Relative affinity constants by electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry: calmodulin binding to peptide analogs of myosin light chain kinase

Abstract

Synthetic RS20 peptide and a set of its point-mutated peptide analogs have been used to analyze the interactions between calmodulin (CaM) and the CaM-binding sequence of smooth-muscle myosin light chain kinase both in the presence and the absence of Ca(2+). Particular peptides, which were expected to have different binding strengths, were chosen to address the effects of electrostatic and bulky mutations on the binding affinity of the RS20 sequence. Relative affinity constants for protein/ligand interactions have been determined using electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry. The results evidence the importance of electrostatic forces in interactions between CaM and targets, particularly in the presence of Ca(2+), and the role of hydrophobic forces in contributing additional stability to the complexes both in the presence and the absence of Ca(2+).

FIGURE 1

ESI-FTICR mass spectrum of CaM with R16L peptide (concentration ration 1:1.5) in 5 mM ammonium acetate buffer, pH 5.9. Insets show the expansions of the 7+ and 8+ charge-states. C represents CaM and P represents peptide, respectively.

FIGURE 2

Expanded ESI-FTICR mass spectra of CaM with (A) RS20, (B) A13L, (C) V11L, (D) V11F, and (E) R16L. The spectra were measured in 5 mM ammonium acetate buffer, pH 5.9, in the absence of CaCl₂. The spectra show peaks originated from the CaM–peptide complexes at the 8+ charge-state and CaM at the 7+ charge-state in their actual intensities relative to each other.

FIGURE 3

Stability in solution of the CaM–peptide complexes. The ratio was calculated using the relative intensities of signals corresponding to CaM–peptide complex at the 8+ charge-state and CaM at the 7+ charge-state. The values are the mean of three repeat experiments ± their standard deviation.

FIGURE 4

ESI-FTICR mass spectrum of CaM with R16L peptide (concentration ration 1:1.5) in 5 mM ammonium acetate buffer, pH 5.9, containing 0.1 mM CaCl₂. Insets show the expansions of the 7+ and 8+ charge-states of CaM–R16L–Ca_n and the 8+ charge-state of CaM.

FIGURE 5

ESI-FTICR mass spectrum of CaM with V11F and RS20 peptides (concentration ratio 1:1.5:1.5) in ammonium acetate buffer, 5 mM, pH 5.9, containing 0.1 mM CaCl₂. Inset shows the expansion of the 8+ charge-state. C represents CaM.

FIGURE 6

Expanded ESI-FTICR mass spectra showing competition reactions of pairs of peptides: (A) RS20 and A13L, (B) RS20 and V11L, (C) RS20 and V11F, (D) V11L and V11F, (E) V11L and A13L, (F) R16L and V11F, (G) R16L and V11L, (H) R16L and A13L, and (I) A13L and V11F. CaM/peptide1/peptide2/CaCl₂ concentration ratio was 1:1.5:1.5:5 in 5 mM ammonium acetate buffer, pH 5.9. The spectra show peaks originated from the CaM–peptide–Ca₄ complexes at the 8+ charge-state.

FIGURE 7

Relative affinities in solution of the RS20, A13L, V11L, V11F, and R16L peptides in the presence of calcium. The ratio of CaM, peptide1, peptide2, and CaCl₂ was 1:1.5:1.5:5 in competition reactions. The relative intensities of CaM–peptide–Ca₄ complexes at the 8+ charge-state were used for calculation. The values are the mean of three repeat experiments ± their standard deviation.

FIGURE 8

ESI-FTICR mass spectra showing the 8+ charge-state of CaM with V11F peptide (concentration ratio 1:1.5) in ammonium acetate buffer, 5 mM, pH 5.9, containing (A) 0.1 mM CaCl₂, (B) 0.1 mM CaCl₂ and 0.05 mM MgCl₂, (C) 0.05 mM CaCl₂ and 0.1 mM MgCl₂, and (D) 0.01 mM CaCl₂ and 0.1 mM MgCl₂. C represents CaM and P peptide, respectively.

FIGURE 9

The ion-source collision-induced-dissociation of CaM–RS20–Ca₄ complex (concentration ratio 1:1.5:5) in 5 mM ammonium acetate buffer, pH 5.9. (A)–(D) were measured on the sample using the ES capillary potentials from 104 V to 224 V. (A) shows the ESI-FTICR mass spectrum of the intact complex. The increase of the capillary potential from 104 V to 152 V (indicated in (B)) results in the decomposition of the complex to CaM–Ca₄ and RS20. Further increase of the capillary potential leads to the decomposition of CaM–Ca₄ (C) and fragmentation of CaM backbone (D). C represents CaM and P peptide, respectively.

FIGURE 10

Stability in the gas-phase of the CaM–peptide–Ca₄ complexes shown as a function of capillary potential. The ratio was calculated using the relative intensities of signals corresponding to CaM–peptide–Ca₄ complex at the 8+ charge-state and CaM–Ca₄ at the 6+ charge-state. C represents CaM and P peptide, respectively.