Native Bovine Pancreatic Trypsin Inhibitor Research Articles

Miniature bovine pancreatic trypsin inhibitors (m-BPTIs) of the West Nile virus NS2B-NS3 protease

The mosquito-borne West Nile virus (WNV) causes a wide range of symptoms ranging from fever to the often fatal viral encephalitis. To date, no vaccine or drug therapy is available. The trypsin-like WNV NS2B-NS3 protease is deemed a plausible drug target and was shown to be inhibited by bovine pancreatic trypsin inhibitor (BPTI), a 58-residue protein isolated from bovine lung. Herein, we report a protein truncation study that resulted in a novel 14-residue cyclic peptide with equipotent inhibitory activity to native BPTI. We believe our truncation strategy can be further applied in the development of peptide-based inhibitors targeting trypsin-like proteases.

The P(2)' residue is a key determinant of mesotrypsin specificity: engineering a high-affinity inhibitor with anticancer activity.

PRSS3/mesotrypsin is an atypical isoform of trypsin, the up-regulation of which has been implicated in promoting tumour progression. Mesotrypsin inhibitors could potentially provide valuable research tools and novel therapeutics, but small-molecule trypsin inhibitors have low affinity and little selectivity, whereas protein trypsin inhibitors bind poorly and are rapidly degraded by mesotrypsin. In the present study, we use mutagenesis of a mesotrypsin substrate, APPI (amyloid precursor protein Kunitz protease inhibitor domain), and of a poor mesotrypsin inhibitor, BPTI (bovine pancreatic trypsin inhibitor), to dissect mesotrypsin specificity at the key P(2)' position. We find that bulky and charged residues strongly disfavour binding, whereas acidic residues facilitate catalysis. Crystal structures of mesotrypsin complexes with BPTI variants provide structural insights into mesotrypsin specificity and inhibition. Through optimization of the P(1) and P(2)' residues of BPTI, we generate a stable high-affinity mesotrypsin inhibitor with an equilibrium binding constant K(i) of 5.9 nM, a >2000-fold improvement in affinity over native BPTI. Using this engineered inhibitor, we demonstrate the efficacy of pharmacological inhibition of mesotrypsin in assays of breast cancer cell malignant growth and pancreatic cancer cell invasion. Although further improvements in inhibitor selectivity will be important before clinical potential can be realized, the results of the present study support the feasibility of engineering protein protease inhibitors of mesotrypsin and highlight their therapeutic potential.

The normal modes of a protein: Native bovine pancreatic trypsin inhibitor

A completely general treatment of normal mode analysis is developed that can be used with any potential energy function and any set of generalized coordinates. The method is applied to the calculation of the normal modes of the small protein bovine pancreatic trypsin inhibitor that has been the subject of many previous theoretical studies. The potential energy function used comprises a torsion angle potential, a van der Waals potential between nonbonded pairs of atoms, and a hydrogen bond potential. Therefore, the generalized coordinates used are the 208 Φ, ϕ, and χ torsion angles about single bonds. This eliminates the difficulties inherent in using internal or Cartesian coordinates for a large molecule. Many dynamic properties of the protein may now be calculated in the normal mode description. In particular, the rms magnitudes and pair correlations of the fluctuations in positions and velocities of the α-carbon atoms and various classes of torsion angles, such as backbone, side chain, β-sheet, and α-helix, are calculated and analyzed to identify the most correlated modes. In addition, the ir intensities are calculated.

Native-like interactions favored in the unfolded bovine pancreatic trypsin inhibitor have different roles in folding.

