Structure Of Bovine Pancreatic Trypsin Inhibitor Research Articles

Charge Transfer and Polarization in Solvated Proteins from Ab Initio Molecular Dynamics

Charge transfer at the Bovine pancreatic trypsin inhibitor (BPTI) protein–water interface was analyzed by means of ab initio Born–Oppenheimer molecular dynamics simulation of the entire protein running on graphical processing units (GPUs). The efficiency of the GPU-based quantum chemistry algorithms implemented in our TeraChem program enables us to perform these calculations on a desktop computer. Mulliken and Voronoi deformation density (VDD) population analysis reveals that between 2.0 and 3.5 electrons are transferred from surrounding water molecules to the protein over the course of the 8.8 ps simulation. Solving for the electronic structure of BPTI in the absence of surrounding water molecules (i.e., in the gas phase) leads to large intraprotein charge transfer, where approximately one electron in total is transferred from neutral to polar residues. Solvation relieves this polarization stress, leading to a neutralization of the excess positive charge of the neutral residues.

Temperature dependence on structure and dynamics of Bovine Pancreatic Trypsin Inhibitor (BPTI): A neutron scattering study

We have studied the influence of temperature on the structure of BPTI in solution by small angle neutron scattering. We have investigated the variation of the radius of gyration and the modification of the shape of BPTI between ambient temperature and 368 K. Results have shown an increase of the radius of gyration from 10.9 Å at ambient temperature up to 13.3 Å at 368 K. Global and internal dynamics of BPTI in solution were studied by quasielastic neutron scattering. The analysis of neutron data in terms of intermediate scattering function reveals two relaxation times τ 1 and τ 2 related respectively to global translational diffusive motions and to internal motions of protein. Motions of protons belonging to lateral chains of residues located at the surface of the protein have been detected. The results are compared to the recently published results concerning the influence of pressure on structure and dynamics of BPTI in solution [Appavou MS et al. Biochimica et Biophysica Acta, 1764, 2006, pp 414–423].

Combining a polarizable force‐field and a coarse‐grained polarizable solvent model: Application to long dynamics simulations of bovine pancreatic trypsin inhibitor

The dynamic coupling between a polarizable protein force field and a particle-based implicit solvent model is described. The polarizable force field, TCPEp, developed recently to simulate protein systems, is characterized by a reduced number of polarizable sites, with a substantial gain in efficiency for an equal chemical accuracy. The Polarizable Pseudo-Particle (PPP) solvent model represents the macroscopic solvent polarization by induced dipoles placed on mobile Lennard-Jones pseudo-particles. The solvent-induced dipoles are sensitive to the solute electric field, but not to each other, so that the computational cost of solvent-solvent interactions is basically negligible. The solute and solvent induced dipoles are determined self-consistently and the equations of motion are solved using an efficient iterative multiple time step procedure. The solvation cost with respect to vacuum simulations is shown to decrease with solute size: the estimated multiplicative factor is 2.5 for a protein containing about 1000 atoms, and as low as 1.15 for 8000 atoms. The model is tested for six 20 ns molecular dynamics trajectories of a traditional benchmark system: the hydrated Bovine Pancreatic Trypsin Inhibitor (BPTI). Even though the TCPEp parameters have not been refined to be used with the solvent PPP model, we observe a good conservation of the BPTI structure along the trajectories. Moreover, our approach is able to provide a description of the protein solvation thermodynamic at the same accuracy as the standard Poisson-Boltzman continuum methods. It provides in addition a good description of the microscopic structural aspects concerning the solute/solvent interaction.

