosome-specific libraries, ideal for sampling Y Chromosome sequences. From these libraries 1566 clones were analyzed. We provide a detailed characterization of 103 clones, generating nearly 50 kb of sequence. For 30 of these clones, we identify regions of homology to known Y chromosomal sequences, confirming the enrichment of the sorted DNA. From the remaining characterized clones, we describe the development of 15 male-specific PCR assays and 19 male-female PCR assays potentially originating from the pseudoautosomal region or other areas of X-Y or autosome-Y homology.     

Viability of a Bac. licheniformis 749/c culture and its formation of penicillinase during storage in the lyophilized state

The amount of the enzyme produced and the colony morphology of Bac. licheniformis 749/C, the penicillinase-producing organism were studied after storage for 3 years in ampoules at a temperature of 4--10 degrees C, in lyophilized form in sodium glutamate, polyvinylpirrolidone, their mixture and horse serum. The highest rate of the culture growth was observed after storage in lyophlized form in sodium glutamate, though the culture was vialable in all other protective media. Two culture types approximately in an equal ratio were observed in the population platings of the culture lyophilized in various protective media.     

The slow step of folding of Staphylococcus aureus PC1 beta-lactamase involves the collapse of a surface loop rate limited by the trans to cis isomerization of a non-proline peptide bond

We wished to test the hypothesis that the non proline cis to trans isomerization of the peptide bond at position 167 in the S. aureus beta-lactamase PC1 exerts a significant controlling effect on the folding pathway of this enzyme. The previous data presented in support of this hypothesis could not rule out the effect of factors unrelated to non-proline cis/trans isomerization. We have used the plasmid pET9d to direct soluble overproduction of the S. aureus beta-lactamase PC1 and a site-directed mutant (Ile 167 to Pro) in Escherichia coli. Following purification the proteins were subjected to a comparative analysis of the kinetics of unfolding and refolding using the techniques of near- and far-UV circular dichroism spectroscopy and fluorescence spectroscopy in conjunction with "double-jump" experiments. Results show that the fully-unfolded I167P mutant enzyme retains 20% of molecules in a fast-refolding form and that slower-refolding molecules fold faster than the recombinant wild-type enzyme. The final stage of folding involves folding of the omega-loop into a conformation essential for enzymatic activity. In support of the original hypothesis, the folding of this omega-loop is rate limited by the isomerization of the Glu 166-Ile 167 peptide bond.     

Kinetic mechanism of a partial folding reaction. 1. Properties Of the reaction and effects of denaturants

The bimolecular association rate constant (kon) and dissociation rate constant (koff) of the complex between fluorescein-labeled S-peptide analogues and folded S-protein are reported. This is the first kinetic study of a protein folding reaction in which most of the starting material is already folded and only a small part (one additional helix) becomes ordered; it provides a folding landscape with a small conformational entropy barrier, and one in which kinetic traps are unlikely. Refolding and unfolding are measured under identical strongly native conditions, and the reaction is found to be two-state at low reactant concentrations. The dissociation constant (Kd) of the complex and the properties of the transition state may be calculated from the rate constants without extrapolation. The folded complex is formed fast (kon = 1.8 x 10(7) M-1 s-1) and is very stable (Kd = 6 pM) at 10 degrees C, 10 mM MOPS, pH 6.7. Charge interactions stabilize the complex by 1.4 kcal mol-1. The charge effect enters in the refolding reaction: increasing the salt concentration reduces kon dramatically and has little effect on koff. Urea and GdmCl destabilize the complex by decreasing kon and increasing koff. The slopes (m-values) of plots of ln Kd vs [cosolvent] are 0.75 +/- 0.04 and 2.8 +/- 0.3 kcal mol-1 M-1 for urea and GdmCl, respectively. The ratio mon/(mon + moff) is 0.54 +/- 0.04 for urea and 0.57 +/- 0.1 for GdmCl, where mon is the m-value for kon and moff is the m-value for koff, indicating that more than half of the sites for interaction with either cosolvent are buried in the ensemble of structures present at the transition state.     

The kinetics and mechanism of the thickening of κ plates in a Cu-Si alloy

Hot stage, polarized light microscopy studies have shown that hcp κ plates, formed in a fcc α Cu-Si matrix, thicken discontinuously. Abrupt, micron-sized increases in thickness take place during the initial minutes of transformation and occasionally thereafter. At all other times, the rate of thickening is, at most, exceedingly small. There are very few misfit dislocations at the broad face of κ plates. The combination of these experimental results and the very small free energy change attending κ precipitation rule out both conventional heterogeneous nucleation and most diffusional growth processes as significant contributors to the development of κ plates. The principal mechanism of this transformation appears to be the formation of bundles of stacking faults, evidently in response to cooling stresses. The fault bundles are then enriched to the composition of the κ phase by the inward diffusion of silicon.     

The femR315 gene from Staphylococcus aureus, the interruption of which results in reduced methicillin resistance, encodes a phosphoglucosamine mutase

The femR315 gene was recently identified by Tn551 insertional mutagenesis as one of the new auxiliary genes, the alteration of which resulted in a drastically reduced methicillin resistance of the Staphylococcus aureus strain COL. femR315 (also known as femD) theoretically encoded a protein of 451 amino acids showing significant amino acid sequence homology with phosphoglucomutases and similar enzymes catalyzing the isomerization of hexoses and hexosamine phosphates (S. Wu, H. de Lencastre, A. Sali, and A. Tomasz, Microb. Drug Resist. 2:277-286, 1996). We describe here the overproduction and purification of the FemR315 protein as well as its identification as the phosphoglucosamine mutase which catalyzes the formation of glucosamine-1-phosphate from glucosamine-6-phosphate, the first step in the reaction sequence leading to the essential peptidoglycan precursor UDP-N-acetylglucosamine. On the basis of these findings, we propose to change the names femR315 and femD to the functionally more appropriate name glmM.     

