Identification of neighboring protein pairs in the Escherichia coli 30 S ribosomal subunit by crosslinking with methyl-4-mercaptobutyrimidate

The 30 S ribosomal subunits of Escherichia coli were treated with methyl-4-mercaptobutyrimidate and oxidized to promote the formation of intermolecular disulfate bonds between neighboring proteins. Attention was focused on protein dimers, which were partially purified either by stepwise extraction of the 30 S particle with LiCl or by polyacrylamide/urea gel electrophoresis of the total crosslinked protein. Protein fractions were then analyzed by polyacrylamide/ sodium dodecyl sulfate diagonal gel electrophoresis. Final identification of the components of crosslinked protein pairs, indicated by molecular weight analysis, was accomplished by two-dimensional polyacrylamide/urea gel electrophoresis. The identification of 21 protein pairs is presented, 14 of which have not been reported previously.     

Analysis of rat serum apolipoproteins by isoelectric focusing. I. Studies on the middle molecular weight subunits.

Analytical isoelectric focusing (IEF) has been applied to the study of the apolipoprotein components of rat serum high density and very low density lipoproteins. The apolipoproteins were separated on 7.5% polyacrylamide gels containing 6.8% urea, with a pH gradient of 4-6. The middle molecular weight range apolipoproteins were identified on IEF gels by the use of apolipoproteins purified by electrophoresis on gels containing sodium dodecyl sulfate (SDS). The A-1 protein focused as 4 to 5 bands from pH 5.46 to 5.82; the A-IV protein and the arginine-rich protein each focused as 4 to 6 bands from pH 5.31 to 5.46. The low molecular weight proteins focused from pH. 4.43 to 4.83 and are the subject of a separate communication. Comparisons of the IEF method with SDS gel electrophoresis, polyacrylamide gel electrophoresis in urea, and Sephadex chromatography are also reported. Additional studies were also carried out that tend to rule out carbamylation or incomplete unfolding of the proteins in the presence of urea as the causes of the observed heterogeneity.     

Cation-induced toroidal condensation of DNA studies with Co3+(NH3)6

The unfolding and refolding of Staphylococcus aureus penicillinase have been followed by urea-gradient electrophoresis. Unfolding of the native state proceeds by an all-or-none transition to fully unfolded protein, with no detectable accumulation of partially unfolded states. In contrast, refolding is complex and proceeds by very rapid, reversible formation of a partially folded state, H, which had been detected and characterized previously, as it is the most stable conformation at intermediate denaturant concentrations. At very low urea concentrations, a more compact conformational state was observed as a transient intermediate in refolding. There was little kinetic heterogeneity of the unfolded protein, as is normally observed with proteins containing proline residues.     

A simple electrophoretic procedure for the determination of the polypeptide composition of the subunits of Fraction 1 protein

A rapid and convenient method is described for resolving the polypeptide composition of Fraction 1 protein. Using crude leaf extracts of a number of Lycopersicon species, Fraction 1 protein was first separated by polyacrylamide gel electrophoresis and the gel slices containing the protein were isoelectrofocused in the presence of 8 m urea. Isoelectric focusing was also applied directly on subunits in gel slices obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polypeptide composition produced is in agreement with previous determinations obtained by more elaborated techniques.     

Isolation of outer membrane proteins of Escherichia coli and their characterization on polyacrylamide gel

Proteins from the outer membrane of Escherichia coli were studied on a ureadodecyl sulfate polyacrylamide gel by electrophoresis. A polyacrylamide gel containing sodium dodecyl sulfate and urea gave an excellent resolution of outer membrane proteins. Seventeen protein bands were reproducibly observed on a gel. By use of Sephadex G-200, DEAE-cellulose and polyacrylamide gel, eight proteins were purified to near homogeneity. Five of them were found to be heat-modifiable proteins. The behavior of these purified proteins was studied on a polyacrylamide gel under three different electrophoretic conditions, which had been used for the analysis of cell envelope proteins. Thus correspondence was made between these purified proteins and envelope proteins reported by other investigators.     