Folding kinetics of a series of bovine pancreatic trypsin inhibitor (BPTI) variants with similar stabilities and structures have been measured. All are strongly destabilized relative to WT. In Y21A, F22A, Y23A, G37A, and F45A, the three native disulfide bonds are retained. In RM(14-38), Cys14 and Cys38 thiols are methylated while C30-C51 and C5-C55 disulfides remain intact. At pH 2 and 20 degrees C, relaxation rate constants of the major kinetic phase range from approximately 10 ms to 0.71 s in the absence of denaturant. All mutants except G37A exhibit standard two-state behavior. Y21A, F22A, and Y23A fold much more slowly than other mutants. The experiments were designed to test the hypothesis that native-like structure detected in the unfolded BPTI is important in folding. Two native-like contacts are implied by NOEs in reduced and unfolded BPTI, between residues Tyr23 and Ala25, and between Gly37 NH and the Tyr35 ring. The results support an earlier hypothesis that formation of the central beta-hairpin, monitored by a local native interaction between Tyr23 and Ala25, is crucial to initiation of BPTI folding. The second native-like contact is important, not in folding initiation, but in preventing a kinetic trap later in the process. Evidence for this comes from mutant G37A, which behaves very differently from the others in displaying a phenomenon called rollover. G37A is, to our knowledge, the first reported case in which a single-site replacement causes rollover, while the wild type and all other known mutants of the same protein show typical two-state chevron plots. The best explanation is that the G37A mutation introduces a kinetic trap of the type described by Chan and Dill [(1998) Proteins 30, 2-33]. In native BPTI, there is an unusual polar interaction between the ring of Tyr35 and the backbone NH of Gly37. Our results suggest that the NH-aromatic interaction between residues 37 and 35 is important throughout folding in stabilizing native-like loop conformations and in preventing the flexible loops from being trapped in nonfunctional conformations during later stages of folding.

Correlative motions and memory effects in molecular dynamics simulations of molecules: principal components and rescaled range analysis suggest that the motions of native BPTI are more correlated than those of its mutants

In this work MD simulations of the native bovine pancreatic trypsin inhibitor (BPTI) and 16 mutants were done in vacuum in order to study memory effects in the mutants using principal component analysis (PCA) and the rescaled range analysis (Hurst exponents). Both PCA and the rescaled range analysis support our previous proposition, based on PCA of lysozyme, that the motions of a native protein are more correlated than those of mutants. The methods are compared, the nature and applications of the rule and the role of the long-range correlations in MD time series (i.e. memory) are discussed in the context of collective motions.

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.

Folding of bovine pancreatic trypsin inhibitor (BPTI) variants in which almost half the residues are alanine

Recent studies indicate that a fraction of the information contained in an amino acid sequence may be sufficient for specifying a native protein structure. An earlier alanine-scanning experiment conducted on bovine pancreatic trypsin inhibitor (BPTI; 58 residues) suggested that if cumulative mutations have additive effects on protein stability, a native protein structure could be built from BPTI sequences that contained many alanine residues distributed throughout the protein. To test this hypothesis, we designed and produced six BPTI mutants containing from 21 to 29 alanine residues. We found that the melting temperature of mutants containing up to 27 alanine residues (48 % of the total number of residues) could be predicted quite well by the sum of the change in melting temperature for the single mutations. Additionally, these same mutants folded into a native-like structure, as judged by their cooperative thermal denaturation curves and heteronuclear multiple quantum correlation (HMQC) NMR spectra. A BPTI mutant containing 22 alanine residues was further shown by 2D and 3D-NMR to fold into a structure very similar to that of native BPTI, and to be a functional trypsin inhibitor. These results provide insight into the extent to which native protein structure and function can be achieved with a highly simplified amino acid sequence.

Leap-dynamics: efficient sampling of conformational space of proteins and peptides in solution

A molecular simulation scheme, called Leap-dynamics, that provides efficient sampling of protein conformational space in solution is presented. The scheme is a combined approach using a fast sampling method, imposing conformational ‘leaps’ to force the system over energy barriers, and molecular dynamics (MD) for refinement. The presence of solvent is approximated by a potential of mean force depending on the solvent accessible surface area. The method has been successfully applied to N-acetyl- L-alanine- N-methylamide (alanine dipeptide), sampling experimentally observed conformations inaccessible to MD alone under the chosen conditions. The method predicts correctly the increased partial flexibility of the mutant Y35G compared to native bovine pancreatic trypsin inhibitor. In particular, the improvement over MD consists of the detection of conformational flexibility that corresponds closely to slow motions identified by nuclear magnetic resonance techniques.

