Structural insights into the non-additivity effects in the sequence-to-reactivity algorithm for serine peptidases and their inhibitors.

Sequence-to-reactivity algorithms (SRAs) for proteins have the potential of being broadly applied in molecular design. Recently, Laskowski et al. have reported an additivity-based SRA that accurately predicts most of the standard free energy changes of association for variants of turkey ovomucoid third domain (OMTKY3) with six serine peptidases, one of which is streptogrisin B (commonly known as Streptomyces griseus peptidase B, SGPB). Non-additivity effects for residues 18I and 32I, and for residues 20I and 32I of OMTKY3 occurred when the associations with SGPB were predicted using the SRA. To elucidate precisely the mechanics of these non-additivity effects in structural terms, we have determined the crystal structures of the unbound OMTKY3 (with Gly32I as in the wild-type amino acid sequence) at a resolution of 1.16 A, the unbound Ala32I variant of OMTKY3 at a resolution of 1.23 A, and the SGPB:OMTKY3-Ala32I complex (equilibrium association constant K(a)=7.1x10(9) M(-1) at 21(+/-2) C degrees, pH 8.3) at a resolution of 1.70 A. Extensive comparisons with the crystal structure of the unbound OMTKY3 confirm our understanding of some previously addressed non-additivity effects. Unexpectedly, SGPB and OMTKY3-Ala32I form a 1:2 complex in the crystal. Comparison with the SGPB:OMTKY3 complex shows a conformational change in the SGPB:OMTKY3-Ala32I complex, resulting from a hinged rigid-body rotation of the inhibitor caused by the steric hindrance between the methyl group of Ala32IA of the inhibitor and Pro192BE of the peptidase. This perturbs the interactions among residues 18I, 20I, 32I and 36I of the inhibitor, probably resulting in the above non-additivity effects. This conformational change also introduces residue 10I as an additional hyper-variable contact residue to the SRA.

Despite having a common P1 Leu, eglin C inhibits alpha-lytic proteinase a million-fold more strongly than does turkey ovomucoid third domain.

Results of the inhibition of alpha-lytic proteinase by two standard mechanism serine proteinase inhibitors, turkey ovomucoid third domain (OMTKY3) and eglin C, and many of their variants are presented. Despite similarities, including an identical P1 residue (Leu) in their primary contact regions, OMTKY3 and eglin C have vastly different association equilibrium constants toward alpha-lytic proteinase, with Ka values of 1.8 x 10(3) and 1.2 x 10(9) M(-1), respectively. Although 12 of the 13 serine proteinases tested in our laboratory for inhibition by OMTKY3 and eglin C are more strongly inhibited by the latter, the million-fold difference observed here with alpha-lytic proteinase is the largest we have seen. The million-fold stronger inhibition by eglin C is retained when the Ka values of the P1 Gly, Ala, Ser, and Ile variants of OMTKY3 and eglin C are compared. Despite the small size of the S1 pocket in alpha-lytic proteinase, interscaffolding additivity for OMTKY3 and eglin C holds well for the four P1 residues tested here. To better understand this difference, we measured Ka values for other OMTKY3 variants, including some that had residues elsewhere in their contact region that corresponded to those of eglin C. Assuming intrascaffolding additivity and using the Ka values obtained for OMTKY3 variants, we designed an OMTKY3-based inhibitor of alpha-lytic proteinase that was predicted to inhibit 10,000-fold more strongly than wild-type OMTKY3. This variant (K13A/P14E/L18A/R21T/N36D OMTKY3) was prepared, and its Ka value was measured against alpha-lytic proteinase. The measured Ka value was in excellent agreement with the predicted one (1.1 x 10(7) and 2.0 x 10(7) M(-1), respectively). Computational protein docking results are consistent with the view that the backbone conformation of eglin C is not significantly altered in the complex with alpha-lytic proteinase. They also show that the strong binding for eglin C correlates well with more favorable atomic contact energy and desolvation energy contributions as compared to OMTKY3.

Functional evolution within a protein superfamily.

