Tryptic Cleavage Research Articles

Retinal Pigment Epithelium Lipofuscin Proteomics

Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

Catecholaminergic polymorphic ventricular tachycardia-related mutations R33Q and L167H alter calcium sensitivity of human cardiac calsequestrin

Two missense mutations, R33Q and L167H, of hCASQ2 (human cardiac calsequestrin), a protein segregated to the lumen of the sarcoplasmic reticulum, are linked to the autosomal recessive form of CPVT (catecholaminergic polymorphic ventricular tachycardia). The effects of these mutations on the conformational, stability and Ca(2+) sensitivity properties of hCASQ2, were investigated. Recombinant WT (wild-type) and mutant CASQ2s were purified to homogeneity and characterized by spectroscopic (CD and fluorescence) and biochemical (size-exclusion chromatography and limited proteolysis) methods at 500 and 100 mM KCl, with or without Ca(2+) at a physiological intraluminal concentration of 1 mM; Ca(2+)-induced polymerization properties were studied by turbidimetry. In the absence of Ca(2+), mutations did not alter the conformation of monomeric CASQ2. For L167H only, at 100 mM KCl, emission fluorescence changes suggested tertiary structure alterations. Limited proteolysis showed that amino acid substitutions enhanced the conformational flexibility of CASQ2 mutants, which became more susceptible to tryptic cleavage, in the order L167H>R33Q>WT. Ca(2+) at a concentration of 1 mM amplified such differences: Ca(2+) stabilized WT CASQ2 against urea denaturation and tryptic cleavage, whereas this effect was reduced in R33Q and absent in L167H. Increasing [Ca(2+)] induced polymerization and precipitation of R33Q, but not that of L167H, which was insensitive to Ca(2+). Based on CASQ2 models, we propose that the Arg(33)-->Gln exchange made the Ca(2+)-dependent formation of front-to-front dimers more difficult, whereas the Leu(167)-->His replacement almost completely inhibited back-to-back dimer interactions. Initial molecular events of CPVT pathogenesis begin to unveil and appear to be different depending upon the specific CASQ2 mutation.

A novel A121T mutation in human cationic trypsinogen associated with hereditary pancreatitis: functional data indicating a loss-of-function mutation influencing the R122 trypsin cleavage site

Background:The understanding of genetic risk factors for chronic pancreatitis increased in the last decade with the discovery of mutations in the cationic trypsinogen gene (PRSS1). The first mutation was detected...

Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics.

Bile acids and bile alcohols in the form of their conjugates are amphipathic end products of cholesterol metabolism with multiple physiological functions. The great variety of bile acids and bile alcohols that are present in vertebrates are tabulated. Bile salts have an enterohepatic circulation resulting from efficient vectorial transport of bile salts through the hepatocyte and the ileal enterocyte; such transport leads to the accumulation of a pool of bile salts that cycles between the liver and intestine. Bile salt anions promote lipid absorption, enhance tryptic cleavage of dietary proteins, and have antimicrobial effects. Bile salts are signaling molecules, activating nuclear receptors in the hepatocyte and ileal enterocyte, as well as an increasing number of G-protein coupled receptors. Bile acids are used therapeutically to correct deficiency states, to decrease the cholesterol saturation of bile, or to decrease the cytotoxicity of retained bile acids in cholestatic liver disease.

Hydrophilic Interaction Chromatography Reduces the Complexity of the Phosphoproteome and Improves Global Phosphopeptide Isolation and Detection

