Fluorine teams up with water to restore inhibitor activity to mutant BPTI
Introducing fluorine into molecules has a wide range of effects on their physicochemical properties, often desirable but in most cases unpredictable. The fluorine atom imparts the C-F bond with low polarizability and high polarity, and significantly affects the behavior of neighboring functional groups, in a covalent or noncovalent manner. Here, we report that fluorine, present in the form of a single fluoroalkyl amino acid side chain in the P1 position of the well-characterized serine-protease inhibitor BPTI, can fully restore inhibitor activity to a mutant that contains the corresponding hydrocarbon side chain at the same site.High resolution crystal structures were obtained for four BPTI variants in complex with bovine b-trypsin, revealing changes in the stoichiometry and dynamics of water molecules in the S1 subsite. These results demonstrate that the introduction of fluorine into a protein environment can result in "chemical complementation" that has a significantly favorable impact on protein-protein interactions.
Introduction<!>Synthesis and characterization of mutant BPTIs<!>High resolution crystal structures of trypsin-BPTI complexes<!>, the water molecules are H-bonded to one another as follows: A-B, B-A and -C, C-B and -D, and D-C (Table S6 ‡).<!>Conclusions<!>Peptide synthesis<!>Native chemical ligation and protein refolding<!>Inhibitor activity assay<!>Protein crystallography
<p>Fluorinated organic compounds are the least abundant among naturally occurring organohalides. In spite of this scarcity, uorine has been recognized as a "magic element" in medicinal chemistry, due to its unique properties. Currently, about 20% of pharmaceuticals and up to 30% of agrochemicals contain at least one uorine atom. 1,2 It has been shown that the incorporation of uorine into small molecules affects numerous physicochemical properties and results in "mimic", "block", and "steric" effects, as well as changes in lipophilicity. 3 In addition, uorine is an excellent biophysical probe, by virtue of the high sensitivity of the 19 F NMR reporter to changes in its environment, and a useful biomedical imaging tool when radioactive 18 F is used as a PET tracer. [4][5][6] A wide variety of amino acid analogues containing uorine atoms have been incorporated into peptides and proteins, 7 but by far the most extensively investigated building blocks are based on those with aliphatic side chains such as leucine (Leu), isoleucine (Ile), and valine (Val) residues, as their synthesis and introduction into polypeptides, in vitro and in vivo, are relatively unproblematic. These building blocks have been shown to, for example, inuence the membrane association characteristics of antimicrobial peptides, the thermal stability of model helical bundles and globular proteins, and the kinetics of b-sheet formation. While these effects can oen be explained based on relative hydrophobicities and secondary structure propensities, they remain exceedingly difficult to predict because they also depend upon the precise microenvironment experienced by the uorinated amino acid side chain, 8 the number of uorine atoms it contains, and the number of unnatural residues present in the sequence. [9][10][11][12][13][14] Studies in our group on coiled-coil peptide model systems were based on the experimentally supported assumption that a triuoromethyl group and an isopropyl group are comparable regarding steric effects. 15 Thus, the unnatural amino acid (2S)-2aminobutanoic acid (Abu) and three of its uorinated derivatives, (2S)-2-amino-4-monouorobutanoic acid (MfeGly), (2S)-2amino-4,4-diuorobutanoic acid (DfeGly), and (2S)-2-amino-4,4,4-triuorobutanoic acid (TfeGly), and the analogue (2S)-2amino-4,4-triuoropentanoic acid (DfpGly) were tested regarding their impact on thermal stability, binding, and folding. 8,[16][17][18] Within the context of a parallel coiled-coil heterodimer, replacement of a central a or d position of the heptad with one of these building blocks was found to be destabilizing, but to different degrees depending on the position of the substitution: the uorinated side chains are more destabilizing at the d position than at the a position (up to a 14 C difference in T M in the case of TfeGly) likely because, as shown by molecular dynamics simulations, the polarized b-methylene groups are oriented toward the hydrophobic core in the former case, and oriented toward solvent in the latter case. 8 Phage display-based screening of this model system to identify preferred canonical interaction partners revealed that the thermal stability of these complexes, in which one uorinated side chain packs against exclusively canonical side chains, can vary up to 10 C (in the case of DfeGly) when the latter are optimized by randomization. 18 DfeGly and TfeGly have also been incorporated into specic sites in model peptides to evaluate the effects that uorine has on the kinetics of amyloid formation and on proteolytic resistance, and the results of these studies are also difficult to express in terms of simple structureactivity relationships. 16,19,20 That is, the unique chemical properties of uorine lead to a situation in which its presence in otherwise natural-like amino acid side chains can have unexpected consequences within a protein environment. Because it is currently not possible to judiciously rationally design uorinated proteins, additional systematic studies are needed to demystify uorine's impact.</p><p>In order to investigate how the unique properties of the C-F bond inuence the characteristics of an otherwise fully natural polypeptide, we chose the well-characterized bovine pancreatic trypsin inhibitor (BPTI), also known as aprotinin. This small globular protein has been extensively investigated structurally and functionally. [21][22][23][24] It has a relatively broad inhibition prole, blocking serine proteases including chymotrypsin, plasmin, and trypsin, as well as metabolic intermediates and ion channels. 25 BPTI is also of clinical importance as an antibrinolytic agent used to reduce bleeding during cardiac surgery, although it was withdrawn from routine administration in 2008 due to increased risk of thrombotic events. 26 In solution, BPTI folds into a compact pear-shaped structure, comprising an antiparallel b-sheet and two helical regions, that is stabilized by three disulde bonds (Cys5/55, Cys14/38, Cys30/ 51). In complex with b-trypsin, residues Pro13 (P3) to Ile19 (P4 0 ) form a binding interface with the S3-S4 0 binding sites. 25,27 In particular, the P1-P3 segment forms an anti-parallel b-sheet with residues 214-216 of trypsin, the main chain nitrogen of Ser195 of the catalytic triad (including also His57 and Asp102) is H-bonded to the carbonyl oxygen of P1, but its side chain hydroxyl is not covalently bonded to the P1 carbonyl carbon at the P1-P1 0 reaction site. Mutational and structural studies of the side chain of Lys15/P1, fully solvent exposed in BPTI in solution, have demonstrated that it plays an important role in inhibitor binding to the protease (Fig. 1a). 28,29 In the current study, we present the total chemical synthesis and characterization of BPTI variants in which Lys15 has been substituted by the noncanonical amino acids Abu, DfeGly, and TfeGly (Fig. 1b). High resolution crystal structures of these mutants of BPTI in complex with trypsin were obtained, revealing how "chemical complementation" involving organic uorine and structural water molecules can restore inhibitor activity to the Lys15Abu BPTI mutant.</p><!><p>Three unnatural variants of BPTI were synthesized by means of solid-phase peptide synthesis and native chemical ligation. [30][31][32] The yields of the C-terminal peptide fragment and the N-terminal peptide-thioester were comparable to literature values. 30,31 The native chemical ligation proceeded with low efficiency due to the poor solubility of the N-terminal peptidethioester. It had been previously reported that approximately 50% of BPTI accumulates as dead intermediates due to a kinetic trap within the refolding pathway. 33 In our hands, protein renaturation on a smaller scale gave yields of 11-17%. The formation of disulde bonds was analyzed by ESI-MS (ESI ‡).