NMR structures of two variants of bovine pancreatic trypsin inhibitor (BPTI) reveal unexpected influence of mutations on protein structure and stability

Here we determined NMR solution structures of two mutants of bovine pancreatic trypsin inhibitor (BPTI) to reveal structural reasons of their decreased thermodynamic stability. A point mutation, A16V, in the solvent-exposed loop destabilizes the protein by 20 degrees C, in contrast to marginal destabilization observed for G, S, R, L or W mutants. In the second mutant introduction of eight alanine residues at proteinase-contacting sites (residues 11, 13, 17, 18, 19, 34, 37 and 39) provides a protein that denatures at a temperature about 30 degrees C higher than expected from additive behavior of individual mutations. In order to efficiently determine structures of these variants, we applied a procedure that allows us to share data between regions unaffected by mutation(s). NOAH/DYANA and CNS programs were used for a rapid assignment of NOESY cross-peaks, structure calculations and refinement. The solution structure of the A16V mutant reveals no conformational change within the molecule, but shows close contacts between V16, I18 and G36/G37. Thus, the observed 4.3kcal/mol decrease of stability results from a strained local conformation of these residues caused by introduction of a beta-branched Val side-chain. Contrary to the A16V mutation, introduction of eight alanine residues produces significant conformational changes, manifested in over a 9A shift of the Y35 side-chain. This structural rearrangement provides about 6kcal/mol non-additive stabilization energy, compared to the mutant in which G37 and R39 are not mutated to alanine residues.     

Thermodynamics of single peptide bond cleavage in bovine pancreatic trypsin inhibitor (BPTI)

A major goal of this paper was to estimate a dynamic range of equilibrium constant for the opening of a single peptide bond in a model protein, bovine pancreatic trypsin inhibitor (BPTI). Ten mutants of BPTI containing a single Xaa-->Met substitution introduced in different parts of the molecule were expressed in Escherichia coli. The mutants were folded, purified to homogeneity, and cleaved with cyanogen bromide to respective cleaved forms. Conformation of the intact mutants was similar to the wildtype, as judged from their circular dichroism spectra. Substantial conformational changes were observed on the chemical cleavage of three single peptide bonds--Met46-Ser, Met49-Cys, and Met53-Thr--located within the C-terminal helix. Cleavage of those peptide bonds caused a significant destabilization of the molecule, with a drop of the denaturation temperature by 56.4 degrees C to 68 degrees C at pH 4.3. Opening of the remaining seven peptide bonds was related to a 10.8 degrees C to 39.4 degrees C decrease in T(den). Free energies of the opening of 10 single peptide bonds in native mutants (Delta G(op,N)) were estimated from the thermodynamic cycle that links denaturation and cleavage free energies. To calculate those values, we assumed that the free energy of opening of a single peptide bond in the denatured state (Delta G(op,D)) was equal to -2.7 kcal/mole, as reported previously. Calculated Delta G(op,N) values in BPTI were in the range from 0.2 to 10 kcal/mole, which was equivalent to a >1 million-fold difference in equilibrium constants. The values of Delta G(op,N) were the largest for peptide bonds located in the C-terminal helix and significantly lower for peptide bonds in the beta-structure or loop regions. It appears that opening constants for single peptide bonds in various proteins span across 33 orders of magnitude. Typical equilibrium values for a single peptide bond opening in a protein containing secondary structure elements fall into negligibly low values, from 10(-3) to 10(-8), and are efficient to ensure stability against proteolysis.     

Reduced BPTI is collapsed. A pulsed field gradient NMR study of unfolded and partially folded bovine pancreatic trypsin inhibitor.

