De novo inference of protein function from coarse‐grained dynamics

Inference of molecular function of proteins is the fundamental task in the quest for understanding cellular processes. The task is getting increasingly difficult with thousands of new proteins discovered each day. The difficulty arises primarily due to lack of high‐throughput experimental technique for assessing protein molecular function, a lacunae that computational approaches are trying hard to fill. The latter too faces a major bottleneck in absence of clear evidence based on evolutionary information. Here we propose a de novo approach to annotate protein molecular function through structural dynamics match for a pair of segments from two dissimilar proteins, which may share even <10% sequence identity. To screen these matches, corresponding 1 µs coarse‐grained (CG) molecular dynamics trajectories were used to compute normalized root‐mean‐square‐fluctuation graphs and select mobile segments, which were, thereafter, matched for all pairs using unweighted three‐dimensional autocorrelation vectors. Our in‐house custom‐built forcefield (FF), extensively validated against dynamics information obtained from experimental nuclear magnetic resonance data, was specifically used to generate the CG dynamics trajectories. The test for correspondence of dynamics‐signature of protein segments and function revealed 87% true positive rate and 93.5% true negative rate, on a dataset of 60 experimentally validated proteins, including moonlighting proteins and those with novel functional motifs. A random test against 315 unique fold/function proteins for a negative test gave >99% true recall. A blind prediction on a novel protein appears consistent with additional evidences retrieved therein. This is the first proof‐of‐principle of generalized use of structural dynamics for inferring protein molecular function leveraging our custom‐made CG FF, useful to all. Proteins 2014; 82:2443–2454. © 2014 Wiley Periodicals, Inc.     

Binding affinity and in vitro cytotoxicity of harmaline targeting different motifs of nucleic acids: An ultimate drug designing approach

The work focuses towards interaction of harmaline, with nucleic acids of different motifs by multispectroscopic and calorimetric techniques. Findings of this study suggest that binding constant varied in the order single‐stranded (ss) poly(A) > double‐stranded calf thymus (CT) DNA > double‐stranded poly(G)·poly(C) > clover leaf tRNA^Phe. Prominent structural changes of ss poly(A), CT DNA, and poly(G)· poly(C) with concomitant induction of optical activity in the bound achiral alkaloid molecule was observed, while with tRNA^Phe, very weak induced circular dichroism perturbation was seen. The interaction was predominantly exothermic, enthalpy driven, and entropy favored with CT DNA and poly(G)·poly(C), while it was entropy driven with poly(A) and tRNA^Phe. Intercalated state of harmaline inside poly(A), CT DNA, and poly(G)·poly(C) was shown by viscometry, ferrocyanide quenching, and molecular docking. All these findings unequivocally pointed out preference of harmaline towards ss poly(A) inducing self‐structure formation. Furthermore, harmaline administration caused a significant decrease in proliferation of HeLa and HepG₂ cells with GI₅₀ of 28μM and 11.2μM, respectively. Nucleic acid fragmentation, cellular ultramorphological changes, decreased mitochondrial membrane potential, upregulation of p53 and caspase 3, generation of reactive oxygen species, and a significant increase in the G₂/M population made HepG₂ more prone to apoptosis than are HeLa cells.     

Transient studies of nutrient uptake, growth, and indole alkaloid accumulation in heterotrophic cultures of hairy roots of Catharanthus roseus

The kinetics of growth, the uptake of macronutrients, and the accumulation of indole alkaloids were investigated in long-term, heterotrophically cultured transgenic ("hairy") roots of Catharanthus roseus.Tabersonine, ajmalicine, and serpentine were monitored over a 70-day period. The doubling time [dry-weight (DW) basis] of C. roseus hairy roots in B5/2 nutrients supplemented with 3% sucrose was 3.6 days. NH(4) (+), NO(3),(-) and P(i) were depleted sequentially from culture medium by hairy roots, while sugars remained undepleted. The growth-limiting nutrient was inorganic nitrogen, NH(4) (+) and NO(3) (-), with exponential-phase overall biomass yields of 34.1 and 5.0 g DW/g nutrient, respectively. Extracellular pH decreased to 4.8 in early exponential phase of culture growth from the initially adjusted value of 5.7, increased subsequently to a maximum of 7.7 in late exponential phase of growth coincident with the maximum of fresh weight (FW)/DW ratio, before decreasing to 5.5-5.0. The organic acids, pyruvate, formate, lactate, and succinate were excreted by hairy roots starting in late phase of exponential growth, possibly resulting in the late-culture pH decrease. Tabersonine accumulation was distinctly growth associated with maximum specific and total yields of 1.15 mg/g DW and 5.6 mg/L, respectively, in late-exponential phase of growth. Serpentine accumulation was non growth associated with increasing specific and total levels in stationary growth phase: 1.3 mg/g DW and 10.5 mg/L, respectively. The accumulation of ajmalicine also appeared growth associated. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 527-534, 1997.     

