The bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor): a milestone protein

The pancreatic Kunitz inhibitor, also known as aprotinin, bovine basic pancreatic trypsin inhibitor (BPTI), and trypsin-kallikrein inhibitor, is one of the most extensively studied globular proteins. It has proved to be a particularly attractive and powerful tool for studying protein conformation as well as molecular bases of protein/protein interaction(s) and (macro)molecular recognition. BPTI has a relatively broad specificity, inhibiting trypsin- as well as chymotrypsin- and elastase-like serine (pro)enzymes endowed with very different primary specificity. BPTI reacts rapidly with serine proteases to form stable complexes, but the enzyme: inhibitor complex formation may involve several intermediates corresponding to discrete reaction steps. Moreover, BPTI inhibits the nitric oxide synthase type-I and -II action and impairs K+ transport by Ca2+-activated K+ channels. Clinically, the use of BPTI in selected surgical interventions, such as cardiopulmonary surgery and orthotopic liver transplantation, is advised, as it significantly reduces hemorrhagic complications and thus blood-transfusion requirements. Here, the structural, inhibition, and bio-medical aspects of BPTI are reported.     

Structural studies of the interactions of normal and abnormal human plasmins with bovine basic pancreatic trypsin inhibitor

Catalytic activity of human plasmin is inhibited by bovine basic pancreatic trypsin inhibitor (BPTI, also known as aprotinin). In spite of increased interest in the function of BPTI as an inhibitor of plasmin, the 3-D structure of the plasmin-BPTI complex has not yet been determined. Therefore, in the present paper, the structure of the plasmin-BPTI complex was constructed by the homology modeling method, which provided information about the high affinity of plasmin for BPTI. Moreover, normal mode analyses of free plasmin, free BPTI and the plasmin-BPTI complex were carried out to investigate the changes in dynamics following complex formation. After study of the plasmin-BPTI interaction, we also investigated the binding of BPTI with abnormal plasmin, theoretically and experimentally. The result showing that BPTI binds to abnormal plasmin in the same way as it does to normal plasmin supports the previous finding that the difference between normal and abnormal plasmins is very small and that the abnormality is localized to the catalytic site.     

13C spin relaxation studies of the basic pancreatic trypsin inhibitor

The longitudinal ¹³C spin relaxation times T₁ and the ¹³C{¹H} nuclear Overhauser enhancement were measured in a concentrated aqueous solution of the basic pancreatic trypsin inhibitor. The correlation time for overall rotational motions of the basic pancreatic trypsin inhibitor molecules was found to be τ_R ≈ 2 × 10^?8 s. In connection with previous ¹H n.m.r. studies of intramolecular motions of the aromatics, we were particularly interested in the correlation times τ_G for intramolecular segmental motions of the aromatic rings. The present experiments revealed no manifestation of intramolecular motions of the aromatics, indicating that τ_G ? 2 × 10^?8 s for the aromatic ring carbon atoms. On the other hand, rapid segmental motions were evidenced for the peripheral carbon atoms of aliphatic amino acid sidechains. Comparison of the ¹H and ¹³C n.m.r. data on the basic pancreatic trypsin inhibitor indicates that the time scale of high resolution ¹H n.m.r. at high fields may in many instances be more appropriate for studies of the molecular dynamics in globular proteins than the time scale of spin relaxation measurements.     

Gas-phase binding of non-covalent protein complexes between bovine pancreatic trypsin inhibitor and its target enzymes studied by electrospray ionization tandem mass spectrometry

