Affinity chromatography of serine proteinases from the bovine intervertebral disc on BPTI-Separon HEMA 1000.

A method for the purification of serine proteinases from the bovine intervertebral disc using affinity chromatography on basic pancreatic trypsin inhibitor (BPTI) immobilized to the hydroxyalkyl methacrylate copolymer Separon HEMA 1000 E is reported. Its advantage is the possibility of obtaining serine proteinases without an artificial alteration in relative molecular mass.     

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.     

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.     

Exploring bovine pancreatic trypsin inhibitor phase transitions

This paper presents an investigation of the phase diagram of BPTI (bovine pancreatic trypsin inhibitor)/350 mM KSCN at pH 4.9 by direct observation and numerical simulations. We report optical microscopy and light and X-ray scattering experiments coupled with theoretical data analysis using numerical tools. The phase diagram is thoroughly determined, as a function of temperature. Two polymorphs are observed by video microscopy and their solubility measured. In this phase diagram, the liquid-liquid phase separation (LLPS) is metastable with respect to the solid-liquid phase separation. Above the T(L-L) boundary curve, solutions are composed of a mixture of BPTI monomers and decamers. Attractive interactions are stronger between decamers than between monomers. Below the T(L-L) boundary curve, the dense phase is highly concentrated in protein and composed of BPTI decamers alone. Thus, the driving force for liquid-liquid or liquid-solid phase separation is the attraction between decamers at low pH. The structure factors of the dense phases are characteristic of repulsive dense phases because of a hard sphere repulsion core, meaning that in the dense phase proteins are actually in contact (interparticle distance of 53 A). In agreement with the Oswald rule of stages, LLPS occurs prior to and impedes the solid nucleation.     

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.     

FAMBE-pH: a fast and accurate method to compute the total solvation free energies of proteins

A fast and accurate method to compute the total solvation free energies of proteins as a function of pH is presented. The method makes use of a combination of approaches, some of which have already appeared in the literature; (i) the Poisson equation is solved with an optimized fast adaptive multigrid boundary element (FAMBE) method; (ii) the electrostatic free energies of the ionizable sites are calculated for their neutral and charged states by using a detailed model of atomic charges; (iii) a set of optimal atomic radii is used to define a precise dielectric surface interface; (iv) a multilevel adaptive tessellation of this dielectric surface interface is achieved by using multisized boundary elements; and (v) 1:1 salt effects are included. The equilibrium proton binding/release is calculated with the Tanford-Schellman integral if the proteins contain more than approximately 20-25 ionizable groups; for a smaller number of ionizable groups, the ionization partition function is calculated directly. The FAMBE method is tested as a function of pH (FAMBE-pH) with three proteins, namely, bovine pancreatic trypsin inhibitor (BPTI), hen egg white lysozyme (HEWL), and bovine pancreatic ribonuclease A (RNaseA). The results are (a) the FAMBE-pH method reproduces the observed pK a's of the ionizable groups of these proteins within an average absolute value of 0.4 p K units and a maximum error of 1.2 p K units and (b) comparison of the calculated total pH-dependent solvation free energy for BPTI, between the exact calculation of the ionization partition function and the Tanford-Schellman integral method, shows agreement within 1.2 kcal/mol. These results indicate that calculation of total solvation free energies with the FAMBE-pH method can provide an accurate prediction of protein conformational stability at a given fixed pH and, if coupled with molecular mechanics or molecular dynamics methods, can also be used for more realistic studies of protein folding, unfolding, and dynamics, as a function of pH.     

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.     

