Related Experiment Videos
Sequential proton NMR resonance assignments, circular dichroism, and structural properties of a 50-residue
G P Mullen1, J B Vaughn, A S Mildvan
1Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Abstract:
Peptide I, a 50-amino acid synthetic peptide based on residues 728 to 777 of DNA polymerase I, binds dNTP substrates and duplex DNA (G. Mullen, P. Shenbagamurthi, and A.S. Mildvan, J. Biol. Chem. 264, 19637-19647, 1988). The structural properties of peptide I at pH 3.9 have been studied by CD spectroscopy and by 2D proton NMR at 600 MHz. The CD spectra are fit by assuming that peptide I contains 17% helix, 17% beta-structure, and 66% coil. The substrate dATP binds tightly to peptide I under these conditions (KD = 0.5 microM) as determined by fluorescence quenching but induces no change in peptide conformation, as detected by CD spectroscopy. Proton resonances of peptide I have been assigned by double quantum filtered correlated spectroscopy, total correlated spectroscopy, and nuclear Overhauser effect spectroscopy. As found with other peptides, peptide I is best characterized by both extended and partially folded secondary structures which equilibrate rapidly on the NMR time scale. A region from residues 3 through 10 displays nuclear Overhauser effects (NOEs) consistent with the rapid equilibration of a nascent helix with a random extended structure. Alternatively this segment of residues is consistent with a series of three opened-out turns. A nonclassical turn is found between residues 14 and 17 and from residues 44 to 47, the latter closing irregular antiparallel strands from residues 42 to 48. The remainder of the peptide is a coil. A residue-by-residue comparison of the best-fit solution structure of the peptide with that of the corresponding sequence in the X-ray structure of the complete enzyme reveals that 36% of the amino acids are found to be in a conformation similar to that in the enzyme. Such partial and transient folding of the peptide indicates that the major role of the remainder of the protein is to provide structural support for the active site region of the enzyme. As detected by interresidue NOEs and NOEs to water protons, the homologous sequence Leu-37-Ile-38-Tyr-39-Gly-40, together with Phe-15 of the peptide, provides an exposed hydrophobic cluster of residues which may constitute the substrate binding site. An exposed cluster of cationic residues consisting of Arg-27, Arg-28, Lys-31, and possibly Arg-48 may provide the binding site for duplex DNA.
Insights
Peptide I, a DNA polymerase I fragment, exhibits partial folding and binds substrates. Its structure, analyzed by NMR and CD spectroscopy, reveals key residues for dNTP and DNA binding, suggesting the full enzyme provides structural support.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Biophysics
Background:
- Peptide I, a synthetic 50-amino acid fragment of DNA polymerase I (residues 728-777), is known to bind deoxynucleoside triphosphates (dNTPs) and duplex DNA.
- Understanding the structural basis of this binding is crucial for elucidating DNA polymerase I function.
Purpose of the Study:
- To investigate the structural properties of Peptide I at pH 3.9 using biophysical techniques.
- To determine the conformational changes upon substrate binding (dATP).
- To identify residues involved in substrate and DNA binding.
Main Methods:
- Circular Dichroism (CD) spectroscopy to assess secondary structure content.
- 2D proton Nuclear Magnetic Resonance (NMR) spectroscopy at 600 MHz for detailed structural analysis.
- Fluorescence quenching to determine binding affinity (dissociation constant, KD).
- Various NMR techniques including double quantum filtered correlated spectroscopy, total correlated spectroscopy, and nuclear Overhauser effect spectroscopy for resonance assignment and structural determination.
Main Results:
- CD spectroscopy indicated Peptide I possesses a mixed secondary structure: 17% helix, 17% beta-structure, and 66% coil.
- dATP bound tightly to Peptide I (KD = 0.5 microM) but did not induce significant conformational changes.
- NMR analysis revealed rapid equilibration between extended and partially folded structures, with specific regions forming turns and antiparallel strands.
- A comparison with the X-ray structure of DNA polymerase I showed 36% residue similarity in conformation.
- A hydrophobic cluster (Leu-37, Ile-38, Tyr-39, Gly-40, Phe-15) and a cationic cluster (Arg-27, Arg-28, Lys-31, Arg-48) were identified as potential binding sites for substrates and DNA, respectively.
Conclusions:
- Peptide I exhibits dynamic, partially folded structures in solution.
- The identified hydrophobic and cationic clusters are likely involved in substrate and DNA binding.
- The findings suggest that the full DNA polymerase I enzyme provides essential structural support to stabilize the active site region.