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Related Experiment Videos

Staphylococcal nuclease: sequential assignments and solution structure.

D A Torchia1, S W Sparks, A Bax

  • 1Bone Research Branch, National Institute of Dental Research, Bethesda, Maryland 20892.

Biochemistry
|June 27, 1989
PubMed
Summary
This summary is machine-generated.

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This study details sequential NMR assignments for staphylococcal nuclease (Nase) in a ternary complex. Isotope-edited NMR methods successfully mapped protein structure and dynamics in solution, revealing flexibility near residue 50.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

  • Staphylococcal nuclease (Nase) is a model enzyme for studying protein folding and dynamics.
  • Understanding the solution structure of enzyme-ligand complexes is crucial for elucidating function.
  • Previous structural studies of Nase have relied heavily on X-ray crystallography.

Purpose of the Study:

  • To determine sequential backbone NMR assignments for the staphylococcal nuclease (Nase) ternary complex.
  • To characterize the solution structure and dynamics of Nase complexed with thymidine 3',5'-diphosphate and Ca2+.
  • To compare solution structures with crystal structures for insights into protein flexibility.

Main Methods:

  • Utilized isotope-edited two-dimensional NMR spectroscopy, including 15N-edited NOESY, 15N-edited COSY, and HMQC.

Related Experiment Videos

  • Employed conventional NMR techniques like NOESY, COSY, and homonuclear Hartmann-Hahn spectroscopy.
  • Applied these methods to a ternary complex of Nase, thymidine 3',5'-diphosphate, and Ca2+.

    • Main Results:

    • Achieved sequential backbone assignments for 127 out of 136 residues in the structured region of Nase.
    • Identified three helical domains and several beta-sheets consistent with crystal structure data.
    • Observed increased flexibility in the solution structure around residue 50 compared to the crystal structure.
    • Correlated amide proton exchange rates with hydrogen bonding observed in the crystal structure.

    Conclusions:

    • Isotope-edited NMR is effective for assigning backbone resonances in large protein complexes like Nase.
    • Solution NMR data largely agrees with crystal structure, but highlights regions of increased flexibility.
    • NMR provides complementary insights into protein dynamics and conformational states not evident from static crystal structures.