</p><p>The overall conformation of the synthetic BPTI variants was analyzed by CD spectroscopy (Fig. 1c). All refolded synthetic mutant BPTIs exhibit CD spectra comparable to the wild-type protein in shape and intensity, indicating that the structural perturbations caused by the Lys15Abu, Lys15DfeGly, and Lys15TfeGly mutations are minimal. In contrast, the unfolded full-length peptides yielded CD spectra that indicate the absence of secondary structure. 34,35 Fig. 1  BPTI is a protein with extremely high thermal stability that is also relatively resistant to common protein denaturing agents such as urea and guanidinium chloride (GdmCl). For example, 6 M GdmCl alone, or heating to 100 C at neutral pH, are conditions that are not sufficient to fully denature the protein. [36][37][38] Therefore, to analyze the effect of P1 mutation on the thermal stability of BPTI, we conducted denaturation experiments with heat treatment in the presence of denaturants under acidic conditions (Table 1). Because Lys15 has a formal positive charge and the analogues studied here are neutral, two distinct sets of conditions, 6 M GdmCl and 8 M urea, were applied. GdmCl is a salt that has an electrostatic masking effect and is therefore a better reporter for hydrophobic contributions to stability, whereas urea is more suitable for monitoring the contributions of electrostatic effects to stability. 39 Under both sets of denaturation conditions, the folded Lys15Abu variant is signicantly less stable than Lys15, Lys15DfeGly, and Lys15TfeGly (Fig. 2). The uorinated side chains have a stabilizing effect that is greater than that of the wild type in the presence of GdmCl, but the opposite is true in the presence of urea, indicating that the positive formal charge of the solvent-exposed Lys contributes to thermal stability to some extent. The Abu data agree well with previous studies of P1 mutants of BPTI, in which differential scanning calorimetry experiments revealed that the presence of hydrophobic aliphatic or aromatic residues at this position is always destabilizing. 35 In contrast, the stabilizing effect of DfeGly and TfeGly suggest that these unnatural building blocks do not behave in a manner that can be explained by hydrophobicity arguments alone. Previous experimental and computational studies have revealed that the introduction of uorine into a methyl group not only leads to an increase in the solvent accessible surface area and hydrophobicity of the group, but also facilitates electrostatic interactions with an aqueous environment due to the highly polarized nature of the C-F bond. 40 Inhibitor activity assay BPTI strongly inhibits serine proteases, including a-chymotrypsin and b-trypsin. In the latter case, it has been shown that the side chain of Lys15/P1 contributes signicantly to the binding enthalpy of the inhibitor-enzyme complex, due to both the H-bond network in which it engages by means of its ammonium group (involving Ser190, Asp189, and Val227, among others), and the burial of the alkyl segment -(CH 2 ) 4of Lys15 in the S1 pocket; e.g., the replacement of Lys15 with Gly leads to a reduction in the K a of BPTI for b-trypsin of 10 9 fold. 29 We conducted standard inhibition assays with bovine b-trypsin 41 for wild-type BPTI and mutants Lys15Abu, Lys15DfeGly, and Lys15TfeGly (Fig. 1d). Substitution of Lys with Abu at P1 dramatically reduces inhibitor activity: at an inhibitor concentration for which the residual activity of the enzyme is 50% in the case of the Lys15 control, this value is 100% in the case of Lys15Abu. Interestingly, both BPTI variants containing uorinated analogues at the P1 site restore inhibitor activity: the association constants determined here are 3.88 x 10 7 M À1 for Lys15DfeGly and 5.20 Â 10 7 M À1 for Lys15TfeGly, similar to the value 5.17 Â 10 7 M À1 obtained for wild-type BPTI (ESI ‡). This remarkable observation suggests that the introduction of two or three uorine atoms into the Abu side chain results in "chemical complementation" that fully restores inhibitor activity to a wild-type level.</p><!><p>To test the above hypothesis and gain insight into the molecular mechanisms by which the uorinated residues are able to restore inhibitor activity to the Lys15Abu BPTI mutant, we determined high-resolution crystal structures of wild-type BPTI and all three variants in complex with bovine b-trypsin: wild-  type at 1.25 Å, Lys15Abu and Lys15DfeGly at 1.37 Å, and Lys15TfeGly at 1.30 Å. The resolution of the wild-type complex is higher than that of other structures currently available in the Protein Data Bank (PDB). 