A hetero-micro-seeding strategy for readily crystallizing closely related protein variants.

Protein crystallization remains difficult to rationalize and screening for optimal crystallization conditions is a tedious and time consuming procedure. Here, we report a hetero-micro-seeding strategy for producing high resolution crystals of closely related protein variants, where micro crystals from a readily crystallized variant are used as seeds to develop crystals of other variants less amenable to crystallization. We applied this strategy to Bovine Pancreatic Trypsin Inhibitor (BPTI) variants, which would not crystallize using standard crystallization practice. Out of six variants in our analysis, only one called BPTI-[5,55]A14G formed well behaving crystals; and the remaining five (A14GA38G, A14GA38V, A14GA38L, A14GA38I, and A14GA38K) could be crystallized only using micro-seeds from the BPTI-[5,55]A14G crystal. All hetero-seeded crystals diffracted at high resolution with minimum mosaicity, retaining the same space group and cell dimension. Moreover, hetero-micro-seeding did not introduce any biases into the mutant's structure toward the seed structure, as demonstrated by A14GA38I structures solved using micro-seeds from A14GA38G, A14GA38L and A14GA38I. Though hetero-micro-seeding is a simple and almost naïve strategy, this is the first direct demonstration of its workability. We believe that hetero-micro-seeding, which is contrasting with the popular idea that crystallization requires highly purified proteins, could contribute a new tool for rapidly solving protein structures in mutational analysis studies.

Immobilization of aprotinin to fibrinogen as a novel method for controlling degradation of fibrin gels.

The goal of this work was to demonstrate that aprotinin conjugated to fibrinogen could (1) maintain its function and (2) control fibrin degradation. Using the chick chorioallantoic membrane (CAM) assay, we found that blood vessels did not directly invade fibrin constructs containing immobilized fibroblast growth factor-2. Because the fibrin quickly degraded within approximately 5 days, we hypothesized that controlling fibrinolysis may improve direct blood vessel invasion. Aprotinin, a protease inhibitor typically added to slow fibrinolysis, is a small protein and can diffuse out of the gel resulting in the loss of fibrinolysis protection. Therefore, using a novel synthesis strategy, aprotinin and a fluorescent reporter, Cy3, were chemically conjugated to fibrinogen. In vitro microplate absorbance assays showed that the conjugated aprotinin was able to inhibit plasmin-mediated fibrin degradation and that its activity was comparable to equimolar levels of soluble, nonconjugated aprotinin. Additionally, we found that fibrinolysis rates could be tuned by varying the level of conjugated aprotinin within the gel. The conjugated aprotinin also demonstrated functionality in vivo. In the chick CAM assay, fibrin gels containing conjugated aprotinin were approximately 5 times larger than gels containing soluble aprotinin after 4 days. Also, in support of our hypothesis, we found that immobilized aprotinin within fibrin gels demonstrated substantial blood vessel invasion.

An improved (99m)Tc-aprotinin kit formulation: quality control analysis of radiotracer stability and cold kit shelf life.

(99m)Tc-aprotinin scintigraphy has been demonstrated to be a useful noninvasive imaging technique for amyloid deposits located in extraabdominal regions of patients. The aim of this study was to develop an improved aprotinin cold kit formulation, to validate the kit for long-term stability, as well as to assess the radiotracer stability by novel quality control methods. The aprotinin cold kit formulation of Trasylol, pyrophosphate (PYP)-chelated stannous reductant and an alkaline buffer, was dispensed into nitrogen-filled vials and aliquots frozen at -20 degrees C. After 0, 1, 2, 3 and 6 months of storage, three samples were reconstituted with 750-850 MBq of (99m)Tc-pertechnetate, followed by quality control analyses by paper chromatography methods at 25, 85 and 265 min postreconstitution (pr). Cation-exchange cartridge quality control methods were also investigated. The cold kits proved to be stable to long-term storage for up to 6 months, and the radiotracer was stable for at least 4 h pr. (99m)Tc-aprotinin was formed at greater than 95% efficiency at all time points tested with (99m)TcO2 present as the major impurity (1-4%) and (99m)Tc-pertechnetate and (99m)Tc-PYP present in trace amounts. An alternative, rapid, safe and reliable method was found in Oasis MCX-BSA-treated cartridges using saline as the eluting solution to assay for (99m)Tc-aprotinin.

Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nalpha-9-fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of side-chain and backbone anchoring strategies, and compatible protection for N-terminal cysteine.

Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C-terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N(alpha)-9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N(alpha)-t-butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side-chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N-terminal cysteine residue have been developed: (i) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-(N-methyl-N-phenylcarbamoyl)sulfenylcysteine [Fmoc-Cys(Snm)-OH], allowing creation of an otherwise fully protected resin-bound intermediate with N-terminal free Cys; (ii) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-triphenylmethylcysteine [Fmoc-Cys(Trt)-OH], generating a stable Fmoc-Cys(H)-peptide upon acidolytic cleavage; and (iii) incorporation of N(alpha)-t-butyloxycarbonyl-S-fluorenylmethylcysteine [Boc-Cys(Fm)-OH], generating a stable H-Cys(Fm)-peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C-termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on-resin, as well as in solution after cleavage/purification.

Native-like conformations are sampled by partially folded and disordered variants of bovine pancreatic trypsin inhibitor.

Partially folded conformational ensembles of bovine pancreatic trypsin inhibitor (BPTI) are accessed by replacing Cys 5, 30, 51, and 55 by alpha-amino-n-butyric acid (Abu) while retaining the disulfide between Cys 14 and 38; the resultant variant is termed [14-38](Abu). Two new analogues with modifications in the beta-turn, P26D27[14-38](Abu) and N26G27K28[14-38](Abu), are compared to partially folded [14-38](Abu), as well as to [R](Abu), the unfolded protein with all six Cys residues replaced by Abu. Structural features of the new analogues of [14-38](Abu) have been determined by circular dichroism (CD), one-dimensional (1)H NMR, and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence experiments. Both analogues are more disordered than the parent [14-38](Abu), but while P26D27[14-38](Abu) has a small population of native-like conformations observed by NMR, no ordered structure is detected for N26G27K28[14-38](Abu). Trypsin inhibition assays were carried out using a modified rat trypsin, C191A/C220A, that minimizes cleavage of unfolded peptides. Both [14-38](Abu) and P26D27[14-38](Abu) significantly inhibit modified trypsin. N26G27K28[14-38](Abu) has low but measurable inhibitor activity, while [R](Abu) has no activity even when in very high molar excess relative to trypsin. ANS fluorescence is enhanced by [14-38](Abu) and by both variants but not by [R](Abu). We conclude that partially folded ensembles of BPTI, even those with little or no CD- or NMR-detectable structure, contain minor populations of native-like conformations. Partially folded [14-38](Abu) and both variants, as well as [R](Abu), have enhanced negative ellipticity in CD spectra acquired in the presence of the osmolyte trimethylamine N-oxide (TMAO). TMAO-induced structure is formed cooperatively, as indicated by thermal unfolding curves. Inhibitor activity as a function of TMAO concentration implies that the osmolyte-induced structure is native-like for [14-38](Abu) and P26D27[14-38](Abu) and is probably native-like for N26G27K28[14-38](Abu). [R](Abu) also shows increased CD-detected structure in the presence of TMAO, but such structure is likely to be collapsed and non-native.

In vitro inhibition of bovine herpes virus 1 reproduction with native and microencapsulated proteinase inhibitor aprotinin.

This study evaluated the antiviral effect of various dosage forms of proteinase inhibitor-aprotinin as a potential remedy for prophylactics and therapy of infectious bovine rhinotracheitis. Formulations of the inhibitor were tested for their influence on bovine herpes virus reproduction in cell cultures. Starch/bovine serum albumin microcapsules with aprotinin were prepared using interfacial cross-linking with terephthaloyl chloride and characterized for their morphology, size and release of the inhibitor. Two types of these microcapsules-impregnated and loaded with the inhibitor-were used in virus infectious studies. Native aprotinin possessed palpable dose-dependent antiviral effect inhibiting the virus reproduction up to 4.0 lg (10000-fold) and delaying the cytopathic effect up to 96 h in the concentration 800-3300 TIU/ml. The bioadhesive, biodegradable aprotinin-loaded microcapsules were the most effective antiviral drug as this formulation allowed to decrease virus infectious titer up to 4.0 lg and a delay in the cytopathic effect of up to 144 h in lesser doses of inhibitor compared with the native form. In comparison the antiviral effect of microcapsules impregnated with aprotinin was not so appreciable. It was interesting to note that the results of the experiments on diverse cultures were very similar. This was because the drugs influenced the fundamental processes of virus replication cycle.

Improved synthesis of C-terminal peptide thioesters on "safety-catch" resins using LiBr/THF.

[reaction: see text] The alkanesulfonamide "safety-catch" resin has proven useful for Fmoc-based synthesis of C-terminal peptide thioesters. We now report that the yield of isolated thioester can increase significantly when the cleavage reaction is carried out in 2 M LiBr/THF rather than DMF or THF. The largest effects are seen with problematic peptides that aggregate or form secondary structures on the resin.