Pulsed field gradient NMR was used to measure the hydrodynamic behavior of unfolded variants of bovine pancreatic trypsin inhibitor (BPTI). The unfolded BPTI species studied were [R]Abu, at pH 4.5 and pH 2.5, and unfolded [14-38]Abu, at pH 2.5. These were prepared by chemical synthesis. [R]Abu is a model for reduced BPTI; all cysteine residues are replaced by alpha-amino-n-butyric acid (Abu). [14-38]Abu retains cysteines 14 and 38, which form a disulfide bond, while the other cysteine residues are replaced by Abu. In the PFG experiments, the diffusion coefficient is measured as a function of protein concentration, and the value of D degree -the diffusion coefficient extrapolated to infinite dilution-is determined. From D degree, a value of the hydrodynamic radius. Rh, is computed from the Stokes-Einstein relationship. At pH 4.5, [R]Abu has an Rh value significantly less than the value calculated for a random coil, while at pH 2.5 the experimental Rh value is the same as for a random coil. In view of the changes in NMR detected structure of [R]Abu at pH 4.5 versus pH 2.5 (Pan H, Barbar E, Barany G, Woodward C. 1995. Extensive non-random structure in reduced and unfolded bovine pancreatic trypsin inhibitor. Biochemistry 34:13974-13981), the collapse of reduced BPTI at pH 4.5 may be associated with the formation of non-native hydrophobic clusters of pairs of side chains one to three amino acids apart in sequence. The diffusion constant of [14-38]Abu was also measured at pH 4.5, where the protein is partially folded. An increase in hydrodynamic radius of partially folded [14-38]Abu, relative to native BPTI, is similar to the increase in radius of gyration measured for other proteins under "molten globule" conditions.     

Electrostatic effect of trypsin binding on the hydrogen exchange rate of bovine pancreatic trypsin inhibitor beta-sheet NH's

The changes of H-D exchange rates upon protein-protein interactions are generally interpreted as a result of the changes of the dynamic properties of the proteins. The effect of trypsin binding on the H-D exchange kinetics of some trypsin inhibitor amide H's was reported (Simon et al., 1984). In this paper the electrostatic potential originating from the trypsin molecule is calculated at the positions of the studied amide H's in the trypsin-trypsin inhibitor complex. We conclude that the observed decrease of the exchange rates is mainly due to the electrostatic field of the trypsin molecule.     

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.     

1H NMR studies at 360 Mhz of the methyl groups in native and chemically modified basic pancreatic trypsin inhibitor (BPTI)

In the ¹H NMR spectra obtained at 360 MHz after digital resolution enhancement, the multiplet resonances of the methyl groups in the basic pancreatic trypsin inhibitor (BPTI) were resolved. With suitable double irradiation techniques the individual methyl resonances were assigned to the different types of aliphatic amino acid residues. Furthermore, from pH titration and comparison of the native protein with chemically modified BPTI, the resonance lines of Ala 16 in the active site and Ala 58 at the C-terminus were identified. Potential applications of the resolved methyl resonances as natural NMR probes for studies of the molecular conformations are discussed.     

Amino-acid substitutions at the fully exposed P1 site of bovine pancreatic trypsin inhibitor affect its stability

It is widely accepted that solvent-exposed sites in proteins play only a negligible role in determining protein energetics. In this paper we show that amino acid substitutions at the fully exposed Lys15 in bovine pancreatic trypsin inhibitor (BPTI) influenced the CD- and DSC-monitored stability: The T(den) difference between the least (P1 Trp) and the most stable (P1 His) mutant is 11.2 degrees C at pH 2.0. The DeltaH(den) versus T(den) plot for all the variants at three pH values (2.0, 2.5, 3.0) is linear (DeltaC(p,den) = 0.41 kcal* mole(-1) * K(-1); 1 cal = 4.18 J) leading to a DeltaG(den) difference of 2.1 kcal*mole(-1). Thermal denaturation of the variants monitored by CD signal at pH 2.0 in the presence of 6 M GdmCl again showed differences in their stability, albeit somewhat smaller (DeltaT(den) =7.1 degrees C). Selective reduction of the Cys14-Cys 38 disulfide bond, which is located in the vicinity of the P1 position did not eliminate the stability differences. A correlation analysis of the P1 stability with different properties of amino acids suggests that two mechanisms may be responsible for the observed stability differences: the reverse hydrophobic effect and amino acid propensities to occur in nonoptimal dihedral angles adopted by the P1 position. The former effect operates at the denatured state level and causes a drop in protein stability for hydrophobic side chains, due to their decreased exposure upon denaturation. The latter factor influences the native state energetics and results from intrinsic properties of amino acids in a way similar to those observed for secondary structure propensities. In conclusion, our results suggest that the protein-stability-derived secondary structure propensity scales should be taken with more caution.     

