Structural and dynamic characteristics of a partially folded state of ubiquitin revealed by hydrogen exchange mass spectrometry.
Structural and dynamic properties of a partially folded conformation (A-state) of ubiquitin are studied using amide hydrogen exchange in solution (HDX) and mass spectrometric detection. A clear distinction between the native state of the protein and the A-state can be made when HDX is carried out in a semicorrelated regime. Convoluted exchange patterns are interpreted with the aid of HDX simulations in a three-state system (highly structured, partially unstructured, and fully unstructured states). The data clearly indicate a highly dynamic character of the non-native state. Furthermore, combination of HDX and protein ion fragmentation in the gas phase [by means of collision-induced dissociation (CAD)] is used to evaluate the conformational stability of various protein segments specifically in the molten globular state. Chain flexibility appears to be distributed very unevenly in this non-native conformation. The highest degree of structural disorder is displayed by the C-terminal segment (Gly(53)-Gly(76)), which was previously suggested to form a transient alpha-helix. The least dynamic segment of ubiquitin in the A-state is Thr(9)-Glu(18) (which was previously suggested to form a stable nativelike beta-strand), with the adjacent segments exhibiting somewhat diminished conformational stability. The study also demonstrates the power of mass spectrometry as a tool in providing conformer-specific information about the structure and dynamics of both native and non-native protein states coexisting in solution under equilibrium.
Hoerner
Joshua K
JK
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Xiao
Hui
H
Kaltashov
Igor A
IA
D000818:Animals; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006859:Hydrogen / Q000737:chemistry*; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D025801:Ubiquitin / Q000737:chemistry*
D025801:Ubiquitin; D006859:Hydrogen