Mutational analysis of the BPTI folding pathway: II. Effects of aromatic-->leucine substitutions on folding kinetics and thermodynamics

The rates of the individual steps in the disulfide-coupled folding and unfolding of eight BPTI variants, each containing a single aromatic to leucine amino acid replacement, were measured. From this analysis, the contributions of the four phenylalanine and four tyrosine residues to the stabilities of the native protein and the disulfide-bonded folding intermediates were determined. While the substitutions were found to destabilize the native protein by 2 to 7 kcal/mol, they had significantly smaller effects on the intermediates that represent the earlier stages of folding, even when the site of the substitution was located within the ordered regions of the intermediates. These results suggest that stabilizing interactions contribute less to conformational stability in the context of a partially folded intermediate than in a fully folded native protein, perhaps because of decreased cooperativity among the individual interactions. The kinetic analysis also provides new information about the transition states associated with the slowest steps in folding and unfolding, supporting previous suggestions that these transition states are extensively unfolded. Although the substitutions caused large changes in the distribution of folding intermediates and in the rates of some steps in the folding pathway, the kinetically-preferred pathway for all of the variants involved intramolecular disulfide rearrangements, as observed previously for the wild-type protein. These results suggest that the predominance of the rearrangement mechanism reflects conformational constraints present relatively early in the folding pathway.     

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.     

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.     

Thermodynamics of aging

The aim of this study is to determine some functions of neutrophil in patients affected by psoriatic arthritis and to compare them to those of patients affected by cutaneous psoriasis and to normal controls. We used a model of experimental cutaneous inflammation allowing to separate a cluster of purified and viable PMN cells. Then we analyzed, within the three groups, the IL-8 concentration in serum and in the supernatant obtained from the inflammatory site to gather data on the possible pathogenic role played by this cytokine in psoriatic arthritis. We studied neutrophil functions in patients with cutaneous psoriasis and psoriatic arthritis, in acute phase, in comparison with healthy control subjects. We investigated in vivo neutrophil migration by Senn's skin window technique and measured adhesion assay and superoxide production in circulating and migrating neutrophils after different stimuli. We also measured IL-8 concentration in serum and in the supernatant obtained from the inflammatory site, artificially created through the skin window scrape. Neutrophil migration in vivo was significantly higher in both groups of patients than in controls. In the presence of fMLP, blood cells showed a burst of superoxide release, which was significantly more pronounced in patients when compared to healthy controls. Neutrophils from skin window scrape showed a much higher response to fMLP as compared to blood cells of all subject groups, but no differences were observed between patients and controls. No correlation was found between the three groups in adhesion ability under basal condition or in response to different stimuli by circulating and migrating neutrophils. Our results also show a great increase of IL-8 in the exudate from patients compared to controls. Our study shows that there is no difference in neutrophil functions between patients with psoriatic arthritis and cutaneous psoriasis; moreover we suggest that the source of high IL-8 levels are neutrophils rather than the keratinocytes.     

Enhanced protein flexibility caused by a destabilizing amino acid replacement in BPTI

A genetically engineered variant of bovine pancreatic trypsin inhibitor (Y35G BPTI) has been shown previously by X-ray crystallography to have a three-dimensional structure dramatically different from that of the wild-type protein, particularly in the protease-binding region of the molecule. Yet, the Y35G variant is a potent trypsin inhibitor. Described here are 15N NMR relaxation studies to compare the backbone dynamics of Y35G BPTI to those of the wild-type protein. The Tyr35 --> Gly substitution increased the transverse relaxation rates of more than one third of all backbone amide groups, but had little effect on the longitudinal relaxation rates, indicating that the substitution facilitates relatively slow backbone motions, estimated to be on the microsecond time-scale. The results indicate that the residues making up the trypsin-binding site undergo large and relatively slow conformational changes in solution, estimated to be on the 5 to 20 micros time-scale. It is thus likely that the crystal structure represents only one of multiple interconverting conformations in solution, only a fraction of which may be competent for binding trypsin. The large thermodynamic destabilization associated with this substitution may arise, in part, from a loss in cooperativity among the multiple stabilizing interactions that are normally favored by the highly ordered structure of the wild-type protein. These results suggest that fully understanding the effects of amino acid replacements on the functional and thermodynamic properties of proteins may often require analysis of the dynamic, as well as the structural, properties of altered proteins.     

Real-time NMR investigations of triple-helix folding and collagen folding diseases

Folding of the collagen triple helix provides an opportunity to look at multichain molecular assembly. This triple helix also offers unique advantages for the study of folding because the process is very slow compared to globular proteins, and the kinetics of folding can be obtained in real time by NMR. Studies on triple-helical peptides illustrate the ability to observe kinetic folding intermediates directly and the ability to propose detailed mechanisms of folding through the use of real-time NMR methods. Defective collagen folding has been implicated in various connective tissue diseases and the capacity of NMR to look at the folding of specific sites provides a tool for obtaining information about altered folding mechanisms. Comparison of folding in peptides that model normal and diseased collagens could shed light on the molecular perturbation and the etiology of disease.     

