Differences in the Unfolding of Procerain Induced by pH, Guanidine Hydrochloride, Urea, and Temperature

Abstract

The structural and functional aspects along with equilibrium unfolding of procerain, a cysteine protease from Calotropis procera, were studied in solution. The energetic parameters and conformational stability of procerain in different states were also estimated and interpreted. Procerain belongs to the alpha + beta class of proteins. At pH 2.0, procerain exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is predominantly a beta-sheet conformation and exhibits strong ANS binding. GuHCl and temperature denaturation of procerain in the molten globule-like state is noncooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts in the molecular structure of procerain, possibly domains, with different stability that unfolds in steps. Moreover, tryptophan quenching studies suggested the exposure of aromatic residues to solvent in this state. At lower pH, procerain unfolds to the acid-unfolded state, and a further decrease in the pH drives the protein to the A state. The presence of 0.5 M salt in the solvent composition directs the transition to the A state while bypassing the acid-unfolded state. GuHCl-induced unfolding of procerain at pH 3.0 seen by various methods is cooperative, but the transitions are noncoincidental. Besides, a strong ANS binding to the protein is observed at low concentrations of GuHCl, indicating the presence of an intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8 M), procerain retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to unfolding by urea at lower pH, and the transitions are cooperative and coincidental. Further, the properties of the molten globule-like state and the intermediate state are different, but both states have the same conformational stability. This indicates that these intermediates may be located on parallel folding routes of procerain.

Biologically Active Novel Conformational State of Botulinum, the Most Poisonous Poison

“…Acrylamide maximally quenches the Trp fluorescence in the light chain at about 40°C, indicating that the peptide segments containing Trp residues are more mobile and accessible at this temperature than at 20°C. Upon further heating, beyond 45°C, there was an increase in the fluorescence intensity, which could be attributed to the release of intrinsic quenching (38). The polypeptide chain in a native protein is folded into a compact structure that strongly limits freedom of molecular movement, thus restricting fluctuations.…”

“…Increase in temperature will enhance the fluidity of a medium since the intramolecular interactions that stabilize the globular structure of the protein are distorted, and the protein becomes loosened, forming pores or pathways for a large molecule like acrylamide to collide with the excited states of internal Trp residues (35). Thus, the measurement of the extent of quenching of Trp fluorescence provides valuable information about the dynamics of the protein and has been powerful in sensing the exposure of Trp residues in the molten-globule state of a protein (38). Increased quenching of Trp fluorescence observed in BoNT/A light chain at 37°C (Fig.…”

Structural characteristics of extra-membrane domains and guanidine hydrochloride-induced denaturation of photosystem 2 core antenna complexes CP43 and CP47

“…Fluorescence spectra provide a sensitive means of characterizing the conformation and function of proteins (Dubey and Jagannadham 2003). CP43 and CP47 are two kinds of membrane protein with pigments, so they can be followed by a number of fluorescence signals, including fluorescence of aromatic amino acids and pigments (Booth et al 2001).…”

“…In contrast, the fluorescence of proteins with both Trp and Tyr will be dominated by the contribution from Trp residues upon excitation at 280 nm because Trp has a higher molar extinction coefficient and fluorescence quantum yield than Tyr and can serve as an energy transfer acceptor for Tyr (Oikawa et al 1985, Barrow et al 1992, Eftink and Shastry 1997. The fluorescence emission maximum (λ max ) of Trp is sensitive to the polarity of the microenvironment around the indole side chain (Eftink and Shastry 1997).…”

“…As a result, the emission spectra of Trp residues can reflect the polarity of their surrounding environment and their location in proteins (Lakowicz 1983). According to Burstein et al (1973), Eftink andShastry (1997), andIsaev-Ivanov et al (2000), there are three main classes of Trp residues in proteins, in which the λ max and location are different: class I (λ max =320-332 nm) includes Trps located in the inner low-polar regions of protein molecules, and classes II (λ max = 340-342 nm) and III (λ max = 350-353 nm) include exposed Trp residues. The class II Trp residues are on the surface of native protein molecules and those of class III in the unfolded denatured ones; they differ only in the extent of perturbation of their electronic structures by water molecules.…”