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Related Experiment Videos

Ca2+ binding and conformational changes in a calmodulin domain

J Evenäs1, A Malmendal, E Thulin

  • 1Physical Chemistry 2, Lund University, Sweden. johan.evenas@fkem2.lth.se

Biochemistry
|September 30, 1998
PubMed
Summary
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Calcium binding to calmodulin

Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Biophysics

Background:

  • Calmodulin's C-terminal domain is crucial for calcium ion (Ca2+) binding and signal transduction.
  • Conserved glutamate residues in Ca2+ binding loops are vital for protein function.
  • Mutations in these residues can alter Ca2+ binding dynamics and structural stability.

Purpose of the Study:

  • To investigate the Ca2+ binding mechanism and structural consequences of mutating a conserved glutamate to glutamine (E104Q) in calmodulin's C-terminal domain.
  • To compare the Ca2+ binding behavior and structural dynamics of the E104Q mutant with wild-type calmodulin and the E140Q mutant.
  • To elucidate the role of specific glutamate residues in stabilizing different calmodulin conformations.

Main Methods:

  • 1H and 15N Nuclear Magnetic Resonance (NMR) spectroscopy.

Related Experiment Videos

  • NMR titration experiments to determine Ca2+ binding constants.
  • Structural characterization of apo, mono-, and di-Ca2+-bound states.
  • Calculation of solution structures for the (Ca2+)2 state of the E104Q mutant.

    • Main Results:

    • The E104Q mutation in calmodulin's C-terminal domain alters the sequential Ca2+ binding order, with Ca2+ binding first to loop IV and then to loop III.
    • Major structural changes in the E104Q mutant occur upon Ca2+ binding to loop IV, indicating differential responses in N- and C-terminal EF-hands.
    • The (Ca2+)1 and (Ca2+)2 states of the E104Q mutant exhibit dynamic behavior, exchanging between conformations resembling closed and open states of wild-type calmodulin.
    • Both E104 and E140 are essential for stabilizing the open conformation in the (Ca2+)2 state, and the calculated ensemble structure for E104Q reflects dynamic averaging rather than a single conformation.

    Conclusions:

    • The conserved glutamate residues E104 and E140 play critical roles in the Ca2+ activation mechanism of calmodulin's C-terminal domain.
    • Mutations at these sites disrupt the normal Ca2+ binding sequence and alter the conformational dynamics.
    • The study highlights the dynamic nature of calmodulin and the importance of specific residues in achieving functional conformations.