Correlation between mutational destabilization of phage T4 lysozyme and increased unfolding rates

The thermodynamics and kinetics of unfolding of 28 bacteriophage T4 lysozyme variants were compared by using urea gradient gel electrophoresis. The mutations studied cause a variety of sequence changes at different residues throughout the polypeptide chain and result in a wide range of thermodynamic stabilities. A striking relationship was observed between the thermodynamic and kinetic effects of the amino acid replacements: All the substitutions that destabilized the native protein by 2 kcal/mol or more also increased the rate of unfolding. The observed increases in unfolding rate corresponded to a decrease in the activation energy of unfolding (delta Gu) at least 35% as large as the decrease in thermodynamic stability (delta Gu). Thus, the destabilizing lesions bring the free energy of the native state closer to that of both the unfolded state and the transition state for folding and unfolding. Since a large fraction of the mutational destabilization is expressed between the transition state and the native conformation, the changes in folding energetics cannot be accounted for by effects on the unfolded state alone. The results also suggest that interactions throughout much of the folded structure are altered in the formation of the transition state during unfolding.     

The conformation and stability of recombinant-derived granulocyte-macrophage colony stimulating factors

The conformation and stability of recombinant-derived human and murine granulocyte-macrophage colony stimulating factors produced in Escherichia coli have been investigated by analytical ultracentrifugation, urea-gradient polyacrylamide gel electrophoresis and several spectroscopic methods. The proteins were demonstrated to be physically homogeneous monomeric proteins with compact globular shapes and shown to have similar secondary structures containing both alpha-helix and beta-sheet structure. The intramolecular disulphide linkages of both proteins were shown to be essential for maintaining native conformation as reduction with dithiothreitol resulted in protein unfolding. Comparison of the human E. coli-derived (non-glycosylated) and mammalian cell culture-derived (glycosylated) proteins by urea-gradient electrophoresis indicated that glycosylation had no major effect on the conformational stability and kinetics of urea induced unfolding and refolding.     

Urea Impedes the Hydrophobic Collapse of Partially Unfolded Proteins

Proteins are denatured in aqueous urea solution. The nature of the molecular driving forces has received substantial attention in the past, whereas the question how urea acts at different phases of unfolding is not yet well understood at the atomic level. In particular, it is unclear whether urea actively attacks folded proteins or instead stabilizes unfolded conformations. Here we investigated the effect of urea at different phases of unfolding by molecular dynamics simulations, and the behavior of partially unfolded states in both aqueous urea solution and in pure water was compared. Whereas the partially unfolded protein in water exhibited hydrophobic collapses as primary refolding events, it remained stable or even underwent further unfolding steps in aqueous urea solution. Further, initial unfolding steps of the folded protein were found not to be triggered by urea, but instead, stabilized. The underlying mechanism of this stabilization is a favorable interaction of urea with transiently exposed, less-polar residues and the protein backbone, thereby impeding back-reactions. Taken together, these results suggest that, quite generally, urea-induced protein unfolding proceeds primarily not by active attack. Rather, thermal fluctuations toward the unfolded state are stabilized and the hydrophobic collapse of partially unfolded proteins toward the native state is impeded. As a result, the equilibrium is shifted toward the unfolded state.     

Sodium dodecyl sulfate polyacrylamide gel electrophoresis as a method for studying the stability of subtilisin

A simple method has been developed to study the stability of subtilisin. Protein incubated at various temperatures in the presence of proteinase inhibitor was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and showed a transition from the intact state to the unfolded state between 55 degrees C and 65 degrees C. Additionally, autolysis was also observed above 65 degrees C. In the absence of inhibitor, similar results were obtained below 55 degrees C; however, above 65 degrees C no protein of any size was observed due to extensive autolysis. These results demonstrate that SDS-PAGE can trap subtilisin in the state in which the protein existed prior to the analysis. It can be used to identify the different forms, including autolysis products, of the protein generated by heat denaturation. This method was used to study SDS-induced unfolding of aprA-subtilisin. When the protein was incubated with 0.25% SDS at different NaCl concentrations, a gradual increase in unfolding was observed with increasing NaCl concentration. This change paralleled a decrease in the critical micelle concentration of SDS, indicating that the rate of unfolding of aprA-subtilisin increases with increasing SDS micelle concentration. No detectable unfolding was observed below the critical micelle concentration.     