The diversity and complexity of proteins and peptides in biological systems requires powerful liquid chromatography-based separations to optimize resolution and detection of components. Proteomics strategies often combine two orthogonal separation modes to meet this challenge. In nearly all cases, the second dimension is a reverse phase separation interfaced directly to a mass spectrometer. Here we report on the use of hydrophilic interaction chromatography (HILIC) as part of a multidimensional chromatography strategy for proteomics. Tryptic peptides are separated on TSKgel Amide-80 columns using a shallow inverse organic gradient. Under these conditions, peptide retention is based on overall hydrophilicity, and a separation truly orthogonal to reverse phase is produced. Analysis of tryptic digests from HeLa cells yielded numbers of protein identifications comparable to that obtained using strong cation exchange. We also demonstrate that HILIC represents a significant advance in phosphoproteomics analysis. We exploited the strong hydrophilicity of the phosphate group to selectively enrich and fractionate phosphopeptides based on their increased retention under HILIC conditions. Subsequent IMAC enrichment of phosphopeptides from HILIC fractions showed better than 99% selectivity. This was achieved without the use of derivatization or chemical modifiers. In a 300-microg equivalent of HeLa cell lysate we identified over 1000 unique phosphorylation sites. More than 700 novel sites were added to the HeLa phosphoproteome.

Absolute quantification of cardiac troponin T by means of liquid chromatography/triple quadrupole tandem mass spectrometry

A liquid chromatography/tandem mass spectrometric method for absolute quantification of cardiac troponin T (cTnT) in mouse heart tissue is presented. Even in such a complex biological sample, the multiple reaction monitoring acquisition mode allowed the selective and sensitive determination of a specific peptide, obtained by cTnT enzymatic digestion. The concentration of this cTnT-specific peptide was considered as a representation of the concentration of its parent protein. Quantification was carried out by means of the matrix-matched calibration curve, constructed by adding the synthetic standard of the target peptide and another synthetic structurally analogous peptide as internal standard. Method identification limit and method quantification limit were estimated as 60 and 110 ng of cTnT per mg of total extracted proteins, respectively. The developed label-free approach has been applied for the absolute quantitation of cTnT because of its diagnostic and prognostic value as cardiac disease marker. However, the method could be of general application, since it requires only the synthesis of two suitable peptides, a protein tryptic cleavage product and an internal standard.

Conserved Asp-137 Imparts Flexibility to Tropomyosin and Affects Function

Tropomyosin (Tm) is an alpha-helical coiled-coil that controls muscle contraction by sterically regulating the myosin-actin interaction. Tm moves between three states on F-actin as either a uniform or a non-uniform semi-flexible rod. Tm is stabilized by hydrophobic residues in the "a" and "d" positions of the heptad repeat. The highly conserved Asp-137 is unusual in that it introduces a negative charge on each chain in a position typically occupied by hydrophobic residues. The occurrence of two charged residues in the hydrophobic region is expected to destabilize the region and impart flexibility. To determine whether this region is unstable, we have substituted hydrophobic Leu for Asp-137 and studied changes in Tm susceptibility to limited proteolysis by trypsin and changes in regulation. We found that native and Tm controls that contain Asp-137 were readily cleaved at Arg-133 with t 1/2 of 5 min. In contrast, the Leu-137 mutant was not cleaved under the same conditions. Actin stabilized Tm, causing a 10-fold reduction in the rate of cleavage at Arg-133. The actin-myosin subfragment S1 ATPase activity was greater for the Leu mutant compared with controls in the absence of troponin and in the presence of troponin and Ca2+. We conclude that the highly conserved Asp-137 destabilizes the middle of Tm, resulting in a more flexible region that is important for the cooperative activation of the thin filament by myosin. We thus have shown a link between the dynamic properties of Tm and its function.

Characterization of the Human Skeletal Muscle Proteome by One-dimensional Gel Electrophoresis and HPLC-ESI-MS/MS

Changes in protein abundance in skeletal muscle are central to a large number of metabolic and other disorders, including, and perhaps most commonly, insulin resistance. Proteomics analysis of human muscle is an important approach for gaining insight into the biochemical basis for normal and pathophysiological conditions. However, to date, the number of proteins identified by this approach has been limited, with 107 different proteins being the maximum reported so far. Using a combination of one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, we identified 954 different proteins in human vastus lateralis muscle obtained from three healthy, nonobese subjects. In addition to a large number of isoforms of contractile proteins, we detected all proteins involved in the major pathways of glucose and lipid metabolism in skeletal muscle. Mitochondrial proteins accounted for 22% of all proteins identified, including 55 subunits of the respiratory complexes I-V. Moreover, a number of enzymes involved in endocrine and metabolic signaling pathways as well as calcium homeostasis were identified. These results provide the most comprehensive characterization of the human skeletal muscle proteome to date. These data hold promise for future global assessment of quantitative changes in the muscle proteome of patients affected by disorders involving skeletal muscle.

