| Autor: |
Raab SA; Department of Chemistry, Indiana University, 800 Kirkwood Avenue, Bloomington, Indiana 47401, United States., El-Baba TJ; Department of Chemistry, Indiana University, 800 Kirkwood Avenue, Bloomington, Indiana 47401, United States., Woodall DW; Department of Chemistry, Indiana University, 800 Kirkwood Avenue, Bloomington, Indiana 47401, United States., Liu W; Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States., Liu Y; Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States., Baird Z; Baxter Healthcare Corporation, 927 South Curry Pike, Bloomington, Indiana 47403, United States., Hales DA; Department of Chemistry, Hendrix College, Conway, Arkansas 72032, United States., Laganowsky A; Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States., Russell DH; Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States., Clemmer DE; Department of Chemistry, Indiana University, 800 Kirkwood Avenue, Bloomington, Indiana 47401, United States. |
| Abstrakt: |
Chymotrypsin inhibitor 2 (CI-2) is a classic model for two-state cooperative protein folding and is one of the most extensively studied systems. Alan Fersht, a pioneer in the field of structural biology, has studied the wild-type (wt) and over 100 mutant forms of CI-2 with traditional analytical and biochemical techniques. Here, we examine wt CI-2 and three mutant forms (A16G, K11A, L32A) to demonstrate the utility of variable-temperature (vT) electrospray ionization (ESI) paired with ion mobility spectrometry (IMS) and mass spectrometry (MS) to map the free energy folding landscape. As the solution temperature is increased, the abundance of each of the six ESI charge states for wt CI-2 and each mutant is found to vary independently. These results require that at least six unique types of CI-2 solution conformers are present. Ion mobility analysis reveals that within each charge state there are additional conformers having distinct solution temperature profiles. A model of the data at ∼30 different temperatures for all four systems suggests the presence of 41 unique CI-2 solution conformations. A thermodynamic analysis of this system yields values of Δ C p as well as Δ G , Δ H , and Δ S for each state at every temperature studied. Detailed energy landscapes derived from these data provide a rare glimpse into Anfinsen's thermodynamic hypothesis and the process of thermal denaturation, normally thought of as a cooperative two-state transition involving the native state and unstructured denatured species. Specifically, as the temperature is varied, the entropies and enthalpies of different conformers undergo dramatic changes in magnitude and relative order to maintain the delicate balance associated with equilibrium. |
