The folding kinetics and thermodynamics of the Fyn-SH3 domain

The equilibrium unfolding and the kinetic folding and unfolding of the 67 residue Fyn-SH3 domain have been investigated. Equilibrium unfolding experiments indicate that, despite the lack of both disulfide bonds and prosthetic groups, Fyn-SH3 is relatively stable with a free energy of folding of -6.0 +/- 0.6 kcal mol-1 at 20 degrees C. Kinetic experiments indicate that the domain refolds in a rapid two-state manner without significant population of intermediates (k = 94.3 s-1 in H2O at 20 degrees C). Despite the presence of two proline residues, the refolding of the domain is monophasic, and no significant proline isomerization-like refolding phase is observed. This can be attributed to an extremely low level of the incorrect (cis) isomer of the structurally important Pro134 residue in the protein denatured in 8 M guanidine hydrochloride. Analysis of the temperature and guanidine hydrochloride dependence of the folding rate suggests that the folding transition state of this protein is relatively well organized. A comparison with the refolding kinetics and thermodynamics of other homologous SH3 domains indicates that these exhibit an equivalent degree of transition state organization. This potentially arises from conservation of key features of the transition state conformation despite sometimes relatively low overall sequence identity. Such a comparison further suggests that relative thermodynamic stability is an important factor in determining the relative folding rates of natural proteins with a common fold, but that specific details of the amino acid sequence can also play a significant role in individual cases.     

The disulfide folding pathway of tick anticoagulant peptide (TAP), a Kunitz-type inhibitor structurally homologous to BPTI

The pathway of oxidative folding of tick anticoagulant peptide (TAP, 60 amino acids and three disulfides) has been analyzed by characterization of the acid and iodoacetate trapped folding intermediates. The results reveal a high degree of heterogeneity of the one- and two-disulfide intermediates and the presence of three-disulfide scrambled species along the folding pathway. The picture of TAP folding differs significantly from the well-documented case of bovine pancreatic trypsin inhibitor (BPTI), despite the fact that both proteins share close structural homology in term of 3-D conformation and disulfide pattern.     

Mutational analysis of the switch II loop of Dictyostelium myosin II

A loop comprising residues 454-459 of Dictyostelium myosin II is structurally and functionally equivalent to the switch II loop of the G-protein family. The consensus sequence of the "switch II loop" of the myosin family is DIXGFE. In order to determine the functions of each of the conserved residues, alanine scanning mutagenesis was carried out on the Dictyostelium myosin II heavy chain gene. Examination of in vivo and in vitro motor functions of the mutant myosins revealed that the I455A and S456A mutants retained those functions, whereas the D454A, G457A, F458A and E459A mutants lost them. Biochemical analysis of the latter myosins showed that the G457A and E459A mutants lost the basal ATPase activity by blocking of the isomerization and hydrolysis steps of the ATPase cycle, respectively. The F458A mutant, however, lost the actin-activated ATPase activity without loss of the basal ATPase activity. These results are discussed in terms of the crystal structure of the Dictyostelium myosin motor domain.     

Titration properties and thermodynamics of the transition state for folding: comparison of two-state and multi-state folding pathways

CI2 folds and unfolds as a single cooperative unit by simple two-state kinetics, which enables the properties of the transition state to be measured from both the forward and backward rate constants. We have examined how the free energy of the transition state for the folding of chymotrypsin inhibitor 2 (CI2) changes with pH and temperature. In addition to the standard thermodynamic quantities, we have measured the overall acid-titration properties of the transition state and its heat capacity relative to both the denatured and native states. We were able to determine the latter by a method analogous to a well-established procedure for measuring the change in heat capacity for equilibrium unfolding: the enthalpy of activation of unfolding at different values of acid pH were plotted against the average temperature of each determination. Our results show that the transition state of CI2 has lost most of the electrostatic and van der Waals' interactions that are found in the native state, but it remains compact and this prevents water molecules from entering some parts of the hydrophobic core. The properties of the transition state of CI2 are then compared with the major folding transition state of the larger protein barnase, which folds by a multi-state mechanism, with the accumulation of a partly structured intermediate (Dphys or I). CI2 folds from a largely unstructured denatured state under physiological conditions via a transition state which is compact but relatively uniformly unstructured, with tertiary and secondary structure being formed in parallel. We term this an expanded pathway. Conversely, barnase folds from a largely structured denatured state in which elements of structure are well formed through a transition state that has islands of folded elements of structure. We term this a compact pathway. These two pathways may correspond to the two extreme ends of a continuous spectrum of protein folding mechanisms. Although the properties of the two transition states are very different, the activation barrier for folding (Dphys-->++) is very similar for both proteins.     