42,43 All structures were rened to low R/R free values with good stereochemistry (Table S3 ‡). Root mean square deviations (RMSD, Table 2) indicate negligible differences in both the global conformation of the mutant complexes compared to the wild-type complex, as well as among the mutant complexes, consistent with our CD spectra (Fig. 1c). All b-trypsin-BPTI complexes can be superimposed well, except at the P1 side chain and the water molecules within the S1 pocket (Fig. 3c). These results are consistent with a study published by Smalås et al. in which nine mutants of BPTI, all containing natural amino acids, were crystallized in complex with this enzyme, and all displayed similar global conformations. 44 The S1 binding pocket of b-trypsin occupied by Abu and its uorinated analogues were analyzed in detail. To clarify the nomenclature that is used throughout this section, the preexisting PDB labels for the chemical groups that are the side chains of Abu, DfeGly, and TfeGly are ABA, OBF, and 3 EG, respectively. Each atom of these functional groups also has its own unique identier, according to the PDB. In agreement with previous studies, the S1 pocket in all complexes is highly polar (Fig. 4b). 29,44 The electron density around the mutated position 15 of BPTI is well dened, indicating a stable conformation within this enzyme subsite (Fig. 4a). Distances were calculated from each hydrogen or uorine substituent of the terminal g C of each side chain and broken down into close contacts to atoms of BPTI or b-trypsin (Fig. 5 and Table S5 ‡) and close contacts to structural waters (Fig. 6 and Table S5 ‡). Distances between these water molecules and their proximity to atoms belonging to b-trypsin or BPTI (other than the side chain at position 15) are given in Table S6. ‡</p><p>The g CH 3 , g CHF 2 , and g CF 3 groups of the side chains of Abu, DfeGly, and TfeGly, respectively, have numerous longer range contacts to the S1 subsite of b-trypsin (Fig. 5 and Table S5 ‡). Atoms ABA HG1 , OBF FG1 , and 3EG FAD , which are similarly oriented, are in the vicinity of the side chain and carbonyl of Ser190 and the main chain atoms of Cys191, and their contact distances are shorter for DfeGly and TfeGly than for Abu. For example, ABA HG1 is 4.0 Å from Ser190 C compared to 3.8 Å and 3.6 Å in the cases of OBF FG1 and 3EG FAD , respectively. In the TfeGly structure, a second conformation (approximately 50% occupancy) is observed for the Ser190 side chain, one in which the H-bond to the hydroxyl of Tyr228 is maintained while the distance between 3EG FAD and Ser190 OG increases from 3.3 to 4.6 Å, likely due to electrostatic repulsion between uorine and oxygen. Atoms ABA HG2 , OBF FG2 , and 3EG FAE map to one another and are nearest the main chain atoms of Cys191 (3.1-4.1 Å) and the side chain amide and main chain nitrogen of Gln192, 3.3-3.9 Å and 3.3-3.7 Å, respectively.</p><p>Considering the studies published by Diederich and coworkers concerning how organic uorine bonds engage in multipolar interactions with protein backbone fragments, H-C a -C]O, and main chain or side chain amides, we determined the relevant angles to describe how each C-F bond in DfeGly and TfeGly approaches the trypsin main chain atoms of residues Ser190 to Gln192 (Table S5 ‡). 1 Although we observed a4 angles (C-F/C C]O ) within the range of 70 to 110 , indicating an orthogonal approach, it is difficult to rule out that this may be a consequence of packing; deconvoluting such   interactions for an sp 3 hybridized group containing multiple uorine atoms is beyond the scope of this study. In contrast, atoms ABA HG3 , OBF HG , and 3EG FAC occupy a region of the S1 pocket in which only two contacts closer than 4.0 Å are observed, to the carbonyl oxygen of Ser210 (3.6-3.7 Å) and to the main chain nitrogen of Trp211 (3.9 Å). These particular atoms of the unnatural side chains at position 15 more closely approach atoms belonging to BPTI itself, namely