Enzyme Kinetics and Characterization of Mouse Pancreatic Elastase

In the present study we have purified and characterized murine pancreatic elastase. The enzyme was extracted from acetone powders of mouse pancreas, fractionally precipitated with ammonium sulfate, and further purified by ion exchange chromatography to a single band on SDS-PAGE. The mouse enzyme exists in a proform, which was activated by removing a signal peptide by tryptic cleavage. The active form of mouse pancreatic elastase was shown by ultracentrifugation to have a molecular weight of 25.9 kDa and a frictional ratio of 1.26. The pH optimum for proteolytic activity was 8.0. Kinetic measurements were made with a variety of substrates and inhibitors and compared with elastases from other sources. The enzymatic properties and kinetic profiles for mouse pancreatic elastase were similar to other known serine elastases.

Zn2+ Enhances the Molecular Chaperone Function and Stability of α-Crystallin

Alpha-crystallin, the major eye lens protein, is a molecular chaperone that plays a crucial role in the suppression of protein aggregation and thus in the long-term maintenance of lens transparency. Zinc is a micronutrient of the eye, but its molecular interaction with alpha-crystallin has not been studied in detail. In this paper, we present results of in vitro experiments that show bivalent zinc specifically interacts with alpha-crystallin with a dissociation constant in the submillimolar range (Kd approximately 0.2-0.4 mM). We compared the effect of Zn2+ with those of Ca2+, Cu2+, Mg2+, Cd2+, Pb2+, Ni2+, Fe2+, and Co2+ at 1 mM on the structure and chaperoning ability of alpha-crystallin. An insulin aggregation assay showed that among the bivalent metal ions, only 1 mM Zn2+ improved the chaperone function of alpha-crystallin by 30% compared to that in the absence of bivalent metal ions. Addition of 1 mM Zn2+ increased the yield of alpha-crystallin-assisted refolding of urea-treated LDH to its native state from 33 to 38%, but other bivalent ions had little effect. The surface hydrophobicity of alpha-crystallin was increased by 50% due to the binding of Zn2+. In the presence of 1 mM Zn2+, the stability of alpha-crystallin was enhanced by 36 kJ/mol, and it became more resistant to tryptic cleavage. The implications of enhanced stability and molecular chaperone activity of alpha-crystallin in the presence of Zn2+ are discussed in terms of its role in the long-term maintenance of lens transparency and cataract formation.

Does Trypsin Cut Before Proline?

Trypsin is the most commonly used enzyme in mass spectrometry for protein digestion with high substrate specificity. Many peptide identification algorithms incorporate these specificity rules as filtering criteria. A generally accepted "Keil rule" is that trypsin cleaves next to arginine or lysine, but not before proline. Since this rule was derived two decades ago based on a small number of experimentally confirmed cleavages, we decided to re-examine it using 14.5 million tandem spectra (2 orders of magnitude increase in the number of observed tryptic cleavages). Our analysis revealed a surprisingly large number of cleavages before proline. We examine several hypotheses to explain these cleavages and argue that trypsin specificity rules used in peptide identification algorithms should be modified to "legitimatize" cleavages before proline. Our approach can be applied to analyze any protease, and we further argue that specificity rules for other enzymes should also be re-evaluated based on statistical evidence derived from large MS/MS data sets.

