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Related Experiment Videos

Lambda cro repressor complex with OR3 DNA: 15N NMR observations.

P Leighton1, P Lu

  • 1Department of Chemistry, University of Pennsylvania, Philadelphia 19104.

Biochemistry
|November 17, 1987
PubMed
Summary
This summary is machine-generated.

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The C-terminal arm of the cro repressor protein is flexible and exposed to solvent, but becomes less mobile and exposed upon DNA binding. This study verifies its role in DNA binding and refines NMR assignments for the protein.

Area of Science:

  • Structural Biology
  • Biophysics
  • Molecular Genetics

Background:

  • The C-terminal residues (63-66) of the coliphage lambda cro repressor were not resolved in initial crystallographic studies.
  • Previous proposals suggested the C-terminus plays a role in DNA binding, but direct experimental evidence was lacking.

Purpose of the Study:

  • To investigate the structure and dynamics of the C-terminal region of the cro repressor protein using 15N Nuclear Magnetic Resonance (NMR) spectroscopy.
  • To directly verify the proposed involvement of the C-terminus in DNA binding.
  • To refine existing NMR assignments and assess potential peptide bond isomerization.

Main Methods:

  • Uniformly 15N-labeled cro repressor protein was used for NMR studies.
  • Resonance assignments were achieved through enzymatic digestion and 13C-15N double-labeling experiments.

Related Experiment Videos

  • Dynamics and solvent accessibility were probed using 15N[1H] Nuclear Overhauser Effects (NOE) and exchange experiments (DEPT).

    • Main Results:

    • Direct resonance assignments for C-terminal residues (56, 62, 63, 66) confirmed their presence and solvent accessibility.
    • The C-terminus exhibits high mobility in solution, acting as a flexible arm.
    • DNA binding, both specific and non-specific, significantly reduced the mobility and solvent exposure of the C-terminal arm.
    • Discrepancies with prior 1H NMR assignments for lysine amides were identified, highlighting the utility of 15N labeling.
    • Isomerization of the Pro-59 peptide bond was shown not to occur.

    Conclusions:

    • The C-terminal arm of the cro repressor is a flexible, solvent-exposed region that directly participates in DNA binding.
    • 15N NMR is a powerful tool for assigning resonances and elucidating the dynamics of biomolecules.
    • The study provides direct experimental evidence supporting the role of the C-terminus in DNA binding and refutes proline isomerization.