their own backbone nitrogen, the upstream carbonyl of Cys14, and the backbone oxygen of Pro13. In this case, the only distance that changes signicantly across the Abu, DfeGly, and TfeGly series is the one from ABA HG3 and 3EG FAC to X aa 15 N (from 2.7 to 3.0 Å), and this difference likely stems from intramolecular electrostatic repulsion in the triuoromethyl variant. The H-bonds involving Pro13 O (with Gly214 N ), Lys15 N (with Ser212 O ), and Lys15 O (with Gly195 N and Ser197 N ) found in the native structure are retained in the mutant complexes. Three water molecules are found in the S1 binding pocket of the wild-type complex, here referred to as A 0 , D 0 , and E 0 , while ve occupy it in the case of all three unnatural complexes, here referred to as A, B, C, D, and E (Fig. 3 and S15 ‡). Thus, when Lys15 is replaced by Abu, DfeGly, or TfeGly, two additional water  molecules occupy the space that is otherwise taken up by the considerably longer side chain of the native P1 residue, a phenomenon that had also been observed in previous work on natural mutants of BPTI, including Lys15Gly and Lys15Thr. 15 In agreement with the literature, our wild-type structure shows that A 0 , D 0 , and E 0 do not interact with one another; instead, A 0 and E 0 mediate H-bonds between the P1 side chain and the enzyme (Fig. S14 and Table S4 ‡). A 0 corresponds to Sol652 44 or DOD1014/E 43 from previous reports, and is equidistant, 2.9 Å, from Lys NZ and Val227 O ; E 0 had been previously published as Sol651 44 or DOD1008/E 43 , and is 2.8 Å away from Lys NZ and Asp189 OD2 . In contrast, water molecule D 0 , earlier referred to as Sol654 44 is not proximal to the Lys15 side chain, but has a total of four H-bond partners from other sources: three from the enzyme (Gln192 NE and Gly214 O ) and one from position P3 of the inhibitor (Pro13 O ).</p><p>Water molecules A, B, C, D, and E are present in the S1 site of each mutant complex, and are virtually superimposable. B and C represent "new" waters that are not found in the wild-type structure (Fig. 3). A, B, C, and D form what can be thought of as a "hydration shell" or "contour line" around the unnatural side chains in the space that is occupied by the longer side chain of lysine in the wild-type structure (Fig. 6); E is distal from the side chain, located equidistantly "above" B and C (3.3-3.5 Å; Tables S5 and S6). Moving from right to le along this "contour line", in accordance with the view depicted in Fig. 5</p><!><p>In all structures, with respect to the enzyme, water molecules A-D have the following environments (Table S6 ‡): A is in the vicinity of Val227, Ser190, and Trp211; B is close to Asp189 and Ser190; C has contacts to Ser190 and Gly214; D is the only structural water that directly contacts BPTI itself, via Pro13, and is also nearby Gln192 and Gly214; E is proximal to Asp189, Lys220, Gly214, and Ser213. In all structures, with respect to the unnatural side chain, water molecules A and B are closest to atoms ABA HG1 , OBF FG1 , and 3EG FAD , substituents of the terminal g C group that map to one another (Fig. 6). In contrast, water C is within 3.7 Å of all three substituents of the terminal g C group in all structures, and water D is proximal to two of the three substituents of the terminal g C group in all structures.</p><p>Interestingly, water molecule C is closer to the uorine atom 3EG FAC in the TfeGly structure (3.4 Å) than it is to the hydrogen atom ABA HG3 in the Abu structure (3.6 Å) or the hydrogen atom OBF HG in the DfeGly structure (3.7 Å). Although it cannot be ruled out that this is a consequence of the slight shi in conformation within the side chain that occurs due to the electrostatic repulsion between 3EG FAC and 3EG N , it may also indicate that a favorable interaction, perhaps a weak OH/FC H-bond, exists between the TfeGly side chain and water C. Certainly, there is no evidence of repulsion as was observed for the Ser190 side chain (occupancies here are unity), and a simple argument based on the greater van der Waals radius of uorine compared to hydrogen would not suffice, as no such phenomenon is seen between water molecule C and atoms OBF FG2 (3.1 Å) or 3EG FAE (3.2 Å), compared to ABA HG2 (3.1 Å); that is, these distances do not decrease when hydrogen is replaced by uorine.