Mutational analysis of the genes encoding the photosystem II proteins in Cyanobacterium synechocystis sp. 6803

Chlorophyll--binding protein CP43 and cytochrome b559, encoded by psbC and psbE/F genes, are the components of photosystem II (PS II). Three psbC- and four psbE/F- mutants were isolated from the collection of PS II-deficient mutants of the cyanobacterium Synechocystis sp. 6803. Restoration of photosynthetic activity was achieved by transformation of psbE/F- mutants with cloned psbE/F gene cluster from wild type cells and each of psbC- mutants--with specific part of wild type psbC gene. DNA fragments carrying the mutations were isolated from mutant cells and sequenced. The mutations which affect PS II activity were identified in psbC gene as "frameshift" mutation, stop-codon formation, or as deletion of three nucleotides resulting in loss of one of three Phe residues in position 422-424 of CP43. Sequence of mutant psbE/F genes revealed single mutations resulting in deletion of Phe-36 or substitution of Pro-63 for Leu in alpha-subunit and Val-29 for Phe in beta-subunit of cytochrome b559.     

Rapid formation of the native 14-38 disulfide bond in the early stages of BPTI folding

Using recombinant variants of BPTI, we have determined the rate constants corresponding to formation of each of the fifteen possible disulfide bonds in BPTI, starting from the reduced, unfolded protein. The 14-38 disulfide forms faster than any of the other 14 possible disulfides. This faster rate results from significantly higher intrinsic chemical reactivities of Cys-14 and Cys-38, in addition to local structure in the reduced protein that facilitates formation of the 14-38 disulfide bond. This disulfide bond is found in native BPTI. Our results suggest that a significant flux of folding BPTI molecules proceed through the one-disulfide intermediate with the 14-38 disulfide bond, denoted [14-38], that has recently been detected on the BPTI folding pathway. In addition to providing a detailed picture of the early events in the folding of BPTI, our results address quantitatively the effect of local structure in the unfolded state on folding kinetics.     

Mutational analysis of the Mu transposase. Contributions of two distinct regions of domain II to recombination

Mu transposase is a member of a protein family that includes many transposases and the retroviral integrases. These recombinases catalyze the DNA cleavage and joining reactions essential for transpositional recombination. Here we demonstrate that, consistent with structural predictions, aspartate 336 of Mu transposase is required for catalysis of both DNA cleavage and DNA joining. This residue, although located 55 rather than 35 residues NH2-terminal of the essential glutamate, is undoubtedly the analog of the second aspartate of the Asp-Asp-35-Glu motif found in other family members. The core domain of Mu transposase consists of two subdomains: the NH2-terminal subdomain (IIA) contains the conserved Asp-Asp-Glu motif residues, whereas the smaller COOH-terminal subdomain (IIB) contains a large positively charged region exposed on its surface. To probe the function of domain IIB, we constructed mutant proteins carrying deletion or substitution mutations within this region. The activity of the deletion proteins revealed that domains IIA and IIB can be provided by different subunits in the transposase tetramer. Substitution mutations at two pairs of exposed lysine residues within the positively charged surface of domain IIB render transposase defective in transposition at a reaction step after DNA cleavage but prior to DNA joining. The severity of this defect depends on the structure of the DNA flanking the cleavage site. Thus, these data suggest that domain IIB is involved in manipulating the DNA near the cleavage site and that this function is important during the transition between the DNA cleavage and the DNA joining steps of recombination.     

Oxidation of cobaltite: Part II. kinetics

A kinetics study has been performed on cobaltite to understand the oxidation processes over a temperature range of 573 to 1173 K using a thermogravimetric method. The results show that oxidation of cobaltite occurs in two stages. In the first stage, which occurs between 823 and 913 K, the majority of the sulfur is removed. However, the arsenic remains in the lattice of the reacted region. A pore-blocking kinetic model yields a satisfactory fit to these experimental data. At higher temperatures, there is a concurrent release of As and S from the crystal lattice of CoAsS. The shrinking-core kinetic model is applicable. Complementary X-ray diffraction and scanning electron microscopic analyses on these partially oxidized samples support the kinetic models. The effects of partial pressure of oxygen and particle size on roasting have been evaluated.     

