An experimentally determined protein folding energy landscape

Abstract

Energy landscapes have been used to conceptually describe and model protein folding but have been difficult to measure experimentally, in large part because of the myriad of partly folded protein conformations that cannot be isolated and thermodynamically characterized. Here we experimentally determine a detailed energy landscape for protein folding. We generated a series of overlapping constructs containing subsets of the seven ankyrin repeats of the Drosophila Notch receptor, a protein domain whose linear arrangement of modular structural units can be fragmented without disrupting structure. To a good approximation, stabilities of each construct can be described as a sum of energy terms associated with each repeat. The magnitude of each energy term indicates that each repeat is intrinsically unstable but is strongly stabilized by interactions with its nearest neighbors. These linear energy terms define an equilibrium free energy landscape, which shows an early free energy barrier and suggests preferred low-energy routes for folding.

Repeat-protein folding: New insights into origins of cooperativity, stability, and topology

“…As long as the interfacial free energy exceeds the intrinsic penalty, adding to the preexisting folded stack of repeats is energetically favorable. This thermodynamic profile is seen in the energy landscape of the Notch ankyrin domain ( Figure 4A), and is reflected in the fitted free energy coefficients of equation 1, which give estimates of ΔG intrinsic and ΔG interface of +6.6 and −9.1 kcal/mol, respectively [76].…”

“…However, the ability of the linear model (eqn 1) to fit to the deletion series suggests that distant repeats can treated as thermodynamically independent [76]. The resolution to this paradox comes from analysis of the coefficients derived from fitting the deletion series.…”

“…As long as the interfacial free energy exceeds the intrinsic penalty, adding to the preexisting folded stack of repeats is energetically favorable. This thermodynamic profile is seen in the energy landscape of the Notch ankyrin domain ( Figure 4A), and is reflected in the fitted free energy coefficients of equation 1, which give estimates of ΔG intrinsic and ΔG interface of +6.6 and −9.1 kcal/mol, respectively [76].There are a number of examples in both biopolymer and material sciences where conformational transitions can be described in terms of nearest-neighbor interactions within a one-dimensional lattice. Such models are a subset of nearest-neighbor "Ising" models ( Although ankyrin and TPR repeats differ in both primary sequence and structure, it is interesting to compare the intrinsic and interfacial energies determined in the Notch and consensus TPR systems.…”

“…In contrast, in bulk studies, destabilization is brought about by a uniformly-acting potential like denaturant or increased temperature, and partial unfolding of one region does not relieve the destabilization resulting on other regions. Because fragments of repeat proteins are known to be stable [74,76], the persistence of stably folded fragments in the wake of partial forced-unfolding transitions should not be unexpected.Since interfaces between ankyrin repeats are highly stabilizing, whereas individual repeats are unstable, the most likely scenario for forced unfolding is one in which individual repeats peel off in steps from a block of folded repeats. In this scenario, forced unfolding studies should identify the minimal number of repeats that can remain folded from the number of single-repeat transitions, as well as from the contour length increment produced in the last transition.…”

Rerouting the Folding Pathway of the Notch Ankyrin Domain by Reshaping the Energy Landscape

“…28 We have used the same analysis to compare how the folding energy landscape of Nank1-5C 2 is altered from that of the Nank1-7Δ. 33 Although Nank1-7Δ and Nank1-5C 2 contain the same number of ankyrin repeats, the unfolding free energy of Nank1-5C 2 is 13 kcal/mol, which is over 5 kcal/mol more stable than Nank1-7Δ ( Figure 4A and 4D).…”