The ability to predict and characterize distributions of reactivities over families and even superfamilies of proteins opens the door to an array of analyses regarding functional evolution. In this article, insights into functional evolution in the Kazal inhibitor superfamily are gained by analyzing and comparing predicted association free energy distributions against six serine proteinases, over a number of groups of inhibitors: all possible Kazal inhibitors, natural avian ovomucoid first and third domains, and sets of Kazal inhibitors with statistically weighted combinations of residues. The results indicate that, despite the great hypervariability of residues in the 10 proteinase-binding positions, avian ovomucoid third domains evolved to inhibit enzymes similar to the six enzymes selected, whereas the orthologous first domains are not inhibitors of these enzymes on purpose. Hypervariability arises because of similarity in energetic contribution from multiple residue types; conservation is in terms of functionality, with "good" residues, which make positive or less deleterious contributions to the binding, selected more frequently, and yielding overall the same distributional characteristics. Further analysis of the distributions indicates that while nature did optimize inhibitor strength, the objective may not have been the strongest possible inhibitor against one enzyme but rather an inhibitor that is relatively strong against a number of enzymes.

Ring-Toss: Capping highly exposed tyrosyl or tryptophyl residues in proteins with beta-cyclodextrin.

We have used UV difference spectroscopy and fluorescence spectroscopy to study the perturbation by beta-cyclodextrin of tyrosyl or tryptophyl residues located at each of the 10 variable consensus contact positions in the third domain of turkey ovomucoid. The goal was to monitor the accessibility of the side chain rings of these residues when located at these positions. The results indicated that the tyrosyl or tryptophyl rings are most highly exposed when located in the P1 position followed by the P4 position. It was possible to determine the association constants for beta-cyclodextrin binding at these positions. When located at the P2, P5, P6 and P3' positions, the rings of the tyrosyl or tryptophyl residues were exposed but less so than at the P1 or P4 positions. By contrast, when located at the P1', P2', P14' and P18' positions, the tyrosyl or tryptophyl residues were insufficiently exposed to be perturbed by beta-cyclodextrin, although they reacted positively to dimethyl sulfoxide solvent perturbation. These findings indicate that beta-cyclodextrin perturbation provides a convenient way to detect highly exposed tyrosyls or tryptophyls in proteins. Furthermore, we evaluated the ability of beta-cyclodextrin to inhibit the interaction of turkey ovomucoid third domain variants with different P1 residues. The results showed that the presence of beta-cyclodextrin had little effect on the association constant when the P1 residue was a glycyl residue, but greatly decreased the association constant when the P1 residue was a tyrosyl or tryptophyl residue. Thus, beta-cyclodextrin may be used to selectively modulate the interaction between proteinase inhibitors and their cognate enzymes.

From the discovery of the reactive site of inhibitors to their sequence to reactivity algorithm.

Michael Laskowski Jr. (1930-2004) was a pioneer in the field of Standard mechanism serine proteinase inhibitors. He made numerous important contributions in the field. This article highlights some of his most important contributions such as the discovery of the reactive site in serine proteinase inhibitors, the proposal of the Standard mechanism of inhibition, and the sequence to reactivity algorithm for the Kazal family of inhibitors.

The role of scaffolding in standard mechanism serine proteinase inhibitors.

In single domain, "standard mechanism" protein inhibitors of serine proteinases, about a dozen residues make contact with the cognate enzyme. The remainder of the molecule, the scaffolding, holds the reactive site region of the inhibitor in a canonical conformation, improves the binding by about six orders of magnitude and protects it from proteolysis. However, the stability and global structure of the scaffolding is irrelevant to inhibition, provided that inhibition is measured much below the melting temperature, Tm.

Structure of the subtilisin Carlsberg-OMTKY3 complex reveals two different ovomucoid conformations.

One of the most studied protein proteinase inhibitors is the turkey ovomucoid third domain, OMTKY3. This inhibitor contains a reactive-site loop (Lys13I-Arg21I) that binds in a nearly identical manner to all studied serine proteinases, regardless of their clan or specificity. The crystal structure of OMTKY3 bound to subtilisin Carlsberg (CARL) has been determined. There are two complete copies of the complexes in the crystallographic asymmetric unit. Whereas the two enzyme molecules are virtually identical [0.16 A root-mean-square difference (r.m.s.d.) for 274 C(alpha) atoms], the two inhibitor molecules show dramatic differences between one another (r.m.s.d. = 2.4 A for 50 C(alpha) atoms). When compared with other proteinase-bound OMTKY3 molecules, these inhibitors show even larger differences. This work facilitates a re-evaluation of the importance of certain ovomucoid residues in proteinase binding and explains why additivity and sequence-based binding-prediction methods fail for the CARL-OMTKY3 complex.

