Bovine Pancreatic Trypsin Inhibitor Research Articles

Protein crystallization: Contribution of small angle X-ray scattering (SAXS)

Small Angle X-ray Scattering (SAXS) experiments together with crystallization experiments have shown the importance of studying macromolecular associations and interactions involved in nucleation and crystal growth mechanisms. In the study by SAXS of Bovine pancreatic trypsin inhibitor (BPTI) and Aspergillus flavus Urate oxidase (UOX) in crystallization conditions, we characterize the crystal growth unit, which is a decamer in the case of BPTI and a tetramer in the case of UOX. We study interactions in solution, showing that salt, added alone to small protein solutions, is efficient to induce attractive interactions leading to crystallization, whereas for large proteins, the addition of non absorbing polymers is necessary to induce attractive interactions, which are characterized by negative values of second virial coefficient (A 2 ).

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.

Accelerated exchange of a buried water molecule in selectively disulfide-reduced bovine pancreatic trypsin inhibitor.

Using magnetic relaxation dispersion (MRD), we have previously shown that the four internal water molecules in bovine pancreatic trypsin inhibitor (BPTI) exchange with bulk water on time scales between 10(-8) and 10(-4) s at room temperature. Because this exchange is controlled by the protein structure, internal water molecules can be used to probe rare conformational fluctuations. Here, we report (2)H and (17)O MRD data at three temperatures for wild-type BPTI and two BPTI variants where the 14-38 disulfide bond has been cleaved by a double Cys --> Ser mutation or by disulfide reduction and carboxamidomethylation. The MRD data show that the internal water molecules are conserved on disulfide cleavage. However, the exchange rate of the water molecule buried near the disulfide bond is enhanced by 2-4 orders of magnitude. The relation of water exchange to other dynamic processes in BPTI is discussed.

Human beta-tryptase: detection and characterization of the active monomer and prevention of tetramer reconstitution by protease inhibitors.

beta-Tryptase is a trypsin-like serine protease stored in mast cell secretory granules primarily as an enzymatically active tetramer. The current study aims to determine whether monomeric beta-tryptase also can exhibit enzyme activity, as suggested previously. At neutral pH beta-tryptase tetramers in the absence of heparin or dextran sulfate spontaneously convert to inactive monomers. Addition of a polyanion to these monomers at neutral pH fails to convert them back to a tetramer or to an enzymatically active state. In contrast, at acidic pH addition of a polyanion resurrects enzyme activity. Whether this activity is associated with tetramers or monomers depends on the concentration of beta-tryptase. Under the experimental conditions employed at pH 6 in the presence of heparin, the monomer concentration at which 50% conversion to tetramers occurs is 193 ng/mL. Activity against tripeptide substrates by monomers is detected at pH 6 but not at pH 7.4, whereas tetramer activity is greater at pH 7.4 than pH 6.0. Active monomers are inhibited by soybean trypsin inhibitor, bovine pancreatic trypsin inhibitor, antithrombin III, and alpha2-macroglobulin, whereas active tetramers are resistant to these inhibitors. Active monomers form complexes with these inhibitors and cleave both antithrombin III and alpha2-macroglobulin. These inhibitors also prevent reconstitution of monomers to tetramers, indicating that inactive monomers become active monomers before becoming active tetramers. The ability of tryptase monomers to become active at acidic pH raises the possibilities of expanded substrate specificities as well as inhibitor susceptibilities where the low-pH environments associated with inflammation or poor vascularity are encountered in vivo.

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.

Alteration of the disulfide-coupled folding pathway of BPTI by circular permutation.

The kinetics of disulfide-coupled folding and unfolding of four circularly permuted forms of bovine pancreatic trypsin inhibitor (BPTI) were studied and compared with previously published results for both wild-type BPTI and a cyclized form. Each of the permuted proteins was found to be less stable than either the wild-type or circular proteins, by 3-8 kcal/mole. These stability differences were used to estimate effective concentrations of the chain termini in the native proteins, which were 1 mM for the wild-type protein and 2.5 to 4000 M for the permuted forms. The circular permutations increased the rates of unfolding and caused a variety of effects on the kinetics of refolding. For two of the proteins, the rates of a direct disulfide-formation pathway were dramatically increased, making this process as fast or faster than the competing disulfide rearrangement mechanism that predominates in the folding of the wild-type protein. These two permutations break the covalent connectivity among the beta-strands of the native protein, and removal of these constraints appears to facilitate direct formation and reduction of nearby disulfides that are buried in the folded structure. The effects on folding kinetics and mechanism do not appear to be correlated with relative contact order, a measure of overall topological complexity. These observations are consistent with the results of other recent experimental and computational studies suggesting that circular permutation may generally influence folding mechanisms by favoring or disfavoring specific interactions that promote alternative pathways, rather than through effects on the overall topology of the native protein.

