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The main challenge in using gemcitabine, a nucleoside analogue anti-cancer, includes resistance of some patients to this compound due to the genetic mutations on deoxycytidine kinase (dCK) that are found in a fraction of the human population. Here, we investigated the dynamics behavior and ligand binding properties of the wild type (WT) dCK and its characterized double mutant using a combination of bioinformatics tools and molecular dynamics (MD) simulation studies. Root-mean-square deviation (RMSD) values of the WT and mutant enzyme in complex with both ligands (deoxycytidine/gemcitabine) demonstrated that the WT enzyme forms a more stable complex with gemcitabine, as compared with its natural ligand (deoxycytidine). However, the stability of the double mutant-deoxycytidine complex is greater, when compared with gemcitabine. It was also found that Arg131 as a critical residue can affect the binding pattern of the enzyme to ligands in a manner that the WT enzyme can interact with both ligands, while, the mutant enzyme cannot establish efficient interaction with gemcitabine, and shows high affinity to deoxycytidine. These data together indicate that gemcitabine can interact with the WT enzyme in a competitive manner, while double mutant can be considered as a resistant variant against gemcitabine. The interaction of anti-cancer gemcitabine with deoxycytidine kinase (dCK) was compared with its natural analogue; deoxycytidine. The dynamics behavior of the representative drug-resistant mutant of deoxycytidine kinase (dCK) was investigated in comparison with the wild type enzyme. The results of this study explain the mechanism of resistance of resistant variant against gemcitabine. [ABSTRACT FROM AUTHOR]

In this study we analyzed the sequence and structure of the human DISC1-Ndel1 complex using bioinformatics tools and molecular dynamics simulation studies. Multiple sequence alignment between the homologue protein sequences in primates shows that corresponding positions of residues in Ndel1 are highly conserved, while the DISC1 has variable conservation lines demonstrating its tolerability against various mutations during evolutionary time scale. In comparison with the mouse variant, structural analysis has shown that the evolutionary inserted charged residues in the human DISC1 (E 8 3 7 -R 8 3 8 ) can establish intra-chain electrostatic interactions with the K 8 1 9 -E 8 2 0 dipeptide that may result in more stability of the DISC1 chain. According to MD simulation studies, the compactness for the human variant of the DISC1-Ndel1 is considerably lower than that of the mouse variant. Analysis of structural fluctuation shows that a fragment at the N-terminus side of the human DISC1 has more residual fluctuation. However, the Ndel1 chain of the human variant has globally more flexibility compared with the mouse variant. Considering the identical amino acid sequence of the Ndel1 chains of human and mouse, it concluded that there is a competition between the inter-chain and intra-chain electrostatic interaction in the human DISC1 that directs the complex to weaker inter-chain interactions with the expense of strengthening the intra-chain stabilizing interaction in complex. Human DISC1-Ndel1complex has important neurobiological roles. The interacting fragment of DISC1 shows a degree of variability within primates, and that of Ndel1 is highly conserved. Occurrence of more charged residues in human DISC1 leads to strengthening the intra-chain electrostatics interaction. [ABSTRACT FROM AUTHOR]

We compared the binding properties and dynamics of three experimentally reviewed isoforms of human dihydrofolate reductase (DHFR). The cytoplasmic variants including isoforms1 and 2 (iso1 and iso2) are produced by alternative splicing; while the mitochondrial form is located in the mitochondria. The iso1 as the canonical sequence contains 187 residues, and iso2 differs from the iso1, where it has 1–52 residues missing at the N-terminus of canonical sequence. Here, the structural models of the iso2 and mitochondrial forms were constructed by the MODELLER program using the crystal structure of the iso1 as the template. Bioinformatics analysis on ligand-bearing structures demonstrates that mitochondrial variant forms more stable complex with ligands compared with iso1 and 2, indicating their different binding properties. The root mean square fluctuation (RMSF) data suggest that C-terminus of iso1 contains two representative highly flexible fragments, while iso2 contains a highly flexible fragment at N-terminus end. Interestingly, both ends of mitochondrial variant have a degree of rigidity. Finally, the observation of differences in structural dynamics and binding properties predicts that the simultaneous existence of enzyme isoforms is a way to increase the speed of the enzyme maneuver in response to various environmental conditions. This prediction needs to be tested experimentally. [ABSTRACT FROM AUTHOR]