Activation Profiles and Regulatory Cascades of the Human Kallikrein-related Peptidases

The human kallikrein (KLK)-related peptidases are the largest family of serine peptidases, comprising 15 members (KLK1-15) and with the majority (KLK4-15) being identified only within the last decade. Members of this family are associated with important diseased states (including cancer, inflammation, and neurodegeneration) and have been utilized or proposed as clinically important biomarkers or therapeutic targets of interest. All human KLKs are synthesized as prepro-forms that are proteolytically processed to secreted pro-forms via the removal of an amino-terminal secretion signal peptide. The secreted inactive pro-KLKs are then activated extracellularly to mature peptidases by specific proteolytic release of their amino-terminal propeptide. Although a key step in the regulation of KLK function, details regarding the activation of the human pro-KLKs (i.e. the KLK "activome") are unknown, to a significant extent, but have been postulated to involve "activation cascades" with other KLKs and endopeptidases. To characterize more completely the KLK activome, we have expressed from Escherichia coli individual KLK propeptides fused to the amino terminus of a soluble carrier protein. The ability of 12 different mature KLKs to process the 15 different pro-KLK peptide sequences has been determined. Various autolytic and cross-activation relationships identified using this system have subsequently been characterized using recombinant pro-KLK proteins. The results demonstrate the potential for extensive KLK activation cascades and, when combined with available data for the tissue-specific expression of the KLK family, permit the construction of specific regulatory cascades. One such tissue-specific cascade is proposed for the central nervous system.

Identification of protein phosphorylation sites within Ser/Thr‐rich cluster domains using site‐directed mutagenesis and hybrid linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry

We describe a method for the analysis of multi-site phosphorylation in serine/threonine (Ser/Thr)-rich protein sequences. Site-specific mutagenesis was used to introduce tryptic cleavage sites in the serine glutamine/threonine glutamine cluster domain (SCD) of the human checkpoint protein kinase (Chk2). The mutant proteins were shown to autophosphorylate on residues that are inducibly phosphorylated when mammalian cells are exposed to ionizing radiation (serine 33/35, serine 516, threonine 68 and threonine 432). Five Ser/Thr clusters within the SCD were flanked by arginine or lysine residues to produce tryptic peptides for nanospray liquid chromatography (nanoLC)/linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry. Phosphorylation sites were assigned using accurate-mass-driven analysis and interpretation of low-energy collision-induced dissociation spectra acquired in the ion trap. In addition to verifying known phosphorylation sites, seventeen novel sites were identified within the SCD of Chk2. The approach should be applicable to other O-linked post-translational modifications that occur in proteins with Ser/Thr-rich sequences.

Proteolytic Cleavage of Ostrich and Turkey Pancreatic Lipases

The aim of this study was to check some biochemical and structural properties of ostrich and turkey pancreatic lipases (OPL and TPL, respectively). Limited proteolysis of OPL and TPL was performed in conditions similar to those reported for porcine pancreatic lipase. In the absence of bile salts and colipase, OPL failed to catalyze the hydrolysis of pure tributyrin or efficiently hydrolyze olive oil emulsion. When bile salts and colipase were preincubated with the substrate, the OPL kinetic behavior remained linear for more than 30 minutes. The enzyme presented a penetration power value into an egg phosphatidylcholine monomolecular film that was comparable to that of HPL and lower than that of TPL. Chymotrypsin, trypsin, and thermolysin were able to hydrolyze OPL and TPL in different ways. In both cases, only N-terminal fragments accumulated during the hydrolysis, whereas no C-terminal fragment was obtained in either case. Tryptic cleavage of OPL and TPL completely degraded the enzymes. Nevertheless, chymotryptic attack generated 35-kd and 43-kd forms for TPL and OPL, respectively. Interestingly, the OPL 43-kd form was inactive, whereas the TPL 35-kd protein conserved its lipolytic activity. OPL, TPL, and mammal pancreatic lipases share a high amino acid sequence homology. Further investigations are, however, needed to identify key residues involved in substrate recognition responsible for biochemical differences between the 2 classes of lipases.