Rigidification of a Flexible Protease Inhibitor Variant upon Binding to Trypsin

The Tyr35 → Gly replacement in bovine pancreatic trypsin inhibitor (BPTI) has previously been shown to dramatically enhance the flexibility of the trypsin-binding region of the free inhibitor and to destabilize the interaction with the protease by about 3 kcal/mol. The effects of this replacement on the enzyme-inhibitor interaction were further studied here by X-ray crystallography and isothermal titration calorimetry (ITC). The co-crystal structure of Y35G BPTI bound to trypsin was determined using 1.65 Å resolution X-ray diffraction data collected from cryopreserved crystals, and a new structure of the complex with wild-type BPTI under the same conditions was determined using 1.62 Å data. These structures reveal that, in contrast to the free protein, Y35G BPTI adopts a conformation nearly identical with that of the wild-type protein, with a water-filled cavity in place of the missing Tyr side-chain. The crystallographic temperature factors for the two complexes indicate that the mutant inhibitor is nearly as rigid as the wild-type protein when bound to trypsin. Calorimetric measurements show that the change in enthalpy upon dissociation of the complex is 2.5 kcal/mol less favorable for the complex containing Y35G BPTI than for the complex with the wild-type inhibitor. Thus, the destabilization of the complex resulting from the Y35G replacement is due to a more favorable change in entropy upon dissociation. The heat capacity changes for dissociation of the mutant and wild-type complexes were very similar, suggesting that the entropic effects probably do not arise from solvation effects, but are more likely due to an increase in protein conformational entropy upon dissociation of the mutant inhibitor. These results define the biophysical role of a highly conserved core residue located outside of a protein-binding interface, demonstrating that Tyr35 has little impact on the trypsin-bound BPTI structure and acts primarily to define the structure of the free protein so as to maximize binding affinity.

Hydrophobic collapse in late-stage folding (in silico) of bovine pancreatic trypsin inhibitor

Hydrophobic collapse is commonly considered as a process of significance for protein folding. Many models have been applied for description of this event. A model introducing an external force field mimicking the hydrophobic environment and simultaneously the driving force for the folding process is presented in this paper. Bovine pancreatic trypsin inhibitor (BPTI) was taken as a test protein. An early-stage folding (in silico) model presented elsewhere was used to create the starting structure for hydrophobic collapse. The resulting structure was energy-refined using molecular dynamics simulation in an explicit solvent. The similarity versus the crystal structure of BPTI is estimated using visual analysis, residue–residue contacts, φ, ψ angle distributions, RMSD, accessible solvent area, radii of gyration and hydrodynamic radii. A program allowing creation of early-stage folding structural forms to be created for any protein is available from http://bioinformatics.cm-uj.krakow.pl/earlystage. The program for late-stage folding simulation is available on request.

Influence of pressure on structure and dynamics of bovine pancreatic trypsin inhibitor (BPTI): Small angle and quasi-elastic neutron scattering studies

We have studied the influence of pressure on structure and dynamics of a small protein belonging to the enzymatic catalysis: the bovine pancreatic trypsin inhibitor (BPTI). Using a copper-beryllium high-pressure cell, we have performed small angle neutron scattering (SANS) experiment on NEAT spectrometer at HMI (Berlin, Germany). In the SANS configuration, the evolution of the radius of gyration and of the shape of the protein under pressures up to 6000 bar has been studied. When increasing pressure from atmospheric pressure up to 6000 bar, the pressure effects on the global structure of BPTI result on a reduction of the radius of gyration from 13.4 Å down to 12.0 Å. Between 5000 and 6000 bar, some transition already detected by FTIR [N. Takeda, K. Nakano, M. Kato, Y. Taniguchi, Biospectroscopy, 4, 1998, pp. 209–216] is observed. The pressure effect is not reversible because the initial value of the radius of gyration is not recovered after pressure release. By extending the range of wave-vectors to high q, we have observed a change of the form factor (shape) of the BPTI under pressure. At atmospheric pressure BPTI exhibits an ellipsoidal form factor that is characteristic of the native state. When the pressure is increased from atmospheric pressure up to 6000 bar, the protein keeps its ellipsoidal shape. The parameters of the ellipsoid vary and the transition detected between 5000 and 6000 bar in the form factor of BPTI is in agreement with the FTIR results. After pressure release, the form factor of BPTI is characteristic of an ellipsoid of revolution with a semi-axis a, slightly elongated with respect to that of the native one, indicating that the pressure-induced structural changes on the protein are not reversible. The global motions and the internal dynamics of BPTI protein have been investigated in the same pressure range by quasi-elastic neutron scattering experiments on IN5 time-of-flight spectrometer at ILL (Grenoble, France). The diffusion coefficients D and the internal relaxation times < τ 2> of BPTI deduced from the analysis of the intermediate scattering functions show a slowing down of protein dynamics when increasing pressure.

