Bovine Pancreatic Trypsin Inhibitor Molecules Research Articles

PH-dependent oligomerization of BPTI in undersaturated and supersaturated solutions studied by dynamic light scattering

Bovine pancreatic trypsin inhibitor (BPTI), a small protein molecule, is reported to form several types of oligomers in solution including a decamer. We investigate in this work the relation between the oligomerization and crystallization. The oligomerization and interaction between BPTI molecules in NaCl and NaH 2PO 4/K 2HPO 4 solutions were investigated by dynamic light scattering (DLS). In the NaCl solutions, we found that strong electrostatic interaction among BPTI and chloride ions led to the decamer formation only at an acidic pH, whereas the protein solutions were monomeric at a neutral or basic pH, where the electrostatic interaction seemed to be unimportant. The single crystals with a hexagonal truncated bipyramidal shape grew only in the decameric solutions at an acidic pH in the presence of NaCl. On the other hand, the electrostatic interaction was weak in the presence of NaH 2PO 4/K 2HPO 4 at all the pH values investigated, and the solutions were always monomeric. In contrast with the NaCl solutions, single crystals with a rod-like shape appeared at a neutral and basic pH in the NaH 2PO 4/K 2HPO 4 solutions. These results suggest that the crystallization mechanism in the presence of NaCl, where electrostatic interactions seem to drive crystallization, is totally different from that in the presence of NaH 2PO 4/K 2HPO 4 where another interaction such as hydrophobic interaction or hydrogen bonding might cause crystallization.

Protein unfolding during reversed-phase chromatography: I. Effect of surface properties and duration of adsorption.

Residue-level features of bovine pancreatic trypsin inhibitor (BPTI) unfolding on reversed-phase chromatography (RPC) surfaces were investigated using hydrogen-deuterium exchange labeling and NMR. A set of silica-based RPC surfaces was used to examine the influence of alkyl chain length and media pore size on adsorbed BPTI conformation. In all cases there was substantial unfolding in the adsorbed state; however, residual protection from exchange was consistently observed. Particle pore size did not influence conformation substantially for C4-alkyl modified silica; however, 120 A pore C18 media produced more hydrogen exchange than any other surface examined. In this case, the radius of curvature inside the pore approaches the size of the BPTI molecule. Generally, the pattern of hydrogen exchange protection was uniform; however, the beta-sheet region was selectively protected on the large-pore C18 media. The beta-sheet region forms a hydrophobic core that forms early when BPTI folds in solution. This suggests that partially unfolded states possessing a native-like structure play an important role in adsorption and elution in RPC. Finally, increased contact time with the surface before elution fostered unfolding and altered chromatographic behavior considerably.

Comparison of Solubility and Interactions of Aprotinin (BPTI) solutions in H2O and D2O

Abstract Small-angle neutron scattering experiments are often performed with proteins solubilized in heavy water because of the large difference in neutron scattering properties of protons and deuterons. In order to characterize the effect of D 2 O on physico-chemical properties of protein solutions, we investigated the effect of D 2 O on the phase diagram and the interactions of bovine pancreatic trypsin inhibitor (BPTI) in solution. We measured the solubility in D 2 O of BPTI solutions in the presence of NaCl (reverse solubility) and KSCN (direct solubility) and compared with the values measured by Lafont et al. in H 2 O under the same conditions [Lafont et al., J. Crystal Growth 173 (1997) 132]. In the two salts, we found that BPTI solubility in D 2 O is significantly lower than in H 2 O. The curves representing the solubility of BPTI in KSCN are shifted by 7.2°C between light and heavy water, a shift obtained previously with lysozyme and representing the difference in the temperature of maximum density of both types of water [Gripon et al., J. Crystal Growth 177 (1997) 238; 178 (1997) 575]. In the case of BPTI in NaCl, we did not find this relationship between the solubility in H 2 O and D 2 O. We found, by dynamic light scattering, that BPTI attractive intermolecular interactions in the presence of NaCl in D 2 O are significantly stronger than in H 2 O. We investigated the association of BPTI molecules in crystallization conditions in the presence of NaCl in H 2 O and D 2 O by small-angle X-ray and neutrons scattering, respectively. In the presence of heavy water, the transition monomer–multimer is observed at about 2 mg/ml of BPTI in 1 M NaCl whereas in light water and in 1.4 M NaCl solution this transition is observed at about 15 mg/ml. These results clearly showed that BPTI in crystallization conditions is a multimer and confirm the importance of the isotopic nature of water in the crystallization of proteins. The replacement of H 2 O by D 2 O decreases the solubility and increases the attractive intermolecular interactions.