Two conformational states of Turkey ovomucoid third domain at low pH: three-dimensional structures, internal dynamics, and interconversion kinetics and thermodynamics.

Turkey ovomucoid third domain (OMTKY3) is shown to exist at low pH as two distinctly folded, interconverting conformations. Activation parameters were determined for the transition, and these were of the type reported previously for cis/trans isomerizations of prolyl peptide bonds. Multidimensional, multinuclear NMR spectroscopy was used to determine the three-dimensional structure of each of the two states of P(5)-Pro(14)Asp OMTKY3 at pH 2.5 and 25 degrees C, under conditions where the two states have equal populations with interchange rates of 0.25 s(-1). The results showed that the two states differ by cis/trans isomerization of the P(8)-Tyr(11)-P(7)-Pro(12) peptide bond, which is cis in the conformer dominant at neutral pH and trans in the conformer appearing at low pH. The major structural differences were found to be in the region of the reactive site loop. The core of the protein, including the antiparallel beta-sheet and a alpha-helix, is preserved in both structures. The state with the cis peptide bond is similar to previously reported structures of OMTKY3 determined by NMR spectroscopy and X-ray crystallography. The cis-to-trans transition results in the relocation of the aromatic ring of P(8)-Tyr(11), disrupts many interactions between the alpha-helix and the reactive-site loop, and leads to more open spacing between this loop and the alpha-helix. In addition, the configurations of two of the three disulfide bonds, P(11)-Cys(8)- P(20)'-Cys(38), and P(3)-Cys(16)- P(17)'-Cys(35), are altered such that the C(alpha)-C(alpha) distances for each disulfide bridge are longer by approximately 1 A in the trans state than in the cis. Mutations at P(1)-Leu(18), P(6)-Lys(13), and P(5)-Pro(14) influence the position of the cis <= => trans equilibrium. In P(1)-Leu(18)Xxx OMTKY3 mutants, the trans state is more favored by P(1)-Gly(18) than by Ala(18) or Leu(18); in P(6)-Lys(13)Xxx OMTKY3 mutants, the trans state is more favored by P(6)-Glu(13) and P(6)-Asp(13) than Lys(13) or His(13). Stabilization of the trans state in P(5)-Pro(14)Xxx OMTKY3 mutants follows the series Xxx = Gly > Asp > Glu > Ala approximately equal His > Pro. In comparing the state with the trans peptide bond to that with the cis, the pK(a) values of P(12)-Asp(7) and P(1)'-Glu(19) are higher and those of P(9)-Glu(10) and P(25)'-Glu(43) are lower. The pK(a) values of other titrating groups in the molecule are similar in both conformational states. These pK(a) changes underlie the pH dependence of the conformational equilibrium and can be explained in part by observed structural differences. (15)N transverse relaxation results indicate that residues P(6)-Lys(13)-P(3)-Cys(16) in the trans state undergo a dynamic process on the microsecond-millisecond time scale not present in the cis state.

Testing of the additivity-based protein sequence to reactivity algorithm.

The standard free energies of association (or equilibrium constants) are predicted for 11 multiple variants of the turkey ovomucoid third domain, a member of the Kazal family of protein inhibitors, each interacting with six selected enzymes. The equilibrium constants for 38 of 66 possible interactions are strong enough to measure, and for these, the predicted and measured free energies are compared, thus providing an additional test of the additivity-based sequence to reactivity algorithm. The test appears to be unbiased as the 11 variants were designed a decade ago to study furin inhibition and the specificity of furin differs greatly from the specificities of our six target enzymes. As the contact regions of these inhibitors are highly positive, nonadditivity was expected. Of the 11 variants, one does not satisfy the restriction that either P(2) Thr or P(1)' Glu should be present and all three measurable results on it, as expected, are nonadditive. For the remaining 35 measurements, 22 are additive, 12 are partially additive, and only one is (slightly) nonadditive. These results are comparable to those obtained for a set of 398 equilibrium constants for natural variants of ovomucoid third domains. The expectation that clustering of charges would be nonadditive is modified to the expectation that major nonadditivity will be observed only if the combining sites in both associating proteins involve large charge clusters of the opposite sign. It is also shown here that an analysis of a small variant set can be accomplished with a smaller subset, in this case 13 variants, rather than the whole set of 191 members used for the complete algorithm.

