Folding kinetics of the S100A11 protein dimer studied by time-resolved electrospray mass spectrometry and pulsed hydrogen-deuterium exchange.
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.
Pan
Jingxi
J
Department of Chemistry, The University of Western Ontario, London, Ontario N6A 5B7, Canada.
Rintala-Dempsey
Anne C
AC
Li
Yu
Y
Shaw
Gary S
GS
Konermann
Lars
L
D000818:Animals; D002118:Calcium / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry*; D019281:Dimerization; D006859:Hydrogen / Q000737:chemistry*; D007700:Kinetics; D011487:Protein Conformation; D017510:Protein Folding; D011817:Rabbits; D011994:Recombinant Proteins; D009418:S100 Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D011994:Recombinant Proteins; D009418:S100 Proteins; D006859:Hydrogen; D003903:Deuterium; D002118:Calcium