Thermal denaturation of Escherichia coli thioredoxin studied by hydrogen/deuterium exchange and electrospray ionization mass spectrometry: monitoring a two-state protein unfolding transition.
Thermally denatured oxidized Escherichia coli thioredoxin (TRX) in 2% acetic acid was examined by electrospray ionization mass spectrometry (ESI-MS) and circular dichroism. Conformational dynamics during thermal unfolding were probed by hydrogen/deuterium (H/D) exchange-in experiments. ESI-MS was used to determine the H/D ratios. TRX shows only a marginal change in negative ellipticity at 222 nm during thermal unfolding, but in the near-UV circular dichroism (240-350 nm) a clear transition is observed (Tm = 61 degrees C), and unfolding goes to completion. ESI mass spectra were recorded as a function of temperature, and the observed bimodal charge state distributions were analyzed assuming a two-state unfolding mechanism which allowed an estimation of the midpoint temperature, Tm = 64 degrees C. Under conditions at which the compact, folded conformational state is only marginally stable (80 degrees C, 2% acetic acid-d1), H/D exchange-in experiments in combination with ESI-MS resulted in mass spectra differing in the number of incorporated deuteriums which indicates the presence of two distinct populations of molecules after short incubation periods. As the exchange-in time increases, the population representing the unfolded state increases and the population which is protected against exchange decreases. The rate of conversion was used to estimate the rate constant of unfolding which was 2.1 +/- 0.2 min-1. The results presented here indicate that thermally denatured TRX under the conditions used may represent a collapsed unfolded state with properties often attributed to molten globule-like states, such as pronounced secondary structure but absence of rigid tertiary structure and, hence, lack of protection against H/D exchange.
Maier
C S
CS
Department of Chemistry, Oregon State University, Corvallis 97331, USA.
Schimerlik
M I
MI
Deinzer
M L
ML
D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D003903:Deuterium; D004926:Escherichia coli; D006358:Hot Temperature; D006859:Hydrogen; D013058:Mass Spectrometry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry*
D001426:Bacterial Proteins; D013879:Thioredoxins; D006859:Hydrogen; D003903:Deuterium