Comparison of solution structures of mutant bovine pancreatic trypsin inhibitor proteins using two-dimensional nuclear magnetic resonance.

Structural perturbations due to a series of mutations at the 30-51 disulfide bond of bovine pancreatic trypsin inhibitor have been explored using NMR. The mutants replaced cysteines at positions 30 and 51 by alanine at position 51 and alanine, threonine, or valine at position 30. Chemical shift changes occur in residues proximate to the site of mutation. NOE assignments were made using an automated procedure, NASIGN, which used information from the wild-type crystal structure. Intensity information was utilized by a distance geometry algorithm, VEMBED, to generate a series of structures for each protein. Statistical analyses of these structures indicated larger averaged structural perturbations than would be expected from crystallographic and other information. Constrained molecular dynamics refinement using AMBER at 900 K was useful in eliminating structural movements that were not a necessary consequence of the NMR data. In most cases, statistically significant movements are shown to be those greater than approximately 1 A. Such movements do not appear to occur between wild type and A30A51, a result confirmed by crystallography (Eigenbrot, C., Randal, M., & Kossiakoff, A.A., 1990, Protein Eng. 3, 591-598). Structural alterations in the T30A51 or V30A51 mutant proteins near the limits of detection occur in the beta-loop (residues 25-28) or C-terminal alpha-helix, respectively.     

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.     

Influence of thermal motion on 1H chemical shifts in proteins: the case of bovine pancreatic trypsin inhibitor.

The possible influence of thermal motion on 1H chemical shifts is discussed for a small stable protein, the bovine pancreatic Kunitz trypsin inhibitor (BPTI). The thermal effects on the aromatic side chains and on the backbone are treated separately. The thermal motion of the aromatic side chains is accounted for in terms of their rotation around the C(alpha)-C(beta) bond and the motion of each individual proton is interpreted as a ratio between the amount of ordered and quite disordered states. The influence of hydrogen bonds is introduced as an extra contribution to the chemical shifts of the bonded proton. Their contribution to the chemical shifts resulting from the polarization of the peptide bond is investigated, as is their influence on local flexibility. Finally, the relative importance of each contribution to the chemical shift information is compared.     

The structure of denatured bovine pancreatic trypsin inhibitor (BPTI)

In the presence of denaturant and thiol initiator, the native bovine pancreatic trypsin inhibitor (BPTI) denatures by shuffling its native disulfide bonds and converts to a mixture of scrambled isomers. The extent of denaturation is evaluated by the relative yields of the scrambled and native species of BPTI. BPTI is an exceedingly stable molecule and can be effectively denatured only by guanidine thiocyanate (GdmSCN) at concentrations higher than 3-4 M. The denatured BPTI consists of at least eight fractions of scrambled isomers. Their composition varies under increasing concentrations of GdmSCN. In the presence of 6 M GdmSCN, the most predominant fraction of scrambled BPTI accounts for 56% of the total structure of denatured BPTI. Structural analysis reveals that this predominant fraction contains the bead-form isomer of scrambled BPTI, bridged by three pairs of neighboring cysteines, Cys5-Cys14, Cys30-Cys38 and Cys51-Cys55. The extreme conformational stability of BPTI has important implications in its distinctive folding pathway.     

