title: 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.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, UNIFESP, Rua Três de Maio, 100, São Paulo 04044-020, Brazil.
authors.forename: Fabiana M
authors.initials: FM
authors.lastname: Alves
authors.forename: Izaura Y
authors.initials: IY
authors.lastname: Hirata
authors.forename: Iuri E
authors.initials: IE
authors.lastname: Gouvea
authors.forename: Marcio F M
authors.initials: MF
authors.lastname: Alves
authors.forename: Morten
authors.initials: M
authors.lastname: Meldal
authors.forename: Dieter
authors.initials: D
authors.lastname: Brömme
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
mesh_terms: D000595:Amino Acid Sequence; D002403:Cathepsins / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry; D003716:Dengue Virus / Q000201:enzymology*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D012697:Serine Endopeptidases / Q000378:metabolism*; D012996:Solutions / Q000737:chemistry; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D000085242:Viral Proteases
chemical_list: D002403:Cathepsins; D003432:Cross-Linking Reagents; D019151:Peptide Library; D010455:Peptides; D011092:Polyethylene Glycols; D012697:Serine Endopeptidases; D012996:Solutions; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C504449:NS3 protease, dengue virus; D000085242:Viral Proteases; C028217:cathepsin S