Controlled Peptide Solvation in Portion-Mixing Libraries of FRET Peptides:  Improved Specificity Determination for Dengue 2 Virus NS2B-NS3 Protease and Human Cathepsin S

Abstract

The solubility of peptides in aqueous buffers used for the enzyme assays is a common limitation for all peptide libraries. In principle, the more water-soluble peptides are, the more susceptible they will be to peptidase hydrolysis. We have demonstrated that this bias can be circumvented in a portion-mixing fluorescence resonance energy transfer (FRET) peptide library by introducing k (lysine in the D-form) in both termini of the peptides. This more solvated library and another one without the k were assayed using trypsin and chymotrypsin as standard peptidases with high selectivity for R and K and for hydrophobic F and Y, respectively. Significantly improved consistency of the information on substrate profiles was obtained from the solvated library. The influence of improved solvation on substrate specificity determination was successfully demonstrated by the difference in specificity observed between the two libraries employing the human cathepsin S (accepts acidic, basic, or neutral amino acids at P1 position) and Dengue 2 virus NS2B-NS3 protease (high specificity to the pair of basic amino acids K-R, R-R, or Q-R/K at P2-P1 positions). In conclusion, hydration of the peptides has a major influence on protease processing, and this bias can be reduced in bound peptide libraries, improving reliability.

Specificity studies on Kallikrein-related peptidase 7 (KLK7) and effects of osmolytes and glycosaminoglycans on its peptidase activity

“…Between the randomized positions, the Fmoc-K-ε-NH 2 -(Abz-Boc), attached to resin and Fmoc-K-ε-NH 2 -(Dnp) at the N-terminus were incorporated. In order to improve the solvation of the peptides attached to the resin bead D-lysine was also added before K-ε-NH 2 -(Abz) and K-ε-NH 2 -(Dnp) [22]. The side chain protecting groups were removed by treatment with a mixture of TFA:thioanisole:ethanedithiol:water (87:5:5:3) for 8 h. For the assays of this library the beads were washed with water (3×) and with the assay buffer (3×) before the addition of protease.…”

“…The identified peptide sequences susceptible to hydrolysis by KLK7 were synthesized as FRET Abz-peptidyl-Q-EDDnp peptides and assayed in solution with KLK7. For more details of the synthesis and assays of this library see reference [22].…”

“…The substrate specificity of KLK7 was also evaluated using support-bound FRET peptide libraries prepared on polyethylene glycol dimethyl acrylamide copolymer (PEGA) resin beads, which were prepared by the split-combine synthesis process that results in a single unique peptide sequence on each of the resin beads. The partial proteolysis of the FRET peptide bound to the solid support, and sequence determination by Edman degradation of the substrates on most of the fluorescent beads provides the sequence of preferred substrates [21,22]. The S 1 specificity was further investigated by FRET peptide libraries with the general formula Abz-GXX-Z-XX-Q-EDDnp, where Z is one of the natural amino acids (except cysteine) and X represents an equimolar mixture of the 19 natural amino acids.…”

Proteome-wide Substrate Analysis Indicates Substrate Exclusion as a Mechanism to Generate Caspase-7 Versus Caspase-3 Specificity

“…However, some constraints limit the global applicability of synthetic peptidebased techniques, such as limited library size for hexa-and heptapeptides and restriction to non-prime cleavage specificities because of C-terminal labeling with fluorophores. Libraries of fluorescence resonance energy transfer peptides and the proteomic identification of protease cleavage sites technique have overcome most of the limitations of library size and non-prime motifs (9,42). Peptide-based studies have yielded valuable insight into the sequence specificity of caspases, although interpretation of the biological significance remains difficult because protease-peptide interactions differ substantially from natural protease-protein interactions (2).…”

The use of Fluorescence Resonance Energy Transfer (FRET) peptidesfor measurement of clinically important proteolytic enzymes

“…More recently, synthetic support-bound peptide libraries have been prepared by the process of split-combination synthesis, which results in a single peptide sequence on each of the resin beads. Using this random synthetic library approach, we improved the specificity studies of Dengue 2 virus NS2B-NS3 protease and human cathepsin S (Alves et al 2007). …”