NMR determination of pKa values for Asp, Glu, His, and Lys mutants at each variable contiguous enzyme-inhibitor contact position of the turkey ovomucoid third domain.

From the larger set of 191 variants at all the variable contact positions in the turkey ovomucoid third domain, we selected a subset that consists of Asp, Glu, His, and Lys residues at eight of the nine contiguous P6-P3' positions (residues 13-21), the exception being P3-Cys16 which is involved in a conserved disulfide bridge. Two-dimensional [1H,1H]-TOCSY data were collected for each variant as a function of sample pH. This allowed for the evaluation of 31 of the 32 pK(a) values for these residues, the exception being that of P5-Lys14, whose signals at high pH could not be resolved from those of other Lys residues in the molecule. Only two of the titrating residues are present in the wild-type protein (P6-Lys13 and P1'-Glu19); hence, these measurements complement earlier measurements by A. D. Robertson and co-workers. This data set was supplemented with results from the pH dependence of NMR spectra of four additional single mutants, P1-Leu18Gly, P1-Leu18Ala, P2-Thr17Val, and P3'-Arg21Ala, and two double mutants, P2-Thr17Val/P3'-Arg21Ala and P8-Tyr11Phe/P6-Lys13Asp. Probably the most striking result was observation of a P2-Thr17...P1'-Glu19 hydrogen bond and a P1'-Glu19-P3'-Arg21 electrostatic interaction within the triad of P2, P1', and P3' (residues 17, 19, and 21, respectively). In several cases, the pK(a) of a particular residue was sensed by resonances not only in that residue but also in residue(s) with which it interacts. Remarkably, in several interacting systems, resonances from different protons within the same residue yielded different pHmid values.

Additivity-based prediction of equilibrium constants for some protein-protein associations.

For many protein families, such as serine proteinases or serine proteinase inhibitors, the family assignment predicts reactivity only in general terms. Both detailed specificity and quantitative reactivity are lacking. We believe that, for many such protein families, algorithms can be devised by defining the subset of n functionally important sequence positions, making the 19n possible single mutants and measuring their reactivity. Given the assumption that the contributions of the n positions are additive, the reactivities of the 20(n) variants can be predicted. This is illustrated by an almost complete algorithm for the Kazal family of protein inhibitors of serine proteinases.

Predicting the reactivity of proteins from their sequence alone: Kazal family of protein inhibitors of serine proteinases.

An additivity-based sequence to reactivity algorithm for the interaction of members of the Kazal family of protein inhibitors with six selected serine proteinases is described. Ten consensus variable contact positions in the inhibitor were identified, and the 19 possible variants at each of these positions were expressed. The free energies of interaction of these variants and the wild type were measured. For an additive system, this data set allows for the calculation of all possible sequences, subject to some restrictions. The algorithm was extensively tested. It is exceptionally fast so that all possible sequences can be predicted. The strongest, the most specific possible, and the least specific inhibitors were designed, and an evolutionary problem was solved.

Contribution of peptide bonds to inhibitor-protease binding: crystal structures of the turkey ovomucoid third domain backbone variants OMTKY3-Pro18I and OMTKY3-psi[COO]-Leu18I in complex with Streptomyces griseus proteinase B (SGPB) and the structure of the free inhibitor, OMTKY-3-psi[CH2NH2+]-Asp19I.

X-ray crystallography has been used to determine the 3D structures of two complexes between Streptomyces griseus proteinase B (SGPB), a bacterial serine proteinase, and backbone variants of turkey ovomucoid third domain (OMTKY3). The natural P1 residue (Leu18I) has been substituted by a proline residue (OMTKY3-Pro18I) and in the second variant, the peptide bond between Thr17I and Leu18I was replaced by an ester bond (OMTKY3-psi[COO]-Leu18I). Both variants lack the P1 NH group that donates a bifurcated hydrogen bond to the carbonyl O of Ser214 and O(gamma) of the catalytic Ser195, one of the common interactions between serine proteinases and their canonical inhibitors. The SGPB:OMTKY3-Pro18I complex has many structural differences in the vicinity of the S1 pocket when compared with the previously determined structure of SGPB:OMTKY3-Leu18I. The result is a huge difference in the DeltaG degrees of binding (8.3 kcal/mol), only part of which can be attributed to the missing hydrogen bond. In contrast, very little structural difference exists between the complexes of SGPB:OMTKY3-psi[COO]-Leu18I and SGPB:OMTKY3-Leu18I, aside from an ester O replacing the P1 NH group. Therefore, the difference in DeltaG degrees, 1.5 kcal/mol as calculated from the measured equilibrium association constants, can be attributed to the contribution of the P1 NH hydrogen bond toward binding. A crystal structure of OMTKY3 having a reduced peptide bond between P1 Leu18I and P'1 Asp19I, (OMTKY3-psi[CH2NH2+]-Asp19I) has also been determined by X-ray crystallography. This variant has very weak association equilibrium constants with SGPB and with chymotrypsin. The structure of the free inhibitor suggests that the reduced peptide bond has not introduced any major structural changes in the inhibitor. Therefore, its poor ability to inhibit serine proteinases is likely due to the disruptions of the canonical interactions at the oxyanion hole.

