334 research outputs found
Human Mitochondrial VDAC Functionality Governs Scaffold Stability
No full textATP transport across the mitochondrial outer membrane occurs through voltage-dependent anion channels (VDACs). The three human VDAC isoforms share a similar primary sequence but differ in their interactome, and thereby, show antagonistic physiological functions. The anti-apoptotic and regulatory role of human VDAC2 (hV2) isoform must therefore originate from subtle variations in its primary sequence. However, the molecular details of how stability and function are regulated in hV2, and its impact on protein-protein interaction, are unknown. Here, we show that the 19-stranded hV2 barrel has evolutionarily retained an energetically suboptimal sequence for superior channel gating and voltage sensing. We find that residues in the N- and C-terminal zones, comprising strands β2-β8 and β17-β18, lower the scaffold stability by 1.0-3.0 kcal/mol. Furthermore, strands β5-β10 and β17 possess sequences that are intrinsically prone to association and aggregation. However, hV2 trades-off stability and aggregation for function. Residues in the N-terminal zone are important for voltage-dependent channel gating characteristics that are essential for metabolite transport. Our results demonstrate how energetic contribution of specific residues in hV2 links inversely to its functional importance. We conclude that the evolutionary selection of hV2 sequence for channel function and regulation is at the expense of innate barrel stability. To our knowledge, this is the first study to establish stability-function trade-off in a human β-barrel membrane protein. We propose that the antagonistic behavior of the three VDAC isoforms resides in subtle differences in the molecular elements that we have identified. These molecular elements decide thermodynamic stability, function, and the association network of the VDAC channels in the mitochondrial outer membrane
Control of human VDAC-2 scaffold dynamics by interfacial tryptophans is position specific
No full textAbstractMembrane proteins employ specific distribution patterns of amino acids in their tertiary structure for adaptation to their unique bilayer environment. The solvent-bilayer interface, in particular, displays the characteristic ‘aromatic belt’ that defines the transmembrane region of the protein, and satisfies the amphipathic interfacial environment. Tryptophan—the key residue of this aromatic belt—is known to influence the folding efficiency and stability of a large number of well-studied α-helical and β-barrel membrane proteins. Here, we have used functional and biophysical techniques coupled with simulations, to decipher the contribution of strategically placed four intrinsic tryptophans of the human outer mitochondrial membrane protein, voltage-dependent anion channel isoform-2 (VDAC-2). We show that tryptophans help in maintaining the structural and functional integrity of folded hVDAC-2 barrel in micellar environments. The voltage gating characteristics of hVDAC-2 are affected upon mutation of tryptophans at positions 75, 86 and 221. We observe that Trp-160 and Trp-221 play a crucial role in the folding pathway of the barrel, and once folded, Trp-221 helps stabilize the folded protein in concert with Trp-75 and Trp-160. We further demonstrate that substituting Trp-86 with phenylalanine leads to the formation of stable barrel. We find that the region comprising strand β4 (Trp-86) and β10-14 (Trp-160 and Trp-221) display slower and faster folding kinetics, respectively, providing insight into a possible directional folding of hVDAC-2 from the C-terminus to N-terminus. Our results show that residue selection in a protein during evolution is a balancing compromise between optimum stability, function, and regulating protein turnover inside the cell
Impacts of Ta Buffer Layer and Cu-Ge-Te Composition on the Reliability of GeSe-Based CBRAM
No full textsponsorship: This work was supported by imec's Core Partner Industrial Affiliation Program on Emerging Memories. The review of this article was arranged by Editor P. Du. (Corresponding author: Janaki Radhakrishnan.) (imec's Core Partner Industrial Affiliation Program on Emerging Memories)status: Publishe
Efficacy of sugarcane bagasse to produce bacterial biofilm in water for fish culture
No full textThe present study has been conducted to understand the efficacy of sugarcane bagasse to produce bacterial biofilm in water. The study period was 90 days. Total Plate count (TPC) in water and substrate was estimated on nutrient agar at room temperature by spread plate method. The TPC in water was the highest (2.10 x 104 ml–1) after 45 days. The average TPC of bacteria on bagasse varied from 140.0 (15d) to 30.25 (90d) x104·g–1. The present study demonstrates that the sugarcane bagasse can produce more bacteria in water thereby the fish can effectively utilize biofilm grown on sugarcane bagasse and provision of a substrate reduces the need for artificial feed.---------------------------------------------------------------------------------------------------------Efficacy of sugarcane bagasse to produce bacterial biofilm in water for fish culture M.V. Radhakrishnan and E.SugumaranDepartment of Zoology, Annamalai University, Annamalainagar – 608 002, Tamilnadu, India *Corresponding Author, Email: [email protected] Cite This Article As: M.V. Radhakrishnan, E. Sugumaran. 2010. Efficacy of sugarcane bagasse to produce bacterial biofilm in water for fish culture. J. Ecobiotechnol. 2(2):41-44
Aromatic interactions in β-hairpin scaffold stability: A historical perspective
No full textNon-covalent interactions between naturally occurring aromatic residues have been widely exploited as scaffold stabilizing agents in de novo designed peptides and in Nature – inspired structures. Our understanding of the factors driving aromatic interactions and their observed interaction geometries have advanced remarkably with improvements in conventional structural studies, availability of novel molecular methods and in silico studies, which have together provided atomistic information on aromatic interactions and interaction strengths. This review attempts to recapitulate the early advances in our understanding of aromatic interactions as stabilizing agents of peptide β-hairpins
Thermodynamic partitioning forces at the membrane protein interface
No full textTransmembrane proteins fold by the orchestrated interplay of membrane chaperones, holdases and the lipid membrane. Upon folding, membrane protein systems attain a thermodynamically stabilized framework through optimized energetics involving protein – membrane – solvent interactions. In particular, the water-bilayer interface demands selectively positioned amino acids to arbitrate interactions between the hydrophobic interior and polar phospholipid headgroups. A fundamental question that arises is the extent to which the amino acid side chain contributes to membrane protein folding and in maintaining protein-lipid interactions at the intricate interface environment of membrane proteins. Our goal is to experimentally deduce the partitioning cost of amino acids at the interface, and the associated physical principles governing the overall scaffold stability.