Structure of tick anticoagulant peptide at 1.6 A resolution complexed with bovine pancreatic trypsin inhibitor.

The structure of tick anticoagulant peptide (TAP) has been determined by X-ray crystallography at 1.6 A resolution complexed with bovine pancreatic trypsin inhibitor (BPTI). The TAP-BPTI crystals are tetragonal, a = b = 46.87, c = 50.35 A, space group P41, four complexes per unit cell. The TAP molecules are highly dipolar and form an intermolecular helical array along the c-axis with a diameter of about 45 A. Individual TAP units interact in a head-to-tail fashion, the positive end of one molecule associating with the distal negative end of another, and vice versa. The BPTI molecules have a uniformly distributed positively charged surface that interacts extensively through 14 hydrogen bonds and two hydrogen bonded salt bridges with the helical groove around the helical TAP chains. Comparing the structure of TAP in TAP-BPTI with TAP bound to factor Xa(Xa) suggests a massive reorganization in the N-terminal tetrapeptide and the first disulfide loop of TAP (Cys5T-Cys15T) upon binding to Xa. The Tyr1(T)OH atom of TAP moves 14.2 A to interact with Asp189 of the S1 specificity site, Arg3(T)CZ moves 5.0 A with the guanidinium group forming a cation-pi-electron complex in the S4 subsite of Xa, while Lys7(T)NZ differs in position by 10.6 A in TAP-BPTI and TAP-Xa, all of which indicates a different pre-Xa-bound conformation for the N-terminal of TAP in its native state. In contrast to TAP, the BPTI structure of TAP-BPTI is practically the same as all those of previously determined structures of BPTI, only arginine and lysine side-chain conformations showing significant differences.

Tyrosine, phenylalanine, and disulfide contributions to the circular dichroism of proteins: circular dichroism spectra of wild-type and mutant bovine pancreatic trypsin inhibitor.

Improved descriptions of the lowest energy excited states of tyrosine and phenylalanine side chains have been developed in order to extend the capabilities of calculating the circular dichroism (CD) spectra of proteins. Four transitions (Lb, La, Bb, and Ba) for each of the side-chain chromophores were considered, and the transition monopole charges were obtained from a CNDO/S calculation on models representing the individual groups. Monopole charges at midpoints of the bonds, corresponding to the maximum transition charge densities in the Lb band, and monopole charges representing the vibronic coupling with the B transitions for the La transition were also included. The aromatic transitions were combined with the peptide transitions (npi, pi0pi n'pi, and pi+pi) and disulfide transitions (n1sigma and n4sigma) in the framework of the origin-independent matrix method to compute the CD spectra of different crystal forms and Y --> L and F --> L mutants of bovine pancreatic trypsin inhibitor (BPTI). The structures of the mutants were obtained by replacing the appropriate tyrosine or phenylalanine residue by leucine in the wild-type crystal structure. The CD calculations were performed on the energy-minimized structures. The CD spectrum calculated for the form II crystal structure of BPTI showed the best agreement with experiment. In the far UV, the calculated and experimental CD spectra agree to various extents for the wild-type and mutant BPTI. Among the mutants, the calculated CD spectra of Y4L, Y10L, Y23L, and F45L showed reasonable agreement with experiment, while those of Y21L and F22L, the two residues interacting with most aromatic groups, showed poor agreement. In the near UV, the negative bands predicted for the wild-type and mutant BPTI have much less intensity than observed experimentally.

Study of protein fluctuation with an effective inter-Cα atomic potential derived from average distances between amino acids in proteins.

