The Location of an Engineered Inter-Subunit Disulfide Bond in Factor for Inversion Stimulation (FIS) Affects the Denaturation Pathway and Cooperativity

Abstract

Two crossed-linked variants of the homodimeric DNA binding protein factor for inversion stimulation (FIS) were created via engineering of single intermolecular disulfide bonds. The conservative S30C and the nonconservative V58C FIS independent mutations resulted in FIS crossed-linked at the A helix (C30-C30) and at the middle of the B helix (C58-C58). This study sought to investigate how the location of an intermolecular disulfide bond may determine the effect on stability and its propagation through the structure to preserve or alter the denaturation cooperativity of FIS. The oxidized and reduced S30C and V58C FIS exhibited a far-UV CD spectrum and DNA binding affinities that were similar to WT FIS, indicating no significant changes in secondary and tertiary structure. However, the reduced and oxidized forms of the mutants revealed significant differences in the stability and equilibrium denaturation mechanism between the two mutants. In the reduced state, S30C FIS had very little effect on FIS stability, whereas V58C FIS was 2-3 kcal/mol less stable than WT FIS. Interestingly, while both disulfide bonds significantly increased the resistance to urea- and guanidine hydrochloride (GuHCl)-induced denaturation, oxidized V58C FIS exhibited a three-state GuHCl-induced transition. In contrast, oxidized S30C FIS displayed a highly cooperative WT-like transition with both denaturants. The three-state denaturation mechanism of oxidized V58C FIS induced by the GuHCl salt was reproduced by urea denaturation at pH 4, suggesting that disruption of a C-terminus salt-bridge network is responsible for the loss of denaturation cooperativity of V58C FIS in GuHCl or urea, pH 4. A second mutation on V58C FIS created to place a single tryptophan probe (Y95W) at the C-terminus further implies that the denaturation intermediate observed in disulfide crossed-linked V58C FIS results from a decoupling of the stabilities of the C-terminus and the rest of the protein. These results show that, unlike the C30-C30 intermolecular disulfide bond, the C58-C58 disulfide bond did not evenly stabilize the FIS structure, thereby highlighting the importance of the location of an engineered disulfide bond on the propagation of stability and the denaturation cooperativity of a protein.

The role of the local environment of engineered Tyr to Trp substitutions for probing the denaturation mechanism of FIS

“…Perhaps the high ionic strength of GuHCl destabilizes the electrostatic network involving the C‐terminus of FIS, thereby decoupling the N 2 ⇆ I 2 and I 2 ⇆ 2U transitions. A similar effect with GuHCl was previously observed for engineered cysteine mutants of FIS 31. This would also be consistent with the 1.6 and 2.2 kcal/mol higher stability of WT and P61A, respectively, when using urea as the denaturant (Table I).…”

“… b Data and fitting of WT (GuHCl), WT (urea), and P61A (urea) denaturation are described in references31,28 and30, respectively. …”

Protein disulfide engineering

“…With regard to false negatives, it has been suggested that geometric models employed in disulfide prediction algorithms may be overly restrictive given the dynamic motion of protein chains and their ability to tolerate some modifications without strain [51]. This indeed may be the case, as there are numerous reports of successfully engineered disulfides that violate geometric constraints of computational models.…”

Geofold: Topology‐based protein unfolding pathways capture the effects of engineered disulfides on kinetic stability

“…We asked whether the changes in topology created by disulfide linkages could explain experimentally determined changes in stability or unfolding rate. Four case studies are presented here: three mutants of barnase22–24 five mutants of T4 lysozyme,25 two of factor for inversion stimulation (FIS),26 and one of E. coli dihydrofolate reductase (DHFR) 27. In each case, biophysical studies were done to determine the stability or unfolding rate.…”

“…Factor for inversion stimulation (FIS, PDB ID:3jrh) is an intertwined, homodimeric DNA‐binding protein. Two single site cysteine mutations were engineered into the dimer, and both mutants formed a disulfide at the two‐fold symmetry interface 26. This created proteins with branched, noncyclic topology, in contrast to the other disulfide linkage mutants presented here, all of which produced a cyclic topology.…”