Fourier transform ion cyclotron resonance mass spectrometric detection of small Ca2+-induced conformational changes in the regulatory domain of humancardiac troponin C

Troponin C (TnC), a calcium-binding protein of the thin filament of muscle, plays a regulatory role in skeletal and cardiac muscle contraction. NMR re veals a small conformational change in the cardiac regulatory N-terminal do main of TnC (cNTnC) on binding of Ca2+ such that the total exposed hydropho bic surface area increases very slightly from 3090 +/- 86 Angstrom(2) for a po-cNTnC to 3108 +/- 71 Angstrom(2) for Ca2+-cNTnC. Here, we show that meas urement of solvent accessibility for backbone amide protons by means of sol ution-phase hydrogen/deuterium (H/D) exchange followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-f ield (9.4 T) Fourier transform Ion cyclotron resonance mass spectrometry is sufficiently sensitive to detect such small ligand binding-induced conform ational changes of that protein. The extent of deuterium incorporation incr eases significantly on binding of Ca2+ for each of four proteolytic segment s derived from pepsin digestion of the apo- and Ca2+-saturated forms of cNT nC. The present results demonstrate that H/D exchange monitored by mass spe ctrometry can be sufficiently sensitive to detect and identify even very sm all conformational changes in proteins, and should therefore be especially informative for proteins too large (or too insoluble or otherwise intractab le) for NMR analysis. (C) 1994 American Society for Mass Spectrometry.