Structure of extracellular tissue factor complexed with factor VIIa inhibited with a BPTI mutant

The event that initiates the extrinsic pathway of blood coagulation is the association of coagulation factor VIIa (VIIa) with its cell-bound receptor, tissue factor (TF), exposed to blood circulation following tissue injury and/or vascular damage. The natural inhibitor of the TF.VIIa complex is the first Kunitz domain of tissue factor pathway inhibitor (TFPI-K1). The structure of TF. VIIa reversibly inhibited with a potent (Ki=0.4 nM) bovine pancreatic trypsin inhibitor (BPTI) mutant (5L15), a homolog of TFPI-K1, has been determined at 2.1 A resolution. When bound to TF, the four domain VIIa molecule assumes an extended conformation with its light chain wrapping around the framework of the two domain TF cofactor. The 5L15 inhibitor associates with the active site of VIIa similar to trypsin-bound BPTI, but makes several unique interactions near the perimeter of the site that are not observed in the latter. Most of the interactions are polar and involve mutated positions of 5L15. Of the eight rationally engineered mutations distinguishing 5L15 from BPTI, seven are involved in productive interactions stabilizing the enzyme-inhibitor association with four contributing contacts unique to the VIIa.5L15 complex. Two additional unique interactions are due to distinguishing residues in the VIIa sequence: a salt bridge between Arg20 of 5L15 and Asp60 of an insertion loop of VIIa, and a hydrogen bond between Tyr34O of the inhibitor and Lys192NZ of the enzyme. These interactions were used further to model binding of TFPI-K1 to VIIa and TFPI-K2 to factor Xa, the principal activation product of TF.VIIa. The structure of the ternary protein complex identifies the determinants important for binding within and near the active site of VIIa, and provides cogent information for addressing the manner in which substrates of VIIa are bound and hydrolyzed in blood coagulation. It should also provide guidance in structure-aided drug design for the discovery of potent and selective small molecule VIIa inhibitors.     

Thermodynamics of slag-metal equilibrium

In a recent publication Elliott, Lynch and Brown (ELB) have criticized the early Flood and Grjotheim (FG) treatment of slag-metal equilibria. The present paper demonstrates that part of the difference between the ELB and FG treatment is in the different choice of standard state. However, the standard state used by ELB was introduced in 1959³ for the treatment of this type of equilibria. A fundamental thermodynamic error is introduced in the ELB derivation by the use of single ion activity coefficients which invalidates their treatment of experimental data.     

Thermodynamics of Ca-Ga alloys

The enthalpies of formation of the intermetallic compounds CaGa₄, Ca₃Ga₈, and CaGa₂, at 298.15 K, were determined by high-temperature liquid gallium solution calorimetric measurements to be −24.9 ± 4.9 kJ·g at.⁻¹, −25.4 ± 2.4 kJ·g at.⁻¹, and −38.8 ± 4.8 kJ·g at.⁻¹, respectively. The enthalpies of formation of CaGa₄ at 988 K and that of Ca₃Ga₈ at 1070 K were determined, using precipitation calorimetry, to be −28.2 ± 1.7 kJ·g at.⁻¹ and −22.5 ± 1.4 kJ·g at.⁻¹, respectively. The integral enthalpy of mixing of the (Ca-Ga) liquid alloys (ΔH ⁰) measured at 1309 K are described by the following Redlich-Kister equation:

Thermodynamics of slag-metal equilibrium

In a recent publication¹ Elliott, Lynch and Brown (ELB) have criticized the early Flood and Grjotheim (FG) treatment² of slag-metal equilibria. The present paper demonstrates that part of the difference between the ELB and FG treatment is in the different choice of standard state. However, the standard state used by ELB was introduced in 1959³ for the treatment of this type of equilibria. A fundamental thermodynamic error is introduced in the ELB derivation by the use of single ion activity coefficients which invalidates their treatment of experimental data.     

Thermodynamics of stacking faults

The Gibbs treatment of interfacial free energy is reviewed and applied to the specific case of stacking faults in alloys. The alternative model of viewing the fault region as a second phase is briefly reviewed. Prior treatments of the topic using the later approach are shown to have led to thermodynamic inconsistencies in some cases. The relation between stacking fault energy and equilibrium separation of partial dislocations is established. The stress required to move partial dislocations or perfect dislocations in alloys is developed on the basis of a virtual displacement from equilibrium approach.     

Thermodynamics of roasting arsenopyrite

Existing thermodynamic data for the Fe-As-S-0 system were evaluted and predominance area diagrams for that system were constructed at 798 and 973 K. Isopleths for the As/S and As/O atomic ratios in the vapor phase have been added to the diagrams by solving the complex equilibria. These modified diagrams were used to evaluate the results of roasting both natural and synthetic arsenopyrite (FeAsS) in inert, reducing, and oxidizing atmospheres at 798 and 873 K. Conditions leading to the retention of As as As₂O₅(s) and FeAsO₄(s) were also reviewed. The experimental results indicate that both reducing and oxidizing atmospheres are more effective in the removal of As than an inert atmosphere. In a reducing atmosphere arsenic sulfides are evolved and the percentage of As removal increases with decreasing P_{O 2}The greatest percent of As removal occurred with highly oxidizing atmospheres which generated As₄O₆ vapor.     

非平衡态热力学分析

为了研究非平衡态热力学理论在具体的冶金过程中的应用,本文用该理论模拟计算了转炉的炼钢过程,并建立了该过程的数学模型。得到了转炉冶炼过程的脱碳速度表达式、钢液中硅锰含量变化表达式、氧含量变化表达式以及温度变化表达式。用C语言对整个冶炼过程进行模拟,结果表明非平衡态热力学模型能够描述整个冶炼过程主要元素的成分变化和熔池温度变化,对转炉炼钢生产实际的过程控制提供了新的理论和方法。