The first glimpse of a complex of nitrogenase component proteins by solution X-ray scattering: conformation of the electron transfer transition state complex of Klebsiella pneumoniae nitrogenase

An essential feature of the mechanism of nitrogenase, the enzyme responsible for biological nitrogen fixation, is the formation of a transient electron transfer complex between the MoFe protein containing the active site at which N2 is reduced, and the Fe protein, which functions as a specific electron donor to the MoFe protein. We have obtained high quality solution X-ray scattering data using synchrotron X-rays of a stable putative electron transfer complex, (MoFe-protein)(Fe-protein.ADP.AIF4)2, of Klebsiella pneumoniae and used the model-independent approach based on the multipole expansion method to provide a stable and unique shape restoration at approximately 15 A resolution. The biological significance of this first molecular structure of a nitrogenase complex is discussed.     

奥氏体不锈钢在含锌PWR一回路水中的均匀腐蚀行为

在模拟压水堆一回路水化学环境中,对主管道316L不锈钢和Stellite 6钴基合金分别开展了0,10,40 μg·L^–1三种Zn质量浓度的均匀腐蚀试验. 试验结束后,采用失重法计算两种材料的腐蚀速率和腐蚀产物释放速率,采用扫描电子显微镜（SEM）、透射电镜能谱仪（TEM-EDS）以及高分辨和傅里叶转变分析氧化膜表面形貌、截面形貌、厚度、元素分布以及双层氧化膜相结构. 结果表明,对于316L不锈钢,1000 h内10 μg·L^–1 Zn的注入对腐蚀速率和释放速率影响不显著,增加Zn质量浓度至40 μg·L^–1后,316L不锈钢的腐蚀速率、腐蚀产物释放速率和氧化膜厚度显著降低,其中氧化膜厚度由250 nm降低至95 nm. 对于具有双相结构的Stellite 6钴基合金,γ-Co基体和碳化物间存在电偶腐蚀效应,γ-Co基体和相界腐蚀更显著. 进一步延长腐蚀时间至3000 h,发现10 μg·L^–1 Zn注入可以显著降低其腐蚀速率和腐蚀产物释放速率,当Zn质量浓度增加至40 μg·L^–1时,钴基合金的腐蚀速率、腐蚀产物释放速和氧化膜厚度进一步降低. 微观分析表明,注锌对两种合金腐蚀抑制机理相似,注入的Zn离子会在金属表面形成含Zn的尖晶石结构,显著提高外层氧化膜的致密性,阻碍金属离子向外扩散及氧离子向内扩散,促进内层氧化膜/基体界面处保护性Cr₂O₃的形成,进而显著降低316L不锈钢和Stellite 6钴基合金的腐蚀速率、腐蚀产物释放速率和氧化膜厚度.

     

The mechanism and kinetics of producing single-crystal powders of titanium carbide via a hydride-calcium method

The mechanism and kinetics of the reduction-carbidization of titanium oxide by calcium hydride and carbide (hydride-calcium method) are studied. This process provides the production of single-crystal powdered titanium carbide. Theoretical analysis and experimental methods is found that the mechanism of the formation of TiC forms is staged and includes two sequential reactions: TiO₂ + 2CaH₂ = Ti + 2CaO + 2H₂↑ and Ti + C = TiC. It is revealed that the formation of titanium carbide proceeds in a calcium melt by the dissolution of titanium and graphite in it with the subsequent crystallization of the TiC particles from the melt. It is noted that the kinetics of the process in the range t = 900?1200°C depends on the temperature and time of isothermal holding. At t > 1100 °C, its abrupt activization is observed. This is due to an increase in the amount of melt based on calcium and the enhancement of the solubility of titanium and carbon in it, which leads to the acceleration of reactions of reduction of titanium oxide to titanium and to the synthesis of titanium carbide. The optimum technological parameters of the process (temperature, time of isothermal holding, and conditions of the calculation of the charge) are determined. It is shown that titanium carbide obtained by this technology is homogeneous and has the content close to stoichiometric one, specifically, TiC_1.0.     

The kinetic mechanism of serpin-proteinase complex formation. An intermediate between the michaelis complex and the inhibited complex

Serine proteinase inhibitors (serpins) form enzymatically inactive, 1:1 complexes (denoted E*I*) with their target proteinases that release free enzyme and cleaved inhibitor only very slowly. The mechanism of E*I* formation is incompletely understood and continues to be a source of controversy. Kinetic evidence exists that formation of E*I* proceeds via a Michaelis complex (E.I) and so involves at least two steps. In this paper, we determine the rate of E*I* formation from alpha-chymotrypsin and alpha1-antichymotrypsin using two approaches: first, by stopped-flow spectrofluorometric monitoring of the fluorescent change resulting from reaction of alpha-chymotrypsin with a fluorescent derivative of alpha1-antichymotrypsin (derivatized at position P7 of the reactive center loop); and second, by a rapid mixing/quench approach and SDS-polyacrylamide gel electrophoresis analysis. In some cases, serpins are both substrates and inhibitors of the same enzyme. Our results indicate the presence of an intermediate between E.I and E*I* and suggest that the partitioning step between inhibitor and substrate pathways precedes P1-P1' cleavage.