Crystal structures of five bovine chymotrypsin complexes with P1 BPTI variants

The bovine chymotrypsin-bovine pancreatic trypsin inhibitor (BPTI) interaction belongs to extensively studied models of protein-protein recognition. The accommodation of the inhibitor P1 residue in the S1 binding site of the enzyme forms the hot spot of this interaction. Mutations introduced at the P1 position of BPTI result in a more than five orders of magnitude difference of the association constant values with the protease. To elucidate the structural aspects of the discrimination between different P1 residues, crystal structures of five bovine chymotrypsin-P1 BPTI variant complexes have been determined at pH 7.8 to a resolution below 2 A. The set includes polar (Thr), ionizable (Glu, His), medium-sized aliphatic (Met) and large aromatic (Trp) P1 residues and complements our earlier studies of the interaction of different P1 side-chains with the S1 pocket of chymotrypsin. The structures have been compared to the complexes of proteases with similar and dissimilar P1 preferences, including Streptomyces griseus proteases B and E, human neutrophil elastase, crab collagenase, bovine trypsin and human thrombin. The S1 sites of these enzymes share a common general shape of significant rigidity. Large and branched P1 residues adapt in their complexes similar conformations regardless of the polarity and size differences between their S1 pockets. Conversely, long and flexible residues such as P1 Met are present in the disordered form and display a conformational diversity despite similar inhibitory properties with respect to most enzymes studied. Thus, the S1 specificity profiles of the serine proteases appear to result from the precise complementarity of the P1-S1 interface and minor conformational adjustments occurring upon the inhibitor binding.     

Structural consequences of accommodation of four non-cognate amino acid residues in the S1 pocket of bovine trypsin and chymotrypsin

Crystal structures of P1 Gly, Val, Leu and Phe bovine pancreatic trypsin inhibitor (BPTI) variants in complex with two serine proteinases, bovine trypsin and chymotrypsin, have been determined. The association constants for the four mutants with the two enzymes show that the enlargement of the volume of the P1 residue is accompanied by an increase of the binding energy, which is more pronounced for bovine chymotrypsin. Since the conformation of the P1 side-chains in the two S1 pockets is very similar, we suggest that the difference in DeltaG values between the enzymes must arise from the more polar environment of the S1 site of trypsin. This results mainly from the substitutions of Met192 and Ser189 observed in chymotrypsin with Gln192 and Asp189 present in trypsin. The more polar interior of the S1 site of trypsin is reflected by a much higher order of the solvent network in the empty pocket of the enzyme, as is observed in the complexes of the two enzymes with the P1 Gly BPTI variant. The more optimal binding of the large hydrophobic P1 residues by chymotrypsin is also reflected by shrinkage of the S1 pocket upon the accommodation of the cognate residues of this enzyme. Conversely, the S1 pocket of trypsin expands upon binding of such side-chains, possibly to avoid interaction with the polar residues of the walls. Further differentiation between the two enzymes is achieved by small differences in the shape of the S1 sites, resulting in an unequal steric hindrance of some of the side-chains, as observed for the gamma-branched P1 Leu variant of BPTI, which is much more favored by bovine chymotrypsin than trypsin. Analysis of the discrimination of beta-branched residues by trypsin and chymotrypsin is based on the complexes with the P1 Val BPTI variant. Steric repulsion of the P1 Val residue by the walls of the S1 pocket of both enzymes prevents the P1 Val side-chain from adopting the most optimal chi1 value.     

Predicted three-dimensional structure of the protease inhibitor domain of the Alzheimer's disease beta-amyloid precursor

Alzheimer's disease is characterized by the deposition of amyloid beta-protein as plaques and tangles in the brains of its victims. The amyloid precursor can be expressed with or without the inclusion of a protease inhibitor domain, the potential role of which in amyloidogenesis has prompted the generation of a model of its three-dimensional structure based on the known structure of a related inhibitor. The model structure predicts that the mutated residues are almost entirely on the surface of the inhibitor domain, while conserved residues constitute the hydrophobic core. In addition, several pairs of structurally complementary, or concerted, mutations are seen. These structural features provide strong evidence for the validity of the modeled structure, and it is suggested that the presence of complementary mutations may be used as a criterion for evaluating protein structures built by homology, in addition to the (spatial) location of the mutations. The terminal residues delimiting the domain are among those furthest from the protease binding site and are in close proximity to one another, thus suggesting the ability of the domain to function as a structural cassette within the context of a larger protein. The electrostatic potentials of the inhibitor and of the related bovine pancreatic trypsin inhibitor reveal how two inhibitors with very different net charges can bind with approximately the same binding constant to trypsin and suggest a mutation of trypsin that might selectively enhance the binding of the amyloid inhibitor domain. The model provides a structural basis for understanding the functional roles of residues in the domain and for designing simpler molecules to test as pharmacologic agents for intervention in Alzheimer's disease.     