Electrostatics of folded and unfolded bovine β-lactoglobulin

We report on electrophoretic, spectroscopic, and computational studies aimed at clarifying, at atomic resolution, the electrostatics of folded and unfolded bovine β-lactoglobulin (BLG) with a detailed characterization of the specific aminoacids involved. The procedures we used involved denaturant gradient gel electrophoresis, isoelectric focusing, electrophoretic titration curves, circular dichroism and fluorescence spectra in the presence of increasing concentrations of urea (up to 8 M), electrostatics computations and low-mode molecular dynamics. Discrepancy between electrophoretic and spectroscopic evidence suggests that changes in mobility induced by urea are not just the result of changes in gyration radius upon unfolding. Electrophoretic titration curves run across a pH range of 3.5–9 in the presence of urea suggest that more than one aminoacid residue may have anomalous pK _a value in native BLG. Detailed computational studies indicate a shift in pKa of Glu44, Glu89, and Glu114, mainly due to changes in global and local desolvation. For His161, the formation of hydrogen bond(s) could add up to desolvation contributions. However, since His161 is at the C terminus, the end-effect associated to the solvated form strongly influences its pK _a value with extreme variation between crystal structures on one side and NMR or low-mode molecular dynamics structures on the other. The urea concentration effective in BLG unfolding depends on pH, with higher stability of the protein at lower pH.     

Effects of lactic acid and NaCl on creatine kinase from rabbit muscle.

The lactic acid induced unfolding and the salt-induced folding of creatine kinase (CK) were studied by enzyme activity, fluorescence emission spectra, circular dichroism spectra, and native polyacrylamide gel electrophoresis. The results showed that the kinetics of CK inactivation was a monophase process. Lactic acid caused inactivation and unfolding of CK with no aggregation during CK denaturation. The unfolding of the whole molecule and the inactivation of CK in solutions of different concentration of lactic acid were compared. Much lower lactic acid concentration values were required to bring about inactivation than were required to produce significant conformational changes of the enzyme molecule. At higher concentrations of lactic acid (more than 0.2 mM) the CK dimers were partially dissociated, as proved by native polyacrylamide gel electrophoresis. NaCl induced the molten globule state with a compact structure after CK was denatured with 0.8 mM lactic acid, and the increasing of anions led to a tight side-chain. The above results suggest that the effect of lactic acid differed from that of other denaturants such as guanidine hydrochloride, HCI, or urea during CK folding, and the molten globule state indicates that intermediates exist during CK folding.     

The subunit interface of the Escherichia coli ribosome. Crosslinking of 30 S protein S9 to proteins of the 50 S subunit.

Purified 50 S ribosomal subunits were found to contain significant amounts of protein coincident with the 30 S proteins S9 and/or S11 on two-dimensional polyacrylamide/urea electropherographs. Peptide mapping established that the protein was largely S9 with smaller amounts of S11. Proteins S5 and L6 were nearly coincident on the two-dimensional polyacrylamide/urea electropherographs. Peptide maps of material from the L6 spot obtained from purified 50 S subunits showed the presence of significant amounts of the peptides corresponding to S5. Experiments in which ³⁵S-labelled 30 S subunits and non-radioactive 50 S subunits were reassociated to form 70 S ribosomes showed that some radioactive 30 S protein was transferred to the 50 S subunit. Most of the transferred radioactivity was associated with two proteins, S9 and S5. Sulfhydryl groups were added to the 50 S subunit by amidination with 2-iminothiolane (methyl 4-mercaptobutyrimidate). These were oxidized to form disulfide linkages, some of which crosslinked different proteins of the intact 50 S ribosomal subunit. Protein dimers were partially fractionated by sequential salt extraction and then by electrophoresis of each fraction in polyacrylamide gels containing urea. Slices of the gel were analysed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Final identification of the constituent proteins in each dimer by two-dimensional polyacrylamide/urea gel electrophoresis showed that 50 S proteins L5 and L27 were crosslinked to S9. The evidence suggests that proteins S5, S9, S11, L5 and L27 are located at the interface region of the 70 S ribosome.     