Deleterious effects of beta-branched residues in the S1 specificity pocket of Streptomyces griseus proteinase B (SGPB): crystal structures of the turkey ovomucoid third domain variants Ile18I, Val18I, Thr18I, and Ser18I in complex with SGPB.

Turkey ovomucoid third domain (OMTKY3) is a canonical inhibitor of serine proteinases. Upon complex formation, the inhibitors fully exposed P1 residue becomes fully buried in the preformed cavity of the enzyme. All 20 P1 variants of OMTKY3 have been obtained by recombinant DNA technology and their equilibrium association constants have been measured with six serine proteinases. To rationalize the trends observed in this data set, high resolution crystal structures have been determined for OMTKY3 P1 variants in complex with the bacterial serine proteinase, Streptomyces griseus proteinase B (SGPB). Four high resolution complex structures are being reported in this paper; the three beta-branched variants, Ile18I, Val18I, and Thr18I, determined to 2.1, 1.6, and 1.7 A resolution, respectively, and the structure of the Ser18I variant complex, determined to 1.9 A resolution. Models of the Cys18I, Hse18I, and Ape18I variant complexes are also discussed. The beta-branched side chains are not complementary to the shape of the S1 binding pocket in SGPB, in contrast to that of the wild-type gamma-branched P1 residue for OMTKY3, Leu18I. Chi1 angles of approximately 40 degrees are imposed on the side chains of Ile18I, Val18I, and Thr18I within the S1 pocket. Dihedral angles of +60 degrees, -60 degrees, or 180 degrees are more commonly observed but 40 degrees is not unfavorable for the beta-branched side chains. Thr18I Ogamma1 also forms a hydrogen bond with Ser195 Ogamma in this orientation. The Ser18I side chain adopts two alternate conformations within the S1 pocket of SGPB, suggesting that the side chain is not stable in either conformation.

What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes?

Proteinases perform many beneficial functions that are essential to life, but they are also dangerous and must be controlled. Here we focus on one of the control mechanisms: the ubiquitous presence of protein proteinase inhibitors. We deal only with a subset of these: the standard mechanism, canonical protein inhibitors of serine proteinases. Each of the inhibitory domains of such inhibitors has one reactive site peptide bond, which serves all the cognate enzymes as a substrate. The reactive site peptide bond is in a combining loop which has an identical conformation in all inhibitors and in all enzyme-inhibitor complexes. There are at least 18 families of such inhibitors. They all share the conformation of the combining loops but each has its own global three-dimensional structure. Many three-dimensional structures of enzyme-inhibitor complexes were determined. They are frequently used to predict the conformation of substrates in very short-lived enzyme-substrate transition state complexes. Turkey ovomucoid third domain and eglin c have a Leu residue at P(1). In complexes with chymotrypsin, these P(1) Leu residues assume the same conformation. The relative free energies of binding of P(1) Leu (relative to either P(1) Gly or P(1) Ala) are within experimental error, the same for complexes of turkey ovomucoid third domain, eglin c, P(1) Leu variant of bovine pancreatic trypsin inhibitor and of a substrate with chymotrypsin. Therefore, the P(1) Leu conformation in transition state complexes is predictable. In contrast, the conformation of P(1) Lys(+) is strikingly different in the complexes of Lys(18) turkey ovomucoid third domain and of bovine pancreatic trypsin inhibitor with chymotrypsin. The relative free energies of binding are also quite different. Yet, the relative free energies of binding are nearly identical for Lys(+) in turkey ovomucoid third domain and in a substrate, thus allowing us to know the structure of the latter. Similar reasoning is applied to a few other systems.

