Synthetic interface peptides as inactivators of multimeric enzymes: inhibitory and conformational properties of three fragments from Lactobacillus casei thymidylate synthase.
Three synthetic peptides corresponding to distinct segments of the subunit interface of the dimeric enzyme thymidylate synthase (residues 17-38, N 22; residues 174-190, M 17; and residues 201-220, C 20) have been investigated for their ability to function as inhibitors by modifying the quaternary structure of the enzyme. A dramatic reduction of enzyme activity is observed following incubation of TS with the C 20 peptide. The N 22 and M 17 peptides were unable to cause any loss of enzymatic activity. Addition of the C 20 peptide results in a loss of fluorescence of TS labeled with a dansyl group at Cys 198, following aggregation and precipitation of the protein. The effects are not observed for the N 22 or M 17 peptides. Loss of enzymatic activity is related to the ability of C 20 to promote protein aggregation. The conformations of the peptides have been studied using CD and NMR in order to correlate the observed function with solution structures. Peptides N 22 and M 17 are largely unstructured in aqueous solution. A population of nascent helical structures or multiple turn conformations has been detected for the C 20 peptide in aqueous solution by NMR. Addition of 50% (v/v) hexafluoroacetone trihydrate (HFA), a structure-stabilizing cosolvent, stabilizes the helical conformation in the C 20 peptide. Under similar conditions, N 22 and M 17 remain largely extended with observations of local beta-turn conformations. Interestingly, the C 20 peptide is a beta-hairpin in the native structure, whereas the other two peptides are individual strand components of a beta-sheet.
Prasanna
V
V
Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
Bhattacharjya
S
S
Balaram
P
P
D000096:Acetone / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D002384:Catalysis; D002942:Circular Dichroism; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D005466:Fluorocarbons; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D012996:Solutions; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000494:pharmacology*; D014867:Water; D006845:Hydrocarbons, Fluorinated
D000096:Acetone; D004791:Enzyme Inhibitors; D005466:Fluorocarbons; D010446:Peptide Fragments; D012996:Solutions; D013940:Thymidylate Synthase; D014867:Water; C017366:hexafluoroacetone; D006845:Hydrocarbons, Fluorinated