NMR, Biophysical, and Biochemical Studies Reveal the Minimal Calmodulin Binding Domain of the HIV-1 Matrix Protein

Solution Structure of Calmodulin Bound to the Binding Domain of the HIV-1 Matrix Protein

“…Extensive loss and/or broadening of the NMR signals precluded determination of the solution structure of the complex. In a subsequent study, we have identified the minimal CaM-binding domain of MA by utilizing a proteolytic digestion assay (61). Analysis of the digestion products by mass spectrometry revealed that the abundant MA species resistant to proteolysis is a peptide spanning residues 8 -43 (61).…”

“…In a subsequent study, we have identified the minimal CaM-binding domain of MA by utilizing a proteolytic digestion assay (61). Analysis of the digestion products by mass spectrometry revealed that the abundant MA species resistant to proteolysis is a peptide spanning residues 8 -43 (61). The formation of the complex between MA-(8 -43) and CaM has led to substantial chemical shift changes for the vast majority of 1 H and 15 N signals in the HSQC spectrum of CaM (Fig.…”

“…Protein Expression and Purification-CaM and MA- (8 -43) samples have been prepared as described (61). The CaM⅐MA-(8 -43) complex was prepared by mixing equimolar amounts of CaM and MA- (8 -43), which was then passed through a gel filtration column (Superdex 75, GE Healthcare).…”

“…We have shown that binding of CaM induces a conformational change in MA, triggering myr exposure (59). By employing NMR, biophysical, biochemical, and mass spectrometry methods we have identified the CaM-binding region to be a region spanning residues 8 -43 (MA-(8 -43)) (61). Within MA, residues 11-19 form an ␣-helix (␣1), residues 20 -30 form a ␤-hairpin, and residues 31-45 form a second ␣-helix (␣2) (11,(62)(63)(64).…”

Structural and Biophysical Characterization of the Interactions between Calmodulin and the Pleckstrin Homology Domain of Akt

“…Expression and purification of the CaM protein were conducted as described (35). CaM samples were stored in a buffer containing 50 mM HEPES or Tris at pH 7.0, 150 mM NaCl, and 5 mM CaCl 2 .…”