Abstract
The complex topologies of large multi-domain globular proteins make the study of their folding and assembly particularly demanding. It is often characterized by complex kinetics and undesired side reactions, such as aggregation. The structural simplicity of tandem-repeat proteins, which are characterized by the repetition of a basic structural motif and are stabilized exclusively by sequentially localized contacts, has provided opportunities for dissecting their folding landscapes. In this study, we focus on the Erwinia chrysanthemi pectin methylesterase (342 residues), an all-β pectinolytic enzyme with a right-handed parallel β-helix structure. Chemicals and pressure were chosen as denaturants and a variety of optical techniques were used in conjunction with stopped-flow equipment to investigate the folding mechanism of the enzyme at 25 °C. Under equilibrium conditions, both chemical- and pressure-induced unfolding show two-state transitions, with average conformational stability (ΔG° = 35 ± 5 kJ·mol−1) but exceptionally high resistance to pressure (Pm = 800 ± 7 MPa). Stopped-flow kinetic experiments revealed a very rapid (τ < 1 ms) hydrophobic collapse accompanied by the formation of an extended secondary structure but did not reveal stable tertiary contacts. This is followed by three distinct cooperative phases and the significant population of two intermediate species. The kinetics followed by intrinsic fluorescence shows a lag phase, strongly indicating that these intermediates are productive species on a sequential folding pathway, for which we propose a plausible model. These combined data demonstrate that even a large repeat protein can fold in a highly cooperative manner.
Highlights
Escherichia coli DH5-α competent cells (Invitrogen, Paisley, UK) were transformed by the pET20bpemA vector and spread out on lysogeny broth (LB) agar plate with 100 μg·mL−1 of ampicillin (Sigma)
The changes in secondary and tertiary structural content were followed as a function of the guanidinium chloride (GdmCl) concentration by circular dichroism (CD) at 218 nm and by tryptophan fluorescence emission at 342 nm
The five tryptophan residues in Pectin methylesterase (PemA), all located in peripheral loops of the β-helix (Figure 1B), give rise to a single broad fluorescence emission band with a maximum at 342 nm, which is consistent with the partial burial of the indole groups into the native structure [49,50]
Summary
The high cooperativity of protein globular structures arises in part from long-range interactions, large proteins often fold and unfold through partially folded intermediate species. The folding of PelC [74,75,76] and pertactin [77,78], two other proteins with a classical β-helix external asparagine stack of three residues is present [50] and a disulfide bridge between structure [59,61], has been studied in some detail. It highlights structural aspects linked to the absence of longrange contacts and the simple topology of repeat proteins, which breaks the correlation between the rate of folding and the density of direct interactions between residues distant in sequence (i.e., contact order), as found for small globular proteins
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