Effects of disulfide bonds on folding behavior and mechanism of the beta-sheet protein tendamistat

Abstract

Tendamistat, a small disulfide-bonded beta-sheet protein, and its three single/double-disulfide mutants are investigated by using a modified Gō-like model, aiming to understand the folding mechanism of disulfide-bonded protein as well as the effects of removal of disulfide bond on the folding process. Our simulations show that tendamistat and its two single-disulfide mutants are all two-state folders, consistent with the experimental observations. It is found that the disulfide bonds as well as three hydrogen bonds between the N-terminal loop-0 and strand-6 are of significant importance for the folding of tendamistat. Without these interactions, their two-state behaviors become unstable and the predictions of the model are inconsistent with experiments. In addition, the effect of disulfide bonds on the folding process are studied by comparing the wild-type tendamistat and its two mutants; it is found that the removal of either of the C11-C27 or C45-C73 disulfide bond leads to a large decrease in the thermodynamical stability and loss of structure in the unfolded state, and the effect of the former is stronger than that of the later. These simulation results are in good agreement with experiments and, thus, validate our model. Based on the same model, the detailed folding pathways of the wild-type tendamistat and two mutants are studied, and the effect of disulfide bonds on the folding kinetics are discussed. The obtained results provide a detailed folding picture of these proteins and complement experimental findings. Finally, the folding nuclei predicted to be existent in this protein tendamistat as well as its mutants are firstly identified in this work. The positions of the nucleus are consistent with those argued in experimental studies. Therefore, a nucleation/growth folding mechanism that can explain the two-state folding manner is clearly characterized. Moreover, the effect by the removal of each disulfide bond on the folding thermodynamics and dynamics can also be well interpreted from their influence on the folding nucleus. The implementation of this work indicates that the modified Gō-like model really describes the folding behavior of protein tendamistat and could be used to study the folding of other disulfide-bonded proteins.

FIGURE 1

The schematic representation (backbone model) of the structure of wild-type tendamistat. The cysteines are displayed with ball-and-stick model and the disulfide bonds are shown with yellow color. The first disulfide bond (Cys-11–Cys-27) is located at the base of the first β-hairpin (composed of strand-1 and strand-2). The second disulfide bond (C45– C73) connects the two outer strands-4 and strand-6.

FIGURE 2

The specific heat C_v as a function of the temperature T. The transition temperatures of the wild-type tendamistat (thick solid line), the C11A/C27S mutant M₁ (dotted line), the C45A/C73A mutant M₂ (thin solid line), and the mutant M₃ removed both of the disulfide bonds (dashed line) are 1.217, 1.179, 1.189, and 1.152, respectively.

FIGURE 3

(a–d) The trajectories of the reaction coordinate Q as a function of time (in unit of 10⁷ molecular dynamics steps), which are obtained from the typical simulations around the respective folding temperature. Each of the model protein has equal probabilities to be found in the unfolded state and in the folded state, which exhibits the two-state folding behavior clearly; (a) for the wild-type tendamistat, (b) for the mutant M₁, (c) for the mutant M₂, and (d) for the mutant M₃. Fig. 3 e shows the distribution of the native contact in the unfolded states for the wild-type tendamistat (thick solid line), the mutant M₁ (dotted line), the mutant M₂ (thin solid line), and the mutant M₃ (dashed line). Q_u is the value of Q at each of the peak of the distribution.

FIGURE 4

Free-energy contour maps as the function of two reaction coordinates: the structural similarity Q and the radius gyration R_g near their respective folding temperatures. The two-state folding processes can be clearly exhibited in the free-energy landscapes of the wild-type tendamistat (a), the mutant M₁ (b), the mutant M₂ (c), and the mutant M₃ (d). The removal of each single-disulfide bond does not take any effects to the two-state folding behavior (refer to the text for more detailed discussion). The free-energy difference between adjacent contour lines is 1.0 k_BT.

FIGURE 5

The probabilities of the unfolded state P_u as a function of the unfolding time τ_u (in unit of 10⁴ molecular dynamic steps) for the wild-type tendamistat (thick solid line), the mutant M₁ (dotted line), the mutant M₂ (thin solid line), and the mutant M₃ (dashed line). Each plot is obtained from the average on >5000 unfolding simulations.

FIGURE 6

The formation probability of different parts of the wild-type tendamistat as a function of the reaction coordinate Q. Different curves correspond to the formation trajectories of different parts: the first β-hairpin S1–2 (thick solid line), β-sheet S1–5 (thin solid line), β-sheet S2–5 (thick dotted line), the second β-hairpin S3–4 (thick dashed line), β-sheet S3–6 (thin dotted line), β-sheet S4–6 (thin dashed line), and the region S0–6 between the N-terminal loop0 and the strand-6 (thin dash-dotted line).

FIGURE 7

The formation probability of each native contact of the wild-type tendamistat at the four typical moments in the folding process that the moment of Q = 0.15 (a), the moment of Q = 0.3 (b), the moment of Q = 0.5 (c), and the moment of Q = 0.8 (d). Different colors indicate different values from 0 to 1, as quantified by the color scale, i.e., the darker of the color corresponds to the bigger of the value of the formation probability.

FIGURE 8

The formation probability of the different parts as a function of the structural similarity Q for the wild-type tendamistat (thick solid line), the mutant M₁ (thin solid line), and the mutant M₂ (dashed line). The different parts are presented, respectively, for S_1–2 (a), for S_3–4 (b), for S_3–6 (c), for S_1–5 (d), for S_2–5 (e), for S_4–6 (f), and for S_0–6 (g). These folding processes are simulated at the temperatures that are lower than their respective folding temperatures.

FIGURE 9

The first appearance time τ_{i, j} of the i–j native contact averaged on 2000 simulations for the wild-type tendamistat (a), the mutant M₁ (b), and the mutant M₂ (c), respectively. The contact color from light gray to black represents the value of the first formation time changing from minimum to maximum gradually.

FIGURE 10

The simple description of the folding pathway for the wild-type tendamistat. The contacts inside the circle regions with thick lines are formed before the transition state; the ones inside the rectangular regions with thick line are formed in the TS; the ones inside the ellipse region with thin lines are formed in the later folding process except several key contacts are found to be formed in the TS; and the ones inside the irregular region with thin lines are formed in the last step of the whole folding process.

FIGURE 11

The probability difference, P_FF–UU, between the P_FF and P_UU, for the native contacts are shown in the contact map. Where P_FF and P_UU represent the formation probability of native contact for the structures at the turning point of the FF and UU events, respectively (see the text for the detailed definition of these two events). (a) For the case of the wild-type tendamistat, most of the contacts within the circles I, II, and III have positive values, while others have negative values; (b) for the case of the mutant M₁ (C11A/C27S mutant), only most of the contacts within the circles I and II have positive values; and (c) for the case of the mutant M₂, only the contacts within the circles I and II have positive values.