Environmental Pathway and Risk Assessment Studies of the Musi River's Heavy Metal Contamination—A Case Study

The Musi River, in Hyderabad, the capital city of Andhra Pradesh state in India, is relatively dry for most of the year except for the four monsoon months when 700–800 mm of rain falls. Throughout the year, sewage, industrial, and hospital waste is released into the river. In the present work the Musi River from Amberpet Bridge to Nallacheruvu (8 km stretch) was assessed and monitored for heavy metal contamination attributable to sewage and industrial effluents. Twelve locations were assessed for Zn, Cr, Cu, Ni, Co, As, Hg, Cd, and Pb in soils, waters, forage grass, milk, and vegetables. A sequential extraction scheme revealed that high levels of Zn, Cr, and Cu were associated with labile fractions, making them more mobile and phytoavailable. Human risk was assessed in people exposed to pollution by analyzing metals concentrations in venous blood and urine. Results showed high amounts of Pb, Zn, Cr, and Ni compared to permissible limits, attributable to the consumption of contaminated food. Metals concentrations were monitored systematically to assess risks and support management decisions to help curtail the possible entry of metals into human food chains. An assessment was also made of a possible analysis of a remediation technology for lead-contaminated soils and water.     

Methyl dynamics for understanding hydrophobic core packing of dynamically different motifs of double-stranded RNA binding domain of protein kinase R

Double-stranded RNA binding domains of human protein kinase R (dsRBD-PKR) regulate distinct cellular functions and the fate of an RNA molecule in the cell. This highly homologous domains present in multiple copies in a number of species, exhibit individual and specific functional specificity. Number of NMR and X-ray crystallographic structural studies reveals that such domains take a common alpha-beta-beta-beta-alpha tertiary fold. However, the functional specificities of these domains could be due to the dynamics of the individual amino acid residues, as has been shown earlier in the case of backbone dynamics of 15N-1H of dsRNA binding motifs (dsRBMs) of human protein kinase R (PKR) (Nanduri S, Rahman F, Williams BRG, Qin J. EMBO J 2000;19:5567-5574). To further investigate if the differences in dynamics of the two dsRBMs are restricted to only backbone, or if the side-chain motions are also different to the extent of influencing their packing of the two hydrophobic cores, we have investigated the methyl group dynamics using 13C-methyl relaxation measurements. The results show that the hydrophobic core of dsRBM1 is more tightly packed than dsRBM2, and it seems to undergo less fast scale motions in the subnanosecond regime.     

Regulatory and structural EF-hand motifs of neuronal calcium sensor-1: Mg 2+ modulates Ca 2+ binding, Ca 2+ -induced conformational changes, and equilibrium unfolding transitions

Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca²⁺ signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca²⁺-specific) and structural (Ca²⁺- or Mg²⁺-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca²⁺ binding using NMR and EF-hand mutants. Ca²⁺ titration, as monitored by [¹⁵N,¹H] heteronuclear single quantum coherence, suggests that Ca²⁺ binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg²⁺ binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca²⁺-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca²⁺ and not Mg²⁺. Ca²⁺ binding induces conformational rearrangements in the protein by reversing Mg²⁺-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg²⁺ with Ca²⁺ reduces the Ca²⁺-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca²⁺-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg²⁺-bound NCS-1 are similar, the early unfolding transitions of Ca²⁺-bound NCS-1 are partially influenced in the presence of Mg²⁺. This study demonstrates the importance of Mg²⁺ as a modulator of calcium homeostasis and active-state behavior of NCS-1.     

