Role of secondary structure in protein-phospholipid surface interactions: reconstitution and denaturation of apolipoprotein C-I:DMPC complexes

Binding of protein to a phospholipid surface is commonly mediated by amphipathic alpha-helices. To understand the role of alpha-helical structure in protein-lipid interactions, we used discoidal lipoproteins reconstituted from dimyristoylphosphatidylcholine (DMPC) and human apolipoprotein C-I (apoC-I, 6 kDa) or its mutants containing single Pro substitutions along the sequence and differing in their alpha-helical content in solution (0-48%) and on DMPC (40-75%). Thermal denaturation revealed that lipoprotein stability correlates weakly with the protein helix content: proteins with higher alpha-helical content on DMPC may form more stable complexes. Lipoprotein reconstitution upon cooling from the heat-denatured state and DMPC clearance studies revealed that protein secondary structure in solution and on DMPC correlates strongly with the maximal temperature of lipoprotein reconstitution: more helical proteins can reconstitute lipoproteins at higher temperatures. Interestingly, at Tc = 24 degrees C of the DMPC gel-to-liquid crystal transition, the clearance rate is independent of the protein helical content. Consequently, if the packing defects at the phospholipid surface are readily available (e.g., at the lipid phase boundary), insertion of protein into these defects is independent of the secondary structure in solution. However, if hydrophobic defects are limited, protein binding and insertion are aided by other surface-bound proteins and depend on their helical propensity: the larger the propensity, the faster the binding and the broader its temperature range. This positive cooperativity in binding of alpha-helices to phospholipid surface, which may result from direct and/or lipid-mediated protein-protein interactions, may be important for lipoprotein metabolism and for protein-membrane binding.     

Biofilm formation on copper and its control by inhibitor/biocide in cooling water environment

The present study has successfully identified the nitrate reducing bacteria present in the cooling water system and also investigated the performance of industrially applied biocide and inhibitor on the bacterial inhibition. In order to carry out the objective of this study, facilities and methods such as 16S rRNA gene sequencing, Lowry assay, SEM, EIS, ICP-MS and weight loss analysis were being utilized. In this study, two out of the five morphologically dis- similar colonies identified through 16S rRNA gene sequencing, namely the Massilia timonae and the Pseudomonas, were being utilized in the biocorrosion study on copper metal. From the surface analysis using SEM demonstrated the phenomenon of biofilm formation on the copper surface. 2-methylbenzimidazole has the addition of methyl group in the diazole ring position of benzimidazole it has create basicity environment and inhibit the metal deterioration. Meanwhile, it is also deducible from the EIS and protein analysis that com- bination of biocide with either of the inhibitors gives rise to better biocorrosion suppression (0.00178 mpy and 0.00171mpy) as compared to the sole effect of either biocide or inhibitor (0.00219 mpy, 0.00162 and 0.00143). Biocorrosion system biocide with MBM was found to exhibit 65% corrosion inhibition efficiency. Moreover, adoption of 2-Methylbenzimidazole seems to display better performance as compared to Multionic 8151, which is adopted in cooling water system.     

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 Å. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.     

Impact of Sugarcane yellow leaf virus (ScYLV) infection on physiological efficiency and growth parameters of sugarcane under tropical climatic conditions in India

Yellow leaf (YL) of sugarcane caused by Sugarcane yellow leaf virus (ScYLV, a Polerovirus of the Luteoviridae family) is a serious disease affecting the crop production and productivity in India. Although impact of the disease on cane growth is observed, no systematic study has been done so far from the tropical Asian region to establish its impact on various physiological parameters, cane yield and juice quality. We have assessed physiological parameters in symptomatic and asymptomatic plants of ten different cultivars and a genotype. In addition, similar comparisons were made between virus-infected and virus-free plants derived through meristem culture. Our studies established that among several physiological parameters, photosynthetic rate (A), stomatal conductance (g _s) and SPAD metre values were significantly reduced in cultivars severely infected with ScYLV. Virus-infected cultivars exhibited significant reduction in growth/yield parameters, viz. stalk height, stalk thickness and number of internodes. Plant growth reductions were found to be 42.9, 42.3 and 38.9 % in susceptible cultivars CoPant 84211, Co 86032 and CoC 671, respectively. In addition to reduction in stalk weight, height and girth, YL disease also reduced juice yield in the affected canes up to 34.15 %. Similarly, comparison of diseased (virus-infected) and virus-free plants derived through meristem culture also revealed a drastic reduction in cane growth/physiological parameters and juice yield due to virus infection. The present study is the first comprehensive report demonstrating that YL disease caused by ScYLV seriously affects cane and juice yield in major sugarcane varieties under tropical climatic conditions (India). Consequently, this situation warrants a massive programme to provide healthy seed material and initiate breeding for YL resistance in sugarcane.     

Effect of uncoupling protein-1 expression on 3T3-L1 adipocyte gene expression

The mitochondrial respiratory uncoupling protein 1 (UCP1) partially uncouples substrate oxidation and oxidative phosphorylation to promote the dissipation of cellular biochemical energy as heat in brown adipose tissue. We have recently shown that expression of UCP1 in 3T3-L1 white adipocytes reduces the accumulation of triglycerides. Here, we investigated the molecular basis underlying UCP1 expression in 3T3-L1 adipocytes. Gene expression data showed that forced UCP1 expression down-regulated several energy metabolism pathways; but ATP levels were constant. A metabolic flux analysis model was used to reflect the gene expression changes onto metabolic processes and concordance was observed in the down-regulation of energy consuming pathways. Our data suggest that adipocytes respond to long-term mitochondrial uncoupling by minimizing ATP utilization.