Effect of Interdomain Linker Length on an Antagonistic Folding–Unfolding Equilibrium between Two Protein Domains

Abstract

Fusion of one protein domain with another is a common event in both evolution and protein engineering experiments. When insertion is at an internal site (e.g., a surface loop or turn), as opposed to one of the termini, conformational strain can be introduced into both domains. Strain is manifested by an antagonistic folding-unfolding equilibrium between the two domains, which we previously showed can be parameterized by a coupling free-energy term (ΔG X ). The extent of strain is predicted to depend primarily on the ratio of the N-to-C distance of the guest protein to the distance between ends of the surface loop in the host protein. Here, we test that hypothesis by inserting ubiquitin (Ub) into the bacterial ribonuclease barnase (Bn), using peptide linkers from zero to 10 amino acids each. ΔG X values are determined by measuring the extent to which Co 2+ binding to an engineered site on the Ub domain destabilizes the Bn domain. All-atom, unforced Langevin dynamics simulations are employed to gain structural insight into the mechanism of mechanically induced unfolding. Experimental and computational results find that the two domains are structurally and energetically uncoupled when linkers are long and that ΔG X increases with decreasing linker length. When the linkers are fewer than two amino acids, strain is so great that one domain unfolds the other. However, the protein is able to refold as dimers and higher-order oligomers. The likely mechanism is a threedimensional domain swap of the Bn domain, which relieves conformational strain. The simulations suggest that an effective route to mechanical unfolding begins with disruption of the hydrophobic core of Bn near the Ub insertion site.

Cytosolic Selection Systems To Study Protein Stability

“…These sandwich fusion-based systems directly link the stability and solubility of model proteins to a phenotype that is easily selectable in E. coli and, in the case of G418, are screenable in the yeast S. cerevisiae. Insertions are usually deleterious, frequently disrupting the protein structure at the insertion site in catastrophic ways (25,33). It is difficult to predict those rare sites within a protein that can accommodate insertions without severe functional disruption (34).…”

“…These types of sites are presumably quite rare, as most insertions are disruptive to protein function (25). We decided to develop a general protocol to find sites within selectable markers that would tolerate insertions and then to find within this set those in which the stability of the inserted protein determined the selectable marker's activity.…”

Chemical Biology Strategies for Posttranslational Control of Protein Function

“…Optimization of insertion site and linker lengths have dramatic effects on the success of allosteric regulation strategies, but are often empirically determined (Cutler et al, 2009). As an alternative to this, Ranganathan and coworkers used statistical coupling analysis (SCA) to identify sectors connected to the surface residues as targets for allosteric modulation(Lee et al, 2008).…”

Influence of Secondary-Structure Folding on the Mutually Exclusive Folding Process of GL5/I27 Protein: Evidence from Molecular Dynamics Simulations

“…[9,10,11]. Mutually exclusive folding protein as domain insertion protein or fusion protein serves to analyze the coupled folding–unfolding equilibrium through the conformational transition of antagonistic folding/unfolding [12,13,14,15]. Some of them can be also potentially regarded as a new class of cytotoxic proteins that can be activated by cell-specific effector molecules [15].…”