The conformational dynamics around the native structure of bovine pancreatic trypsin inhibitor (BPTI) in both the oxidized and reduced forms was analyzed by a Monte Carlo method using an approximate residue-residue potential derived from the statistics of average distances between Cα atoms of residues as proposed by the present author (T. Kikuchi, J Comput Chem 1996, 17, 226-237). The results from using this effective potential are similar to those from molecular dynamics simulations, taking all atoms into account, and are consistent with temperature factors from an X-ray analysis and disulfide formation from a kinetic experiment. This agreement suggests that the essential nature of the potential energy surface formed by the potential around the native structure closely mimics the actual energy landscape within the resolution of Cα atomic fluctuation. Furthermore, it is expected that the potential we found can describe the basic properties of folding kinetics. Examination of the fluctuation property of the native structure of BPTI threaded by a sequence from cytochrome b562 reveals differences specific to the sequence and this result also shows that the dynamical properties obtained in our calculations are not only ascribed to the geometrical constraints of the initial conformation but also the force field specifically produced by a sequence. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 713-719, 1999.

A folded protein can be transported across the chloroplast envelope and thylakoid membranes.

Many thylakoid lumenal proteins are nuclear encoded, cytosolically synthesized, and reach their functional location after posttranslational targeting across two chloroplast envelope membranes and the thylakoid membrane via proteinaceous transport systems. To study whether these transmembrane transport machineries can translocate folded structures, we overexpressed the 17-kDa subunit of the oxygen-evolving complex of photosystem II (prOE17) that had been modified to contain a unique C-terminal cysteine. This allowed us to chemically link a terminal 6.5-kDa bovine pancreatic trypsin inhibitor (BPTI) moiety to prOE17 to create the chimeric protein prOE17-BPTI. Redox reagents and an irreversible sulfhydryl-specific cross-linker, bis-maleimidohexane, were used to manipulate the structure of BPTI. Import of prOE17-BPTI into isolated chloroplasts and thylakoids demonstrates that the small tightly folded BPTI domain is carried across both the chloroplast envelopes and the delta pH-dependent transmembrane transporter of the thylakoid membrane when linked to the correctly targeted OE17 precursor. Transport proceeded even when the BPTI moiety was internally cross-linked into a protease-resistant form. These data indicate that unfolding is not a ubiquitous requirement for protein translocation and that at least some domains of targeted proteins can maintain a nonlinear structure during their translocation into and within chloroplasts.

Protein Structure in the Lyophilized State: A Hydrogen Isotope Exchange/NMR Study with Bovine Pancreatic Trypsin Inhibitor

Protein Structure in the Lyophilized State: A Hydrogen Isotope Exchange/NMR Study with Bovine Pancreatic Trypsin Inhibitor

Simultaneous Refinement of the Structure of BPTI Against NMR Data Measured in Solution and X-ray Diffraction Data Measured in Single Crystals

The structure of the bovine pancreatic trypsin inhibitor (BPTI) has been determined to high resolution by both NMR spectroscopy in solution and X-ray diffraction in crystals. The root-mean-square difference calculated between the two structures for the polypeptide backbone is 0·9 Å. Several amino acid side-chains, of which all but one are charged or polar, have different conformations. We find that by refining one structure simultaneously against both the NMR and crystallographic data sets, it can accommodate both. Different starting configurations were used, including the X-ray structure 5pti, an NMR conformer, and the X-ray structure in the full unit cell with extra solvent placed in the bulk solvent region. The X-ray structures quickly converged to accommodate the NMR data in addition to the crystallographic data. Starting from an NMR conformer, however, the convergence to accommodate the more abundant X-ray data in addition to the NMR data is much slower.

The loop problem in proteins: A monte carlo simulated annealing approach

A Monte Carlo simulated annealing (MCSA) algorithm was used to generate the conformations of local regions in bovine pancreatic trypsin inhibitor (BPTI) starting from random initial conformations. In the approach explored, only the conformation of the segment is computed; the rest of the protein is fixed in the known native conformation. Rather than follow a single simulation exhaustively, computer time is better used by performing multiple independent MCSA simulations in which different starting temperatures are employed and the number of conformations sampled is varied. The best computed conformation is chosen on the basis of lowest total energy and refined further. The total energy used in the annealing is the sum of the intrasegment energy, the interaction energy of the segment with the local surrounding region, and a distance constraint to generate a smooth connection of the initially randomized segment with the rest of the protein. The rms deviations between the main-chain conformations of the computed segments in BPTI and those of the native x-ray structure are 0.94 A for a 5-residue alpha-helical segment, 1.11 A for a 5-residue beta-strand segment, and 1.03, 1.61, and 1.87 A for 5-, 7-, and 9-residue loop segments. Side-chain deviations are comparable to the main-chain deviations for those side chains that interact strongly with the fixed part of the protein. A detailed view of the deviations at an atom-resolved level is obtained by comparing the predicted segments with their known conformations in the crystal structure of BPTI. These results emphasize the value of predetermined fixed structure against which the computed segment can nest.