Expression and characterization of elastase inhibitors from the ascarid nematodes Anisakis simplex and Ascaris suum.

Two elastase inhibitors, ASPI-1 and ASPI-2, from the parasitic nematode Anisakis simplex, have been isolated and characterized. Because these inhibitors are similar in size (60 amino acids in length) and primary sequence (52 and 47% identical) to the Ascaris suum chymotrypsin/elastase inhibitor-1 (AsC/E-1), we suggest that these Anisakis elastase inhibitors belong to the same unique class of canonical inhibitors formed by the family of Ascaris inhibitors (Huang K, Strynadka NCJ, Bernard VD, Peanasky RJ, James MG. Structure 1994;2:679-689). To compare ASPI-1 with AsC/E-1, we expressed both inhibitors in Pichia pastoris and found that: (1) the association constant of rASPI-1 with porcine pancreatic elastase (PPE) is similar to native inhibitor (Ka = 4.5 x 10(9) and 6.4 x 10(9) M(-1), respectively); (2) rASPI-1 is a potent inhibitor of PPE and human leukocyte elastase (Ka = 1.6 x 10(9) M(-1)); and (3) it is only a very weak inhibitor of chymotrypsin (CHYM) (Ka = 1.2 x 10(6) M(-1)). In contrast to the Anisakis inhibitor, however, rAsC/E inhibitor-1 is a very strong inhibitor of both PPE (Ka = 3.5 x 10(10) M(-1)) and CHYM (Ka = 3.6 x 10(12) M(-1)). We also found that the determined reactive sites (P1-P'1) of rASPI-1 and rAsC/E-1, as recognized by PPE, are Ala 28-Met 29 and Leu 31-Met 32, respectively. These P1-P'1 residues of AsC/E-1 constitute the same reactive site as that also recognized by CHYM (Peanasky RJ, Bentz Y, Homandberg GA, Minor ST, Babin DR. Arch Biochem Biophys 1994;232:135-142). The difference in specificities of ASPI-1 and AsC/E-1 toward their cognate serine proteases may be attributed to the P1 and P'3 residues in the inhibitors. Elastase, which recognizes both alanine and leucine, canaccommodate both ascarid inhibitors, whereas chymotrypsin, which prefers bulky, hydrophobic residues, only recognizes the Ascaris C/E inhibitor-1.

Use of fluorescence enhancement technique to study bilirubin-albumin interaction.

Bilirubin albumin solution gave an emission spectrum in the wavelength range 500-600 nm with emission maxima at 528 nm when excited at 487 nm. The magnitude of fluorescence intensity increased on increasing bilirubin/albumin molar ratio. At three different albumin concentrations, namely, 1.0, 2.5 and 10.0 microM, there was an initial linear increase in fluorescence up to a molar ratio 1.0 in all cases beyond which it sloped off or decreased. This fluorescence enhancement was used to calculate the binding parameters of bilirubin-albumin interaction and the value of binding constant was found to be 1.72 x 10(7) l/mol similar to the published values obtained with other methods. Different serum albumins, namely, human (HSA), goat (GSA), pig (PSA) and dog serum albumins (DSA) bound bilirubin with almost the same affinity when studied by the technique of fluorescence enhancement. Bilirubin albumin interaction was also studied at different pH and ionic strengths. There was a decrease in bilirubin-albumin complex formation on either decreasing the pH from 9.0 to 7.0 or increasing the ionic strength from 0.15 to 1.0. These results suggest that the technique of fluorescence enhancement can be used successfully to study the bilirubin-albumin interaction.

Molecular basis of indomethacin-human serum albumin interaction.

Studies on the strength and extent of binding of the non-steroidal anti-inflammatory drug indomethacin to human serum albumin (HSA) have provided conflicting results. In the present work, the serum-binding of indomethacin was studied in 55 mM sodium phosphate buffer (pH 7.0) at 28 degrees C, by using a fluorescence quench titration technique. The interaction of indomethacin with human serum albumin has been studied as a function of temperature, ionic strength and pH. The results suggest that electrostatic interaction plays a major role in the binding. The possible role of lysine residues in this interaction was studied by modifying exposed and buried lysine residues of HSA with potassium cyanate and studying indomethacin binding with the modified HSA. The data suggest that the interaction takes place via a salt bridge formation between the carboxylate group of indomethacin and a buried lysine residue of HSA. A technique involving fluorescence enhancement of bilirubin upon its interaction with HSA was used to study its displacement by indomethacin. The displacement, although apparently competitive in nature, was not strong suggesting that the primary sites of interaction of bilirubin and indomethacin are different.

