title: Functional and structural characterization of a synthetic peptide representing the N-terminal domain of prokaryotic pyruvate dehydrogenase.
abstract: A synthetic peptide (Nterm-E1p) is used to characterize the structure and function of the N-terminal region (amino acid residues 4-45) of the pyruvate dehydrogenase component (E1p) from the pyruvate dehydrogenase multienzyme complex (PDHC) from Azotobacter vinelandii. Activity and binding studies established that Nterm-E1p specifically competes with E1p for binding to the dihydrolipoyl transacetylase component (E2p) of PDHC. Moreover, the experiments show that the N-terminal region of E1p forms an independent folding domain that functions as a binding domain. CD measurements, two-dimensional (2D) (1)H NMR analysis, and secondary structure prediction all indicate that Nterm-E1p has a high alpha-helical content. Here a structural model of the N-terminal domain is proposed. The peptide is present in two conformations, the population of which depends on the sample conditions. The conformations are designated "unfolded" at pH > or =6 and "folded" at pH <5. The 2D (1)H TOCSY spectrum of a mixture of folded and unfolded Nterm-E1p shows exchange cross-peaks that "link" the folded and unfolded state of Nterm-E1p. The rate of exchange between the two species is in the range of 0.5-5 s(-1). Sharp resonances in the NMR spectra of wild-type E1p demonstrate that this 200 kDa enzyme contains highly flexible regions. The observed dynamic character of E1p and of Nterm-E1p is likely required for the binding of the E1p dimer to the two different binding sites on E2p. Moreover, the flexibility might be essential in sustaining the allosteric properties of the enzyme bound in the complex.
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands. Hengveld@chem.vu.nl
authors.forename: Annechien F
authors.initials: AF
authors.lastname: Hengeveld
authors.forename: Carlo P M
authors.initials: CP
authors.lastname: van Mierlo
authors.forename: Henno W
authors.initials: HW
authors.lastname: van den Hooven
authors.forename: Antonie J W G
authors.initials: AJ
authors.lastname: Visser
authors.forename: Aart
authors.initials: A
authors.lastname: de Kok
mesh_terms: D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology*; D019281:Dimerization; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000502:physiology*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011768:Pyruvate Dehydrogenase Complex / Q000138:chemical synthesis / Q000737:chemistry* / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
chemical_list: D010446:Peptide Fragments; D011768:Pyruvate Dehydrogenase Complex; D019791:Guanidine