Solid‐phase synthesis of bovine pancreatic trypsin inhibitor BPTI) and two analogues

The linear sequence of bovine pancreatic trypsin inhibitor (BPTI) has been assembled by stepwise Fmoc solid-phase peptide synthesis on a polyethylene glycol-polystyrene (PEG-PS) graft support with p-alkoxybenzyl ester anchoring. Similar methods were used to prepare two analogues, the first with all six half-cystine (Cys) residues replaced by alpha-amino-n-butyric acid (Abu), and the second with replacement of Abu at four Cys positions while retaining the native pairing between positions 14 and 38. Following cleavage from the support, the linear molecules (reduced form) were purified by semipreparative reversed-phase high performance liquid chromatography (HPLC). The native structure of BPTI was then formed by oxidation of a dilute solution of the protein at pH 8.7 in the presence of oxidized glutathione. The BPTI analogue with one disulfide bridge was obtained following treatment with dimethyl sulfoxide (DMSO)-pH 6 buffer (1:9). Overall yields of homogeneous proteins were 2-4%, and further characterization was provided by amino acid analysis, sequencing, ion electrospray mass spectrometry, analytical HPLC, and capillary zone electrophoresis (CZE). Purified synthetic BPTI with the native sequence was indistinguishable from natural material by the analytical and biophysical criteria applied, including circular dichroism (CD) spectra and inhibition of trypsin action. Studies are in progress to evaluate conformational features of the analogues which respectively lack two, or all three, of the native disulfide bridges.

Sequential NMR resonance assignment and structure determination of the Kunitz-type inhibitor domain of the Alzheimer's .beta.-amyloid precursor protein

Certain precursor proteins (APP751 and APP770) of the amyloid beta-protein (AP) present in Alzheimer's disease contain a Kunitz-type serine protease inhibitor domain (APPI). In this study, the domain is obtained as a functional inhibitor through both recombinant (APPIr) and synthetic (APPIs) methodologies, and the solution structure of APPI is determined by 1H 2D NMR techniques. Complete sequence-specific resonance assignments (except for P13 and G37 NH) for both APPIr and APPIs are achieved using standard procedures. Ambiguities arising from degeneracies in the NMR resonances are resolved by varying sample conditions. Qualitative interpretation of short- and long-range NOEs reveals secondary structural features similar to those extensively documented by NMR for bovine pancreatic trypsin inhibitor (BPTI). A more rigorous interpretation of the NOESY spectra yields NOE-derived interresidue distance restraints which are used in conjunction with dynamic simulated annealing to generate a family of APPI structures. Within this family, the beta-sheet and helical regions are in good agreement with the crystal structure of BPTI, whereas portions of the protease-binding loops deviate from those in BPTI. These deviations are consistent with those recently described in the crystal structure of APPI (Hynes et al., 1990). Also supported in the NMR study is the hydrophobic patch in the protease-binding domain created by side chain-side chain NOE contacts between M17 and F34. In addition, the NMR spectra indicate that the rotation of the W21 ring in APPI is hindered, unlike Y21 in BPTI, showing a greater than 90% preference for one orientation in the hydrophobic groove.

A hybrid approach to theoretical analysis of bovine pancreatic trypsin inhibitor

A static analysis of bovine pancreatic trypsin inhibitor (BPTI) is presented based on a new discrete/continuum approach to modeling the dynamics of biomolecules. This hybrid method utilizes knowledge of the intramolecular potential and molecular configuration to generate a field of elastic modulus tensors. These tensors, which relate the local stress and strain for each atom in the biomolecule, can be used to judge the local rigidity as well as indicate regions of high stress. Comparing the tensor fields for an unrelaxed and a relaxed configuration, the microscopic structure of BPTI is found to be anisotropic and to have regions of stress even when it is relaxed in the potential field. However, when these fields are averaged over the whole protein or over individual residues the structure becomes more isotropic and the stressed regions vanish. Using these averaged tensors, we calculated bulk properties such as Young's modulus and the Lamé constants and they agreed with previously reported values.

