Effect of Preformed Correct Tertiary Interactions on Rapid Two-State Tendamistat Folding:  Evidence for Hairpins as Initiation Sites for β-Sheet Formation

Abstract

The role of preformed correct side chain interactions, such as disulfide bonds, on protein folding kinetics is still not well understood. We investigated the effect of disulfide bond replacements on folding and stability of the small beta-sheet protein tendamistat. Tendamistat folds very fast (tau = 10 ms at pH 7 in water) and without detectable intermediates, which facilitates molecular interpretation of the kinetic data. Tendamistat contains two disulfide bonds, one between cysteines 11 and 27, which connects the ends of a beta-hairpin, and a second one between cysteines 45 and 73, which brings together the two outer strands of a three-stranded beta-sheet. Two single-disulfide variants of the protein were prepared by site-directed mutagenesis (tendamistat C11A/C27S and tendamistat C45A/C73A), and the effects on stability and on folding were monitored. Replacement of either disulfide bond leads to a large decrease in protein stability (DeltaDeltaG0 = 6.0 kcal/mol for the C11A/C27S variant and 5.1 kcal/mol for the C45A/C73A variant). This effect is caused both by entropic stabilization of the unfolded state and by enthalpic destabilization of the native structure. Kinetic experiments show that the main effect of fixed side chain contacts is on the unfolding rate. For both single-disulfide variants, unfolding is strongly accelerated (4250 times in the C11A/C27S variant and 250 times in the C45A/C73A variant) whereas the refolding rate constants are only slightly decreased. The activation parameters show that the observed small effect on the refolding reaction in the C11A/C27S variant is a consequence of large and compensating changes in the entropy and enthalpy of activation. Structural interpretation of the kinetic data suggests that formation of the beta-hairpin stabilized by the C11-C27 disulfide bond forms in the rate-limiting step of the refolding process. The interactions between the outer strands of the beta-sheet connected by the C45-C73 disulfide bond, in contrast, are made late in refolding. These results support the idea that beta-hairpins are initiation sites for beta-sheet formation and that additional strands are added late in the folding process.

Protein folding simulations: From coarse‐grained model to all‐atom model

“…Our model showed that the disulfide bonds as well as the three hydrogen bonds between the N-terminal loop-0 and strand-6 are of significant importance for the folding of tendamistat (79); without either interaction, the two-state behaviors become unstable or the folding pathway changes significantly. The simulation based on the modified model showed that tendamistat and its two singledisulfide mutants are all two-state folders, consistent with the experimental observations (76,79). By comparing the folding behaviors of the wild-type tendamistat with its two mutants, it was found that the removal of either C11-C27 or C45-C73 disulfide bond leads to a large decrease in the thermodynamical stability and the loss of structure in the unfolded state; and the effect of the former is stronger than that of the latter, as shown in Fig.…”

“…It is an allb-sheet protein with 74 amino acids and has two disulfide bonds, C11-C27 and C45-C73. There are extensive experimental studies on this protein (72)(73)(74)(75)(76)(77)(78)(79). As a brief summary of these works, tendamistat exhibits a two-state folding behavior that is unique among all the disulfide bonded proteins discovered so far, and this behavior sustains if either disulfide bond is removed (76).…”

“…There are extensive experimental studies on this protein (72)(73)(74)(75)(76)(77)(78)(79). As a brief summary of these works, tendamistat exhibits a two-state folding behavior that is unique among all the disulfide bonded proteins discovered so far, and this behavior sustains if either disulfide bond is removed (76). A surprising feature is that the removal of the C11-C27 disulfide bond leads to a larger effect on the stability than the removal of the C45-C73, since the latter involves a longer sequence stretch thus is presumed to have a larger entropic effect (Fig.…”

Identification and Characterization of an Equilibrium Intermediate in the Unfolding Pathway of an All β-Barrel Protein

“…Molten globule intermediates usually display a significant exposure of hydrophobic cores to the solvent. Hence, ANS binds strongly to MG and fluoresces intensely (29,30). The dye generally exhibits weak binding affinity to the native and unfolded states of proteins (29).…”

Cross-Strand Coupling and Site-Specific Unfolding Thermodynamics of a Trpzip β-Hairpin Peptide Using 13C Isotopic Labeling and IR Spectroscopy

“…Studies of isolated, water-soluble -hairpins can provide valuable insight into properties of -sheet structure and folding since -hairpins are considered to act as possible nucleation sites for protein folding. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] Additionally, their detailed structural interactions can be relevant in developing an understanding of the mechanism for forming various -sheet structures such as found in many (amyloid-like) neurodegenerative diseases in which protein aggregation is an important pathology. [15][16][17][18] During the past decade, there have been many reports discussing de novo designed, water-soluble -hairpin peptide systems, as well documented in several reviews.…”