Coherent Experimental and Simulation Approach To Explore the Underlying Mechanism of Denaturation of Stem Bromelain in Osmolytes

Abstract

Characterization of a protein in the context of its environment is of crucial importance for a complete understanding of its function. Although biophysical techniques provide powerful tools for studying the stability and activity of the enzyme in the presence of various cosolvents, an approach of combining both experimental techniques and molecular dynamic (MD) simulations may lead to the mechanistic insight into the interactions governing the stability of an enzyme. The knowledge of these interactions can be further utilized for range of modifications in the wild form of an enzyme for various pharmaceutical applications. Herein, we employed florescence, UV-visible, circular dichroism (CD), dynamic light scattering (DLS) study, and MD simulations for comprehensive understanding of stem bromelain (BM) in the presence of betaine, sarcosine, arginine, and proline. The thermal stability of BM in the presence of 1 M of osmolytes is found to be in order: proline > betaine > buffer > arginine > sarcosine. BM gets more preferentially hydrated in the presence of betaine and proline than in sarcosine and arginine. Nonetheless, MD simulations suggest that betaine, sarcosine, and arginine at 1 M interact with the active site of BM through H-bonding except proline which are responsible for more disruption of active site. The distances between the catalytic site residues are 1.6, 1.9, 4.3, 5.0, and 6.2 Å for BM in proline, buffer, betaine, arginine, and sarcosine at 1 M, respectively. To the best of our knowledge, this is the first report on detailed unequivocal evidence of denaturation and deactivation of BM in the presence of methylamines and amino acids.

A Vibrant Accelerating Strategic Solvent System for Sustainability and Functionality for Proteins: Unvarying Structure of Stem Bromelain in Hybrid Ionic Fluids

“…The obtained Tm values of SB in the presence of IL, DES, and HIF are presented in Figure 3 and also provided in Table S1. For native SB, the transition temperature (T m ) was found to be 64.1 °C, which is consistent with the literature value; 36 later, the T m values decrease, which significantly decreases with increase in the concentration of IL (Figure 3). Also, the thermal transition curves have been provided in Figure S5.…”

Effect of osmolytes on the thermal stability of proteins: replica exchange simulations of Trp-cage in urea and betaine solutions

“…[32][33][34][35] While it has been found that denaturing action of urea is generally enhanced by temperature, 36,37 the effect of temperature on protein stabilization by osmoprotectants is more complex. 33,35,38 In particular, it has been observed that at room temperatures betaine and similar protectants (sarcosine, glycine and proline) usually act as relatively weak stabilizers and may even slightly destabilize some proteins. However, as temperature increases, their stabilizing effect is also markedly enhanced (e.g., the effect of betaine on the unfolding free energy of cytochrome c changes from E0 kcal mol À1 at 293 K to 0.6 kcal mol À1 at 353 K).…”

Implications of Imidazolium-Based Ionic Liquids as Refolding Additives for Urea-Induced Denatured Serum Albumins

“…Thus, the addition of ILs initiated the refolding of the protein structure, which eventually leads to movement of Trp residues toward the hydrophobic core of the protein. 41 As depicted in Figure 1a,c, the IL-1 is a more efficient refolding agent for both the serum albumins, namely BSA and HSA, respectively. Based on these results, refolding of ureadenatured serum albumins was successfully achieved under optimized IL concentration (i.e., 10 −5 M for BSA and 10 −4 M for HSA).…”

“…Addition of ILs to the denatured protein leads to enhanced fluorescence intensity accompanied by a blue shift in the emission maxima. Thus, the addition of ILs initiated the refolding of the protein structure, which eventually leads to movement of Trp residues toward the hydrophobic core of the protein …”