The potential of electrospray ionization (ESI) mass spectrometry (MS) to detect non-covalent protein complexes has been demonstrated repeatedly. However, questions about correlation of the solution and gas-phase structures of these complexes still produce vigorous scientific discussion. Here, we demonstrate the evaluation of the gas-phase binding of non-covalent protein complexes formed between bovine pancreatic trypsin inhibitor (BPTI) and its target enzymes over a wide range of dissociation constants. Non-covalent protein complexes were detected by ESI-MS. The abundance of the complex ions in the mass spectra is less than expected from the values of the dissociation constants of the complexes in solution. Collisionally activated dissociation (CAD) tandem mass spectrometry (MS/MS) and a collision model for ion activation were used to evaluate the binding of non-covalent complexes in the gas phase. The internal energy required to induce dissociation was calculated for three collision gases (Ne, Ar, Kr) over a wide range of collision gas pressures and energies using an electrospray ionization source. The order of binding energies of the gas-phase ions for non-covalent protein complexes formed by the ESI source and assessed using CAD-MS/MS appears to differ from that of the solution complexes. The implication is that solution structure of these complexes was not preserved in the gas phase.     

Balancing conformational and oxidative kinetic traps during the folding of bovine pancreatic trypsin inhibitor (BPTI) with glutathione and glutathione disulfide

The oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) has served as a paradigm for the folding of disulfide-containing proteins from their reduced form, as well as for protein folding in general. Many extracellular proteins and most pharmaceutically important proteins contain disulfide bonds. Under traditional conditions, 0.125 mM glutathione disulfide (GSSG) and no glutathione (GSH), the folding pathway of BPTI proceeds through a nonproductive route via N* (a two disulfide intermediate), or a productive route via N' (and other two disulfide intermediates which are in rapid equilibrium with N'). Both routes have the rearrangement of disulfide bonds as their rate-determining steps. However, the effects of the composition of the redox buffer, GSSG and GSH, on folding has not been extensively investigated. Interestingly, BPTI folds more efficiently in the presence of 5 mM GSSG and 5 mM GSH than it does under traditional conditions. These conditions, which are similar to those found in vivo, result in a doubly mixed disulfide between N' and glutathione, which acts as an oxidative kinetic trap as it has no free thiols. However, with 5 mM GSSG and 5 mM GSH the formation of the double mixed disulfide is compensated for by N* being less kinetically stable and the more rapid conversion of the singly mixed disulfides between N' and glutathione to native protein (N). Thus a major rate-determining step becomes the direct conversion of a singly mixed disulfide to N, a growth-type pathway. Balancing the formation of N* and its stability versus the formation of the doubly mixed disulfide and its stability results in more efficient folding. Such balancing acts may prove to be general for other disulfide-containing proteins.     

Specific ion effects at protein surfaces: a molecular dynamics study of bovine pancreatic trypsin inhibitor and horseradish peroxidase in selected salt solutions

The distribution of sodium, choline, sulfate, and chloride ions around two proteins, horseradish peroxidase (HRP) and bovine pancreatic trypsin inhibitor (BPTI), is investigated by means of molecular dynamics simulations with the aim to elucidate ion adsorption at the protein surface. Although the two proteins under investigation are very different from each other, the ion distributions around them are remarkably similar. Sulfate is always strongly attached to the proteins, choline shows a significant, but unspecific, propensity for the protein surfaces, and sodium ions have a weak surface affinity, while chloride has virtually no preference for the protein surface. In mixtures of all four ion species in protein solutions, the resulting distributions are almost a superposition of the distributions of sodium sulfate and choline chloride, except that sodium partially replaces choline close to the proteins. The present simulations support a picture of ions interacting with individual ionic and polar amino acid groups rather than with an averaged protein surface. The results thus show how subtle the so-called Hofmeister and electroselectivity effects are in salt solution of proteins, making all simplified interaction models questionable.     

Binding of oxovanadium(V) anion to bovine serum albumin,human serum albumin and bovine pancreatic trypsin

The binding of vanadium(V) to bovine serum albumin (BSA), human serum albumin (HSA), and bovine pancreatic trypsin in the absence and presence of urea has been studied at different pH values and temperatures by spectrophotometric and equilibrium dialysis methods. The binding data were found to be pH and temperature dependent. The binding data at pH 5.57, studied by the absorbance method, were found approximately identical with those obtained from the equilibrium dialysis method at this pH. The enthalpy change at pH 5.57 for vanadium(V)-protein was −368.4 cal Mole⁻¹ for BSA, −328.8 cal Mole⁻¹ for HSA and −1372 cal Mole⁻¹ for trypsin respectively. The association constants and the number of binding sites were calculated from Scatchard plots and found to be at maximum at lower pH and at lower temperature. The free energy of the combining sites was lowest at higher pH and highest at low pH. Therefore, a lower temperature and a lower pH offered more sites in the protein molecule for interaction with vanadium(V) ions. Statistical effects seem to be more significant at lower vanadium(V) ion concentrations, and electrostatic effects more significant at higher concentrations.     