Engineering an unnatural N α-anchored disulfide into BPTI by total chemical synthesis: structural and functional consequences

A disulfide-engineered analogue of bovine pancreatic trypsin inhibitor (BPTI), ((N α-(CH 2) 2S-)Gly 38)BPTI, has been prepared using a thioester-mediated auxiliary functional group chemical ligation of a N α-ethanethiol-containing peptide segment with a peptide- αCOSR segment. In this study, N α-(ethanethiol)Gly 38 replaces the native Cys 38, providing the sulfhydryl group required for ligation and folding. Comparisons between ((N α-(CH 2) 2SH)Gly 38)BPTI, synthetic native BPTI and reference BPTI purchased from Sigma were made using mass spectroscopy, enzyme inhibitor association constant determination ( K a) and 1H-nuclear magnetic resonance total correlated spectroscopy ( 1H-NMR TOCSY) measurements. The K a value for ((N α-(CH 2) 2SH)Gly 38)BPTI was approximately 20-fold lower than synthetic and reference BPTI, which was attributed to perturbations in the binding loop of the protein (near Cys 14). This hypothesis was confirmed by two-dimensional (2D) 1H-NMR TOCSY experiments. The data reported here demonstrate that total chemical synthesis by auxiliary functional group chemical ligation is a practical method for the synthesis of a novel class of biologically active protein analogues containing additional functional groups linked to the protein backbone.

Hydroiodic acid attachment kinetics as a chemical probe of gaseous protein ion structure: bovine pancreatic trypsin inhibitor

The kinetics of attachment of hydroiodic acid (HI) to the (M + 6H) 6+ ions of native and reduced forms of bovine pancreatic trypsin inhibitor (BPTI) in the quadrupole ion trap environment are reported. Distinctly nonlinear (pseudo first-order) reaction kinetics are observed for reaction of the native ions, indicating two or more noninterconverting structures in the parent ion population. The reduced form, on the other hand, shows very nearly linear reaction kinetics. Both forms of the parent ion attach a maximum of five molecules of hydroiodic acid. This number is expected based on the amino acid composition of the protein. There is a total of 11 strongly basic sites in the protein (i.e., six arginines, four lysines, and one N-terminus). An ion with protons occupying six of the basic sites has five available for hydroiodic acid attachment. The kinetics of successive attachment of HI to the native and reduced forms of BPTI also differ, particularly for the addition of the fourth and fifth HI molecules. A very simple kinetic model describes the behavior of the reduced form reasonably well, suggesting that all of the neutral basic sites in the reduced BPTI ions have roughly equal reactivity. However, the behavior of the native ion is not well-described by this simple model. The results are discussed within the context of differences in the three-dimensional structures of the ions that result from the presence or absence of the three disulfide linkages found in native BPTI. The HI reaction kinetics appears to have potential as a chemical probe of protein ion three-dimensional structure in the gas phase. Hydroiodic acid attachment chemistry is significantly different from other chemistries used to probe three-dimensional structure and hence, promises to yield complementary information.