Binding of native and [homoserine lactone-52]-52,53-seco-bovine basic pancreatic trypsin inhibitor (kunitz inhibitor) to porcine pancreatic β-kallikrein-B and bovine α-chymtorpsin: Thermodynaic study

Values of the association equilibrium constant (K_a) for the binding of the native and of the cyanogen bromide-cleaved bovine basic pancreatic trypsin inhibitor (native BPTI and [Hse lactone-52]-52,53-seco-BPTI, respectively) to neuraminidase-treated porcine pancreatic β-Kallikrein-B (kallikrein) and bovine α-chymotrypsin (chymotrypsin) have been determined between pH4.0 and 9.0, and 20.0°C. Over the whole pH range explored, native BPTI and [Hse lactone-52]-52,53-seco-BPTI show the same affinity for kallikrein. On the other hand, the affinity of [se lactone-52]-52,53-seco-BPTI for chymotrypsin is high4er, around neutrality, than that found for native BPTI by about one order of magnitude, coverging in the acidic pH limb. The simplest mechanism accounting for the observed data implies that, on lowering the pH from 9.0 to 4.0 (i) the decrease in affinity for the binding of native BPTI to kalikrein and chymotrypsin, as well as for the association of [Hse lactone-52]-52,53-seco-BPTI to kalikrein, reflects the acidic pK shift, upon inhibitor association, of a single inozing group; and (ii) the decrease of K_a values for [Hse lactone-52]-52,53-seco-BPTI binding to chymotrypsin appears to be modulated by the acidic pK shift, upon inhibitor association, of two non-equivalent proton-binding residues. On the basis of the stereochemistry of the serine proteinase/inhibitor contact region(s), these data indicate that long-rang structural changes in [Hse lactone-52]-52,53-seco-BPTI are energetically linked to the chymotrypsin: inhibitor complex formation. This observation represents an important aspect for the mechanism of molecular recognition and regulation in BPTI.     

Effects of N-terminal extension peptides on the structure and stability of bovine pancreatic trypsin inhibitor studied by H n.m.r.

Four N-terminal extended species of the wild-type bovine pancreatic trypsin inhibitor (WT-BPTI), Arg-BPTI (1-BPTI), Met-Glu-Ala-Glu-BPTI (4-BPTI), Ser-Ile-Glu-Gly-Arg-BPTI (5-BPTI) and Gly-Ser-Ile-Glu-Gly-Arg-BPTI (6-BPTI) have been studied by 1H n.m.r. The overall structure of the protein is largely unaffected by the addition of extension peptides. pH titration effects on the C-terminal Ala 58 H beta chemical shift indicate that the structure of 1-BPTI at neutral pH is very similar to that of the WT protein, with a salt bridge between the main chain terminal charges. A salt bridge interaction is prevented by addition of the longer extension peptides. Temperature stabilities are measured by high temperature hydrogen isotope exchange and by microcalorimetry. The stability of 1-BPTI is equal to that of WT-BPTI. A slight decrease in stability is observed for longer extensions, following the order WT-BPTI = 1-BPTI < 5-BPTI = 6-BPTI < 4-BPTI. Small changes in chemical shift are observed for 30 invariant resonances in 4-, 5- and 6-BPTI and for a subset of this group in 1-BPTI. These protons are distributed over about half of the BPTI molecule. The size of the chemical shift changes for many resonances follow the same ranking as the temperature stability. The chemical shift effects are attributed to charge and dielectric effects from extension peptides that probably share a common orientation on the surface of BPTI.     