Structure and dynamics of the solvation of bovine pancreatic trypsin inhibitor in explicit water: a comparative study of the effects of solvent and protein polarizability

To isolate the effects of the inclusion of polarizability in the force field model on the structure and dynamics of the solvating water in differing electrostatic environments of proteins, we present the results of molecular dynamics simulations of the bovine pancreatic trypsin inhibitor (BPTI) in water with force fields that explicitly include polarization for both the protein and the water. We use three model potentials for water and two model potentials for the protein. Two of the water models and one of the protein models are polarizable. A total of six systems were simulated representing all combinations of these polarizable and nonpolarizable protein and water force fields. We find that all six systems behave in a similar manner in regions of the protein that are weakly electrostatic (either hydrophobic or weakly hydrophilic). However, in the vicinity of regions of the protein with relatively strong electrostatic fields (near positively or negatively charged residues), we observe that the water structure and dynamics are dependent on both the model of the protein and the model of the water. We find that a large part of the dynamical dependence can be described by small changes in the local environments of each region that limit the local density of non-hydrogen-bonded waters, precisely the water molecules that facilitate the dynamical relaxation of the water-water hydrogen bonds. We introduce a simple method for rescaling for this effect. When this is done, we are able to effectively isolate the influence of polarizability on the dynamics. We find that the solvating water's relaxation is most affected when both the protein and the water models are polarizable. However, when only one model (or neither) is polarizable, the relaxation is similar regardless of the models used.     

Photoinduced phase transitions

Employing actinic light to alter/stabilise a particular thermodynamic phase via the photo-isomerisation of the constituent molecules is an interesting tool to investigate soft matter from a new dimension. This article focuses on our recent results on several aspects of these non-equilibrium phase transitions, which are isothermal in nature. We specifically discuss (i) the influence of different parameters, such as confinement, applied electric field, pressure etc., on the dynamics associated with both the photochemical transition driving the equilibrium nematic to the non-equilibrium isotropic phase and the thermal back relaxation recovering the nematic phase, (ii) unique light-driven disorder–order transition in a reentrant system, (iii) dynamic self-assembly of the smectic A phase, which is stabilised only in the presence of actinic light, (iv) novel temperature-intensity phase diagrams and an example of primary and secondary photo-ferroelectric effects in an antiferroelectric smectic C system. These results highlight the fact that the actinic light can be used as a new tool to study phase transitions and the associated critical phenomena that could also bring about effects that are not seen in equilibrium situations.     

Complexation of bovine serum albumin and sugar beet pectin: structural transitions and phase diagram

The complexation between bovine serum albumin (BSA) and sugar beet pectin (SBP) was studied in situ by coupling glucono-δ-lactone (GDL) induced acidification with dynamic light scattering and turbidity measurements. Individual measurements at specific pHs and mixing ratios were also carried out using zeta potentiometry, gel permeation chromatography-multiangle laser light scattering (GPC-MALLS), and isothermal titration calorimetry (ITC). These investigations together enabled the establishment of a phase diagram of BSA/SBP and the identification of the molecular events during protein/polysaccharide complexation in relation to the phase diagram, which showed five regions: (I) a stable region of mixed individual soluble polymers, (II) a stable region of intramolecular soluble complexes, (III) a quasi-stable region of intermolecular soluble complexes, (IV) an unstable region of intermolecular insoluble complexes, and (V) a second stable region of mixed individual soluble polymers, on lowering pH. We found for the first time that the complexation could take place well above the critical pH(c), the value that most previous studies had regarded as the onset occurrence of complexation. A model of structural transitions between the regions was proposed. The borderline between region II and region III represents the BSA/SBP stoichiometry for intramolecular soluble complex at a specific pH, while that between region III and region IV identifies the composition of the intermolecular insoluble complex. Also studied was the effect of NaCl and CaCl(2) on the phase diagram and structural transitions.     