Protein folding in the landscape perspective: chevron plots and non-Arrhenius kinetics

We use two simple models and the energy landscape perspective to study protein folding kinetics. A major challenge has been to use the landscape perspective to interpret experimental data, which requires ensemble averaging over the microscopic trajectories usually observed in such models. Here, because of the simplicity of the model, this can be achieved. The kinetics of protein folding falls into two classes: multiple-exponential and two-state (single-exponential) kinetics. Experiments show that two-state relaxation times have "chevron plot" dependences on denaturant and non-Arrhenius dependences on temperature. We find that HP and HP+ models can account for these behaviors. The HP model often gives bumpy landscapes with many kinetic traps and multiple-exponential behavior, whereas the HP+ model gives more smooth funnels and two-state behavior. Multiple-exponential kinetics often involves fast collapse into kinetic traps and slower barrier climbing out of the traps. Two-state kinetics often involves entropic barriers where conformational searching limits the folding speed. Transition states and activation barriers need not define a single conformation; they can involve a broad ensemble of the conformations searched on the way to the native state. We find that unfolding is not always a direct reversal of the folding process.     

Perturbations of the denatured state ensemble: modeling their effects on protein stability and folding kinetics

By considering the denatured state of a protein as an ensemble of conformations with varying numbers of sequence-specific interactions, the effects on stability, folding kinetics, and aggregation of perturbing these interactions can be predicted from changes in the molecular partition function. From general considerations, the following conclusions are drawn: (1) A perturbation that enhances a native interaction in denatured state conformations always increases the stability of the native state. (2) A perturbation that promotes a non-native interaction in the denatured state always decreases the stability of the native state. (3) A change in the denatured state ensemble can alter the kinetics of aggregation and folding. (4) The loss (or increase) in stability accompanying two mutations, each of which lowers (or raises) the free energy of the denatured state, will be less than the sum of the effects of the single mutations, except in cases where both mutations affect the same set of partially folded conformations. By modeling the denatured state as the ensemble of all non-native conformations of hydrophobic-polar (HP) chains configured on a square lattice, it can be shown that the stabilization obtained from enhancement of native interactions derives in large measure from the avoidance of non-native interactions in the D state. In addition, the kinetic effects of fixing single native contacts in the denatured state or imposing linear gradients in the HH contact probabilities are found, for some sequences, to significantly enhance the efficiency of folding by a simple hydrophobic zippering algorithm. Again, the dominant mechanism appears to be avoidance of non-native interactions. These results suggest stabilization of native interactions and imposition of gradients in the stability of local structure are two plausible mechanisms involving the denatured state that could play a role in the evolution of protein folding and stability.     

The influence of solutes on kinetics and thermodynamics of liquid indium-oxygen systems

The influence of small additions of the solutes Sn, Pb, Cu, Ag, and Ti on the diffusivity, solubility, activity, and the activity coefficient of oxygen in liquid indium was studied for temperatures between 750 and 950 °C, using solid state electrochemical techniques. A sharp increase in diffusivity and a decrease in solubility of oxygen were observed in all cases when small amounts of these solutes were added to liquid indium. Further additions of these solutes moderately increased the oxygen diffusivity and decreased the oxygen solubility. The thermodynamics of liquid binary alloys of indium, In-Sn, In-Pb, In-Cu, and In-Ag were also studied up to 10 at pct of these solutes. There is evidence that the large increase in diffusivity and decrease in solubility of oxygen in indium is due to cluster formations in liquid metal. Formerly with the Union Carbide Corporation,     

Influences of non-stoichiometry on thermodynamics and kinetics of iron oxides reduction processes

Abstract

Non-stoichiometry influences both the thermodynamic and kinetic analyses of the iron oxide redox processes. The thermochemical data of iron oxide redox reactions in various textbooks are not consistent, and the kinetic characteristics are not well understood because of the non-stoichiometry. To clarify such confusions, some famous thermodynamic data are compared, and highly precise experimental work conducted for verification. It is shown that the thermodynamic data for the pure iron oxide reduction reactions from JANAF agree well with the experimental results; the eutectoid temperature of iron oxides was proven to be 576°C; Dieckmann’s defect model of magnetite was proven in good agreement with the experimental results only at high oxygen activities but not low oxygen activities; and the dependences of iron deficiency on Δwt-% (weight loss ratio) and Fe²⁺% (ferrous ratio) were calculated and experimentally verified in pure iron oxides reduction processes.     

Protein folding: how the mechanism of GroEL action is defined by kinetics

We propose a mechanism for the role of the bacterial chaperonin GroEL in folding proteins. The principal assumptions of the mechanism are (i) that many unfolded proteins bind to GroEL because GroEL preferentially binds small unstructured regions of the substrate protein, (ii) that substrate protein within the cavity of GroEL folds by the same kinetic mechanism and rate processes as in bulk solution, (iii) that stable or transient complexes with GroEL during the folding process are defined by a kinetic partitioning between formation and dissociation of the complex and the rate of folding and unfolding of the protein, and (iv) that dissociation from the complex in early stages of folding may lead to aggregation but dissociation at a late stage leads to correct folding. The experimental conditions for refolding may play a role in defining the function of GroEL in the folding pathway. We propose that the role of GroES and MgATP, either binding or hydrolysis, is to regulate the association and dissociation processes rather than affecting the rate of folding.     