</p><p>To summarize the distance analysis of the crystal data (Fig. S16 ‡), the atoms that are oriented toward the enzyme's main chain (ABA HG1 /OBF FG1 /3EG FAD and ABA HG2 /OBF FG2 / 3EG FAE ) certainly show a trend in which the uorine atoms (DfeGly and TfeGly) are closer to the enzyme backbone than the hydrogen atoms (Abu). Considering the fact that this is observed for all nearby main chain atoms (Ser190 CB , Ser190 C , Cys191 N , Cys191 CA , Cys191 C , Gln192 N , and Gln192 CA ), there seems to be evidence for the type of previously described "uorophilic environment" provided by protein backbone fragments, in this case those of Ser190, Cys191, and Gln192, rather than particular potential hydrogen bonding interactions. For example, the distances between uorine atoms and the nearby hydrogen atoms of the enzyme were also determined (Table S9 in ESI ‡). The closest intermolecular CH/FC contacts are OBF FG2 and 3EG FAE to Cys191 HA (2.7 and 2.6 Å, respectively), and 3EG FAC to Trp211 HA (2.6 Å). Even if these contacts may be considered to fall within a weak hydrogen-bonding regime, it is unlikely that these specic interactions account for the greater proximity between the side chain constituents of BPTI residue 15 and the enzyme backbone, especially considering that trypsin residue Trp211 is distal to residues Cys191 and Gln192 (i.e. these interactions would not "pull" in the same direction).</p><p>As presented in Table 3, the average B-factor of all water molecules in the four complexes is >30 Å2 , and the B-factors of the individual structural water molecules in the S1 pocket are much lower, excluding Lys15Abu, 13-23 Å2 . The following comparisons are meaningful because of the uniformity in  Previous crystallographic studies of trypsin-mutant BPTI complexes containing canonical amino acids at the P1 position showed that although the incorporation of bulkier hydrophobic amino acids results in tighter binding than observed for the polar and charged P1 side chain mutants, signicantly lower B-factors for structural waters were only observed in the latter category. 44 These results suggest that the uorine atoms in DfeGly and TfeGly interact with these water molecules in such a way that they are more tightly held than they are by the hydrogens in the Abu side chain, an argument that is strengthened by the observation that waters B and C are slightly further away from each other and from water E in the Abu inhibitor variant compared to the uorinated variants; thus, it seems unlikely that stronger water-water contacts are exclusively responsible for this apparent reduction in dynamics. In light of previous reports that have described the important role of trapped water molecules in protein-ligand interactions, 45 and a recent study describing surface uorination and hydration dynamics in proteins, 46 a mechanism in which uorine interacts with water molecules B and C, which in turn can be thought of as extensions of the enzyme, to restore inhibitor activity, is plausible.</p><!><p>The high resolution crystal structures of the BPTI-trypsin complexes investigated here reveal an intriguing mechanism by which uorinated amino acids can engage in noncovalent interactions within the context of a protein environment. When the Lys15 residue of wild-type BPTI is substituted with the nonuorinated hydrophobic amino acid Abu, its inhibitor activity is dramatically reduced and the B-factors of the two new proximal structural waters in the S1 binding site are comparatively high, indicating that the waters are more loosely held. In contrast, when DfeGly or TfeGly are used to replace the P1 residue, the inhibitor activity is restored to the wild-type level and these B-factors are relatively low, indicating that the waters are more tightly held. Our results demonstrate what can be described as "chemical complementation" involving partially uorinated amino acids and structural water molecules that is only possible due to uorine's unique impact on an otherwise hydrophobic aliphatic side chain. Such a role for the bioorthogonal element uorine has not yet been described, and our observations lend support not only to the view that uorinated amino acids constitute a truly unique family of building blocks for protein engineering, but provide important information that will nd applications in peptide and protein engineering, peptide drug design, organocatalysis, and biomaterial development.