The Implications of Proteolytic Background for Shotgun Proteomics

The analysis by liquid chromatography coupled to tandem mass spectrometry of complex peptide mixtures, generated by proteolysis of protein samples, is the main proteomics method used today. The approach is based on the assumption that each protein present in a sample reproducibly and predictably generates a relatively small number of peptides that can be identified by mass spectrometry. In this study this assumption was examined by a targeted peptide sequencing strategy using inclusion lists to trigger peptide fragmentation attempts. It was found that the number of peptides observed from a single protein is at least one order of magnitude greater than previously assumed. This unexpected complexity of proteomics samples implies substantial technical challenges, explains some perplexing results in the proteomics literature, and prompts the need for developing alternative experimental strategies for the rapid and comprehensive analysis of proteomes.

Chymotrypsin C (caldecrin) promotes degradation of human cationic trypsin: Identity with Rinderknecht's enzyme Y

Digestive trypsins undergo proteolytic breakdown during their transit in the human alimentary tract, which has been assumed to occur through trypsin-mediated cleavages, termed autolysis. Autolysis was also postulated to play a protective role against pancreatitis by eliminating prematurely activated intrapancreatic trypsin. However, autolysis of human cationic trypsin is very slow in vitro, which is inconsistent with the documented intestinal trypsin degradation or a putative protective role. Here we report that degradation of human cationic trypsin is triggered by chymotrypsin C, which selectively cleaves the Leu(81)-Glu(82) peptide bond within the Ca(2+) binding loop. Further degradation and inactivation of cationic trypsin is then achieved through tryptic cleavage of the Arg(122)-Val(123) peptide bond. Consequently, mutation of either Leu(81) or Arg(122) blocks chymotrypsin C-mediated trypsin degradation. Calcium affords protection against chymotrypsin C-mediated cleavage, with complete stabilization observed at 1 mM concentration. Chymotrypsin C is highly specific in promoting trypsin degradation, because chymotrypsin B1, chymotrypsin B2, elastase 2A, elastase 3A, or elastase 3B are ineffective. Chymotrypsin C also rapidly degrades all three human trypsinogen isoforms and appears identical to enzyme Y, the enigmatic trypsinogen-degrading activity described by Heinrich Rinderknecht in 1988. Taken together with previous observations, the results identify chymotrypsin C as a key regulator of activation and degradation of cationic trypsin. Thus, in the high Ca(2+) environment of the duodenum, chymotrypsin C facilitates trypsinogen activation, whereas in the lower intestines, chymotrypsin C promotes trypsin degradation as a function of decreasing luminal Ca(2+) concentrations.

Use of Biotin Derivatives to Probe Conformational Changes in Proteins

Sulfosuccinimidyl-6-(biotinamido) hexanoate and derivatives thereof covalently bind to the epsilon-amino group of lysine residues. Our observation that access of the biotin derivative to specific lysine residues depends on conformational properties of the entire polypeptide chain prompted us to investigate whether differential biotinylation patterns of a protein can be used as indicators for conformational changes. Bovine serum albumin is a soluble protein with characteristic unfolding kinetics upon exposure to high temperature. First, we show that biotinylation patterns of proteins are highly reproducible. Second, we demonstrate by mass spectrometry and tandem mass spectrometry that unfolding of the protein correlates with the accessibility of the biotin derivative to specific lysine residues. We have applied this experimental strategy to the analysis of a cell-surface protein, viz. the human band 3 anion exchanger of erythrocytes infected with the malaria parasite Plasmodium falciparum. We found that Lys(826) in a highly flexible loop can be biotinylated in non-infected (but not infected) erythrocytes, confirming earlier observations (Winograd, E., and Sherman, I. W. (2004) Mol. Biochem. Parasitol. 138, 83-87) based on epitope-specific monoclonal antibodies suggesting that this region undergoes a conformational change upon infection.