Global analysis of the effects of temperature and denaturant on the folding and unfolding kinetics of the N-terminal domain of the protein L9

The folding and unfolding kinetics of the N-terminal domain of the ribosomal protein L9 have been measured at temperatures between 7 and 85 degrees C and between 0 and 6 M guanidine deuterium chloride. Stopped-flow fluorescence was used to measure rates below 55 degrees C and NMR lineshape analysis was used above 55 degrees C. The amplitudes and rate profiles of the stopped-flow fluorescence experiments are consistent with a two-state folding mechanism, and plots of ln(k) versus guanidine deuterium chloride concentration show the classic v-shape indicative of two-state folding. There is no roll over in the plots when the experiments are repeated in the presence of 400 mM sodium sulfate. Temperature and denaturant effects were fit simultaneously to the simple model k=D exp(-DeltaG*/RT) where DeltaG* represents the change in apparent free energy between the transition state and the folded or unfolded state and D represents the maximum possible folding speed. DeltaG* is assumed to vary linearly with denaturant concentration and the Gibbs-Helmholtz equation is used to model stability changes with temperature. Approximately 60% of the surface area buried upon folding is buried in the transition state as evidenced by changes in the heat capacity and m value between the unfolded state and the transition state. The equilibrium thermodynamic parameters, DeltaCp degrees, m and DeltaG degrees, all agree with the values calculated from the kinetic experiments, providing additional evidence that folding is two-state. The folding rates at 0 M guanidine hydrochloride show a non-Arrhenius temperature dependence typical of globular proteins. When the folding rates are examined along constant DeltaG degrees/T contours they display an Arrhenius temperature dependence with a slope of -8600 K. This indicates that for this system, the non-Arrhenius temperature dependence of folding can be accounted for by the anomalous temperature dependence of the interactions which stabilize proteins.     

Mutational analysis of the major loop of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases. Effects on protein stability and substrate binding

The carbohydrate-binding cleft of Bacillus licheniformis 1,3-1, 4-beta-D-glucan 4-glucanohydrolase is partially covered by the surface loop between residues 51 and 67, which is linked to beta-strand-(87-95) of the minor beta-sheet III of the protein core by a single disulfide bond at Cys61-Cys90. An alanine scanning mutagenesis approach has been applied to analyze the role of loop residues from Asp51 to Arg64 in substrate binding and stability by means of equilibrium urea denaturation, enzyme thermotolerance, and kinetics. The DeltaDeltaGU between oxidized and reduced forms is approximately constant for all mutants, with a contribution of 5.3 +/- 0.2 kcal.mol-1 for the disulfide bridge to protein stability. A good correlation is observed between DeltaGU values by reversible unfolding and enzyme thermotolerance. The N57A mutant, however, is more thermotolerant than the wild-type enzyme, whereas it is slightly less stable to reversible urea denaturation. Mutants with a <2-fold increase in Km correspond to mutations at residues not involved in substrate binding, for which the reduction in catalytic efficiency (kcat/Km) is proportional to the loss of stability relative to the wild-type enzyme. Y53A, N55A, F59A, and W63A, on the other hand, show a pronounced effect on catalytic efficiency, with Km > 2-fold and kcat < 5% of the wild-type values. These mutated residues are directly involved in substrate binding or in hydrophobic packing of the loop. Interestingly, the mutation M58A yields an enzyme that is more active than the wild-type enzyme (7-fold increase in kcat), but it is slightly less stable.     

