Fourier transform ion cyclotron resonance mass spectrometric detection of small Ca(2+)-induced conformational changes in the regulatory domain of human cardiac 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 reveals a small conformational change in the cardiac regulatory N-terminal domain of TnC (cNTnC) on binding of Ca2+ such that the total exposed hydrophobic surface area increases very slightly from 3090 +/- 86 A2 for apo-cNTnC to 3108 +/- 71 A2 for Ca(2+)-cNTnC. Here, we show that measurement of solvent accessibility for backbone amide protons by means of solution-phase hydrogen/deuterium (H/D) exchange followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform Ion cyclotron resonance mass spectrometry is sufficiently sensitive to detect such small ligand binding-induced conformational changes of that protein. The extent of deuterium incorporation increases significantly on binding of Ca2+ for each of four proteolytic segments derived from pepsin digestion of the apo- and Ca(2+)-saturated forms of cNTnC. The present results demonstrate that H/D exchange monitored by mass spectrometry can be sufficiently sensitive to detect and identify even very small conformational changes in proteins, and should therefore be especially informative for proteins too large (or too insoluble or otherwise intractable) for NMR analysis.
Wang
F
F
Center for Interdisciplinary Magnetic Resonance, Florida State University, Tallahassee 32310, USA.
Li
W
W
Emmett
M R
MR
Marshall
A G
AG
Corson
D
D
Sykes
B D
BD
D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry*; D020134:Catalytic Domain; D017357:Cyclotrons; D005583:Fourier Analysis; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry*; D010434:Pepsin A / Q000737:chemistry; D010449:Peptide Mapping; D011487:Protein Conformation; D012997:Solvents; D019209:Troponin C / Q000737:chemistry*
D012997:Solvents; D019209:Troponin C; D006859:Hydrogen; D010434:Pepsin A; D002118:Calcium