Functional and Structural Characterization of a Synthetic Peptide Representing the N-Terminal Domain of Prokaryotic Pyruvate Dehydrogenase
| Autor: | and Antonie J. W. G. Visser, Annechien F. Hengeveld, Carlo P. M. van Mierlo, Aart de Kok, Henno W. van den Hooven |
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| Rok vydání: | 2002 |
| Předmět: |
Models
Molecular Protein Denaturation Protein Folding Protein Conformation Stereochemistry Molecular Sequence Data Population Allosteric regulation Biochemie Pyruvate Dehydrogenase Complex Biochemistry Protein Structure Secondary Structure-Activity Relationship Life Science Amino Acid Sequence Dihydrolipoyl transacetylase Binding site education Nuclear Magnetic Resonance Biomolecular Protein secondary structure Guanidine Azotobacter vinelandii education.field_of_study biology Chemistry Hydrogen-Ion Concentration biology.organism_classification Pyruvate dehydrogenase complex Peptide Fragments Protein Structure Tertiary Molecular Weight Crystallography Spectrometry Fluorescence Dimerization Binding domain |
| Zdroj: | Biochemistry 41 (2002) Biochemistry, 41, 7490-7500 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi012172u |
| Popis: | 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 pHor =6 and "folded" at pH5. 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. |
| Databáze: | OpenAIRE |
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