Portion-mixing peptide libraries of quenched fluorogenic substrates for complete subsite mapping of endoprotease specificity.

Abstract

A solid-phase assay for the complete subsite mapping doftheactive site ofeldop eaes has been developed.A lbrary of resin-bound ptase ubstrates was sytsed both on kieselguhr-supported polyamide resin and on a polyethylene glycol-poly-(NN-dlnethylacryhmlde) copolymer type of resin that alows proteas to diffuse into the interior and perform their catalytic activity. An lic acid and 3-it rosine were used a an efclet donor-acceptor p for the resonance energy transfer. The synthesis was performed in a manual library generator that allows simple wet ming of the bead and ll washing p eus. After treatment wit subtiAsin Carlsberg, beads were c and subjected to peptide seq , affording the preferred s quences, their cleavage bond, and a esiqstimation of the turnover. A satitcal dbution of prefed amino acids was obtain for each subsite. The result was compaed with data from kinetic studies In solution.In early studies of the activity of isolated proteolytic enzymes, polypeptides were subjected to digestion with the enzyme (1) and the preferred cleavage site, if any, was determined by isolation of the fragments and subsequent Edman degradation. This often tedious work provided lead amino acid sequences that could be used for determination of the optimal substrates. These were prepared by chemical synthesis and the kinetic constants were measured by determination ofthe rate ofproduct formation by HPLC (2), NMR spectroscopy (3), or spectrophotometric monitoring when chromogenic or fluorogenic substrates could be used (4, 5). More recently the development of internally quenched fluorogenic substrates of the resonance energy transfer type (6, 7) has facilitated the complete subsite mapping (5,(8)(9)(10)(11)(12) for endoproteases. In these substrates, it is important that efficient long-range energy transfer is observed between the donor and the acceptor to span the entire active site of the endopeptidase, thus minimizing the interaction between the enzyme and the chromophoric probes. The donor-acceptor amino acid pair o-aminobenzamide (ABz)-3-nitrotyrosine [Tyr(NO2)] (13) for which excellent quenching of fluorescence is observed has recently been described (5). This donor-acceptor pair is conveniently introduced in parallel multiple-column peptide synthesis (MCPS) (14) of numerous substrates and has been used for subsite mapping of a variety of proteases (6,(15)(16)(17)(18)(19)(20)(21). However, substantial effort is still required to identify the optimal substrates.The use ofcombinatorial peptide libraries (22) and portionmixing libraries (23, 24) is widely accepted as the method of choice for defining binding motifs and unknown biological activities. The portion-mixing library is particularly convenient for the presentation of millions of substrates to a protease with unknown specificity. The problem is, however, to detect, isolate, and characterize the active substances. Due to the high quantum yield of the ABz group, the fluorescence can easily be observed visually in the absence of Tyr(N02), whereas peptides containi...

An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl‐prolyl cis/trans isomerase activity

“…The linkages between protein domains are often particularly susceptible to proteolysis. Subtilisin is known as a protease having a broad subsite specificity [36,37]. Therefore, it is comprehensible that proteins possibly are readily digested when employed in subtilisin treatment.…”

Altered -3 Substrate Specificity of Escherichia coli Signal Peptidase 1 Mutants as Revealed by Screening a Combinatorial Peptide Library

“…Furthermore, it cannot give individual sequences or reveal any sequence coverage. The previous FRET method (34) can determine the preferred sequences that are cleaved by proteases but Edman sequencing is expensive and cannot determine the site of cleavage.…”

“…The peptide library was synthesized on a solid support using a split-pool methodology to generate a one bead-one sequence library (26). A polyethylene glycol and acrylamide-based amino PEGA 1900 resin was chosen as the solid support due to its ability to swell in hydrophilic conditions thus allowing relatively large biomolecules such as enzymes to easily permeate the resin (34).…”

Highly Sensitive Quenched Fluorescent Substrate of Legionella Major Secretory Protein (Msp) Based on Its Structural Analysis

“…In this formula, Pya (L-pyrenylalanyl) is the fluorescent moiety; Nop (L-4-NO 2 -phenylalanyl) the quencher moiety; X indicates either a single natural amino acid chosen from Ala, Leu, Ile, Glu, Thr, Gln, Phe, Trp, Pro, or Lys or a mixture of these 10 amino acids, and n varies from 0 to 3. This library has been prepared by solid phase synthesis, using the split and mix method (22). When n ϭ 0, the sub-library is constituted by one peptide; when n ϭ 1, the sub-library is a batch of 10 peptides; when n ϭ 2, the sub-library corresponds to ten batches of 10 peptides, and for n ϭ 3 the sub-library corresponds to 100 batches of 10 peptides.…”