Interaction between the basic inhibitor of bovine pancreas and chymotrypsin and trypsin. Selective anthraniloylation of the maleylated inhibitor

The interaction between the maleylated basic pancreatic inhibitor, anthraniloylated on its lysine-15 residue, and chymotrypsin is studied by fluorescence intensity, fluorescence polarization, circular dichroism, circular polarization of fluorescence and sedimentation. These measurements show that the interaction takes place through the entrance of the anthraniloyl group into an asymmetric environment in which it is rigidly held. The dissociation constant of the complex is 2.5 × 10^?8m. The interaction between the modified inhibitor and trypsin takes place through a site which is not the anthraniloylated lysine-15 side-chain, yet not far from it.     

Crevice-forming mutants of bovine pancreatic trypsin inhibitor: stability changes and new hydrophobic surface.

Four mutants of bovine pancreatic trypsin inhibitor (BPTI) with replacements in the rigid core result in the creation of deep crevices on the surface of the protein. Other than crevices at the site of the mutation, few other differences are observed in the crystal structures of wild-type BPTI and the mutants F22A, Y23A, N43G, and F45A. These mutants are highly destabilized relative to wild type (WT). The differences between WT and mutants in the free energy change associated with cooperative folding/unfolding, delta delta G0 (WT-->mut), have been measured by calorimetry, and they are in good agreement with delta delta G0(WT-->mut) values from hydrogen exchange rates. For F22A the change in free energy difference is about 1.7 kcal/mol at 25 degrees C; for the other three mutants it is in the range of 5-7 kcal/mol at 25 degrees C. The experimental delta delta G0(WT-->mut) values of F22A, Y23A, and F45A are reasonably well accounted for as the sum of two terms: the difference in transfer free energy change, and a contribution from exposure to solvent of new surface (Eriksson, A.E., et al., 1992, Science 255, 178-183), if the recently corrected transfer free energies and surface hydrophobicities (De Young, L. & Dill, K., 1990, J. Phys. Chem. 94, 801-809; Sharp, K.A., et al., 1991a, Science 252, 106-109) are used and only nonpolar surface is taken into account. In N43G, three protein-protein hydrogen bonds are replaced by protein-water hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein conformational landscapes: Energy minimization and clustering of a long molecular dynamics trajectory

Using energy minimization and cluster analysis, we have analyzed a 1020 ps molecular dynamics trajectory of solvated bovine pancreatic trypsin inhibitor. Elucidation of conformational sub states in this way both illustrates the degree of conformational convergence in the simulation and reduces the structural data to a tractable subset. The relative movement of structures upon energy minimization was used to estimate the sizes of features on the protein potential energy surface. The structures were analyzed using their pairwise root-mean-square C_α deviations, which gave a global measure of conformational changes that would not be apparent by monitoring single degrees of freedom. At time scales of 0.1 ps, energy minimization detected sharp transitions between energy minima separated by 0.1 Å rms deviation. Larger conformational clusters containing these smaller minima and separated by 0.25 Å were seen at 1 ps time scales. Both of these small features of the conformational landscape were characterized by movements in loop regions associated with small, correlated backbone dihedral angle shifts. On a nanosecond time scale, the main features of the protein energy landscape were clusters separated by over 0.7 Å rms deviation, with only seven of these sub states visited over the 1 ns trajectory. These substates, discernible both before and after energy minimization, differ mainly in a monotonic pivot of the loop residues 11–18 over the course of the simulation. This loop contains lysine 17, which specifically binds to trypsin in the active site. The trajectory did not return to previously visited clusters, indicating that this trajectory has not been shown to have completely sampled the conformational substates available to it. Because the apparent convergence to a single region of conformation space depends on both the time scale of observation and the size of the conformational features examined, convergence must be operationally defined within the context of the simulation. © 1995 Wiley-Liss, Inc.