In order to determine the preference or avoidance of the first and second positions of individual dipeptides for adopting different structural conformations, we randomly select defined structural groups of proteins from protein data bank and statistically analyzed the distribution of all 400 possible dipeptides in different secondary structural elements. Considering different combinations of α-helix (α), β strand (β) and coil (c) including αα, αβ, αc, ββ, βα, βc, cc, cα, cβ conformations, we found that some dipeptides are randomly distributed in these conformations, while others have non-random distribution for a given conformation. Finally, we provide new set of data containing preference and avoidance tendencies that originate from the neighbor effect for each amino acid according to the context of secondary structural element. The output of current work can provide novel data for different fields of structural bioinformatics as well as experiments involving site-directed mutagenesis. [ABSTRACT FROM AUTHOR]

We used a combination of experimental and bioinformatic studies to elucidate the importance of Serine 128 and Valine 183 on the activity and thermal stability of mnemiopsin 1 by substitution of S 128 and V 183 with glycine and threonine, respectively (S128G and V183T mutants). Luminescence emissions of S128G and V183T were reduced to 71.6% and 46.6% with respect to the original activity of the wild type protein. According to circular dichroism (CD) measurements, compactness of mutants decreased in comparison with wild type (WT) protein. Differential scanning calorimetry (DSC) indicated that T m values of thermal unfolding are not changed significantly upon mutation. Herein, we suggest that the protein variants unfold through molecular association and intermediate states. Bioinformatic studies revealed that local fluctuation of residues in S128G increased with respect to WT protein. However, S128G mutation leads to increment of the accessible surface area of lysine188. Therefore, this change is thermodynamically favorable. Finally, both experimental and theoretical studies showed a delicate balance between all structural alterations, determining total conformational stability of the protein. [ABSTRACT FROM AUTHOR]

A series of single and double mutants generated on residues of a surfaced-exposed helix at the C-terminal domain of chondroitinase ABC I (cABC I) from proteus vulgaris. M886A, G887E, and their respective double mutant, MA/GE were inspired by the sequence of a similar helix segment in 30S ribosomal protein S1. Additionally, M889I, Q891K, and the corresponding double mutant, MI/QK, were made regarding the sequence of a similar helix in chondroitin lyase from Proteus mirabilis. Circular dichroism spectra in the far-UV region, demonstrate that the ordered structure of wild-type (WT), and double mutants are the same; however, the helicity of the ordered structures in MI/QK is higher than that of the WT enzyme. When compared with the single mutants, the double mutants showed higher activity, and that the activity of MI/QK is higher than that of the WT enzyme. Heat-induced denaturation experiments showed that the stability of the tertiary structure of double mutants at moderate temperatures is higher compared with the WT, and single mutants. It concluded that this helix can be considered as one of the hot spots region that can be more manipulated to obtain improved variants of cABC I. • A surfaced-exposed helix connects two organized sheets in chondroitinase ABC I. • The helix was replaced with similar variants found in other proteins. • Structural features of the mutants are changed compared with the WT enzyme. • It is a hot spot region, and can be manipulated to obtain improved enzyme variants. [ABSTRACT FROM AUTHOR]

Cyclomaltodextrinase (CDase) is a member of the alpha-amylase family GH13, the subfamily GH13_20. In addition to CDase and neopullulanase, this subfamily also contains maltogenic amylase. They have common structural features, but different substrate specificity. In current work, a combination of bioinformatics and experimental tools were used for designing and constructions of single and double mutants of a new variant of CDase from Anoxybacillus flavithermus. Considering the evolutionary variable positions 123 and 127 at the dimer interface of subunits in the alpha-amylase family, these positions in CDase were modified and three mutants, including A123V, C127Q and A123V/C127Q were constructed. The tertiary structure of WT and mutants were made with the MODELLER program, and the phylogenetic tree of homologous protein sequences was built with selected programs in Phylip package. Enzyme kinetic studies revealed that the catalytic efficiency of mutants, especially double one, is lower than the WT enzyme. Heat-induced denaturation experiments were monitored by measuring the UV/Vis signal at 280 nm, and it was found that WT protein is structurally more stable at 25 °C. However, it is more susceptible to changes in temperature compared to the double mutant. It was concluded that the positions 123 and 127 at the dimeric interface of CDase, not only could affect the conformational stability; but also; the catalytic properties of the enzyme by setting up the active site configuration in the dimeric state. Unlabelled Image • Positions 123 and 127 in cyclomaltodextrinase (CDase) were changed the sequence conditions of other Glycoside hydrolases. • Thermal stability of WT protein at 25 °C is greater than mutants. • WT protein is more sensitive to changes in temperature, • The loop in the dimeric interface has critical role in catalytic efficiency and conformational stability. [ABSTRACT FROM AUTHOR]