Effects of aspartate on rabbit muscle creatine kinase and the salt induced molten globule state

The aspartate (Asp)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, circular dichroism (CD) spectra, native polyacrylamide gel electrophoresis and ultraviolet difference spectra. The results showed that Asp caused inactivation and unfolding of CK, with no aggregation during CK denaturation. The kinetics of CK unfolding followed a one phase process. At higher concentrations of Asp (>2.5mM), the CK dimers were partially dissociated. Inactivation occurred before noticeable conformational change during CK denaturation. Asp denatured CK was mostly reactivated and refolded by dilution. KCl induced the molten globule state with compact structure after CK was denatured with 10mM Asp. These results suggest that the effect of Asp differed from that of other denaturants such as guanidine, HCl or urea during CK unfolding. Asp is a reversible protein denaturant and the molten globule state indicates that intermediates exist during CK folding.     

The subunit interface of the Escherichia coli ribosome: Identification of proteins at the interface between the 30 S and 50 S subunits by crosslinking with 2-iminothiolane

The 70 S ribosomes of Escherichia coli were treated with 2-iminothiolane with the resultant addition of 110 sulfhydryl groups per ribosome. The modified ribosomes were oxidized to promote disulfide bond formation, some of which formed intermolecular crosslinks. About 50% of the crosslinked 70 S ribosomes did not dissociate when exposed to low concentrations of magnesium in the absence of reducting agent. Dissociation took place in the presence of reducing agents, which indicated that the subunits had become covalently linked by disulfide linkages. Proteins extracted from purified crosslinked 70 S ribosomes were first fractionated by polyacrylamide/urea gel electrophoresis. The proteins from sequential slices of these gels were analyzed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Monomeric proteins derived from crosslinked dimers appeared below the diagonal containing non-crosslinked proteins, since the second electrophoresis, but not the first, is run under reducing conditions to cleave the crosslinked species. Final identification of the proteins in each dimer was made by radioiodination of the crosslinked proteins, followed by two-dimensional polyacrylamide/urea gel electrophoresis in the presence of non-radioactive total 70 S proteins as markers. This paper describes the identification of 23 protein dimers that contained one protein from each of the two different ribosomal subunits. The proteins implicated must have some part of their structure in proximity to the other ribosomal subunit and are therefore defined as “interface proteins”. The group of interface proteins thus defined includes 50 S proteins that are part of the 5 S RNA: protein complex and 30 S proteins at the initiation site. Correlations between the crosslinked interface proteins and other functional data are discussed.     

Thermodynamic and Kinetic Characterization of a Germ Line Human λ6 Light-Chain Protein: The Relation between Unfolding and Fibrillogenesis

Proteins encoded by the gene segment 6a of the λ variable light-chain repertoire are strongly associated with amyloid deposition. 6aJL2 is a model protein constructed with the predicted sequences encoded by the 6a and JL2 germ line genes. In this work, we characterized the urea- and temperature-induced unfolding of 6aJL2. In the short time scale, spectroscopic, hydrodynamic and calorimetric experiments were compatible with a two-state transition. Furthermore, ΔG, m and the midpoint urea concentration obtained from equilibrium experiments were compatible with those obtained from kinetic experiments. Since fibril formation is a slow process, samples were also incubated for longer times. After incubation for several hours at 37 °C, spectroscopic, hydrodynamic and calorimetric experiments revealed the presence of a partially unfolded off-pathway intermediate around the midpoint urea concentration (1.5-3.0 M urea). In vitro fibrillogenesis assays show that the maximum growth rate for fibril formation and the minimum lag time were obtained at urea concentrations where the partially unfolded state was populated (2.5 M urea at 37 °C). This indicates that this partially unfolded state is critical for in vitro fibril formation. Concentration-dependent kinetics and hydrodynamic properties of the intermediate were consistent with a soluble oligomeric state. The intermediate is formed around the midpoint urea concentration, where the native and unfolded states are equally populated and their rate of interconversion is the slowest. This situation may promote the slow accumulation of an intermediate state that is prone to aggregate.     

Isolation and characterization of an inhibitory subunit of the Mg2+-Ca2+-ATPase of Escherichia coli

1. Stimulation of the Escherichia coli ATPase activity by urea and trypsin shows that the ATPase activity both in the membrane-bound and the solubilized form is partly masked.2. A protein, inhibiting the ATPase activity of Escherichia coli, can be isolated by sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified ATPase. The inhibitor was identified with the smallest of the subunits of E. coli ATPase.3. The molecular weight of the ATPase inhibitor is about 10 000, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and deduced from the amino acid composition.4. The inhibitory action is independent of pH, ionic strength or the presence of Mg²⁺ or ATP.5. The ATPase inhibitor is heat-stable, insensitive to urea but very sensitive to trypsin degradation.6. The Escherichia coli ATPase inhibitor does not inhibit the mitochondrial or the chloroplast ATPase.     

