Proteolytic stability of β-peptide bonds probed using quenched fluorescent substrates incorporating a hemoglobin cleavage site

AbstractA set of designed internally quenched fluorescence peptide substrates has been used to probe the effects of insertion of β-peptide bonds into peptide sequences. The test sequence chosen corresponds to a proteolytically susceptible site in hemoglobin α-chain, residues 32–37. Fluorescence and mass spectral measurements demonstrate that the insertion of an β-residues at the potential cleavage sites completely abolishes the action of proteases; in addition, the rate of cleavage of the peptide bond preceding the site of modification is also considerably reduced

Proteolytic stability of beta-peptide bonds probed using quenched fluorescent substrates incorporating a hemoglobin cleavage site

A set of designed internally quenched fluorescence peptide substrates has been used to probe the e¡ects of insertion of beta-peptide bonds into peptide sequences. The test sequence chosen corresponds to a proteolytically susceptible site in hemoglobin K-chain, residues 32-37. Fluorescence and mass spectral measurements demonstrate that the insertion of an beta-residues at the potential cleavage sites completely abolishes the action of proteases; in addition, the rate of cleavage of the peptide bond preceding the site of modification is also considerably reduced

Stage-specific profiling of Plasmodium falciparum proteases using an internally quenched multispecificity protease substrate

Novel internally quenched fluorescence peptide substrates containing sequence specific sites for cleavage by multiple proteases were designed and synthesized. The 28 and 29 residue peptides contain an N-terminal fluorescence acceptor group, 4-(4-dimethylaminophenylazo) benzoic acid (DABCYL), and a C-terminal fluorescence donor group, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (EDANS). Efficient energy transfer between the donor and acceptor groups .anking the peptide sequence was achieved by incorporation of a central DPro-Gly segment, which serves as a conformation nucleating site, inducing hairpin formation. This multispecificity protease substrate was used to profile the proteolytic activities in the malarial parasite Plasmodium falciparum in a stage dependent manner using a combination of fluorescence and MALDI mass spectrometry. Cysteine protease activity was shown to be dominating at neutral pH, whereas aspartic protease activity contributed predominantly to the proteolytic repertoire at acidic pH. Maximum proteolysis was observed at the trophozoite stage followed by the schizonts and the rings

Stage-specific profiling of Plasmodium falciparum proteases using an internally quenched multispecificity protease substrate

Novel internally quenched fluorescence peptide substrates containing sequence specific sites for cleavage by multiple proteases were designed and synthesized. The 28 and 29 residue peptides contain an N-terminal fluorescence acceptor group, 4-(4-dimethylaminophenylazo) benzoic acid (DABCYL), and a C-terminal fluorescence donor group, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (EDANS). Efficient energy transfer between the donor and acceptor groups .anking the peptide sequence was achieved by incorporation of a central DPro-Gly segment, which serves as a conformation nucleating site, inducing hairpin formation. This multispecificity protease substrate was used to profile the proteolytic activities in the malarial parasite Plasmodium falciparum in a stage dependent manner using a combination of fluorescence and MALDI mass spectrometry. Cysteine protease activity was shown to be dominating at neutral pH, whereas aspartic protease activity contributed predominantly to the proteolytic repertoire at acidic pH. Maximum proteolysis was observed at the trophozoite stage followed by the schizonts and the rings

Structural and spectral response of Aequorea victoria green fluorescent proteins to chromophore fluorination?

Global replacements of tyrosine by 2- and 3-fluorotyrosine in “enhanced green” and “enhanced yellow” mutants of Aequorea victoria green fluorescent proteins (avGFPs) provided protein variants with novel biophysical properties. While crystallographic and modeled structures of these proteins are indistinguishable from those of their native counterparts (i.e., they are perfectly isomorphous), there are considerable differences in their spectroscopic properties. The fluorine being an integral part of the avGFP chromophore induces changes in the titration curves, variations in the intensity of the absorbance and fluorescence, and spectral shifts in the emission maxima. Furthermore, targeted fluorination in close proximity to the fluorinated chromophore yielded additional variants with considerably enhanced spectral changes. These unique spectral properties are intrinsic features of the fluorinated avGFPs, in the context of the rigid chromophore−microenvironment interactions. The availability of the isomorpohous crystal structures of fluorinated avGFPs allowed mapping of novel, unusual interaction distances created by the presence of fluorine atoms. In addition, fluorine atoms in the ortho position of the chromophore tyrosyl moiety exhibit a single conformation, while in the meta position two conformer states were observed in the crystalline state. Such global replacements in chromophores of avGFPs and similar proteins result in “atomic mutations” (i.e., H → F replacements) in the structures, offering unprecedented opportunities to understand and manipulate the relationships between protein structure and spectroscopic properties

Structural and spectral response of Aequorea victoria green fluorescent proteins to chromophore fluorination?

Global replacements of tyrosine by 2- and 3-fluorotyrosine in “enhanced green” and “enhanced yellow” mutants of Aequorea victoria green fluorescent proteins (avGFPs) provided protein variants with novel biophysical properties. While crystallographic and modeled structures of these proteins are indistinguishable from those of their native counterparts (i.e., they are perfectly isomorphous), there are considerable differences in their spectroscopic properties. The fluorine being an integral part of the avGFP chromophore induces changes in the titration curves, variations in the intensity of the absorbance and fluorescence, and spectral shifts in the emission maxima. Furthermore, targeted fluorination in close proximity to the fluorinated chromophore yielded additional variants with considerably enhanced spectral changes. These unique spectral properties are intrinsic features of the fluorinated avGFPs, in the context of the rigid chromophore−microenvironment interactions. The availability of the isomorpohous crystal structures of fluorinated avGFPs allowed mapping of novel, unusual interaction distances created by the presence of fluorine atoms. In addition, fluorine atoms in the ortho position of the chromophore tyrosyl moiety exhibit a single conformation, while in the meta position two conformer states were observed in the crystalline state. Such global replacements in chromophores of avGFPs and similar proteins result in “atomic mutations” (i.e., H → F replacements) in the structures, offering unprecedented opportunities to understand and manipulate the relationships between protein structure and spectroscopic properties

Sodium channel modulating activity in a N-conotoxin from an Indian marine snail

Abstract A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH 2 has been isolated and puri¢ed from the venom of the molluscivorous snail, Conus amadis, collected o¡ the southeastern coast of India. Chemical modi¢cation and mass spectrometric studies establish that Am 2766 has three disul¢de bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the N N-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na + channel, stably expressed in Chinese hamster ovary cells. Unlike N N-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.

Sodium channel modulating activity in a delta-conotoxin from an Indian marine snail

A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH2 has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the delta-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na+ channel, stably expressed in Chinese hamster ovary cells. Unlike delta-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels