Protein intermediates in equilibrium with native states may play important roles in protein dynamics but, in cases, can initiate harmful aggregation events. Investigating equilibrium protein intermediates is thus important for understanding protein behaviour (useful or pernicious) but it is hampered by difficulties in gathering structural information. We show here that the phi-analysis techniques developed to investigate transition states of protein folding can be extended to determine low-resolution three-dimensional structures of protein equilibrium intermediates. The analysis proposed is based solely on equilibrium data and is illustrated by determination of the structure of the apoflavodoxin thermal unfolding intermediate. In this conformation, a large part of the protein remains close to natively folded, but a 40 residue region is clearly unfolded. This structure is fully consistent with the NMR data gathered on an apoflavodoxin mutant designed specifically to stabilise the intermediate. The structure shows that the folded region of the intermediate is much larger than the proton slow-exchange core at 25 degrees C. It also reveals that the unfolded region is made of elements whose packing surface is more polar than average. In addition, it constitutes a useful guide to rationally stabilise the native state relative to the intermediate state, a far from trivial task.
Submitter: Shu-Ching Ou
Submission Date: Nov. 22, 2018, 10:41 p.m.
|Number of data points||408|
|Assays/Quantities/Protocols||Experimental Assay: Stability: m value ; Experimental Assay: Stability: ΔH: Native-to-Intermediate Equilibrium ; Experimental Assay: Stability: Tm: Native-to-Intermediate Equilibrium ; Experimental Assay: Stability: ΔH: Intermediate-to-Denatured Equilibrium ; Experimental Assay: Stability: Tm: Intermediate-to-Denatured Equilibrium ; Experimental Assay: Urea50 ; Derived Quantity: Stability: ΔΔG_fold ; Derived Quantity: Stability: ΔΔG_fold using <m> ; Derived Quantity: Thermal Stability: ΔΔG: Native-to-Intermediate ; Derived Quantity: Thermal Stability: ΔΔG: Intermediate-to-Denatured ; Derived Quantity: ϕ Value|
|Libraries||Variants of Apoflavodoxin|