title: HX-ESI-MS and optical studies of the unfolding of thioredoxin indicate stabilization of a partially unfolded, aggregation-competent intermediate at low pH.
abstract: Hydrogen exchange monitored by mass spectrometry (HX-MS), in conjunction with multiple optical probes, has been used to characterize the unfolding of thioredoxin. Equilibrium and kinetic studies have been carried out at pH 7 and 3. The HX-MS measurements are shown to be capable of distinguishing between native (N) and unfolded (U) protein molecules when both are present together, and their application in kinetic experiments allows the unfolding reaction to be delineated from the proline isomerization reaction to which it is coupled. At pH 7, equilibrium unfolding studies monitored by three optical probes, intrinsic fluorescence at 368 nm, ellipticity at 222 nm, and ellipticity at 270 nm, as well as by HX-MS, indicate that no intermediate is populated at pH 7, the unfolding reaction is slower than the proline isomerization reaction that follows it, and the three optical probes yield identical kinetics for unfolding, which occurs in a single kinetic phase. The fractional change in any of the three optical signals at any time of unfolding predicts the fraction of the molecules that have become U, as determined by HX-MS. Hence, unfolding at pH 7 appears to occur via a two-state N <==> U mechanism. In contrast at pH 3, HX-MS as well as optical measurements indicate that an unfolding intermediate is stabilized and hence accumulates in equilibrium with N and U, at concentrations of denaturant that define the transition zone of the equilibrium unfolding curve. The intermediate has lost the near-UV signal characteristic of N and possesses fewer amide hydrogen sites that are stable to exchange than does N. Kinetic experiments at pH 3, where unfolding is much faster than proline isomerization, show that more than one intermediate accumulates transiently during unfolding. Thus, the unfolding of thioredoxin occurs via an N <==> I <==> U mechanism, where I is a partially unfolded intermediate that is stabilized and hence populated at pH 3 but not at pH 7. It is shown that transient aggregation of this intermediate results in a deceleration of the kinetics of unfolding at high protein concentrations at pH 3 but not at pH 7.
authors.affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India.
authors.forename: Ajazul
authors.initials: A
authors.lastname: Hamid Wani
authors.forename: Jayant B
authors.initials: JB
authors.lastname: Udgaonkar
mesh_terms: D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D055095:Optics and Photonics; D011487:Protein Conformation; D017510:Protein Folding; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D013879:Thioredoxins