Exploring the “intensity fading” phenomenon in the study of noncovalent interactions by MALDI-TOF mass spectrometry

The difficulties to detect intact noncovalent complexes involving proteins and peptides by MALDI-TOF mass spectrometry have hindered a widespread use of this approach. Recently, "intensity fading MS" has been presented as an alternative strategy to detect noncovalent interactions in solution, in which a reduction in the relative signal intensity of low molecular mass binding partners (i.e., protease inhibitors) can be observed when their target protein (i.e., protease) is added to the sample. Here we have performed a systematic study to explore how various experimental conditions affect the intensity fading phenomenon, as well as a comparison with the strategy based on the direct detection of intact complexes by MALDI MS. For this purpose, the study is focused on two different protease-inhibitor complexes naturally occurring in solution, together with a heterogeneous mixture of nonbinding molecules derived from a biological extract, to examine the specificity of the approach, i.e., those of carboxypeptidase A (CPA) bound to potato carboxypeptidase inhibitor (PCI) and of trypsin bound to bovine pancreatic trypsin inhibitor (BPTI). Our results show that the intensity fading phenomenon occurs when the binding assay is carried out in the sub-muM range and the interacting partners are present in complex mixtures of nonbinding compounds. Thus, at these experimental conditions, the specific inhibitor-protease interaction causes a selective reduction in the relative abundance of the inhibitor. Interestingly, we could not detect any gaseous noncovalent inhibitor-protease ions at these conditions, presumably due to the lower high-mass sensitivity of MCP detectors.     

Dynamics of a protein and water molecules surrounding the protein: hydrogen-bonding between vibrating water molecules and a fluctuating protein

Internal and rigid-body motions of bovine pancreatic trypsin inhibitor (BPTI) and of water molecules surrounding the BPTI are studied in a vicinity of an energy minimum using a normal mode analysis proposed as the independent molecule model. Water's rigid-body motion is predominant in comparison to its internal motions. We have derived information about the relationship between the magnitude of a thermal ellipsoid of an H-bonding atom and the anisotropy of its ellipsoid, and the relationship between the magnitude of the ellipsoid and the H-bond strength. We see a relationship between vibrational frequencies (assuming rigid-body motion of the water molecules) and the H-bond strength of the water taking part in this H-bonding. Analyzing the H-bond strength, we found that a hydrogen in water is likely to H-bond to oxygen in the protein, whereas an oxygen in water has a less strong preference to H-bond to the protein. For water molecules acting as the hydrogen acceptor, strong H-bonding has longer lifetimes than weak H-bonding.     

Substrate turnover and inhibitor binding as selection parameters in directed evolution of blood coagulation factor Xa

A library of blood coagulation factor Xa (FXa)-trypsin hybrid proteases was generated and displayed on phage for selection of derivatives with the domain "architecture" of trypsin and the specificity of FXa. Selection based on binding to soybean trypsin inhibitor only provided enzymatically inactive derivatives, due to a specific mutation of serine 195 of the catalytic triad to a glycine, revealing a significant selection pressure for proteolytic inactive derivatives. By including a FXa peptide substrate in the selection mixture, the majority of the clones had retained serine at position 195 and were enzymatically active after selection. Further, with the inclusion of bovine pancreatic trypsin inhibitor, in addition to the peptide substrate, the selected clones also retained FXa specificity after selection. This demonstrates that affinity selection combined with appropriate deselection provides a simple strategy for selection of enzyme derivatives that catalyse a specific reaction.     

Hydration free energies and entropies for water in protein interiors

Free energy calculations for the transfer of a water molecule from the pure liquid to an interior cavity site in a protein are presented. Two different protein cavities, in bovine pancreatic trypsin inhibitor (BPTI) and in the I76A mutant of barnase, represent very different environments for the water molecule: one which is polar, forming four water-protein hydrogen bonds, and one which is more hydrophobic, forming only one water-protein hydrogen bond. The calculations give very different free energies for the different cavities, with only the polar BPTI cavity predicted to be hydrated. The corresponding entropies for the transfer to the interior cavities are calculated as well and show that the transfer to the polar cavity is significantly entropically unfavorable while the transfer to the nonpolar cavity is entropically favorable. For both proteins an analysis of the fluctuations in the positions of the protein atoms shows that the addition of a water molecule makes the protein more flexible. This increased flexibility appears to be due to an increased length and weakened strength of protein-protein hydrogen bonds near the cavity.     