Interface residues are important contributors for membrane protein folding, and additionally serve as post-folding membrane anchors. In this study, we have quantified the free energy change associated with the chemical nature of the residue at the membrane interface. We achieve this by studying the unassisted folding equilibrium of the 8-stranded transmembrane β-barrel enzyme PagP in phosphocholine lipidic micelles and lipid vesicles. We present the first experimentally measured whole-protein free energy scale for side chain partitioning at the membrane interface, for all naturally abundant amino acids. We obtain differential contributions of the protein- and lipid-facing interface residues to the barrel folding pathway and stability of the folded scaffold. The most favorable transfer at the amphiphilic lipid-facing interface is for hydrophobic amino acids, whereas we observe the highest energetic cost of transfer for charged and polar groups. On the contrary, we find that small and polar residues are most favorably transferred to the protein-facing interface, whereas hydrophobic and aromatic residues promote alternate folding pathways in PagP.
Our studies establish that the side chain non-polar accessible surface area shows a direct correlation with the partitioning free energy change at the lipid-facing interface residue. We also find that the non-polar accessible surface correlates inversely with the thermodynamic partitioning free energy change when the interface residue is protein-facing. We demonstrate how PagP maintains a balance between concerted folding and hydrophobic collapse by evolutionary choice of small polar residues at the protein-facing interface. We also identify the importance of interface polar residues to the folding pathway of β-barrel membrane proteins
Oxidative Thiol Modifications as Molecular Redox Sensors in Human Mitochondria
No full textNeurodegenerative diseases and ageing are intricately linked with biological cellular levels of reactive oxygen species (ROS). The highest levels of ROS are produced during mitochondrial respiration, and an optimal balance of ROS levels for physiological signaling over cellular damage is regulated by ROS scavengers. In the mitochondrial outer membrane, voltage-dependent anion channels (VDACs) are believed to function in close conjunction with the molecular ROS sensors in the mitochondrial intermembrane space, to balance ROS transport into the cytosolic milieu versus the trigger of mitochondria-mediated apoptosis. However, the role of mitochondrial VDACs, and the molecular elements involved in ROS scavenging, has not yet been identified. In particular, the anti-apoptotic function of human VDAC2 isoform and the unusual abundance of thiolate ions - ¬presented as cysteines in VDAC2 - allowed us to hypothesize that redox homeostasis is effected by VDAC2 cysteines. Using molecular measurements of VDAC2 energetics in native-like environments, single-channel functional studies and insights from oxidative cysteine modifications in vitro and in vivo, we demonstrate that VDAC2 has evolutionary role as a molecular redox regulator. Cysteine enrichment in human VDAC2 is not stochastic, and is essential to counter cellular oxidative challenges and redox stress. For the first time in mitochondrial bioenergetics, our findings demonstrate both a vital role of VDAC2 as a metabolite flux regulator and how this protein is indispensable for oxidative homeostasis and the trigger of mitophagy
Baclofen-induced neurotoxicity in a patient with end-stage renal disease
No full textBaclofen, predominantly excreted by the kidneys is accumulated in patients with renal insufficiency leading to the central nervous system toxicity. Here the author reports a patient with end-stage renal disease on maintenance hemodialysis (HD) who developed drowsiness and became unresponsive within a day after taking single 10 mg dose of baclofen. Patient improved completely after two sessions of HD
Do folding elements trade‐off with function in the human mitochondrial metabolite transporter?
No full textIn humans, transmembrane β-barrels are found exclusively in the outer mitochondrial membrane (OMM). They comprise metabolite transporters, translocases, and chaperones, which together control cellular homeostasis. The metabolite transporters — known as voltage-dependent anion channels (VDACs) – are the most abundant OMM barrels. They are 19-stranded β-barrel structures that also form important pharmacological targets due to their roles in metabolite flux, steroidogenesis, gametogenesis, and ROS regulation. Despite long-standing efforts, molecular factors that regulate VDAC function and folding at the atomistic level are not known. Here, we map the folding nucleus and the assembly pathway of human VDAC isoform 2 in phosphocholine vesicles. We carry out a comprehensive measurement of hVDAC2 folding kinetics, and equilibrium thermodynamic contribution, of each of the 270 non-Ala residues of the 294-residue barrel. Interestingly, we obtain a two-state thermodynamic equilibrium that is attained through two detectable on-pathway early folding intermediates, which are assembled in milliseconds – seconds. These intermediates undergo slow conformational restructuring to form the folded VDAC2 barrel. We find that specific charged residues that form a part of these early intermediates also positively regulate the gating characteristics of VDAC2. This observation is unprecedented, as directed evolution of protein sequences in favor of function usually place a stability burden on its structure that additionally impedes protein assembly. Our findings provide molecular insight on the assembly pathway of human VDAC2, and directly link these folding elements with VDAC2 function as the metabolite transporter of human mitochondria
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