Chemical shift based editing of CH<Subscript>3</Subscript> groups in fractionally <Superscript>13</Superscript>C-labelled proteins using GFT (3, 2)D CT-H<Emphasis Type="Underline">CC</Emphasis>H-COSY: stereospecific assignments of CH<Subscript>3</Subscript> groups of Val and Leu residues

We propose a (3, 2)D CT-HCCH-COSY experiment to rapidly collect the data and provide significant dispersion in the spectral region containing (13)C-(1)H cross peaks of CH(3) groups belonging to Ala, Ile, Leu, Met, Thr and Val residues. This enables one to carry out chemical shift based editing and grouping of all the (13)C-(1)H cross peaks of CH(3) groups belonging to Ala, Ile, Leu, Met, Thr and Val residues in fractionally (10%) (13)C-labelled proteins, which in turn aids in the sequence-specific resonance assignments in general and side-chain resonance assignments in particular, in any given protein. Further, we demonstrate the utility of this experiment for stereospecific assignments of the pro-R and pro-S methyl groups belonging to the Leu and Val residues in fractionally (10%) (13)C-labelled proteins. The proposed experiment opens up a wide range of applications in resonance assignment strategies and structure determination of proteins.     

Overexpression, on-column refolding and isotopic labeling of Hahellin from Hahella chejuensis, a putative member of the betagamma-crystallin superfamily

A gene which encodes a hypothetical protein of 40 kDa has been identified in the genome of a marine bacterium Hahella chejuensis, as a putative member of βγ-crystallin superfamily. This hypothetical protein contains a putative βγ-crystallin-like domain, along with other domains for carbohydrate binding regions. It is named as Hahellin. A PCR amplified stretch of 92-amino acid residue long protein was cloned into pET21a vector and overexpressed in Escherichia coli strain BL21(DE3)pLysS cells. The recombinant Hahellin, produced as inclusion bodies, was estimated to be around 50% of the total cellular protein content which was solubilized in 8 M urea. The protein was purified and refolded using an anion exchange column. The MALDI-TOF mass spectrometry revealed the purity and monomeric nature of the protein. Further, a method to prepare isotopically (¹⁵N/¹³C) labeled protein with high yield for NMR studies is reported. The uniformly ¹⁵N-labeled Hahellin thus produced has been characterized by recording a sensitivity enhanced 2D [¹⁵N]–[¹H] HSQC spectrum. The well, dispersed peaks in the spectrum confirm that the protein is indeed well folded and suitable for further studies by NMR.     

Characterization of low-energy excited states in the native state ensemble of non-myristoylated and myristoylated neuronal calcium sensor-1

Information on the low-energy excited states of a given protein is important as this controls the structural adaptability and various biological functions of proteins such as co-operativity, response towards various external perturbations. In this article, we characterized individual residues in both non-myristoylated (non-myr) and myristoylated (myr) neuronal calcium sensor-1 (NCS-1) that access alternate states by measuring nonlinear temperature dependence of the backbone amide-proton (1H(N)) chemical shifts. We found that ~20% of the residues in the protein access alternative conformations in non-myr case, which increases to ~28% for myr NCS-1. These residues are spread over the entire polypeptide stretch and include the edges of α-helices and β-strands, flexible loop regions, and the Ca2(+)-binding loops. Besides, residues responsible for the absence of Ca2(+)-myristoyl switch are also found accessing alternative states. The C-terminal domain is more populated with these residues compared to its N-terminal counterpart. Individual EF-hands in NCS-1 show significantly different number of alternate states. This observation prompts us to conclude that this may lead to differences in their individual conformational flexibility and has implications on the functionality. Theoretical simulations reveal that these low-energy excited states are within an energy band of 2-4 kcal/mol with respect to the native state.     

A simulation model of <Emphasis Type="Italic">Escherichia coli</Emphasis> osmoregulatory switch using E-CELL system

Background  

Bacterial signal transduction mechanism referred to as a "two component regulatory systems" contributes to the overall adaptability of the bacteria by regulating the gene expression. Osmoregulation is one of the well-studied two component regulatory systems comprising of the sensor, EnvZ and the cognate response regulator, OmpR, which together control the expression of OmpC and OmpF porins in response to the osmolyte concentration.