Monitoring of unfolding of metallo-proteins by electrospray ionization mass spectrometry.
An electrospray ionisation (ESI) mass spectrometric method for the determination of the equilibrium constant and free energy (DeltaG) of protein unfolding was used to monitor the denaturation process at different pH of three metallo-proteins, i.e. wild-type copper azurin, zinc azurin and wild-type amicyanin. The time course of the unfolding process was followed by dissolving the proteins under denaturing conditions (methanol-water (1 : 1, v/v)) at different pH (2.5, 3.0, 3.5) and recording ESI spectra at time intervals. The spectra showed two series of peaks, corresponding to the native holo-protein and the unfolded apo-protein. From the intensity ratio of these two series of peaks at increasing time and at equilibrium, the equilibrium constants for the unfolding process for the three proteins could be determined. From these equilibrium constants a DeltaG degrees derivation was attempted. The DeltaG degrees values obtained decrease with decrease in pH, in agreement with the expected reduction of conformational stability of proteins at lower pH. The results obtained confirm that ESI-MS can be used for monitoring of unfolding process and to derive quantitative thermodynamic data.
Cunsolo
Vincenzo
V
Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy.
Foti
Salvatore
S
La Rosa
Carmelo
C
Saletti
Rosaria
R
Canters
G W
GW
Verbeet
M Ph
MP
D001400:Azurin / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry
D001426:Bacterial Proteins; D008667:Metalloproteins; C017079:mauC protein, Methylobacterium extorquens; D001400:Azurin; D003300:Copper; D015032:Zinc