Determinants of backbone dynamics in native BPTI: cooperative influence of the 14–38 disulfide and the tyr35 side-chain

15Nitrogen relaxation experiments were used to characterize the backbone dynamics of two modified forms of bovine pancreatic trypsin inhibitor (BPTI). In one form, the disulfide between Cys14 and Cys38 in the wild-type protein was selectively reduced and methylated to generate an analog of the final intermediate in the disulfide-coupled folding pathway. The second form was generated by similarly modifying a mutant protein in which Tyr35 was replaced with Gly (Y35G). For both selectively reduced proteins, the overall conformation of native BPTI was retained, and the relaxation data for these proteins were compared with those obtained previously with the native wild-type and Y35G proteins. Removing the disulfide from either protein had only small effects on the observed longitudinal relaxation rates (R1) or heteronuclear cross relaxation rates (nuclear Overhauser effect), suggesting that the 14–38 disulfide has little influence on the fast (ps to ns) backbone dynamics of either protein. In the wild-type protein, the pattern of residues undergoing slower (μs to ms) internal motions, reflected in unusually large transverse relaxation rates (R2), was also largely unaffected by the removal of this disulfide. It thus appears that the large R2 rates previously observed in native wild-type protein are not a direct consequence of isomerization of the 14–38 disulfide. In contrast with the wild-type protein, reducing the disulfide in Y35G BPTI significantly decreased the number of backbone amides displaying large R2 rates. In addition, the frequencies of the backbone motions in the modified protein, estimated from R2 values measured at multiple refocusing delays, appear to span a wider range than those seen in native Y35G BPTI. Together, these observations suggest that the slow internal motions in Y35G BPTI are more independent in the absence of the 14–38 disulfide and that formation of this bond may lead to a substantial loss of conformational entropy. These effects may account for the previous observation that the Y35G substitution greatly destabilizes the disulfide. The results also demonstrate that the disulfide and the buried side-chain influence the dynamics of the folded protein in a highly cooperative fashion, with the effects of removing either being much greater in the absence of the other.

Selective Bridging of Bis-Cysteinyl Residues by Arsonous Acid Derivatives as an Approach to the Characterization of Protein Tertiary Structures and Folding Pathways by Mass Spectrometry

Bis-cysteine selective modifications were successfully applied with melarsen oxide (MEL), an arsonous acid derivative, for tertiary structural studies of peptides and a model protein. The arsonous acid modified peptides and proteins were amenable to direct characterizations by mass spectrometry, e.g., direct molecular weight determinations and mass spectrometric peptide mapping that identified stoichiometry and sites of modification, respectively. Proteolytic digestion and mass spectrometric fragmentation of modified oxytocin showed that MEL-bridged peptide derivatives are structural homologues to the disulfide-bonded macrocyclic peptides. Mass spectrometric analyses determined the MEL modification site in partially reduced and selectively modified bovine pancreatic trypsin inhibitor (BPTI) bridging Cys-14 and Cys-38. The BPTI · MEL derivative was resistant to proteolysis by both Lys-C and trypsin and thus represented a rigid structure like native BPTI. MEL exhibited several advantageous features such as (i) cross-linking two closely spaced thiol groups, providing detailed tertiary structure information; (ii) high solubility as monomeric ortho acid in aqueous and organic solutions; (iii) adding a relatively large mass increment to proteins upon single modification; (iv) enabling UV monitoring of the derivatization due to a strong chromophor; and (v) performing fast and specific modifications of bis-thiol groups in proteins to form stable structures without any side reactions even with a high molar excess of MEL. The investigated physical and chemical properties of MEL suggest general applicability for selective bis-thiol modifications, enabling protein structure–function studies in both soluble and membrane proteins and the study of protein-folding reactions.

Conformation of native, reduced and [5-55]Ala bovine pancreatic trypsin inhibitor in the gas phase

Collision cross sections have been measured for gas phase ions of native oxidized bovine pancreatic trypsin inhibitor (BPTI), native reduced BPTI and a mutant form of BPTI containing a single disulphide bond between residues 5 and 55 ([5-55]Ala BPTI). Cross sections for [5-55]Ala BPTI and reduced BPTI were 9% and 17% greater respectively than those for native BPTI. Cross sections for native BPTI were smaller than previous estimates from the crystal structure but in reasonable agreement with values calculated from the radius of gyration determined by x-ray scattering from solution BPTI. The increase in cross section for reduced BPTI over native BPTI is similar to that seen in molecular dynamics simulations. The results show that the disulphide bonds of BPTI contribute to the folding of the gas phase ions, but that even in the absence of disulphide bonds the protein ions maintain compact structures. Comparisons to relative areas calculated from hydrodynamic radii of BPTI in solution suggest that when disulphide linkages are removed, BPTI in the gas phase unfolds less than BPTI in solution.

