Refolding of lysozyme by quasistatic and direct dilution reaction paths: A first-order-like state transition

A first-order-like state transition model is considered to be a global reaction mechanism to directly folded proteins from an unfolded state to its native form. In order to verify the general applicability of this mechanism, we used lysozyme as a model protein. It was fully unfolded by $4.5\mathrm{M}$ urea, $0.1\mathrm{M}$ dithiothreitol (DTT) in $p\mathrm{H}3$ and refolded to its native form by way of an overcritical reaction path (a quasistatic process) or directly crossing transition boundary path (a directly dilution process). In addition to the two states coexisting in the direct folding path, lyzosyme might be trapped in a glassy state. However, it can escape from the glassy state by concentration twice. This indicates the existence of a state transition line or boundary in the direct folding reaction. However, lysozyme can continuously fold from unfolded to native by an overcritical reaction path. During the overcritical path, four stable folding intermediates and native lysozyme were obtained. The secondary structures, particle size distributions, thermal stabilities, and oxidation state of disulfide bonds of folding intermediates were analyzed by circular dichroism spectra, dynamic light scattering, differential scanning calorimetry, and Raman spectra, respectively. According to the data, the intermediates of both the overcritical reaction and the direct crossing transition boundary paths can be described by a common concept pertaining to a model that undergoes collapse, sequential, and first-order-like state transition. This indicated that protein folding by way of different reaction paths might follow a similar folding mechanism—i.e., a mechanism of overcritical folding of intermediates. A protein folding reaction diagram is postulated and discussed. In spite of a global interaction mechanism the $\alpha$-helix is formed prior to the $\beta$-sheet, which may indicate that protein folding is initiated by local interactions.

Figure 1

Figure 2

The CD profiles analysis by SELCON3 of overcritical reaction path and direct folding intermediates of lysozyme. The secondary structure Hr: regular $\alpha$-helix, Hd: distorted $\alpha$-helix, Br: regular $\beta$-strand, Bd: distorted $\beta$-strand, Tu: turns, and Un: unordered. The % of folding intermediates distributed in each secondary structure are represented by histograms: $U$, mosaic with white background; $M_1$, slash from upper left to lower right with gray background; $M_2$, horizontal line with white background; $M_3$, filled in solid gray; $M_4$, slash from upper left to lower right with white background; $M_5$, slash from lower left to upper right with gray background; and $I$, filled in solid back.

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Figure 4

Raman shift of lysozyme folding intermediates. (a) The S-S $\left({\nu }_{\text{S-S}}\right)$ stretching region. (b) The S-H $\left({\nu }_{\text{S-H}}\right)$ stretching region. The arrow indicates the S-S stretching frequency. Stars denote the NCN and NCO bending signals around 546 and $560{\text{cm}}^{-1}$ of high-concentration urea, respectively. All profiles, excluding the denature lysozyme, in (a) were normalized with their intensity and shifted at $510{\text{cm}}^{-1}$, magnified to normal count. “Arb. units” means arbitrary unit of intensity.

Figure 5

Proposed first-order-like state transition model of the protein folding reaction diagram. $\Phi \left(n_1,n_2,\dots \right)$ denotes the folding state of protein. The parameters of $n,n_1$, and $n_2$ denote the folding state variables such as temperature, concentration of denaturants, etc.