Calculation of Protein Conformation by the Build-up Procedure. Application to Bovine Pancreatic Trypsin Inhibitor Using Limited Simulated Nuclear Magnetic Resonance Data

Low-energy conformations of a set of tetrapeptides derived from the small protein bovine pancreatic trypsin inhibitor (BPTI) were generated by a build-up procedure from the low-energy conformations of single amino acid residues. At each stage, various-size fragments were built up from all combinations of smaller ones, the total energies were then minimized, and the low-energy conformations were retained for the next stage. The energies of the tetrapeptides were re-ordered by including the effects of hydration. No information other than the amino acid sequence was used to obtain the low-energy conformations of the hydrated tetrapeptides. The latter were then supplemented with a limited set of simulated NMR distance information, derived from the X-ray structure of BPTI, to provide a basis for building the rest of the whole protein molecule by the same procedure. A total of 189 upper bounds, plus 12 pairs of upper and lower bounds pertaining to the location of the three disulfide bonds in this molecule, were used. Four sets of conformations of the entire molecule were generated by utilizing different combinations of smaller fragments. It was possible to obtain low-energy conformations with small rms deviations, 1.1 to 1.4 A for the alpha-carbons, from the structure derived by X-ray diffraction. The average deviations of the backbone dihedral angles were also low, viz. 23 degrees to 26 degrees.

Estimation of intramolecular distance distributions in bovine pancreatic trypsin inhibitor by site-specific labeling and nonradiative excitation energy-transfer measurements

A series of four bovine pancreatic trypsin inhibitor (BPTI) derivatives, site specifically labeled by (2-methoxy-1-naphthyl)methyl (MNA) at the N-terminal amino group and by [7-(dimethylamino)-coumarin-4-yl]acetyl (DA-coum) at one of the four epsilon-amino groups, was prepared. The four derivatives, N alpha-MNA-Arg1-N epsilon-DA-coum-Lysn-BPTI [(1-n)BPTI] (n = 15, 26, 41, and 46), were purified by affinity chromatography and high-performance liquid chromatography (HPLC). The homogeneity of each derivative and its site of labeling were characterized by HPLC tryptic peptide mapping. Nonradiative energy transfer from MNA (donor) to DA-coum (acceptor) was measured by monitoring donor emission and acceptor excitation spectra. Transfer efficiencies between 45% and 85% were observed. The fluorescence decay of MNA in MNA-BPTI, a derivative labeled by a donor without an acceptor, is monoexponential, with a lifetime of 6.8 +/- 0.15 ns. The decay kinetics of MNA fluorescence measured for derivatives labeled both by donor and acceptor showed a small deviation from monoexponential decay with shorter average lifetimes. Analysis of the experimental decay curves yielded the detailed intramolecular distance distribution functions for each pair of labeled sites. The averages of the calculated distance distribution functions are close to the values expected from the known structure of BPTI in the crystalline state. The derivatives thus obtained are suitable for investigation of conformational transitions of the labeled protein and for monitoring localized changes such as those involved in the folding or unfolding transitions.

Laser photo-CIDNP as a surface probe for proteins in solution.

APPLICATIONS of chemically induced dynamic nuclear polarisation (CIDNP) have been limited so far to reactions of small organic molecules1,2. We report here the observation of CIDNP in the 360-MHz NMR spectrum of a protein. The effect is generated by a cyclic reaction of a photo-excited dye with certain amino acid residues that are accessible to the dye. Of the common amino acids polarisation has been observed in tyrosine, histidine and tryptophan. The method distinguishes between exposed and buried groups and thus yields valuable information on the solution structure of proteins. Here we discuss the CIDNP observed in tyrosine residues of bovine pancreatic trypsin inhibitor (BPTI). The three-dimensional structure of BPTI (molecular weight 6,500) has been studied extensively by X-ray crystallography3,4 and by NMR5,6.