Structure of single-disulfide variants of bovine pancreatic trypsin inhibitor (BPTI) as probed by their binding to bovine β-trypsin

Native bovine pancreatic trypsin inhibitor (BPTI) contains three disulfide bonds: Cys5–Cys55, Cys14–Cys38 and Cys30–Cys51. Correct cysteine pairing, native structure, and full anti-proteinase activity can be restored in the process of oxidative refolding of reduced BPTI. Oxidative refolding starts with the formation of single disulfide intermediates. All 15 single-disulfide variants of BPTI (three native and 12 non-native combinations) have been expressed in Escherichia coli . In each variant the remaining four cysteine residues were replaced by alanine. Four of these variants are shown here to inhibit bovine β-trypsin: three of them contain native and one non-native (Cys5–Cys51) disulfide. All but one (Cys5–Cys55) variant are slowly digested by the enzyme, therefore measurements were performed at pH 4.0, at which trypsin activity is low. Binding constants of these four single disulfide variants were at least two orders of magnitude lower than for native BPTI. Remarkably, in some of the variants the bonding constants were found to be higher for the reduced rather than for the oxidized form of the variant. Also for the fully reduced native BPTI, determined here, the binding constant is of considerable value. Two sets of control experiments demonstrated that the binding of reduced native BPTI to trypsin is specific. In the first, mutation of Lys15 (P1 position) in the binding loop abolished binding of the reduced forms to trypsin. In the second, the binding of reduced native BPTI to anhydrotrypsin yielded the expected UV difference spectra. In general, the results obtained indicate that the inhibitor activity can be induced even in the reduced protein. This activity is not a local effect, such as the nature of residues surrounding the binding loop, but rather is induced by residual structure in the unfolded protein. This structure has been shown to consist of a set of hydrophobic residues and the data presented here indicate that reduced cysteine residues provide further stabilization of such a hydrophobic cluster. On the other hand, improper pairing of the cysteine residues in non-native single disulfide variants destabilizes the enzyme-inhibitor complex by inducing deformations of the binding loop region.

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.

Comparison of the (30-51, 14-38) Two-disulphide Folding Intermediates of the Homologous Proteins Dendrotoxin K and Bovine Pancreatic Trypsin Inhibitor by Two-dimensional 1H Nuclear Magnetic Resonance

The disulphide folding pathway of bovine pancreatic trypsin inhibitor (BPTI) revealed that the native conformation is still stable in each intermediate state with two native disulphide linkages, in the absence of each of the corresponding third disulphide bonds. This is thought to be a consequence of the extreme stability of the native BPTI conformation. The current study addresses the question of whether the native-like conformation would be populated significantly at the two-disulphide stage in disulphide refolding if the final native structure is less stable than in the case of BPTI. Dendrotoxin K from black mamba venom provides a good model to test this, since it contains the BPTI fold and was shown to fold predominantly viathe same pathway, but its native conformation is less stable than that of BPTI. The conformation of a chemically trapped two-disulphide intermediate in the disulphide refolding of dendrotoxin K, with blocking groups on Cys5 and Cys55 and disulphide bonds between Cys30 and Cys51, and Cys14 and Cys38, respectively, has been determined by 1H NMR spectroscopy and compared to those of the native protein and of the corresponding intermediate in BPTI. The analysis reveals that the dendrotoxin K intermediate adopts a partly-folded conformation, in contrast to the quasi-native conformation of the corresponding BPTI intermediate. It is similar to the partly-folded conformation of the BPTI intermediate with just the Cys30-Cys51 disulphide bond, but with a more fixed conformation in the region of the Cys14-Cys38 disulphide bond. The destabilisation of the fully native conformation of the dendrotoxin K intermediate, relative to BPTI, appears to reduce the cooperativity of the folding process.