</p><!><p>The peptide fragments 1-X aa 15-37 and 38-58 of BPTI were synthesized according to standard Fmoc/tBu chemistry by means of an automated SyroXP-I peptide synthesizer or an Activo P11 synthesizer. The noncanonical amino acids were incorporated into the protein sequence by manual coupling with 1.25 equivalents of the Fmoc-L-amino acid, HOBt, and DIC relative to resin loading. The fully protected peptide fragment of BPTI 1-X aa 15-37 was cleaved from the resin by treatment with triuoroethanol/HOAc/DCM (v/v/v, 1 : 1 : 8) for two hours, and thioesterication was achieved by incubation with ve equivalents of each benzotriazol-1-yl-oxytripyrrolidinophosphonium hexauorophosphate (PyBOP), p-acetamidothiophenol, and DIEPA in DCM overnight. All peptide fragments were puried by means of RP-HPLC with a C18 column, and purity was veried by analytical RP-HPLC coupled with ESI-MS (ESI ‡). Global structural analysis and thermal stability determination CD spectra were recorded on a Jasco J-715 spectropolarimeter at 25 C. Overall peptide concentrations were 20 mM in 10 mM tris(hydroxymethyl)aminomethane (Tris) buffer at pH 7.4. CD-spectra were obtained in the far-UV range (190-240 nm) using 0.1 cm Quartz Suprasil cuvettes equipped with a stopper. The nitrogen ow rate was set to $3.0 L min À1 . The spectra were recorded at 0.2 nm intervals with a 2 nm bandwidth and response time of 3 seconds. Aer baseline correction, the measured ellipticities were converted to molar ellipticities per residue (degree cm 2 per dmol per residue) by normalizing for the concentration of peptide, the number of amino acids in the sequence, and the path length.</p><!><p>The thermal stability of BPTI variants was performed by means of CD measurement. Protein samples were dissolved in buffer containing either 10 mM Tris-HCl, pH 2.0, with 6 M GdmCl or 10 mM Tris-HCl, pH 2, with 8 M urea. Melting curves were recorded by monitoring the absorbance at 222 nm while heating at a rate of 3 K min À1 , from 20 C to 100 C. Melting curves were obtained in triplicate and averaged. Thermodynamic parameters were determined by means of nonlinear least square tting of the normalized CD melting curves (ESI ‡).</p><!><p>A standard spectrophotometric inhibitor assay was carried out to determine the inhibitor activity of each BPTI variant (ESI ‡). Na-Benzoyl-L-arginine 4-nitroanilide hydrochloride was used as b-trypsin substrate. The assay was carried out in a buffer containing 0.046 M Tris-HCl and 0.0115 M CaCl 2 , pH 8.1.</p><!><p>b-Trypsin-BPTI complexes were puried by dialysis. Protein crystallization was conducted by means of sitting drop vapor diffusion technique with a commercially available kit (ESI ‡). Well-formed crystals were soaked in 30% glycol plus reservoir solution and frozen in liquid nitrogen. Data was collected at Berlin BESSY II, beamline 14-2. X-ray data collection was performed at 100 K. Diffraction data were processed with the XDS 47 in space group I222 (Table S3 ‡). The structure of the wild-type trypsin/BPTI complex was solved by molecular replacement with the coordinates of single polypeptide chains of b-trypsin and BPTI (PDB: 2FTL) 41 as search models using PHASER. 48 The structure of 4Y0Z, 4Y10, and 4Y11 were determined by isomorphous replacement. For calculation of the free R-factor, a randomly generated set of 5% of the reections from the diffraction data set was used and excluded from the renement. The structure was initially rened by applying a simulated annealing protocol and in later renement cycles by maximumlikelihood restrained renement using PHENIX followed by iterative, manual model building cycles with COOT. 49 Model quality was evaluated with MolProbity. 50 Figures were prepared using PyMOL. 51 The atomic coordinates and structure factor amplitudes have been deposited in the Protein Data Bank under the accession codes 4Y0Y, 4Y0Z, 4Y10, and 4Y11.</p>