Spectral-fluorescent properties of maize grain trypsin inhibitor

The trypsin inhibitor isolated from maize grain has been investigated by the methods of IR, UV, fluorescence, derivative, and differential spectroscopy. Its spectral characteristics have been determined and the influence of the temperature and of a detergent on the structure of the protein has been studied.Scientific-Research Institute of Biology, Dnepropetrovsk State University, Dnepropetrovsk. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 375–381, May–June, 1989.     

Model for phase coexistence in phase transitions

We propose a general model for describing the phenomena of phase coexistence in relation to pressure induced phase transformations by means of the T–P distribution in statistical thermodynamics. Using the well‐known B1–B2 transition in NaCl as a prototype, we demonstrate how phase coexistence gives rise to the changes in the bulk modulus and the equation‐of‐state across the transition. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2004     

Hydroiodic acid attachment kinetics as a chemical probe of gaseous protein ion structure: Bovine pancreatic trypsin inhibitor

The kinetics of attachment of hydroiodic acid (HI) to the (M + 6H)6+ ions of native and reduced forms of bovine pancreatic trypsin inhibitor (BPTI) in the quadrupole ion trap environment are reported. Distinctly nonlinear (pseudo first-order) reaction kinetics are observed for reaction of the native ions, indicating two or more noninterconverting structures in the parent ion population. The reduced form, on the other hand, shows very nearly linear reaction kinetics. Both forms of the parent ion attach a maximum of five molecules of hydroiodic acid. This number is expected based on the amino acid composition of the protein. There is a total of 11 strongly basic sites in the protein (i.e., six arginines, four lysines, and one N-terminus). An ion with protons occupying six of the basic sites has five available for hydroiodic acid attachment. The kinetics of successive attachment of HI to the native and reduced forms of BPTI also differ, particularly for the addition of the fourth and fifth HI molecules. A very simple kinetic model describes the behavior of the reduced form reasonably well, suggesting that all of the neutral basic sites in the reduced BPTI ions have roughly equal reactivity. However, the behavior of the native ion is not well-described by this simple model. The results are discussed within the context of differences in the three-dimensional structures of the ions that result from the presence or absence of the three disulfide linkages found in native BPTI. The HI reaction kinetics appears to have potential as a chemical probe of protein ion three-dimensional structure in the gas phase. Hydroiodic acid attachment chemistry is significantly different from other chemistries used to probe three-dimensional structure and hence, promises to yield complementary information.     

Toluene-induced phase transitions in blue phase liquid crystals

ABSTRACT

We report phase transitions in blue phase-forming liquid crystals (LCs) that are triggered by exposure to toluene vapours. Specifically, we reveal that room-temperature cholesteric phase mixtures of MLC-2142 and S-811 form blue phases (BP I, II and III) with increasing vapour pressure of toluene. To probe the mechanism underlying this observation, we investigated the phase behaviour of mixtures of BP-forming LCs containing a range of non-volatile aromatic compounds (e.g. pyrene). We interpret our observations to indicate that the principal effect of small aromatic compounds is to decrease the energy penalty associated with the formation of disclination lines in BPs. We also conclude that the absorption of toluene into the BP-forming LCs lowers the energy required for the formation of disclination cores in the BP phase, thus allowing the elastically favoured double-twist cylinders to form at lower temperatures. We demonstrate that BP-forming LCs containing pyrene can be used to detect toluene at concentrations below 200 ppm at room temperature. Overall, these results guide the design of LC-based materials that respond to VOCs at concentrations relevant to occupational settings.