Effects of trifluoroethanol on the conformations of peptides representing the entire sequence of bovine pancreatic trypsin inhibitor.

The effects of the cosolvent trifluoroethanol on the conformations of four peptides representing the entire sequence of bovine pancreatic trypsin inhibitor (BPTI) have been measured by CD and NMR. No substantial amounts of helical conformations were induced in one peptide with four proline residues dispersed throughout its sequence, and there were no substantial effects on its average conformational properties or on the interactions between neighboring residues that are normally evident. The other three peptides became helical, although not completely, over their entire lengths. There was a reasonable correlation between the induced content of alpha-helix and the predicted helical propensities of all four peptides. Only one of these peptides is helical in native BPTI; the other two are extended beta-strands. The latter two have an intrinsic propensity for helix formation, but a greater propensity for beta-sheet formation in folded proteins.

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.

Contribution of individual side-chains to the stability of BPTI examined by alanine-scanning mutagenesis

Bovine pancreatic trypsin inhibitor (BPTI) serves as an important model system for the examination of almost all aspects of protein structure. Systematic studies of the effects of mutation on the thermodynamic stability of BPTI, however, have been limited by the extreme stability of the protein. A derivative of BPTI containing only the 5-55 disulfide bond, termed [5-55]Ala, has been shown previously to fold into a structure very similar to that of native BPTI and to be a functional trypsin inhibitor. [5-55]Ala undergoes a reversible thermal unfolding transition with a melting temperature of 39 degrees C, and is therefore well suited for stability studies. Using an alanine-scanning mutagenesis approach, we have examined the contribution to stability of each side-chain in the [5-55]Ala derivative of BPTI. These studies demonstrate the importance of the two hydrophobic cores composed largely of clusters of aromatic residues, as well as the internal hydrogen-bonding network, in stabilizing BPTI. Overall, there is a strong relationship between change in buried surface area and stability for both polar and hydrophobic residues, with proportionality constants of 50 and 20 cal/A2, respectively. None of the alanine substitutions substantially stabilized [5-55]Ala. Nonetheless, approximately 60% (28/46) of the alanine mutants were destabilized by less than 10 degrees C, suggesting that a form of BPTI with up to half of its residues being alanine could fold into a stable structure resembling the native one.

The stabilizing effects of hydrophobic cores on peptide folding of bovine-pancreatic-trypsin-inhibitor folding-intermediate model.

A synthetic peptide model composed of alpha-helical and beta-sheet portions (P alpha P beta) is a crucial folding intermediate in the folding of bovine pancreatic trypsin inhibitor (BPTI), which contains 30 amino acid residues, and provides a good model for studying the folding structure. Using this model the roles of hydrophobic cores, such as non-polar amino acids and a short beta strand, in the folding structure stability were investigated by deleting the hydrophobic amino acids or substituting with Ala. As a first step, a mutant peptide, P alpha 6 P beta 1, was made by removing the four residues (NNFK46) from the N-terminus of P alpha which make a short beta strand in native BPTI, and each of the hydrophobic cores, Phe45 of P alpha and Tyr21 of P beta, were substituted by Ala. Without the short beta strand the peptide still folds in spite of its low solubility, suggesting that a simple alpha helix and central antiparallel beta sheet contain sufficient information to direct the folding of this region of molecule. The peptide without Tyr21 was much more unstable than P alpha 6 P beta 1, such that most of the folding structure collapsed even at 0 degree C, whereas without Phe45 the structure was more stable than P alpha 6 P beta 1. This indicates that the hydrophobic interactions of Tyr21 in P beta are more important in BPTI folding.