Toward new designed proteins derived from bovine pancreatic trypsin inhibitor (BPTI): covalent cross-linking of two 'core modules' by oxime-forming ligation.

A 25-residue disulfide-cross-linked peptide, termed 'oxidized core module' (OxCM), that includes essentially all of the secondary structural elements of bovine pancreatic trypsin inhibitor (BPTI) most refractory to hydrogen exchange, was shown previously to favor nativelike beta-sheet structure [Carulla, N., Woodward, C., and Barany, G. (2000) Synthesis and Characterization of a beta-Hairpin Peptide That Represents a 'Core Module' of Bovine Pancreatic Trypsin Inhibitor (BPTI). Biochemistry 39, 7927-7937]. The present work prepares to explore the hypothesis that the energies of nativelike conformations, relative to other possible conformations, could be decreased further by covalent linkage of two OxCMs. Optimized syntheses of six approximately 50-residue OxCM dimers are reported herein, featuring appropriate monomer modifications followed by oxime-forming ligation chemistry to create covalent cross-links at various positions and with differing lengths. Several side reactions were recognized through this work, and modified procedures to lessen or mitigate their occurrence were developed. Particularly noteworthy, guanidine or urea denaturants that were included as peptide-solubilizing components for some reaction mixtures were proven to form adducts with glyoxylyl moieties, thus affecting rates and outcomes. All six synthetic OxCM dimers were characterized by 1D (1)H NMR; three of them showed considerable chemical shift dispersion suggestive of self-association and mutual stabilization between the monomer units.

13C NMR, X-ray, and differential scanning calorimetry investigations of truncated BPTI (aprotinin) analogues.

Truncated BPTI missing residues 1 and 2 is investigated together with variants thereof (Lys-15, Arg-17, and Arg-42 are replaced by other residues in various combinations). A comparison of the X-ray structure of BPTI with that of 3-58BPTI(K15R,R17A,R42S) shows only minor variations for the backbone, but the lack of salt bridge between the terminals and the lack of two N-terminal residues provide a structure open at one end. Comparisons of amide exchange rates show a dramatic increase for the most slowly exchanging NH protons of 3-58BPTI and the analogues thereof, as compared to those of the wild-type despite only small differences in the structures. The amide exchange rates for truncated analogues increase with decreasing TTEP (temperature top endothermic peak) values. On the basis of the known structural changes comparisons to 13C chemical shifts are made. 13C chemical shifts are assigned using the D-isotope and HMBC techniques. Excellent resolution is obtained in these 1D natural abundance spectra. 13C NMR chemical shifts are shown to be able to gauge structural changes. A comparison of 13C chemical shifts of WT BPTI (aprotinin) and 3-58BPTI reveals effects caused by (i) the removal of the salt bridge of the terminii, (ii) the charge of the N-terminus, and (iii) the increased mobility of the side chain of Tyr-23. Small effects are also seen due to a conformational change of the aromatic ring of Phe-4. Ring current shifts at 13C chemical shifts are calculated. The difference in the calculated ring current effects are small comparing the wild-type with 3-58BPTI(K15R,R17A,R42S) provided the structures are relaxed. Protein unfolding as a function of pH and temperature is studied by DSC. Unfolding occurs at lower temperature with N-terminally truncated analogues, and the maximum is shifted toward higher pH.

Reduction of the infectivity of scrapie agent as a model for BSE in the manufacturing process of Trasylol.

The Trasylol manufacturing process was investigated with respect to its capacity for the inactivation/removal of infectivity causing bovine spongiform encephalopathy (BSE). Four process steps were selected for this investigation and scaled down to laboratory scale. Authentic samples of bovine lungs used in the Trasylol manufacturing plant were taken and spiked in laboratory scale experiments with high infectious titres of the rodent adapted scrapie strain ME 7 which served as model for BSE. After performing the respective process steps the output samples collected were tested in C57BL mice carrying the Sinc gene. An overall reduction of the infectious agent in the order of 18 log10 was observed, indicating a very high capacity of the Trasylol process for the inactivation/removal of the BSE/scrapie agent. The discussed safety strategy for the product leads to the conclusion that Trasylol is BSE safe.

Unfolded BPTI variants with a single disulfide bond have diminished non-native structure distant from the crosslink.

