Abstract
A crucial mechanism to the formation of native, fully functional, 3D structures from local secondary structures is unraveled in this study. Through the introduction of various amino acid substitutions at four canonical β-turns in a three-fingered protein, Toxin α from Naja nigricollis, we found that the release of internal entropy to the external environment through the globally synchronized movements of local substructures plays a crucial role. Throughout the folding process, the folding species were saturated with internal entropy so that intermediates accumulated at the equilibrium state. Their relief from the equilibrium state was accomplished by the formation of a critical disulfide bridge, which could guide the synchronized movement of one of the peripheral secondary structure. This secondary structure collided with a core central structure, which flanked another peripheral secondary structure. This collision displaced the internal thermal fluctuations from the first peripheral structure to the second peripheral structure, where the displaced thermal fluctuations were ultimately released as entropy. Two protein folding processes that acted in succession were identified as the means to establish the flow of thermal fluctuations. The first process was the time-consuming assembly process, where stochastic combinations of colliding, native-like, secondary structures provided candidate structures for the folded protein. The second process was the activation process to establish the global mutual relationships of the native protein in the selected candidate. This activation process was initiated and propagated by a positive feedback process between efficient entropy release and well-packed local structures, which moved in synchronization. The molecular mechanism suggested by this experiment was assessed with a well-defined 3D structure of erabutoxin b because one of the turns that played a critical role in folding was shared with erabutoxin b.
Highlights
The three-fingered protein domain (TFPD) is a small, functional module composed of 60 to 90 amino acid residues that form three successive fingers [1,2,3,4,5]
The structure of the TFPD was initially established in erabutoxin b [6,7], from Laticauda semifasciata, which binds to the nicotinic acetylcholine receptor
Protein folding is motivated by a need to release the inevitably accumulated internal thermal fluctuations to the external environment
Summary
The three-fingered protein domain (TFPD) is a small, functional module composed of 60 to 90 amino acid residues that form three successive fingers [1,2,3,4,5]. (5) Three disulfide bonds are located close to each other, and an invariant asparagine residue near the C-terminus (N61 in erabutoxin b) is essential to hold their relative orientation [1]. The significant structural attributes of the TFPD fold are as follows: (1) The N-terminus half of the domain is primarily stabilized by inter-strand hydrogen bonds to form rigid βpleated sheets. These three disulfide bonds are essentially buried in the folded state, while the fourth disulfide bond (S43-S54 in erabutoxin b) is solvent-exposed [10]. This result has been understood to correlate with the conformational variability of the intermediates [8,10]
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