Substitutions at the P1′ position in BPTI strongly affect the association energy with serine proteinases

The role of the S1′ subsite in trypsin, chymotrypsin and plasmin has been examined by measuring the association with seven different mutants of bovine pancreatic trypsin inhibitor (BPTI); the mutants contain Gly, Ala, Ser, Val, Leu, Arg, and Trp at the P′1 position of the reactive site. The effects of substitutions at the P′1 position on the association constants are very large, comprising seven orders of magnitude for trypsin and plasmin, and over five orders for chymotrypsin. All mutants showed a decrease of the association constant to the three proteinases in the same order: Ala > Gly > Ser > Arg > Val > Leu > Trp. Calorimetric and circular dichroism methods showed that none of the P1′ substitutions, except the P1′-Val mutant, lead to destabilisation of the binding loop conformation. The X-ray structure of the complex formed between bovine β-trypsin and P′1-Leu BPTI showed that the P′1-Leu sterically conflicts with the side-chain of P3′-Ile, which thereby is forced to rotate approximately 90°. Ile18 (P3′) in its new orientation, in turn interacts with the Tyr39 side-chain of trypsin. Introduction of a large side-chain at the P1′ position apparently leads to a cascade of small alterations of the trypsin-BPTI interface that seem to destabilise the complex by it adopting a less optimized packing and by tilting the BPTI molecule up to 15° compared to the native trypsin-BPTI complex.

Comparison of solubility and interactions of aprotinin (BPTI) solutions in H 2O and D 2O

Small-angle neutron scattering experiments are often performed with proteins solubilized in heavy water because of the large difference in neutron scattering properties of protons and deuterons. In order to characterize the effect of D 2O on physico-chemical properties of protein solutions, we investigated the effect of D 2O on the phase diagram and the interactions of bovine pancreatic trypsin inhibitor (BPTI) in solution. We measured the solubility in D 2O of BPTI solutions in the presence of NaCl (reverse solubility) and KSCN (direct solubility) and compared with the values measured by Lafont et al. in H 2O under the same conditions [Lafont et al., J. Crystal Growth 173 (1997) 132]. In the two salts, we found that BPTI solubility in D 2O is significantly lower than in H 2O. The curves representing the solubility of BPTI in KSCN are shifted by 7.2°C between light and heavy water, a shift obtained previously with lysozyme and representing the difference in the temperature of maximum density of both types of water [Gripon et al., J. Crystal Growth 177 (1997) 238; 178 (1997) 575]. In the case of BPTI in NaCl, we did not find this relationship between the solubility in H 2O and D 2O. We found, by dynamic light scattering, that BPTI attractive intermolecular interactions in the presence of NaCl in D 2O are significantly stronger than in H 2O. We investigated the association of BPTI molecules in crystallization conditions in the presence of NaCl in H 2O and D 2O by small-angle X-ray and neutrons scattering, respectively. In the presence of heavy water, the transition monomer–multimer is observed at about 2 mg/ml of BPTI in 1 M NaCl whereas in light water and in 1.4 M NaCl solution this transition is observed at about 15 mg/ml. These results clearly showed that BPTI in crystallization conditions is a multimer and confirm the importance of the isotopic nature of water in the crystallization of proteins. The replacement of H 2O by D 2O decreases the solubility and increases the attractive intermolecular interactions.

