Unassisted refolding of urea‐denatured arginine kinase from shrimp Feneropenaeus chinensis: Evidence for two equilibrium intermediates in the refolding pathway

Abstract

The refolding process and the equilibrium intermediates of urea-denatured arginine kinase (AK) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) intrinsic fluorescence, far-UV circular dichroism (CD), size-exclusion chromatography (SEC), and enzymatic activity. In dilute denaturant, two equilibrium refolding intermediates (I and NЈ) were discovered, and a refolding scheme of urea-denatured AK was proposed. During the refolding of urea-denatured AK, the fluorescence intensity increased remarkably, accompanied by a significant blue shift of the emission maximum and a pronounced increase in molar ellipticity of CD at 222 nm. The first folding intermediate (I) was inactive in urea solution ranging between 2.4 and 3.0 M. The second (NЈ) existed between a 0.4-and 0.8-M urea solution, with slightly increased activity. Neither the blue shift emission maximum nor the molar ellipticity of CD at 222 nm showed significant changes in these two regions. The two intermediates were characterized by monitoring the ANS binding ability in various residual urea solutions, and two peaks of the emission intensity were observed in urea solutions of 0.6 and 2.8 M, respectively. The SEC results indicated that a distribution coefficient (K D ) platform existed in urea solutions ranging between 2.4 and 3.0 M urea, suggesting that there was a similarly apparent protein profile and size in the urea solution region. The refolding kinetics showed that the urea-denatured AK was in two-phase refolding. Proline isomerization occurred in the unfolding process of AK, which blocked the slow phase of refolding. These results suggested that the refolding process of urea-denatured AK contained at the least two equilibrium refolding intermediates.

Polyvinylpyrrolidone 40 Assists the Refolding of Bovine Carbonic Anhydrase B by Accelerating the Refolding of the First Molten Globule Intermediate

“…The activities of refolding samples were measured after 1 h of equilibration. The details regarding the kinetic experiments have been described elsewhere (44). In brief, standard buffer was added to the GdnHCl-denatured CAB to initiate the refolding of the protein.…”

The roles of C-terminal loop residues of dimeric arginine kinase from sea cucumber Stichopus japonicus in catalysis, specificity and structure

“…As a member of the phosphagen kinases which have been used as models of protein folding [1][2][3] and a paradigm for understanding the mechanisms of bimolecular reactions [4][5][6][7][8], AK plays a central role in energy metabolism of invertebrates by means of the temporary buffering of ATP level in accordance with the energy requirement [9][10][11]. Although the function of AK is analogous to that of creatine kinase (CK) in vertebrates, it has not been investigated as AK from sea cucumber Stichopus japonicus is a homologous dimer, and each subunit has 371 amino acids and a molecular weight of about 42 kDa [11].…”

Effects of aspartic acid and potassium chloride on arginine kinase from shrimp

“…More and more evidences have shown that a molten globular state exists as an intermediate between the native and the fully unfolded state in general protein folding. Recent evidence has shown that during the refolding of denatured AK there were at least two refolding intermediates [4,5]. In the molten globule state the molecule is compact with a native-like secondary structure, but a poorly defined tertiary structure [20,21].…”

“…It is known that AK from shrimp Fenneropenaeus chinensis is a monomer protein with molecular mass of 40 kDa [3]. The activity and conformational changes of AK in some denaturants have been extensively studied, and upon diluting the denaturant under suitable conditions, both the conformation and the catalytic activity can be quantitatively recovered [4,5]. AK aggregation depends on protein Abbreviations: AK, arginine kinase; ANS, 1-anilino-8-naphthalenesulfonate; Asp, aspartic acid; CD, circular dichroism; GuHCl, guanidine hydrochloride; KCl, potassium chloride * Corresponding author.…”