Determination of aprotinin by titration with bovine trypsin with end-point detection by high-performance liquid chromatography

A method for the determination of aprotinin (bovine pancreatic trypsin inhibitor, BPTI) is described. The procedure involves the formation of the BPTI-trypsin complex in the presence of an excess of BPTI, quantitative separation of the residual BPTI from the mixture by affinity chromatography and identification and evaluation of the residual BPTI by reversed-phase high-performance liquid chromatography. The method is precise with a mean coefficient of variation of 4.0 and 4.3% for intra- and inter-assay runs, respectively, and has a limit of determination of 3.0 micrograms of aprotinin. The proposed method can be applied to commercial samples, even in very dilute solutions, for the standardisation of aprotinin.     

Hydrogen exchange rates in proteins from water (1)H transverse magnetic relaxation

Access to the fast exchange kinetics of labile protein hydrogens in solution is provided by exchange broadening of the water 1H NMR line. We analyzed the chemical shift modulation contribution of labile hydrogens in bovine pancreatic trypsin inhibitor (BPTI) to the transverse 1H spin relaxation rate, R2, of the bulk solvent. Both the experimental pH dependence and the CPMG dispersion of R2 could be quantitatively accounted for on the basis of known chemical shifts, exchange rates, and ionization constants for BPTI. This analysis provided, for the first time, the hydrogen exchange rate constants for Lys and Arg side chains in a protein and pointed to an internal catalysis of the N-terminal amino protons in BPTI by a salt bridge. The method can be used for mapping the hydrogen exchange rates in protein solutions and biomaterials, which may be important for the control of relaxation-weighted contrast in biological MRI.     

Harmonic analysis of large systems. III. Comparison with molecular dynamics

Atomic motions in bovine pancreatic trypsin inhibitor (BPTI), derived from molecular dynamics, harmonic analysis, and quasiharmonic analysis, are compared when a single protein model, energy parameters, and environment are employed. Molecular dynamics (MD) was carried out for 2 nanoseconds. An average structure was determined from the last nanosecond of the MD simulation, when no major structural changes were observed. This structure was used for several harmonic analysis calculations as well as for a reference structure for the quasiharmonic analysis, for both full basis and reduced basis sets. In contrast to the harmonic analysis results, the quasiharmonic reduced basis calculation using a spherical harmonics reduced basis provided good agreement with the full basis calculation, suggesting that when anharmonic effects are considered, BPTI can behave as a homogeneous object. An extensive analysis of the normal modes from a diverse set of 201 minimized MD simulation frames was performed. On only the sub-picosecond time scale were energy minima revisited after a transition to another state. This analysis shows that the dynamics average structure is not representative of the simulation frames in terms of energy and vibrational frequencies. For this model of BPTI, 42% of the motion (mean-squared fluctuation) can be attributed to harmonic limit behavior. A spectral analysis of the correlation function of deformation for a particular normal mode or quasiharmonic mode can be used to determine the time scales of motions which correspond to harmonic vibration, large-scale drift, or sharp transitions between local substrates. © 1995 John Wiley & Sons, Inc.     

Stochastic dynamic simulation of a protein

The internal motions of a small protein, the bovine pancreatic trypsin inhibitor (BPTI) in solution, are investigated in the framework of the Langevin equation. In this approach, the effects of the solvent molecules are incorporated by suitably defining the friction and random forces. The friction coefficients are determined from a molecular dynamics simulation. The details of the rapid fluctuations of protein atoms obtained by stochastic and molecular dynamics simulation techniques are compared by calculating the generalized density of states obtained via an incoherent neutron scattering. Presently, our stochastic dynamics simulation is one order of magnitude faster than the molecular dynamics simulation with the explicit inclusion of the water molecules. Generalizations of the present stochastic dynamics approach for studying the large-scale motion in proteins are briefly outlined and the probability of a further speedup by an additional order of magnitude is discussed.     

