Enhanced picture of protein-folding intermediates using organic solvents in H/D exchange and quench-flow experiments.
Hydrogen/deuterium exchange followed by trapping of the labeled species in the aprotic solvent DMSO has been used to elucidate structure in both the burst-phase molten globule-folding intermediate of apomyoglobin and in an equilibrium intermediate that models the kinetic intermediate. Precise estimates can be made of exchange times in an interrupted exchange-out experiment at pH 4 followed by analysis in DMSO solution, giving extensive sequence-specific information about the structure of the equilibrium intermediate. In addition, the use of DMSO as a solvent for NMR measurements after quench-flow pH-pulse labeling experiments gives a greatly increased data set for the elucidation of the kinetic folding pathway. Interestingly, differences are observed in some regions of apomyoglobin between the equilibrium and kinetic intermediates. These differences are quantitative rather than qualitative; that is, the overall patterns of labeling and secondary structure formation remain similar between the two species. However, local differences are observed, which probably reflect the difference in the solution conditions for the equilibrium experiment (pH 4) vs. the kinetic experiment (pH 6) and the change in the status of the stabilizing hydrogen bond between the side chains of His-24 and His-119.
Nishimura
Chiaki
C
Department of Molecular Biology and The Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Dyson
H Jane
HJ
Wright
Peter E
PE
D000818:Animals; D001059:Apoproteins / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D004121:Dimethyl Sulfoxide; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009211:Myoglobin / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D014907:Whales / Q000378:metabolism*
D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin; D004121:Dimethyl Sulfoxide