Alcohol and Temperature Induced Conformational Transitions in Ervatamin B: Sequential Unfolding of Domains

Abstract

The structural aspects of ervatamin B have been studied in different types of alcohol. This alcohol did not affect the structure or activity of ervatamin B under neutral conditions. At a low pH (3.0), different kinds of alcohol have different effects. Interestingly, at a certain concentration of non-fluorinated, aliphatic, monohydric alcohol, a conformational switch from the predominantly α-helical to β-sheeted state is observed with a complete loss of tertiary structure and proteolytic activity. This is contrary to the observation that alcohol induces mostly the α-helical structure in proteins. The O-state of ervatamin B in 50% methanol at pH 3.0 has enhanced the stability towards GuHCl denaturation and shows a biphasic transition. This suggests the presence of two structural parts with different stabilities that unfold in steps. The thermal unfolding of ervatamin B in the O-state is also biphasic, which confirms the presence of two domains in the enzyme structure that unfold sequentially. The differential stabilization of the structural parts may also be a reflection of the differential stabilization of local conformations in methanol. Thermal unfolding of ervatamin B in the absence of alcohol is cooperative, both at neutral and low pH, and can be fitted to a two state model. However, at pH 2.0 the calorimetric profiles show two peaks, which indicates the presence of two structural domains in the enzyme with different thermal stabilities that are denatured more or less independently. With an increase in pH to 3.0 and 4.0, the shape of the DSC profiles change, and the two peaks converge to a predominant single peak. However, the ratio of van't Hoff enthalpy to calorimetric enthalpy is approximated to 2.0, indicating non-cooperativity in thermal unfolding.

Adopting Selected Hydrogen Bonding and Ionic Interactions fromAspergillus fumigatusPhytase Structure Improves the Thermostability ofAspergillus nigerPhyA Phytase

“…Because the temperature-induced denaturing of proteins is affected by these noncovalent interactions (24), the enhanced thermotolerance, including the 7°C increase in melting temperature, in mutant A58E P65S Q191R T271R could be attributed to the introduced hydrogen bonding and salt bridges by the four amino acid substitutions. The two peaks seen in the thermogram of the mutant may represent independent folding of the two protein domains, the ␣ domain and the ␣/␤ domain (11). The increased melting temperature of mutant A58E P65S Q191R T271R will help the enzyme to resist heat inactivation during feed pelleting (18).…”

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

“…Extensive studies on the folding of papain as well as other related proteins have been carried out in our laboratory ( − ). Two other cysteine proteases, ervatamin C and B, isolated in our laboratory, have also been used as model systems ( − ) in understanding the folding of cysteine proteases. Folding studies on similar proteins from different sources and proteins within a family with individual variations would certainly help in generalizing the folding behavior of cysteine proteases as well as complementing the mechanisms proposed thereafter.…”

Antigen Retrieval Causes Protein Unfolding

“…When formaldehyde is dissolved in buffer to prepare formalin, it reacts instantaneously with water and exists predominately in the form of methylene glycol (Fox et al 1985;Le Botlan et al 1983). Methylene glycol, like other low molecular weight alcohols, can alter protein structure (Brandts et al 1989;Kundu et al 2002;Naseem and Khan 2003) through at least two mechanisms. The first mechanism involves promotion of protein unfolding through weakening of the hydrophobic stabilization within the protein core (Brandts et al 1989;Kundu et al 2002).…”

“…Methylene glycol, like other low molecular weight alcohols, can alter protein structure (Brandts et al 1989;Kundu et al 2002;Naseem and Khan 2003) through at least two mechanisms. The first mechanism involves promotion of protein unfolding through weakening of the hydrophobic stabilization within the protein core (Brandts et al 1989;Kundu et al 2002). The second mechanism involves the energetically unfavorable interaction of alcohols with the peptide backbone (Pace et al 2004).…”