The BPTI decamer observed in acidic pH crystal forms pre-exists as a stable species in solution

Bovine pancreatic trypsin inhibitor (BPTI) crystallizes under acidic pH conditions in the presence of thiocyanate, chloride and sulfate ions, yielding three different polymorphs in P2 1, P6 422 and P6 322 space groups, respectively. In all three crystal forms, the same decamer is found in the packing (ten BPTI molecules organized through two perpendicular 2-fold and 5-fold axes as a well-defined and compact object) in contrast to the monomeric crystal forms observed at basic pH conditions. The crystallization of BPTI under acidic conditions (pH 4.5) was investigated by small angle X-ray scattering with both under- and supersaturated BPTI solutions. Data showed the oligomerization of BPTI molecules under all investigated conditions. Accordingly, various mixtures of discrete oligomers ( n=1 to 10) were considered. Calculated scattering curves were obtained using models based on the crystallographic structures, and the experimental patterns were analyzed as a linear combination of the model curves using a non-linear curve fitting procedure. The results, confirmed by gel filtration experiments, unambiguously demonstrate the co-existence of two different BPTI particles in solution: a monomer and a decamer, with no evidence of any other intermediates. Moreover, using both approaches, the fraction of decamers was found to increase with increasing salt concentration, even beyond the solubility curve. We therefore propose that at acidic pH, BPTI crystallizes following a two step process: decamers are first built in under- and supersaturated solutions, upon which crystal growth proceeds by decamer stacking. Indeed, those BPTI crystals should best be described as “BPTI decamer” crystals.

Diffusion in supersaturated solutions: Application to the case of supersaturated protein solutions

The derivation of solute diffusion coefficient from the quasiequilibrium probability density function of the underlying stochastic quantity-characteristic size of subcritical solute clusters, was carried out within the classical formalism of metastable state relaxation (nucleation). It has been demonstrated that (a) the solute diffusion coefficient is necessarily concentration dependent; (b) the concentration dependent solute diffusivity is the function of only two parameters, solute diffusivity at saturation and total surface energy of a solute molecule. This energy, in principle, can be also a function of solute concentration for supersaturated solutions such as protein supersaturated solutions. In addition to that it has been demonstrated for supersaturated solutions of the proteins such as lysozyme and Bovine Pancreatic Trypsin Inhibitor (BPTI) solutions that (a) the total surface energy of lysozyme and BPTI molecules in the metastable state is concentration dependent. (b) The solute diffusivity as a function of solute concentration, observed in supersaturated protein solutions such as lysozyme and BPTI, can be accurately reproduced (with error less than 1.2%) on the base of classical theory of the metastable state relaxation, given that the surface energy of the protein molecules is concentration dependent.

High-resolution structure of bovine pancreatic trypsin inhibitor with altered binding loop sequence

A mutant of bovine pancreatic trypsin inhibitor (BPTI) has been constructed and expressed in Escherichia coli in order to probe the kinetic and structural consequences of truncating the binding loop residues to alanine. In addition to two such mutations (Thr11Ala and Pro13Ala), it has a conservative Lys15Arg substitution at position P 1 and an unrelated Met52Leu change. In spite of the binding loop modification, the affinity for trypsin is only 30 times lower than that of the wild-type protein. At pH 7.5 the protein can be crystallized on the time-scale of hours, yielding very stable crystals of a new (tetragonal) form of BPTI. Conventional source X-ray data collected to 1.4 Å at room temperature allowed anisotropic structure refinement characterized by R = 0.1048. The structure reveals all 58 residues, including the complete C terminus, which is in a salt-bridge contact with the N terminus. The Cys14-Cys38 disulfide bridge is observed in two distinct chiralities. This bridge, together with an internal water molecule, contributes to the stabilization of the binding loop. The Ala mutations have only an insignificant and localized effect on the binding loop, which retains its wild-type conformation (maximum deviation of loop C α atoms of 0.7 Å at Ala13). Four (instead of the typical three) additional water molecules are buried in an internal cleft and connected to the surface via a sulfate anion. Three more SO 4 2− anions are seen in the electron density, one of them located on a 2-fold axis. It participates in the formation of a dimeric structure between symmetry-related BPTI molecules, in which electrostatic and hydrogen bonding interactions resulting from the mutated Lys15Arg substitution are of central importance. This dimeric interaction involves direct recognition loop-recognition loop contacts, part of which are hydrophobic interactions of the patches created by the alanine mutations. Another 2-fold symmetric interaction between the BPTI molecules involves the formation of an antiparallel intermolecular β-sheet that, together with the adjacent intramolecular β-hairpin loops, creates a four-stranded structure.

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.