Analysis and Quantification of Diagnostic Serum Markers and Protein Signatures for Gaucher Disease

Novel approaches for the qualitative and quantitative proteomics analysis by nanoscale LC-MS applied to the study of protein expression response in depleted and undepleted serum of Gaucher patients undergoing enzyme replacement therapy are presented. Particular emphasis is given to the method reproducibility of these LC-MS experiments without the use of isotopic labels. The level of chitotriosidase, an established Gaucher biomarker, was assessed by means of an absolute concentration determination technique for alternate scanning LC-MS generated data. Disease associated proteins, including fibrinogens, complement cascade proteins, and members of the high density lipoprotein serum content, were recognized by various clustering methods and sorting and intensity profile grouping of identified peptides. Condition-unique LC-MS protein signatures could be generated utilizing the measured serum protein concentrations and are presented for all investigated conditions. The clustering results of the study were also used as input for gene ontology searches to determine the correlation between the molecular functions of the identified peptides and proteins.

The hinge region operates as a stability switch in cGMP‐dependent protein kinase Iα

The molecular mechanism of cGMP-dependent protein kinase activation by its allosteric regulator cyclic-3',5'-guanosine monophosphate (cGMP) has been intensely studied. However, the structural as well as thermodynamic changes upon binding of cGMP to type I cGMP-dependent protein kinase are not fully understood. Here we report a cGMP-induced shift of Gibbs free enthalpy (DeltaDeltaGD) of 2.5 kJ.mol-1 as determined from changes in tryptophan fluorescence using urea-induced unfolding for bovine PKG Ialpha. However, this apparent increase in overall stability specifically excluded the N-terminal region of the kinase. Analyses of tryptic cleavage patterns using liquid chromatography-coupled ESI-TOF mass spectrometry and SDS/PAGE revealed that cGMP binding destabilizes the N-terminus at the hinge region, centered around residue 77, while the C-terminus was protected from degradation. Furthermore, two recombinantly expressed mutants: the deletion fragment Delta1-77 and the trypsin resistant mutant Arg77Leu (R77L) revealed that the labile nature of the N-terminus is primarily associated with the hinge region. The R77L mutation not only stabilized the N-terminus but extended a stabilizing effect on the remaining domains of the enzyme as well. These findings support the concept that the hinge region of PKG acts as a stability switch.

Mass spectrometric identification of the trypsin cleavage pathway in lysyl‐proline containing oligotuftsin peptides

Trypsin cleaves specifically peptide bonds at the C-terminal side of lysine and arginine residues, except for -Arg-Pro- and -Lys-Pro- bonds which are normally resistant to proteolysis. Here we report evidence for a -Lys-Pro- tryptic cleavage in modified oligotuftsin derivatives, Ac-[TKPKG]4-NH2) (1), using high-resolution mass spectrometry and HPLC as primary methods for analysis of proteolytic reactions. The proteolytic susceptibility of -Lys-Pro- bonds was strongly dependent on flanking residues, and the flexibility of the peptide backbone might be a prerequisite for this unusual cleavage. While -Lys-Gly- bonds in 1 were rapidly cleaved, the modification of these Lys residues by the attachment of a ss-amyloid(4-10) epitope to yield -Lys(X)-Gly derivatives prevented cleavage of this bond, and provided trypsin cleavage of -Lys-Pro- bonds, the pathway of this degradation being independent on the type of Lys-N(epsilon)-side chains (acetyl group, amino acid, peptide). Substitution of the Lys residues by Ala at the P'2 positions decreased the tryptic cleavage, while replacement of the bulky side chain of Thr at the P2 positions strongly increased the cleavage of -Lys-Pro- bonds. Circular dichroism (CD) data of the modified oligotuftsin derivatives are in accord with enhanced flexibility of the peptide backbone, as a prerequisite for increased susceptibility to cleavage of -Lys-Pro- bonds. These results obtained of oligotuftsin derivatives might have implications for the proteolytic degradation of target peptides that require specific conformational preconditions.