Atypical Kunitz-type serine proteinase inhibitors produced by the ruminant placenta.

Recently, an unusual family of genes was identified with expression confined to the trophoblast of ruminant ungulate species. The members of this family (the trophoblast Kunitz domain proteins, or TKDPs) are characterized by the presence of one or more similar, approximately 80-residue repeat sequences placed ahead of a Kunitz serine proteinase-inhibitor domain. To examine the specificity of the Kunitz moiety, the Kunitz domains of selected TKDPs and a control Kunitz protein, bovine pancreatic trypsin inhibitor (BPTI), were produced as glutathione S-transferase fusions, and their abilities to inhibit six serine proteinases were examined. Circular dichroism spectroscopy confirmed that the Kunitz fold was intact. Three of the TKDPs had unusual residues at their P1 "warhead" (ovine TKDP-1, Asn; bovine TKDP-3, Thr; and bovine TKDP-5, Ile) and exhibited no measurable inhibitory activity toward any of the proteinases. Three (ovine TKDP-3, bovine TKDP-3, and bovine TKDP-4) lacked the conserved cysteines at residues 14 and 38 that form one of the highly conserved disulfide bonds that are structurally important in all known mammalian Kunitz proteins. Ovine TKDP-3 and bovine TKDP-4 had P1 lysines and inhibited trypsin and plasmin with K(i) values only approximately 10-fold higher than that of BPTI. Bovine TKDP-2 had a P1 lysine and the three conserved disulfides, but it possessed an unusual residue (Asp) at P2. It exhibited no inhibitory activity. These data suggest that the function of the TKDP, like certain Kunitz proteins found in snake venoms, may not be in proteinase inhibition.

Degradation of Mutated Bovine Pancreatic Trypsin Inhibitor in the Yeast Vacuole Suggests Post-endoplasmic Reticulum Protein Quality Control

The rate-limiting step in protein secretion is folding, which occurs in the endoplasmic reticulum (ER) lumen, and almost all secreted proteins contain disulfide bonds that form in the ER and stabilize the native state. Secreted proteins unable to fold may aggregate or they may be subject to ER-associated protein degradation. To examine the fate of aberrant forms of a well characterized, disulfide-bonded secreted protein, we expressed bovine pancreatic trypsin inhibitor in yeast. Bovine pancreatic trypsin inhibitor is a single domain, 58-amino acid polypeptide containing three disulfide bonds, and yeast cells secrete the wild type protein. In contrast, the Y35L mutant, which folds rapidly but is unstable, remains soluble and is not secreted. Surprisingly, the proteolysis of Y35L is unaffected in yeast containing mutations in genes encoding factors required for ER-associated protein degradation and is stable if artificially retained in the ER. Rather, Y35L is diverted from the Golgi to the vacuole and degraded. Because only the mutant protein is quantitatively proteolyzed these data suggest that a post-ER quality control check-point diverts unstable proteins to the vacuole for degradation.

Structure-Function Analysis of the Reactive Site in the First Kunitz-type Domain of Human Tissue Factor Pathway Inhibitor-2

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type proteinase inhibitor that regulates a variety of serine proteinases involved in coagulation and fibrinolysis through their non-productive interaction with a P(1) residue (Arg-24) in its first Kunitz-type domain (KD1). Previous kinetic studies revealed that TFPI-2 was a more effective inhibitor of plasmin than several other serine proteinases, but the molecular basis for this specificity was unclear. In this study, we employed molecular modeling and mutagenesis strategies to produce several variants of human TFPI-2 KD1 in an effort to identify interactive site residues other than the P(1) Arg that contribute significantly to its inhibitory activity and specificity. Molecular modeling of KD1 based on the crystal structure of bovine pancreatic trypsin inhibitor revealed that KD1 formed a more energetically favorable complex with plasmin versus trypsin and/or the factor VIIa-tissue factor complex primarily due to strong ionic interactions between Asp-19 (P(6)) and Arg residues in plasmin (Arg-644, Arg-719, and Arg-767), Arg-24 (P(1)) with Asp-735 in plasmin, and Arg-29 (P(5)') with Glu-606 in plasmin. In addition, Leu-26 through Leu-28 (P(2)'-P(4)') in KD1 formed strong van der Waals contact with a hydrophobic cluster in plasmin (Phe-583, Met-585, and Phe-587). Mutagenesis of Asp-19, Tyr-20, Arg-24, Arg-29, and Leu-26 in KD1 resulted in substantial reductions in plasmin inhibitory activity relative to wild-type KD1, but the Asp-19 and Tyr-20 mutations revealed the importance of these residues in the specific inhibition of plasmin. In addition to the reactive site residues in the P(6)-P(5)' region of KD1, mutation of a highly conserved Phe at the P(18)' position revealed the importance of this residue in the inhibition of serine proteinases by KD1. Thus, together with the P(1) residue, the nature of other residues flanking the P(1) residue, particularly at P(6) and P(5)', strongly influences the inhibitory activity and specificity of human TFPI-2.