Aberrant processing of wild-type and mutant bovine pancreatic trypsin inhibitor secreted by Aspergillus niger

Bovine pancreatic trypsin inhibitor (BPTI) was secreted by Aspergillus niger at yields of up to 23 mg l −1 using a protein fusion strategy. BPTI was linked to part of the fungal glucoamylase protein (GAM) with a dibasic amino acid (KEX2) processing site at the fusion junction. Electrospray ionisation mass spectrometry and N-terminal protein sequencing revealed that, although biologically active in vitro, the purified products from a number of independent transformants consisted of a mixture of BPTI molecules differing at the N-terminus. Approximately 35–60% of this mixture was processed correctly. Aberrant processing of the GAM–BPTI fusion protein by the A. niger KEX2-like endoprotease was the most likely cause of this variation although the involvement of other fungal endoproteases could not be ruled out. In vitro studies have highlighted a weak interaction between BPTI and the Saccharomyces cerevisiae KEX2 endoprotease, suggesting that BPTI is not a potent inhibitor of KEX2p. A small proportion of the recombinant BPTI (10%) showed `nicking' of the K15–A16 bond, indicating an interaction with a fungal trypsin-like enzyme. Mutant BPTI homologues designed to have anti-elastase activity, BPTI(K15V), BPTI(K15V,P13I) and BPTI(K15V,G12A), have also been expressed and secreted by A. niger. They also showed a similar spectrum of aberrant N-terminal processing but no `nicking' of the K15–V16 bond was observed. Comparison of A. niger with other expression systems showed that it is an effective system for producing BPTI and its homologues, although not all molecules were correctly processed. This variation in processing efficiency may be useful in understanding the important determinants of protein processing in this fungus.

"Designing out" disulfide bonds: thermodynamic properties of 30-51 cystine substitution mutants of bovine pancreatic trypsin inhibitor.

We have used a combination of spectroscopic and calorimetric techniques to assess the thermodynamic and extrathermodynamic consequences of paired amino acid substitutions at positions 30 and 51 in bovine pancreatic trypsin inhibitor (BPTI). Correctly folded, wild type BPTI contains a disulfide at the 30-51 positions, with the nonbackbone atoms of this cystine being relatively solvent inaccessible. Mutants missing this buried 30-51 disulfide adopt a conformation very similar to that of the native state of wild type BPTI (Eigenbrot et al., 1990, 1992), although they are severely destabilized relative to the wild type molecule (Hurle et al., 1990). We have conducted a systematic effort to find the energetically most favorable substitution for this buried 30-51 disulfide in BPTI. To this end, we have studied and characterized the thermally induced and guanidine hydrochloride-induced denaturation transitions for a family of mutants in which the amino acid residue(s) at positions 30 and/or 51 have been systematically altered. Specifically, we studied the unfolding transitions of the following series of residue 30/residue 51 paired substitution mutants: C30A/C51A, C30V/C51A, C30G/C51A, C30S/C51A, C30T/C51A, C30A/C51S, C30S/C51S, and C30G/C51M. For this series of mutants, comparisons between the relative stabilization free energies, derived from analysis of the denaturation profiles, allow us to reach the following conclusions: (a) side chains containing polar moieties (Ser and Thr) are destabilizing, with this effect being position dependent (i.e., a serine substitution is more destabilizing at position 51 than at position 30); (b) the destabilizing effects of two serine substitutions are approximately additive, suggesting that side chain-side chain hydrogen bonds between the two serine hydroxyl groups probably are weak or nonexistent; (c) the thermodynamic impact of a Gly30 substitution is consistent with a glycine-induced increase in the configurational entropy of the unfolded state; (d) the C30G/C51M mutant is highly destabilized relative to the C30A/C51A mutant despite the fact that, based on considerations of hydrophobicity and steric fit, substitution of a buried disulfide by Gly30 and Met51 would be expected to be optimal. Methionine may be particularly ill-suited as a buried disulfide substitute due to the large loss of side chain conformational entropy it undergoes during the transition from the unfolded to the native state. In the aggregate, our data provide insight into the residue-, position-, and context-dependent consequences on protein stability of "designing out" the buried 30-51 disulfide bond in the BPTI molecule. These data also suggest that a previously unrecognized component of disulfide bridge stabilization of proteins is the relatively minor penalty in side chain conformational entropy incurred by cystine residues during folding due to their severely restricted rotation even in the unfolded state.