脲梯度电泳方法的技术关键

介绍在应用丙烯酸胺－脲梯度电泳技术进行蛋白质折叠、去折叠研究工作中的实验步骤和技术关键,并在文献方法的基础上作了改进。通过加入15％～0％的甘油,抵消在凝胶中由于脲浓度不同而引起的溶液粘度变化,保证在凝胶上脲浓度不同的部位对蛋白质保持同样的电泳阻力,防止前沿偏斜．采用核黄素光照催化合脲凝胶的聚合,以防止凝胶在梯度灌制完成前发生聚合．加浓缩胶和样品梳于脲梯度胶上可较好地克服边缘效应,获得好的样品迁移图谱．     

Exploring Early Stages of the Chemical Unfolding of Proteins at the Proteome Scale

After decades of using urea as denaturant, the kinetic role of this molecule in the unfolding process is still undefined: does urea actively induce protein unfolding or passively stabilize the unfolded state? By analyzing a set of 30 proteins (representative of all native folds) through extensive molecular dynamics simulations in denaturant (using a range of force-fields), we derived robust rules for urea unfolding that are valid at the proteome level. Irrespective of the protein fold, presence or absence of disulphide bridges, and secondary structure composition, urea concentrates in the first solvation shell of quasi-native proteins, but with a density lower than that of the fully unfolded state. The presence of urea does not alter the spontaneous vibration pattern of proteins. In fact, it reduces the magnitude of such vibrations, leading to a counterintuitive slow down of the atomic-motions that opposes unfolding. Urea stickiness and slow diffusion is, however, crucial for unfolding. Long residence urea molecules placed around the hydrophobic core are crucial to stabilize partially open structures generated by thermal fluctuations. Our simulations indicate that although urea does not favor the formation of partially open microstates, it is not a mere spectator of unfolding that simply displaces to the right of the folded←→unfolded equilibrium. On the contrary, urea actively favors unfolding: it selects and stabilizes partially unfolded microstates, slowly driving the protein conformational ensemble far from the native one and also from the conformations sampled during thermal unfolding.     

Gel electrophoresis in studies of protein conformation and folding

Electrophoresis through polyacrylamide gels is a useful method for distinguishing conformational states of proteins and analyzing the thermodynamic and kinetic properties of transitions between conformations. Although the relationship between protein conformation and electrophoretic mobility is quite complex, relative mobilities provide qualitative estimates of compactness. Conformational states which interconvert slowly on the time scale of the electrophoretic separation can often be resolved, and the rates of interconversion can be estimated. If the transitions are more rapid, then the electrophoretic mobility represents the equilibrium distribution of conformations. Protein unfolding transitions induced by urea are readily studied using slab gels containing a gradient of urea concentration perpendicular to the direction of electrophoresis. Protein applied across the top of such a gel migrates in the presence of continuously varying urea concentrations, and a profile of the unfolding transition is generated directly. Transitions induced by other agents could be studied using analogous gradient gels. Electrophoretic methods are especially suited for studying small quantities of protein, and complex mixtures, since the different components can be separated during the electrophoresis.     

绿脓杆菌apoazurin在溶液中存在两种天然构象

如何解释绿脓杆菌apoazurin变性过程的复杂机制是一个有争议的问题.最近的研究表明apoazurin的复杂变性机制可以归结为其天然态存在着至少两种构象.利用内源荧光发射谱和圆二色谱进一步研究了apoazurin的脲变性机制,发现其稳态脲变性符合表观的二态过程,但其动力学为双相过程.在高浓度脲中快反应在几秒钟内完成,而慢反应要经过几个小时.快反应和慢反应的mu值分别为2.24和2.45kJ·mol-1·M-1,去折叠活化能的差值为22kJ·mol-1.时间分辨的荧光发射谱和圆二色谱可以用天然态和完全变性态的谱图通过一个固定的比例参数进行重建.结果表明,过去被广泛接受的存在着变性中间体的机制是不正确的,而apoazurin在天然态存在至少两种构象的假设是合理的.