BACKGROUND: NMR studies of denatured states, both fully unfolded and partially folded, give insight into the conformations and interactions favored in initial stages of folding, and in early intermediates formed during folding. We have characterized non-random structures favored in unfolded, reduced BPTI [1], and in partially folded BPTI [2]. Here, we report NMR-detected structure of two analogs of unfolded BPTI with one native 14-38 disulfide bond. RESULTS: Analogs Y21A[14-38]Abu and Y23A[14-38]Abu, obtained by chemical synthesis of [14-38]Abu with Y21 or Y23 replaced by alanine, are models for unfolded BPTI with 14-38 the only disulfide. Compared to unfolded BPTI with all three disulfides broken, the unfolded 14-38 BPTI analogs have numerous differences, including loss of non-native, turn-like conformations for beta 2 residues, diminished non-native aromatic-aliphatic NOEs, and increased intermediate chemical exchange of residues that have native-like conformations in partially folded BPTI. Although the Y21A and Y23A analogs have similar CD and NMR properties, specific differences in NOE patterns and in exchange broadening are observed. CONCLUSIONS: Changes in unfolded BPTI associated with formation of the 14-38 disulfide bond are consistent with less non-native structure, and more native-like structure, in residues composing the stable core of antiparallel beta-sheet in partially folded BPTI. Specific differences between Y21A[14-38]Abu and Y23A[14-38]Abu indicate that replacement of Y23 results in less ordered structure than replacement of Y21.

BPTI backbone variants and implications for inhibitory activity.

Structural variants of BPTI were synthesized en route an enzymatic-chemical semisynthesis. The P1-P2 amide bond of the inhibitor molecule, which, as donor, contributes a hydrogen bond towards trypsin in the enzyme-inhibitor complex, was replaced by either a ketomethylene function or an ester bond yielding molecules with inhibitory activity. The two backbone-mutated BPTI derivatives showed increased dissociation constants of their respective trypsin complexes, obviously due to the lack of a single hydrogen-bond interaction in the enzyme-inhibitor complex.

Solid-phase synthesis of bovine pancreatic trypsin inhibitor (BPTI) and two analogues. A chemical approach for evaluating the role of disulfide bridges in protein folding and stability.

The linear sequence of bovine pancreatic trypsin inhibitor (BPTI) has been assembled by stepwise Fmoc solid-phase peptide synthesis on a polyethylene glycol-polystyrene (PEG-PS) graft support with p-alkoxybenzyl ester anchoring. Similar methods were used to prepare two analogues, the first with all six half-cystine (Cys) residues replaced by alpha-amino-n-butyric acid (Abu), and the second with replacement of Abu at four Cys positions while retaining the native pairing between positions 14 and 38. Following cleavage from the support, the linear molecules (reduced form) were purified by semipreparative reversed-phase high performance liquid chromatography (HPLC). The native structure of BPTI was then formed by oxidation of a dilute solution of the protein at pH 8.7 in the presence of oxidized glutathione. The BPTI analogue with one disulfide bridge was obtained following treatment with dimethyl sulfoxide (DMSO)-pH 6 buffer (1:9). Overall yields of homogeneous proteins were 2-4%, and further characterization was provided by amino acid analysis, sequencing, ion electrospray mass spectrometry, analytical HPLC, and capillary zone electrophoresis (CZE). Purified synthetic BPTI with the native sequence was indistinguishable from natural material by the analytical and biophysical criteria applied, including circular dichroism (CD) spectra and inhibition of trypsin action. Studies are in progress to evaluate conformational features of the analogues which respectively lack two, or all three, of the native disulfide bridges.

Resolution of proline acylation problem for thiol capture strategy by use of a chloro-dibenzofuran template.

The acyl transfer rate for proline, in the prior thiol capture strategy, was enhanced by changing the electronic character of the dibenzofuran template. The rate of amide bond formation between proline and cysteine by the 1-chloro-4-hydroxy-6-mercaptodibenzofuran was measured to be 0.012 min-1, which translates to a half-life of 53 min. Further enhancement of the reaction rate was accomplished by the use of a 1,3-dichloro-dibenzofuran template. The k1 for the reaction was measured to be 0.093 min-1, and the half-life was calculated to be 7 min. To test the applicability of the activated template, 1-chloro-4-hydroxy-6-mercaptodibenzofuran, in peptide synthesis, the 34 amino acid long peptide, H-RPDFCLEPPYTGPCRKARNNFKSADECMRTCGGA-OH, was synthesized. This peptide represents the condensation of the N-terminal 13-mer and the C-terminal 21-mer of the basic pancreatic trypsin inhibitor.

Chemical semisynthesis of aprotinin homologues and derivatives mutated in P' positions.