Stringent response in Vibrio cholerae: genetic analysis of spoT gene function and identification of a novel (p)ppGpp synthetase gene

RelA and SpoT of Gram-negative organisms critically regulate cellular levels of (p)ppGpp. Here, we have dissected the spoT gene function of the cholera pathogen Vibrio cholerae by extensive genetic analysis. Unlike Escherichia coli , V. cholerae Δ relA Δ spoT cells accumulated (p)ppGpp upon fatty acid or glucose starvation. The result strongly suggests RelA-SpoT-independent (p)ppGpp synthesis in V. cholerae . By repeated subculturing of a V. cholerae Δ relA Δ spoT mutant, a suppressor strain with (p)ppGpp⁰ phenotype was isolated. Bioinformatics analysis of V. cholerae whole genome sequence allowed identification of a hypothetical gene ( VC1224 ), which codes for a small protein (∼29 kDa) with a (p)ppGpp synthetase domain and the gene is highly conserved in vibrios; hence it has been named relV . Using E. coli Δ relA or Δ relA Δ spoT mutant we showed that relV indeed codes for a novel (p)ppGpp synthetase. Further analysis indicated that relV gene of the suppressor strain carries a point mutation at nucleotide position 676 of its coding region (Δ relA Δ spoT relV676 ), which seems to be responsible for the (p)ppGpp⁰ phenotype. Analysis of a V. cholerae Δ relA Δ spoT Δ relV triple mutant confirmed that apart from canonical relA and spoT genes, relV is a novel gene in V. cholerae responsible for (p)ppGpp synthesis.     

BALB/Mtv-null mice responding to strong mouse mammary tumor virus superantigens restrict mammary tumorigenesis

The absence of endogenous mouse mammary tumor viruses (MMTVs) in the congenic mouse strain, BALB/Mtv-null, restricts the early steps of exogenous C3H MMTV infection, preventing the superantigen (Sag) response and mammary tumorigenesis. Here we demonstrate that BALB/Mtv-null mice also resist tumor induction by FM MMTV, which encodes a stronger Sag compared to C3H MMTV. In contrast to infections with C3H MMTV, Mtv-null mice show FM-MMTV Sag-specific responses comparable to those observed in susceptible BALB/c mice. Neither virus shows significant replication in the spleen or mammary gland. Thus, Mtv-null mice restrict MMTV replication and mammary tumorigenesis even after a robust Sag response.     

Mutational analysis of the (p)ppGpp synthetase activity of the Rel enzyme of Mycobacterium tuberculosis

Rel_Mtb, a GTP pyrophosphokinase encoded by the Mycobacterium tuberculosis (Mtb) genome, catalyzes synthesis of (p)ppGpp from ATP and GDP(GTP) and its hydrolysis to GDP(GTP) and pyrophosphate to mediate stringent response, which helps bacteria to survive during nutrient limitation. Like other members of Rel_Spo homologs, Rel_Mtb has four distinct domains: HD, Rel_Spo (RSD), TGS and ACT. The N-terminal HD and RSD are responsible for (p)ppGpp hydrolysis and synthesis, respectively. In this study, we have dissected the rel _Mtb gene function and determined the minimal region essential for (p)ppGpp synthetic activity. The Rel_Mtb and its truncated derivatives were expressed from an arabinose inducible promoter (P _BAD ), and in vivo functional analyses were done in a (p)ppGpp null Escherichia coli strain. Our results indicate that only 243 amino acids (188–430 residues) containing fragment are sufficient for Rel_Mtb (p)ppGpp synthetic activity. The results were further confirmed by in vitro assays using purified proteins. We further characterized the RSD of Rel_Mtb by substituting several conserved amino acids with structurally related residues and identified six such residues, which appeared to be critical for maintaining its catalytic activity. Furthermore, we have also extended our analysis to an RSD encoding gene rv1366 of Mtb, and experimental results indicated that the encoded protein Rv1366 is unable to synthesize (p)ppGpp.