A semi-implicit solvent model for the simulation of peptides and proteins.

We present a new model of biomolecules hydration based on macroscopic electrostatic theory, that can both describe the microscopic details of solvent-solute interactions and allow for an efficient evaluation of the electrostatic hydration free energy. This semi-implicit model considers the solvent as an ensemble of polarizable pseudoparticles whose induced dipole describe both the electronic and orientational solvent polarization. In the presented version of the model, there is no mutual dipolar interaction between the particles, and they only interact through short-ranged Lennard-Jones interactions. The model has been integrated into a molecular dynamics code, and offers the possibility to simulate efficiently the conformational evolution of biomolecules. It is able to provide estimations of the electrostatic solvation free energy within short time windows during the simulation. It has been applied to the study of two small peptides, the octaalanine and the N-terminal helix of ribonuclease A, and two proteins, the bovine pancreatic trypsin inhibitor and the B1 immunoglobin-binding domain of streptococcal protein G. Molecular dynamics simulations of these biomolecules, using a slightly modified Amber force field, provide stable and meaningful trajectories in overall agreement with experiments and all-atom simulations. Correlations with respect to Poisson-Boltzmann electrostatic solvation free energies are also presented to discuss the parameterization of the model and its consequences.

A Natural Prothrombin Mutant Reveals an Unexpected Influence of A-chain Structure on the Activity of Human α-Thrombin

We have recently identified in two unrelated patients with bleeding tendency a homozygous mutation causing a deletion of one of the two contiguous Lys(9)/Lys(10) residues in the A-chain of alpha-thrombin (DeltaK9). We used in vitro expression analysis to clarify the role of the deletion of Lys(9) or Lys(10) in the thrombin function. The k(cat)/K(m) value of the hydrolysis by DeltaK9 of the synthetic substrate Phe-Pip-Arg-p-nitroanilide (where Pip represents l-pipecolyl) and fibrinopeptide A was 18- and 60-fold lower, respectively, compared with wild type (WT). Interaction with antithrombin was also reduced in the mutant, the association rate being about 20-fold lower than in the WT thrombin. The sensitivity to sodium ion of DeltaK9 was found significantly attenuated compared with the WT form. DeltaK9 has a very weak platelet-activating capacity, attributed to a severely defective PAR1 interaction, whereas the binding to the platelet glycoprotein Ibalpha was unaffected. Likewise, the interaction with protein C was severely impaired, whereas interaction with thrombomodulin had a normal K(d) value. At variance with these findings, both low affinity (basic pancreatic trypsin inhibitor) and high affinity (N-alpha-[2-naphthylsulfonyl-glycyl]-4-amidinophenylalanine-piperidide) thrombin inhibitors displayed a better binding to DeltaK9 than to the WT form, indicating a better accommodation of these inhibitors into the catalytic pocket of DeltaK9. A molecular dynamics simulation of the DeltaK9 thrombin in full explicit water solvent provided support to the role of the A-chain in affecting conformation and catalytic properties of the B-chain, especially in some insertion loops of the enzyme, such as the 60-loop, as well as in the geometry of the catalytic triad residues.

Exosite occupation by heparin enhances the reactivity of blood coagulation factor IXa.

Blood coagulation factor IXa (fIXa) is a trypsin-like serine protease with low inherent activity that is greatly enhanced in the factor X activation complex. Molecular details of the conversion of fIXa from an inactive enzyme into a fully functional procoagulant are unclear. Recent studies have identified a heparin-binding exosite in the protease domain of fIXa. Effects of exosite occupation on fIXa activity are unclear. We used the Kunitz-type inhibitor bovine pancreatic trypsin inhibitor (BPTI) to probe fIXa reactivity in the absence and in the presence of heparin. While fIXa alone was poorly reactive with BPTI (K(i) approximately 0.7 mM), this reactivity was increased roughly 20-fold (K(i) = 37 +/- 6 microM) by heparin. This was reproducible with low-molecular-weight heparin (enoxaparin; K(i) = 70 +/- 12 microM). Surface plasmon resonance studies of the interaction between heparin and BPTI indicated an unstable interaction with very low affinity (K(d) = 172 microM). In contrast, kinetic studies revealed a high-affinity interaction between heparin and fIXa (K(d) = 128 +/- 26 nM) and showed that the enhancement of BPTI inhibition of fIXa by heparin was well described by a competitive inhibition model where heparin acts as an affecter of fIXa reactivity with inhibitor. Fluorescence studies with dansyl-EGR-fIXa supported the high-affinity interaction between heparin and fIXa and suggested an altered environment in the fIXa active-site region upon heparin binding. This modulating effect of heparin was supported by the observation of a heparin-induced increase in reactivity of fIXa toward a pentapeptide substrate. When viewed together, the results imply that specific physiological exosite interactions with heparin can induce alterations in the environment of the extended fIXa active site that can result in increased reactivity.

