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This study was conducted to examine the role of the central domain of cyclomaltodextrinase in terms of stability, substrate specificity, becoming dodecameric form, and enzyme activity. To this end, H403R/L309V double-point mutation and T280Q single-point mutation were performed at the central domain and (β/α)8-barrel. The results indicated that the activity of the H403R/L309V mutant at the optimal pH and temperature increased by about 25% and 40%, respectively. Plus, the irreversible thermal inactivation of the H403R/L309V mutant at 60 °C and 160 min was approximately twice of the enzyme without mutation. Both mutants underwent significant structural change relative to the wild enzyme and subsequently a significant catalytic activity. However, the catalytic efficiency (kcat/Km) of the H403R/L309V mutant increased in the presence of beta- and gamma-cyclomaltodextrin substrates compared to the wild enzyme and T280Q mutant. As a result, by applying the L309V mutant and given the smaller size of the valine, leucine spatial inhibition in the wild protein seems to decline, and also it facilitates the substrate access to active site amino acids. Moreover, as gamma substrate is larger, eliminating the effect of spatial inhibition on this substrate has a greater effect on improving the catalytic activity of this enzyme.

AbstractIt has been found that the development of schizophrenia and some other psychiatric disorders is related to defects in the normal functioning of Disrupted-In-Schizophrenia 1 (DISC1). It is a large-sized protein containing 855 residues and acts as an active hub at the core of many interactions with various proteins. On the other hand, NudE Neurodevelopment Protein 1 Like 1 (Ndel1) plays a role in nervous system development via interaction with the DISC1. It was shown that some point mutations on DISC1 have clinical implications. In line with these reports, here we have used the NMR structure of the wild-type (WT) C-terminal tail of DISC1 in complex with the N-terminal fragment of Ndel1, and have constructed the three-dimensional structures of L62Q and L29Q mutants, as the pathologic variants of the complex. The time-dependent interaction of DISC1 with Ndel1 in the WT complex and mutants was simulated by performing molecular dynamics (MD) simulation using programs in the GROMACS package. It was found that the flexibility of residues in some regions of the protein chains increases, and secondary structural changes from ordered toward unordered one leads to destabilizing of the complex in mutants. Destabilization of the complex upon substitution of Leu by Gln was also confirmed by analysis of the contact map plot.Communicated by Ramaswamy H. Sarma

The stabilities of Ca2+-regulated ctenophore and coelenterate apo-photoproteins, apo-mnemiopsin (apo-Mne) and apo-aequorin (apo-Aeq), respectively, were compared biochemically, biophysically, and structurally. Despite high degrees of structural and functional conservation, drastic variations in stability and structural dynamics were found between the two proteins. Irreversible thermoinactivation experiments were performed upon incubation of apo-photoproteins at representative temperatures. The inactivation rate constants (kinact) at 50 °C were determined to be 0.001 and 0.004 min–1for apo-Mne and apo-Aeq, respectively. Detailed analysis of the inactivation process suggests that the higher thermostability of apo-Mne is due to the higher activation energy (Ea) and subsequently higher values of ΔH* and ΔG* at a given temperature. According to molecular dynamics simulation studies, the higher hydrogen bond, electrostatic, and van der Waals energies in apo-Mne can validate the relationship between the thermal adaptation of apo-Mne and the energy barrier for the inactivation process. Our results show that favorable residues for protein thermostability such as hydrophobic, charged, and adopted α-helical structure residues are more frequent in the apo-Mne structure. Although the effect of acrylamide on fluorescence quenching suggests that the local flexibility in regions around Trp and Tyr residues of apo-Aeq is higher than that of apo-Mne, which results in it having a better ability to penetrate acrylamide molecules, the root-mean-square fluctuation of helix A in apo-Mne is higher than that in apo-Aeq. It seems that the greater flexibility of apo-Mne in these regions may be considered as a determining factor, affecting the thermal stability of apo-Mne through a balance between structural rigidity and flexibility.

AbstractTo correlate the structural features of enzymes to temperature adaptation, we studied psychrophile, mesophile, and thermophile adenylate kinases as model enzymes using bioinformatics and computational tools. Phylogenetic analysis revealed that mesophile and thermophile variants are clustered in one stem of phylogenetic tree and are close to contemporary time, while psychrophile enzyme is more close to their common ancestor. This finding is in good agreement with the process of environmental changes from ice age toward current warm conditions on the earth. We also performed Molecular Dynamics simulation at corresponding temperatures of all enzyme variants including 308, 318, and 328 K. It was found that mesophile enzyme has no distinct deviation of Root Mean Square Deviation (RMSD) and Radius of Gyration (Rg) values from equilibrium states at operating temperature of thermophile enzyme as well as its own optimum temperature. However, psychrophile enzyme undergoes more fluctuations with higher amplitude of change; particularly at 328 K. It was also found that initial increasing of RMSD and Rg for Psychrophile enzyme at all temperatures is occurred gradually; while, the increment of this structural parameters for thermophile enzyme at 328 K is occurred in a highly cooperative and switching manner demonstrating snap structural change of thermophile enzyme in its own temperature. By analysis of Root Mean Square Fluctuation values at different temperatures, we identified two flexible fragments in adenylate kinases so that different dynamic behavior of these regions in mesophile enzyme against operating temperatures of psychrophile and thermophile variants is critical in compensation of flexibility challenges at respective temperatures.

The protein folding kinetics of hen egg white lysozyme (HEWL) was studied using experimental and bioinformatics tools. The structure of the transition state in the unfolding pathway of lysozyme was determined with stopped-flow kinetics using intact HEWL and its chemically modified derivative, in which six lysine residues have been modified. The overall consistency of φ-value (φ ≈ 1) indicates that lysine side chains interactions are subject to breaking in the structure of the transition state. Following experimental evidences, multiple sequence alignment of lysozyme family in vertebrates and exact structural examination of lysozyme, showed that the α-helix in the structure of lysozyme has critical role in the unfolding kinetics.