Folding Kinetics of the S100A11 Protein Dimer Studied by Time-Resolved Electrospray Mass Spectrometry and Pulsed Hydrogen−Deuterium Exchange

Abstract

This study reports the application of electrospray ionization (ESI) mass spectrometry (MS) with on-line rapid mixing for millisecond time-resolved studies of the refolding and assembly of a dimeric protein complex. Acid denaturation of S100A11 disrupts the native homodimeric protein structure. Circular dichroism and HSQC nuclear magnetic resonance measurements reveal that the monomeric subunits unfold to a moderate degree but retain a significant helicity and some tertiary structural elements. Following a rapid change in solution conditions to a slightly basic pH, the native protein reassembles with an effective rate constant of 6 s(-)(1). The ESI charge state distributions measured during the reaction suggest the presence of three kinetic species, namely, a relatively unfolded monomer (M(U)), a more tightly folded monomeric reaction intermediate (M(F)), and dimeric S100A11. These three forms exhibit distinct calcium binding properties, with very low metal loading levels for M(U), up to two calcium ions for M(F), and up to four for the dimer. Surprisingly, on-line pulsed hydrogen-deuterium exchange (HDX) reveals that each of the monomeric forms of the protein comprises two subspecies that can be distinguished on the basis of their isotope exchange levels. As the reaction proceeds, the more extensively labeled species are depleted. The exponential nature of the measured intensity-time profiles implies that the rate-determining step of the overall process is a unimolecular event. The kinetics are consistent with a sequential folding and assembly mechanism involving two increasingly nativelike monomeric intermediates en route to the native S100A11 dimer.

A New Approach to Explore the Impact of Freeze-Thaw Cycling on Protein Structure: Hydrogen/Deuterium Exchange Mass Spectrometry (HX-MS)

“…HX-MS has been widely used in solution phase for analysis of protein conformation and dynamics (23)(24)(25), as well as structural changes upon a variety of state transitions, such as folding/unfolding (26), ligand binding (27), and self-association (28,29). Further, Li et al carried out hydrogen/deuterium exchange in protein powders to measure the effects of lyophilization and excipients on protein structure (30).…”

The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two‐state transition

“…The analysis of different ion charge state distribution (CSD) envelopes derived from different protein conformations is one of the methods for monitoring protein structural transition by ESI-MS (Chowdhury et al 1990;Loo et al 1991). ESI-MS has proven particularly useful for detection and characterization of folding/unfolding intermediates (Grandori 2002;Haq et al 2005;Wintrode et al 2005) and for kinetic studies of protein structural transitions (Konermann 2004;Furdui et al 2006;Pan et al 2006). It has been well documented that an unfolded protein molecule can accommodate more protons upon desolvation than can a compact native protein molecule because of the increased solvent-exposed surface area (Mohimen et al 2003).…”

Temporal Development of Protein Structure during S100A11 Folding and Dimerization Probed by Oxidative Labeling and Mass Spectrometry

“…45 The acid-denatured state of S100A11 has been characterized by electrospray ionization (ESI) MS, CD, and NMR spectroscopy. 46 These techniques revealed that acid exposure induces complete dimer dissociation. The free subunits exhibit a greatly diminished NMR chemical shift dispersion at pH 2, indicating major perturbations in tertiary structure.…”

“…Time-resolved ESI-MS has previously been used to examine the mechanism of this process at the intact protein level. 46 It was found that the reaction proceeds through a burst-phase monomeric intermediate. Pulsed HDX suggested the formation of additional transient species, although the isotope exchange data were convoluted and did not provide clear insights into the reaction mechanism.…”

“…Pulsed HDX suggested the formation of additional transient species, although the isotope exchange data were convoluted and did not provide clear insights into the reaction mechanism. 46 These difficulties may be attributable to the retention of native-like secondary structure in acid-denatured S100A11, which makes it challenging to differentiate kinetic species on the basis of their hydrogen-bonding characteristics.…”