Modeling of serine protease prototype reactions with the flexible effective fragment potential quantum mechanical/molecular mechanical method

A complete cycle of chemical transformations for the serine protease prototype reaction is modeled following calculations with the flexible effective fragment quantum mechanical/molecular mechanical (QM/MM) method. The initial molecular model is based on the crystal structure of the trypsin–bovine pancreatic trypsin inhibitor complex including all atoms of the enzyme within approximately 15–18 A of the oxygen center Oγ of the catalytic serine residue. Several selections of the QM/MM partitioning are considered. Fractions of the side chains of the residues from the catalytic triad (serine, histidine and aspartic acid) and a central part of a model substrate around the C–N bond to be cleaved are included into the QM subsystem. The remaining part, or the MM subsystem, is represented by flexible chains of small effective fragments, whose potentials explicitly contribute to the Hamiltonian of the QM part, but the corresponding fragment–fragment interactions are described by the MM force fields. The QM/MM boundaries are extended over the Cα–Cβ bonds of the peptides assigned to the QM subsystem in the enzyme, C–C and C–N bonds in model substrates. Multiple geometry optimizations have been performed by using the RHF/6-31G method in the QM part and OPLSAA or AMBER sets of MM parameters, resulting in a series of stationary points on the complex potential-energy surfaces. All structures generally accepted for the serine protease catalytic cycle have been located. Energies at the stationary points found have been recomputed at the MP2/6-31+G* level for the QM part in the protein environment. Structural changes along the reaction path are analyzed with special attention to hydrogen-bonding networks. In the case of a model substrate selected as a short peptide CH3(NHCO-CH2)2 – HN–CO–(CH2–NHCO)CH3 the computed energy profile for the acylation step shows too high activation energy barriers. The energetics of this rate-limiting step is considerably improved, if more realistic model for the substrate is considered, following the motifs of the ThrI11–GlyI12–ProI13-–CysI14–LysI15–AlaI16–ArgI17–IleI18–IleI19 sequence of the bovine pancreatic trypsin inhibitor.

NMR R1 rho rotating-frame relaxation with weak radio frequency fields.

NMR spin relaxation in the rotating frame (R(1 rho)) is one of few methods available to characterize chemical exchange kinetic processes occurring on micros-ms time scales. R(1 rho) measurements for heteronuclei in biological macromolecules generally require decoupling of (1)H scalar coupling interactions and suppression of cross-relaxation processes. Korzhnev and co-workers demonstrated that applying conventional (1)H decoupling schemes while the heteronuclei are spin-locked by a radio frequency (rf) field results in imperfect decoupling [Korzhnev, Skrynnikov, Millet, Torchia, Kay. J. Am. Chem. Soc. 2002, 124, 10743-10753]. Experimental NMR pulse sequences were presented that provide accurate measurements of R(1 rho) rate constants for radio frequency field strengths > 1000 Hz. This paper presents new two-dimensional NMR experiments that allow the use of weak rf fields, between 150 and 1000 Hz, in R(1 rho) experiments. Fourier decomposition and average Hamiltonian theory are employed to analyze the spin-lock sequence and provide a guide for the development of improved experiments. The new pulse sequences are validated using ubiquitin and basic pancreatic trypsin inhibitor (BPTI). The use of weak spin-lock fields in R(1 rho) experiments allows the study of the chemical exchange process on a wider range of time scales, bridging the gap that currently exists between Carr-Purcell-Meiboom-Gill and conventional R(1 rho) experiments. The new experiments also extend the capability of the R(1 rho) technique to study exchange processes outside the fast exchange limit.

The van der Waals interaction between protein molecules in an electrolyte solution.