Thermodynamic criterion for the conformation of P1 residues of substrates and of inhibitors in complexes with serine proteinases.

Eglin c, turkey ovomucoid third domain, and bovine pancreatic trypsin inhibitor (Kunitz) are all standard mechanism, canonical protein inhibitors of serine proteinases. Each of the three belongs to a different inhibitor family. Therefore, all three have the same canonical conformation in their combining loops but differ in their scaffoldings. Eglin c (Leu45 at P1) binds to chymotrypsin much better than its Ala45 variant (the difference in standard free energy changes on binding is -5.00 kcal/mol). Similarly, turkey ovomucoid third domain (Leu18 at P1) binds to chymotrypsin much better than its Ala18 variant (the difference in standard free energy changes on binding is -4.70 kcal/mol). As these two differences are within the +/-400 cal/mol bandwidth (expected from the experimental error), one can conclude that the system is additive. On the basis that isoenergetic is isostructural, we expect that within both the P1 Ala pair and the P1 Leu pair, the conformation of the inhibitor's P1 side chain and of the enzyme's specificity pocket will be identical. This is confirmed, within the experimental error, by the available X-ray structures of complexes of bovine chymotrypsin Aalpha with eglin c () and with turkey ovomucoid third domain (). A comparison can also be made between the structures of P1 (Lys+)15 of bovine pancreatic trypsin inhibitor (Kunitz) ( and ) and of the P1 (Lys+)18 variant of turkey ovomucoid third domain (), both interacting with chymotrypsin. In this case, the conformation of the side chains is strikingly different. Bovine pancreatic trypsin inhibitor with (Lys+)15 at P1 binds to chymotrypsin more strongly than its Ala15 variant (the difference in standard free energy changes on binding is -1.90 kcal/mol). In contrast, turkey ovomucoid third domain variant with (Lys+)18 at P1 binds to chymotrypsin less strongly than its Ala18 variant (the difference in standard free energies of association is 0.95 kcal/mol). In this case, P1 Lys+ is neither isostructural nor isoenergetic. Thus, a thermodynamic criterion for whether the conformation of a P1 side chain in the complex matches that of an already determined one is at hand. Such a criterion may be useful in reducing the number of required X-ray crystallographic structure determinations. More importantly, the criterion can be applied to situations where direct determination of the structure is extremely difficult. Here, we apply it to determine the conformation of the Lys+ side chain in the transition state complex of a substrate with chymotrypsin. On the basis of kcat/KM measurements, the difference in free energies of activation for Suc-AAPX-pna when X is Lys+ and X is Ala is 1.29 kcal/mol. This is in good agreement with the corresponding difference for turkey ovomucoid third domain variants but in sharp contrast to the bovine pancreatic trypsin inhibitor (Kunitz) data. Therefore, we expect that in the transition state complex of this substrate with chymotrypsin, the P1 Lys+ side chain is deeply inserted into the enzyme's specificity pocket as it is in the (Lys+)18 turkey ovomucoid third domain complex with chymotrypsin.

Interaction of bromocresol green with different serum albumins studied by fluorescence quenching.

The binding of bromocresol green to bovine serum albumin at micromolar concentrations leads to quenching of protein fluorescence. This property has been used here to study interaction of bromocresol green with bovine serum albumin as a function of pH and ionic strength. The transformation of fluorescence quench data obtained with bromocresol green into Scatchard plots yielded an association constant of 3.06 x 10(7) 1 M-1 and a binding capacity of about 1.0. The affinity of bromocresol green for bovine serum albumin remains virtually unchanged between pH 4.0 and 8.0 but decreases by about 7 fold with increase in ionic strength from 0.01 to 1.0. Six other serum albumins obtained from cat, dog, human, pig and sheep have also been studied for bromocresol green binding. Although all the albumins studied bind bromocresol green, they show considerable differences in their affinities towards the dye. It appears that despite a great degree of overall similarity in their structure and conformation, serum albumins from different species differ in their ligand binding properties.