A third native one-disulphide intermediate in the folding of bovine pancreatic trypsin inhibitor.

A previously unidentified intermediate has been detected in the early stages of the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI). The intermediate contains one disulphide bond between residues 14 and 38 and is denoted [14-38]. The 14-38 disulphide bond is also found in native BPTI. Although the other native one-disulphide intermediates, [30-51] and [5-55], are thermodynamically more stable, [14-38] can be populated substantially at the early stages of BPTI folding. Moreover, initial characterization of the kinetic properties of this intermediate strongly suggest that a substantial fraction of BPTI molecules fold by way of the [14-38] intermediate. Our results emphasize the importance of native-like tendencies in protein folding.

The pro region of BPTI facilitates folding

The in vitro folding pathway of bovine pancreatic trypsin inhibitor (BPTI) has been described previously in terms of the disulfide-bonded intermediates that accumulate during folding of the protein. Folding is slow, occurring in hours at pH 7.3, 25°C. In addition, approximately half of the BPTI molecules become trapped as a dead-end, native-like intermediate. In vivo, BPTI is synthesized as a precursor protein that includes a 13 residue amino-terminal pro region. This pro region contains a cysteine residue. We find that, in vitro, both the rate of formation and the yield of properly folded BPTI are increased substantially in a recombinant model of pro-BPTI. The cysteine residue is necessary for this effect. Moreover, a single cysteine residue, tethered to the carboxy-terminal end of BPTI with a flexible linker of repeating Ser-Gly-Gly residues, is sufficient to assist in disulfide formation. Thus, the pro region appears to facilitate folding by providing a tethered, solvent-accessible, intramolecular thiol-disulfide reagent.

Intermolecular interaction between bovine pancreatic trypsin inhibitor molecules probed by Brownian dynamics simulation

Intermolecular interaction between bovine pancreatic trypsin inhibitor molecules probed by Brownian dynamics simulation

Intracellular expression of BPTI fusion proteins and single column cleavage/affinity purification by chymotrypsin

The novel and efficient expression system described here produces formerly poorly expressed, proteolytically unstable mutants of bovine pancreatic trypsin inhibitor (BPTI). A new, single column method for the cleavage of a recombinant fusion to BPTI and affinity purification of the BPTI moiety by immobilized chymotrypsin is an integral part of the system. Wild-type and mutant BPTI molecules are expressed in Escherichia coli as fusion proteins forming intracellular inclusion bodies. Transcription initiation is under the control of the E. coli trp promoter. The expressed protein is tripartite fusion comprising (i) a portion of the TrpLE leader peptide, (ii) a synthetic IgG binding domain derived from protein A and (iii) the BPTI variant. Solubilization of the inclusion bodies and refolding of the fusion proteins in a thiol-disulfide shuffling system yields correctly folded inhibitor molecules. In the single column purification and cleavage procedure, immobilized chymotrypsin cleaves the refolded fusion protein and releases affinity purified active BPTI mutants with correct N-termini. Mutant BPTI molecules which do not fold into active inhibitors are also stably expressed in inclusion bodies but cannot be purified by this method. Unlike previously described secretion systems for the production of BPTI, expression levels in this system appear to be independent of both the mutation in the BPTI gene and the activity of the expressed protein. Mutants poorly expressed in secretion systems can now be produced in sufficient quantities for protein folding studies and structural analysis using X-ray crystallography and NMR spectroscopy.

On the relevance of non-random polypeptide conformations for protein folding

The possibility that any non-random conformation in reduced bovine pancreatic trypsin inhibitor (BPTI) and ribonuclease A might be significant for folding has been considered, using the experimental data available on forming the first disulphide bond in each. It is a thermodynamic necessity that whatever conformation stabilises a particular disulphide bond be stabilised to the same extent by the presence of the disulphide. The stabilising effects of disulphides are known approximately, so the stability of any non-random conformation found in a one-disulphide intermediate can be estimated in the absence of the disulphide bond. The non-random conformation in the BPTI intermediates is sufficiently labile to indicate that it would be expected to be present in no more than 5% of the reduced BPTI molecules. There is much less non-random conformation apparent in ribonuclease A. Whatever conformations are represented in the bulk of these two reduced proteins cannot favour disulphide formation and further productive folding.