In this report we present a general formulation to calculate the van der Waals interaction between two protein molecules in an electrolyte solution using boundary element method of solving linearized Poisson-Boltzmann equation. Our formulation is based upon an inhomogeneous dielectric model of proteins at the residue level. Our results for bovine pancreatic trypsin inhibitor at various relative orientations indicate that the anisotropy of the interaction can be tens of kBT.

Binding of bovine pancreatic trypsin inhibitor to trypsinogen: spectroscopic and volumetric studies.

We have investigated the binding of bovine pancreatic trypsin inhibitor (BPTI) to bovine trypsinogen by combining ultrasonic velocimetry, high precision densimetry, and fluorescence spectroscopy. We report the changes in volume, adiabatic compressibility, van't Hoff enthalpy, entropy, and free energy that accompany the association of the two proteins at 25 degrees C and pH 8.0. We have used the measured changes in volume and compressibility in conjunction with available structural data to characterize the binding-induced changes in the hydration properties and intrinsic packing of the two proteins. Our estimate reveals that 110 +/- 40 water molecules become released to the bulk from the hydration shells of BPTI and trypsinogen. Furthermore, we find that the intrinsic coefficient of adiabatic compressibility of the two proteins decreases by 14 +/- 2%, which is suggestive of the binding-induced rigidification of the proteins' interior. BPTI-trypsinogen association is an entropy-driven event which proceeds with an unfavorable change in enthalpy. The favorable change in entropy results from partial compensation between two predominant terms. Namely, a large favorable change in hydrational entropy slightly prevails over a close in magnitude but opposite in sign change in configurational entropy. The reduction in configurational entropy and, consequently, protein dynamics is consistent with the observed decrease in intrinsic compressibility. In general, results of this work emphasize the vital role that water plays in modulating protein recognition events.

Internal hydration of protein cavities: studies on BPTI

In this paper, we present a theoretical method to calculate the hydration free energies of the protein cavities and clefts. This method is also used to compute the binding probabilities of buried water molecules. Our approach considers an ensemble of potential binding sites located within the protein and water molecules as ligand particles. The free energy to transfer a water molecule from the gas phase to the protein is calculated from the thermodynamic cycle. The protein is viewed as a dielectric continuum and a water molecule is embedded in it. Electrostatic contributions to the energies of bound water molecules and their interaction energies are computed from the solution of the Poisson equation. The nonpolar contribution and the binding entropy change are assumed to be the same for every bound water molecule. The obtained energies are used in the binding polynomial to calculate the constants for the hydration reactions and the hydration free energies. Calculated values are compared with available measurements for the initial steps of the hydration of BPTI (bovine pancreatic trypsin inhibitor) in the gas phase. The obtained energies are also used to calculate the population probabilities of the hydration sites and compute their titration curves. The results of the studies on BPTI demonstrate that an isolated buried water in the small protein cavity binds to the protein with a lower affinity than a cluster of three water molecules in a large protein cleft.

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.

Thermodynamics of Protein Hydration Computed by Molecular Dynamics and Normal Modes

Thermodynamic properties of bovine pancreatic trypsin inhibitor (BPTI) hydrated by up to 1600 water molecules, i.e., a ratio of the weight of water to that of protein, h, from 0 to ∼4.5, are studied by molecular dynamics (MD) simulation and normal-mode analysis (NMA). Temperatures range from 100 to 300 K to include the hydrated protein glass transition, Tg ≈ 200 K. The partial specific heat of BPTI computed by MD reaches fully hydrated values around h ≈ 0.7 below and above Tg. For smaller h, distinctly different variations in the apparent specific heat of BPTI with h below and above Tg are found and related to the respective decrease and increase in protein flexibility with h. Changes upon varying h in the energy landscape of hydrated BPTI near its native configuration are examined. The partial entropy of BPTI is computed with NMA up to Tg. Above 200 K, an increase in the partial entropy of BPTI is computed with specific heats of hydrated BPTI obtained by MD. The difference between the partial entropy of ...

Modelling free energy of a rigid protein in solid water: Comparison between rigid-body motions and harmonic oscillators

We test two methods to estimate a partition function of a system consisting of multi-atom molecules with intermolecular interaction. Our test case is a protein in water. The first method is based on rigid-body motions. The space in which the protein (bovine pancreatic trypsin inhibitor) would be moving is limited, so by making a correction to a partition function of a rigid body, we can obtain the function. The function depends on temperature and space. The second method is based on harmonic oscillators. Under the potential field produced by surrounding water molecules, a protein behaves like a set of harmonic oscillators. We obtain the partition function for the oscillators within a harmonic approximation. The function also depends on temperature and the strength of potential energy between the protein and waters. Comparison of the two methods indicates that the second method is better for estimating a partition function for a protein in water.