Optimization of DTD-tRNA code and mito-tRNA(Gly) discriminator base is important for emergence of mitochondria.

We have demonstrated earlier that protein microenvironments were conserved around disulfide-bridged cystine motifs with similar functions, irrespective of diversity in protein sequences. Here, cysteine thiol modifications were characterized based on protein microenvironments, secondary structures and specific protein functions. Protein microenvironment around an amino acid was defined as the summation of hydrophobic contributions from the surrounding protein fragments and the solvent molecules present within its first contact shell. Cysteine functions (modifications) were grouped into enzymatic and non-enzymatic classes. Modifications studied were-disulfide formation, thio-ether formation, metal-binding, nitrosylation, acylation, selenylation, glutathionylation, sulfenylation, and ribosylation. 1079 enzymatic proteins were reported from high-resolution crystal structures. Protein microenvironments around cysteine thiol, derived from above crystal structures, were clustered into 3 groups-buried-hydrophobic, intermediate and exposed-hydrophilic clusters. Characterization of cysteine functions were statistically meaningful for 4 modifications (disulfide formation, thioether formation, sulfenylation, and iron/zinc binding) those have sufficient amount of data in the current dataset. Results showed that protein microenvironment, secondary structure and protein functions were conserved for enzymatic cysteine functions, in contrast to the same function from non-enzymatic cysteines. Disulfide forming enzymatic cysteines were tightly packed within intermediate protein microenvironment cluster, have alpha-helical conformation and mostly belonged to CxxC motif of electron transport proteins. Disulfide forming non-enzymatic cysteines did not belong to conserved motif and have variable secondary structures. Similarly, enzymatic thioether forming cysteines have conserved microenvironment compared to non-enzymatic cystienes. Based on the compatibility between protein microenvironment and cysteine modifications, more efficient drug molecules could be designed against cysteine-related diseases.

Homochirality of biomacromolecules is a prerequisite for their proper functioning and hence essential for all life forms. This underscores the role of cellular chiral checkpoints in enforcing homochirality during protein biosynthesis. d‐Aminoacyl‐tRNA deacylase (DTD) is an enzyme that performs ‘chirality‐based proofreading’ to remove d‐amino acids mistakenly attached to tRNAs, thus recycling them for further rounds of translation. Paradoxically, owing to its l‐chiral rejection mode of action, DTD can remove glycine as well, which is an achiral amino acid. However, this activity is modulated by discriminator base (N73) in tRNA, a unique element that protects the cognate Gly‐tRNAGly. Here, we review our recent work showing various aspects of DTD and tRNAGly coevolution and its key role in maintaining proper translation surveillance in both bacteria and eukaryotes. Moreover, we also discuss two major optimization events on DTD and tRNA that resolved compatibility issues among the archaeal and the bacterial translation apparatuses. Importantly, such optimizations are necessary for the emergence of mitochondria and successful eukaryogenesis.

In our previous study, we have shown that the microenvironments around conserved amino acids are also conserved in protein families (Bandyopadhyay and Mehler, Proteins 2008; 72:646-659). In this study, we have hypothesized that amino acids perform similar functions when embedded in a certain type of protein microenvironment. We have tested this hypothesis on the microenvironments around disulfide-bridged cysteines from high-resolution protein crystal structures. Although such cystines mainly play structural role in proteins, in certain enzymes they participate in catalysis and redox reactions. We have performed and report a functional annotation of enzymatically active cystines to their respective microenvironments. Three protein microenvironment clusters were identified: (i) buried-hydrophobic, (ii) exposed-hydrophilic, and (iii) buried-hydrophilic. The buried-hydrophobic cluster encompasses a small group of 22 redox-active cystines, mostly in alpha-helical conformations in a -C-x-x-C- motif from the Oxido-reductase enzyme class. All these cystines have high strain energy and near identical microenvironments. Most of the active cystines in hydrolase enzyme class belong to buried hydrophilic microenvironment cluster. In total there are 34 half-cystines detected in buried hydrophilic cluster from hydrolases, as a part of enzyme active site. Even within the buried hydrophilic cluster, there is clear separation of active half-cystines between surface exposed part of the protein and protein interior. Half-cystines toward the surface exposed region are higher in number compared to those in protein interior. Apart from cystines at the active sites of the enzymes, many more half-cystines were detected in buried hydrophilic cluster those are part of the microenvironment of enzyme active sites. However, no active half-cystines were detected in extremely hydrophilic microenvironment cluster, that is, exposed hydrophilic cluster, indicating that total exposure of cystine toward the solvent is not favored for enzymatic reactions. Although half-cystines in exposed-hydrophilic clusters occasionally stabilize enzyme active sites, as a part of their microenvironments. Analysis performed in this work revealed that cystines as a part of active sites in specific enzyme families or folds share very similar protein microenvironment regions, despite of their dissimilarity in protein sequences and position specific sequence conservations. Proteins 2016; 84:1576-1589. © 2016 Wiley Periodicals, Inc.

Proteins involved in proton-/electron-transfer processes often possess “functional” aspartates/aspartic acids (Asp) with variable protonation states. The mechanism of Asp protonation–deprotonation within proteins is unclear. Two questions were askedthe possible types of determinants responsible for Asp protonation–deprotonation and the spatial arrangements of the determinants leading to selective stabilization. The questions were analyzed using nine different solvent models, which scanned the complete protein dielectric range, and four protein models, which illustrated the spatial arrangements around Asp, termed as “molecular association”. The methods employed were quantum chemical calculations and constant pH simulations. The types of the determinants identified were charge–charge interaction, H bonding, dipole−π interaction, extended electronic conjugation, dielectric effect, and solvent accessibility. All solvent-exposed Asp [buried fraction (BF) less than 0.5] were aspartates, and buried Asp were either aspartic acids or aspartates, each having a different “molecular association”. The exposed aspartates were stabilized via a H-bonding network with bulk water, buried aspartates via salt bridge or, minimum, two intramolecular H bonds, and buried aspartic acids via, minimum, one intramolecular H bond. An “acid–alcohol pair” (involving Ser/Thr/Tyr) was a common determinant to any “functional” buried aspartate/aspartic acid. Higher energy “molecular associations” observed within proteins compared to those within water, presumably, indicated easy molecular restructuring and alteration of the Asp protonation states during a protein-mediated proton/electron transfer.