Probing the disulfide folding pathway of insulin-like growth factor-I

The crucial step of folding of recombinant proteins presents serious challenges to obtaining the native structure. This problem is exemplified by insulin-like growth factor (IGF)-I which when refolded in vitro produces the native three-disulfide structure, an alternative structure with mispaired disulfide bonds and other isomeric forms. To investigate this phenomenon we have examined the refolding properties of an analog of IGF-I which contains a 13-amino acid N-terminal extension and a charge mutation at position 3 (Long-[Arg3]IGF-I). Unlike IGF-I, which yields 45% of the native structure and 24% of the alternative structure when refolded in vitro, Long-[Arg3]IGF-I yields 85% and 10% of these respective forms. To investigate the interactions that affect the refolding of Long-[Arg3]IGF-I and IGF-I, we acid-trapped folding intermediates and products for inclusion in a kinetic analysis of refolding. In addition to non-native intermediates, three native-like intermediates were identified, that appear to have a major role in the in vitro refolding pathway of Long-[Arg3]IGF-I; a single-disulfide Cys18-Cys61 intermediate, an intermediate with Cys18-Cys61 and Cys6-Cys48 disulfide bonds and another with Cys18-Cys61 and Cys47-Cys52 disulfide bonds. Furthermore, from our kinetic analysis we propose that the Cys18-Cys61, Cys6-Cys48 intermediate forms the native structure, not by the direct formation of the last (Cys47-Cys52) disulfide bond, but by rearrangement via the Cys18-Cys61 intermediate and a productive Cys18-Cys61, Cys47-Cys52 intermediate. In this pathway, the last disulfide bond to form involves Cys6 and Cys48. Finally, we apply this pathway to IGF-I and conclude that the divergence in the in vitro folding pathway of IGF-I is caused by non-native interactions involving Glu3 that stabilize the alternative structure.     

基于数值模拟的Cu-Al复合粉体内氧化热力学与动力学研究

本文采用数值模拟研究了Cu-Al复合粉体的内氧化热力学和动力学过程,分析了时间、温度、粉体粒度和Al含量对内氧化动力学的影响。结果表明:Cu-Al复合粉体内氧化反应的氧分压要控制为低于上临界值,下限氧分压是一个极小量,对于内氧化控制无实际意义;Cu-Al粉体的内氧化反应主要在最初较短时间内完成,其内氧化程度、速率主要取决于温度、粉末粒度、Al含量和时间,在较高的温度下有利于提高内氧化的程度和速度。实验证明Cu-0.5%Al复合粉在900℃下、30 min内即可完成内氧化生成Cu-Al2O3复合粉体,与数值模拟的结果相一致。     

Transient kinetics and thermodynamics of anthroylouabain binding to Na/K-ATPase

In this study the antiarrhythmic and the proarrhythmic activities of almokalant, a selective class III antiarrhythmic agent, were compared. The antiarrhythmic effect of the drug was tested in pentobarbital-anaesthetised rabbits. Arrhythmia was evoked by occluding and releasing the left circumflex coronary artery. Almokalant in a dose of 250 nmol/kg i.v., significantly decreased the incidence of reperfusion induced ventricular fibrillation (21% vs. 75% in the control group) and increased the proportion of surviving animals during reperfusion (86% vs. 42%). The proarrhythmic effect of almokalant was examined during alpha1-adrenoceptor stimulation in chloralose-anaesthetised rabbits. Almokalant (75 nmol/kg per min) triggered torsade de pointes arrhythmias in 8 animals out of 11. The dose of almokalant (mean+/-S.E.M.) required to produce this effect was 1181+/-519 nmol/kg. It is concluded that, although almokalant is an effective antiarrhythmic agent against ischaemia-reperfusion induced arrhythmias, it has marked proarrhythmic activity during alpha1-adrenoceptor stimulation.     

Growth kinetics of solid-liquid Ga interfaces: Part II. Theoretical

The faceted (111) and (001) Ga interfaces grow at low supercoolings with either of the lateral growth mechanisms, two-dimensional nucleation growth (2DNG) or screw dislocation-assisted growth (SDG), depending on the perfection of the interface. The classical theories regarding the growth kinetics of smooth interfaces describe the results qualitatively but not quantitatively. The latter is due to the inadequacy of the assumptions made in the classical theories, which treat the interfacial atomic migration the same as the liquid bulk diffusion process and the step edge energy as independent of the supercooling. Beyond a threshold supercooling, the results show that the faceted interfaces gradually become kinetically rough as the supercooling increases. The step energy, treated as a function of the supercooling, is shown to diverge exponentially with the supercooling at the faceted/nonfaceted transition. At supercoolings exceeding the transition value, dislocations do not affect the growth rate. Furthermore, beyond the transition, the growth rates are linearly dependent on the supercooling, which implies that the growth mode changes from lateral to normal. A generalized lateral growth equation, which includes the interfacial diffusivity and supercooling-dependent step edge free energy, is given to describe the growth kinetics of both interfaces up to supercoolings marking the kinetic roughening transition. This paper is based on a presentation made in the symposium “The Role of Ledges in Phase Transformations” presented as part of the 1989 Fall Meeting of TMS-MSD, October 1–5, 1989, in Indianapolis, IN, under the auspices of the Phase Transformations Committee of the Materials Science Division, ASM INTERNATIONAL.