An extended concept for the replacement of amino acids in the P' region of aprotinin by chemical semisynthesis is presented. Either fragment condensation with dipeptides protected as tert-butyl ester or stepwise introduction of two single amino acid-tert-butyl esters into a partially esterified aprotinin derivative (with free Lys15-carboxyl group) lacking the amino acids Ala16 and Arg17 leads to aprotinin homologues and derivatives mutated in the P'1 and P'2 position. This method may complement the recently reported enzymatic synthesis by enabling access to aprotinin homologues and derivatives, which cannot be prepared enzymatically. The synthesis of [Ala17]BPTI and [seco-17/18]BPTI is described in detail.

Enzymatic semisynthesis of aprotinin homologues mutated in P' positions.

The replacement of amino acids in the P'1 and P'2 position of aprotinin, the bovine pancreatic trypsin inhibitor, is described. Using the "modified" inhibitor as starting material, with the hydrolyzed reactive-site peptide bond Lys15-Ala16, the residues P'1 (Ala16) and P'2 (Arg17) were split off by the action of aminopeptidase K. Incorporation of suitable dipeptides containing a basic residue (Lys or Arg) in the C-terminal position was carried out in a "one pot" reaction involving trypsin-catalyzed coupling. In this way, the native fragment Ala16-Arg17 was reintroduced and also replaced by Gly-Arg, Ala-Lys, and Leu-Arg yielding intact inhibitor molecules. The mechanism for incorporation of dipeptides was investigated by treating the aprotinin derivative with the Arg17-Ile18 peptide bond hydrolyzed with trypsin under proteosynthetic conditions. We established that only inhibitor molecules cleaved between Lys15 and Xaa16 are intermediates leading to the desired products. The inhibitory properties of the new aprotinin homologues were tested, and the significance of the P'1 residue for the inhibition of trypsin, kallikrein, and chymotrypsin was deduced.

Enzymatic fragment substitution as a tool in protein design.

An easy and rapid enzymatic method is described which allows replacement of P'-residues in bovine pancreatic trypsin inhibitor. Insertion of Xaa-Arg or Xaa-Lys into a BPTI fragment lacking P1' = Ala16 and P2' = Arg17 was carried out in a "one pot" reaction catalysed by trypsin in the presence of 80% 1,4 butanediol.

Semisynthetic aprotinin derivatives with specific alterations at the reactive-site peptide bond can be used to study structure-function relationships.

Aprotinin derivatives with decarboxylated lysine, arginine or valine at position 15, the P1 position of modified aprotinin, were produced semisynthetically. Modified aprotinin with oxidatively deaminated Arg1 and Ala16 was also synthesized. Specific reduction of this derivative yielded a modified aprotinin with lactic acid at position 16, the P'1 position. Only the aprotinin derivatives with decarboxylated Lys15 or Arg15 showed moderate inhibitory activity against trypsin and kallikrein, despite the absence of the carboxyl group. The KD values measured were in the range of 10(-7) M. The aprotinin derivative with decarboxylated valine showed no inhibitory activity; neither against trypsin, kallikrein and chymotrypsin, nor against the human leukocyte elastase. From these data it was concluded that the ion-pair interaction of the Lys15, or the Arg15 inhibitor side-chain with the aspartate in the trypsin specificity pocket is important for the inhibitory activity. Furthermore, the KD values indicated that the interaction of the reactive-site's carbonyl group with the enzyme's oxyanion hole also contributes to the inhibitory activity. These two interactions are important, but not essential for inhibitory activity. In contrast to these findings, the existence of an alpha-amino group at the P'1 position seems to be essential for inhibitory activity. The synthesized aprotinin derivatives lacking an alpha-amino group at this position were without any inhibitory activity against serine proteinases.

Semisynthesis of Arg15, Glu15, Met15, and Nle15-aprotinin involving enzymatic peptide bond resynthesis.

The semisynthesis of homologues of aprotinin, the bovine pancreatic trypsin inhibitor, is described. The P1 lysine15 residue was replaced by two methods. The first procedure, which consisted of two enzymatic steps for the incorporation of other amino acids has previously been described. The second approach consisted of six steps of both enzymatic and chemical nature. The modified inhibitor, in which the lysine15-alanine16 peptide bond is hydrolyzed, was used as the starting material. All carboxyl groups of the modified inhibitor were esterified with methanol; the lysine15 methylester group was then selectively hydrolyzed. Afterward, lysine15 itself was split off. Arginine, glutamic acid, methionine, and L-2-aminohexanoic acid (norleucine, Nle) were incorporated using water-soluble carbodiimide combined with an acylation catalyst. The methylester group was used to prevent polymerization. The reactive-site peptide bonds were resynthesized using either chymotrypsin or trypsin.