The small alpha/beta protein ubiquitin has been used as a model system for experimental and computational studies on protein folding for many years. Here, we present a comprehensive phi-value analysis and characterize the structure and energetics of the transition state ensemble (TSE). Twenty-seven non-disruptive mutations are made throughout the structure and a range of phi-values from zero to one are observed. The values cluster such that medium and high values and found only in the N-terminal region of the protein, whilst the C-terminal region has consistently low phi-values. In the TSE, the main alpha-helix appears to be fully formed (two phi-values which specifically probe helical structure are one) and the helix is stabilized by packing against the first beta-turn, which is partially structured. In striking comparison, the phi-values in the C-terminal region are all very low, suggesting that this region of the protein is largely unstructured in the TSE. Data are consistent with a nucleation-condensation mechanism in which there is a highly polarized folding nucleus comprising the first beta-hairpin and the alpha-helix. Data presented from the protein engineering study and phi-value analysis are compared with results from other experimental studies and also computational studies.
Submitter: Connie Wang
Submission Date: Aug. 9, 2017, 12:43 p.m.
|Number of data points||390|
|Assays/Quantities/Protocols||Experimental Assay: mF ; Experimental Assay: lnk4MGdnHCl ; Experimental Assay: lnk (H20) U ; Experimental Assay: lnk (H20) F ; Experimental Assay: ∆∆G(H20) ; Experimental Assay: mU ; Derived Quantity: phi-unfolding ; Derived Quantity: phi-folding|
|Libraries||Kinetic and thermodynamic parameters for the unfolding and folding of wild-type and mutant ubiquitin|