Variable-Target-Function and Build-up Procedures for the Calculation of Protein Conformation. Application to Bovine Pancreatic Trypsin Inhibitor Using Limited Simulated Nuclear Magnetic Resonance Data

An implementation of the variable-target-function procedure, first introduced by Braun and Go [W. Braun and N. Go, J. Mol. Biol. 186, 611-626 (1985)], has been used to generate conformations of the small protein bovine pancreatic trypsin inhibitor (BPTI), given a limited set of simulated data that could be obtained by nuclear magnetic resonance (NMR) techniques. A hybrid strategy was also used to calculate conformations of BPTI, given the same information. In the hybrid strategy, low-energy structures of medium-size fragments (decapeptides) of BPTI were generated using the variable-target-function method, followed by restrained energy optimization. The low-energy conformations were used as a basis to build up the complete fifty-eight-residue BPTI molecule. By using the variable-target-function approach, in which energy considerations were not introduced until full conformations of the entire BPTI molecule had been generated, it was not possible to obtain calculated structures with rms deviations from the X-ray conformation of less than 1.6 A for the alpha-carbons. On the other hand, with the hybrid strategy, which involved the consideration of realistic energy terms in the early stages of the calculations, it was possible to calculate low-energy conformations of BPTI with rms deviations from the X-ray structure of 1.06 to 1.50 A for the alpha-carbons. When the rms deviations were computed along the amino acid sequence, it was found that there was a good correlation between deviations among the calculated structures and deviations from the X-ray structure.

Production of native, correctly folded bovine pancreatic trypsin inhibitor by Escherichia coli.

A gene for bovine pancreatic trypsin inhibitor (BPTI) was fused to the coding sequence for the Escherichia coli alkaline phosphatase signal peptide and expressed in E. coli under the control of the alkaline phosphatase promoter. When induced in phosphate-depleted medium such cells produced a trypsin inhibitor that was indistinguishable from native, properly folded BPTI. In particular, the BPTI produced by E. coli had three disulfide bonds that appeared to be identical to those found in native BPTI, as assayed by sensitivity to iodoacetate, dithiothreitol, and urea. This expression/secretion system will make possible the production of variant BPTI molecules, thus allowing the perturbing effects of amino acid substitutions on BPTI folding, structure, and function to be assessed.

Prediction of a low-frequency mode in bovine pancreatic trypsin inhibitor molecule

One of the frontiers today in molecular biology is to measure, identify and go further to predict the low-frequency internal motion of biological macromolecules, which is crucially important for understanding the dynamic mechanism of various biological functions occurring in such molecules. Based on the theory of continuity model developed recently for dealing with the internal low-frequency motion of a biological macromolecule, it is predicted that the low-frequency phonons with wave number of about 23 cm −1 might be excited in BPTI molecule.

Efficient Monte Carlo method for simulation of fluctuating conformations of native proteins.

AbstractA powerful Monte Carlo method is described to simulate thermal conformational fluctuations in native proteins by using an empirical conformational energy function in which bond lengths and bond angles are kept fixed and only dihedral angles are independent variables. In this method, collective variables corresponding to eigenvectors of the second‐derivative matrix of the energy function at its minimum point are scaled according to corresponding eigenvalues in such a way that the energy function in terms of the scaled collective variables is isotropic at the minimum point. Simulation is carried out with an isotropic step size in the space of these scaled collective variables. This simulation method is applied to a small protein, bovine pancreatic trypsin inhibitor (BPTI), and its model harmonic system defined by a quadratic energy function with the same second‐derivative matrix as that of BPTI at its minimum point. Efficiency of the simulation method with an isotropic step size in the space of the scaled collective variables is found to be about 500–50 times greater than the conventional method with with an isotropic step in the space of the usual nonscaled variables. One step of this new method generates conformational changes that occur in the real‐time range of 0.05 ps. In a record of 5 × 105 step simulation, the BPTI molecule is observed to migrate beyond a single minimum‐energy region.