Improvements on the protein–dipole Langevin–dipole model

The protein–dipole Langevin–dipole (PDLD) model developed by Warshel and co-workers is an approach to evaluate electrostatic interactions in protein systems from microscopic sights. This model grasped the main physical factors and required little computations. But it might need the tests from every aspect. In the present work, we have chosen the solvation energies of Asp3, Glu7, Glu49, and Asp50 in bovine pancreatic trypsin inhibitor (BPTI) as a calibration to discuss the influences of parameters and conditions on the simulation results in the PDLD model. Some improvements have been proposed. The calculated solvation energies associated with ionizing the four acidic groups in BPTI and aspartic acid in solution are found in good agreement with the corresponding observed results if the improved PDLD approach and computational methods are used. © 1992 by John Wiley & Sons, Inc.     

Disulfide bond isomerization in basic pancreatic trypsin inhibitor: multisite chemical exchange quantified by CPMG relaxation dispersion and chemical shift modeling

Conformational changes occurring on the microsecond-millisecond time scale in basic pancreatic trypsin inhibitor (BPTI) are investigated using nuclear magnetic resonance spectroscopy. The rczz CPMG experiment (Wang, C.; Grey, M. J.; Palmer, A. G. J. Biomol. NMR 2001, 21, 361-366) is used to record (15)N spin relaxation dispersion data, R(ex)(1/tau(cp)), in which 1/tau(cp) is the pulsing rate in the CPMG sequence, at two static magnetic fields, 11.7 and 14.1 T, and three temperatures, 280, 290, and 300 K. These data are used to characterize the kinetics and mechanism of chemical exchange line broadening of the backbone (15)N spins of Cys 14, Lys 15, Cys 38, and Arg 39 in BPTI. Line broadening is found to result from two processes: the previously identified isomerization of the Cys 38 side chain between chi(1) rotamers (Otting, G.; Liepinsh, E.; Wüthrich, K. Biochemistry 1993, 32, 3571-3582) and a previously uncharacterized process on a faster time scale. At 300 K, both processes contribute significantly to the relaxation dispersion for Cys 14 and an analytical expression for a linear three-site exchange model is used to analyze the data. At 280 K, isomerization of the Cys 38 side chain is negligibly slow and the faster process dominates the relaxation dispersion for all four spins. Global analysis of the temperature and static field dependence of R(ex)(1/tau(cp)) for Cys 14 and Lys 15 is used to determine the activation parameters and chemical shift changes for the previously uncharacterized chemical exchange process. Through an analysis of a database of chemical shifts, (15)N chemical shift changes for Cys 14 and Lys 15 are interpreted to result from a chi(1) rotamer transition of Cys 14 that converts the Cys 14-Cys 38 disulfide bond between right- and left-handed conformations. At 290 K, isomerization of Cys 14 occurs with a forward and reverse rate constant of 35 s(-1) and 2500 s(-1), respectively, a time scale more than 30-fold faster than the Cys 38 chi(1) isomerization. A comparison of the kinetics and thermodynamics for the transitions between the two alternative Cys 14-Cys 38 conformations highlights the factors that affect the contribution of disulfide bonds to protein stability.     

Harmonic analysis of large systems. II. Comparison of different protein models

A series of normal mode analyses of bovine pancreatic trypsin inhibitor (BPTI) has been performed. The results of modifying the long-range truncation of electrostatics, reducing the conformational space of the system (reduced basis normal mode analysis), and using different parameter sets and models for the potential function are reported. Both explicit (904 atoms) and polar hydrogen (580 atoms) representations of BPTI were examined and produced nearly identical normal mode vectors but slightly modified vibrational frequencies. The truncation methods—no cutoff, shift, and switch—were examined, and the use of a short switching function was found to alter harmonic motion greatly. A table relating the different cutoff methods to several previously published frequencies for BPTI indicates that the diversity of published lowest frequencies is due to the use of different electrostatic models rather than to inherent differences in the models or energy parameters. Examining reduced basis results demonstrates that a dihedral basis yields similar normal mode vectors, though the vibrational frequencies are shifted to higher values. The analysis of BPTI harmonic dynamics using a spherical harmonic reduced basis set yields significantly altered dynamics, indicating that BPTI is not well represented as a homogeneous object at low temperatures. © 1995 John Wiley & Sons, Inc.

1 This article is a U.S. Government work and, as such, is in the public domain in the United States of America.

     

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.