NMR Structural and Biophysical Analysis of the Disease-Linked Inner Mitochondrial Membrane Protein MPV17

MPV17 is an integral inner mitochondrial membrane protein, whose loss-of-function is linked to the hepatocerebral form of the mitochondrial-DNA-depletion syndrome, leading to a tissue-specific reduction of mitochondrial DNA and organ failure in infants. Several disease-causing mutations in MPV17 have been identified and earlier studies with reconstituted protein suggest that MPV17 forms a high conductivity channel in the membrane. However, the molecular and structural basis of the MPV17 functionality remain only poorly understood. In order to make MPV17 accessible to high-resolution structural studies, we here present an efficient protocol for its high-level production in E. coli and refolding into detergent micelles. Using biophysical and NMR methods, we show that refolded MPV17 in detergent micelles adopts a compact structure consisting of six membrane-embedded α-helices. Furthermore, we demonstrate that MPV17 forms oligomers in a lipid bilayer that are further stabilized by disulfide-bridges. In line with these findings, MPV17 could only be inserted into lipid nanodiscs of 8–12 nm in diameter if intrinsic cysteines were either removed by mutagenesis or blocked by chemical modification. Using this nanodisc reconstitution approach, we could show that disease-linked mutations in MPV17 abolish its oligomerization properties in the membrane. These data suggest that, induced by oxidative stress, MPV17 can alter its oligomeric state from a properly folded monomer to a disulfide-stabilized oligomeric pore which might be required for the transport of metabolic DNA precursors into the mitochondrial matrix to compensate for the damage caused by reactive oxygen species.     

The influence of different lipid environments on the structure and function of the hepatitis C virus p7 ion channel protein

Abstract

The hepatitis C virus (HCV) encodes the p7 protein that oligomerizes to form an ion channel. The 63 amino acid long p7 monomer is an integral membrane protein predominantly found in the endoplasmic reticulum (ER). Although it is currently unknown whether p7 is incorporated into secreted virions, its presence is crucial for the release of infectious virus. The molecular and biophysical mechanism employed by the p7 ion channel is largely unknown, but in vivo it is likely to be embedded in membranes undergoing changes in lipid composition. In this study we analyze the influence of the lipid environment on p7 ion channel structure and function using electrophysiology and synchrotron radiation circular dichroism (SRCD) spectroscopy. We incorporated chemically synthesized p7 polypeptides into artificial planar membranes of various lipid compositions. A lipid bilayer composition comprising phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (4:1 PC:PE) led to burst-like patterns in the channel recordings with channel openings lasting up to 0.5 s. The reverse ratio of PC:PE (1:4) gave rise to individual channels continuously opening for up to 8 s. SRCD spectroscopy of p7 embedded into liposomes of corresponding lipid compositions suggests there is a structural effect of the lipid composition on the p7 protein.     

Role of electrostatics in the binding of charged metallophthalocyanines to neutral and charged phospholipid membranes

Binding of the cationic tetra(tributylammoniomethyl)-substituted hydroxoaluminum phthalocyanine (AlPcN₄) to bilayer lipid membranes was studied by fluorescence correlation spectroscopy (FCS) and intramembrane field compensation (IFC) methods. With neutral phosphatidylcholine membranes, AlPcN₄ appeared to bind more effectively than the negatively charged tetrasulfonated aluminum phthalocyanine (AlPcS₄), which was attributed to the enhancement of the coordination interaction of aluminum with the phosphate moiety of phosphatidylcholine by the electric field created by positively charged groups of AlPcN₄. The inhibitory effect of fluoride ions on the membrane binding of both AlPcN₄ and AlPcS₄ supported the essential role of aluminum-phosphate coordination in the interaction of these phthalocyanines with phospholipids. The presence of negative or positive charges on the surface of lipid membranes modulated the binding of AlPcN₄ and AlPcS₄ in accord with the character (attraction or repulsion) of the electrostatic interaction, thus showing the significant contribution of the latter to the phthalocyanine adsorption on lipid bilayers. The data on the photodynamic activity of AlPcN₄ and AlPcS₄ as measured by sensitized photoinactivation of gramicidin channels in bilayer lipid membranes correlated well with the binding data obtained by FCS and IFC techniques. The reduced photodynamic activity of AlPcN₄ with neutral membranes violating this correlation was attributed to the concentration quenching of singlet excited states as proved by the data on the AlPcN₄ fluorescence quenching.     

A world of cytochrome P450s

The world we live in is a biosphere influenced by all organisms who inhabit it. It is also an ecology of genes, with some having rather startling effects. The premise put forth in this issue is cytochrome P450 is a significant player in the world around us. Life and the Earth itself would be visibly different and diminished without cytochrome P450s. The contributions to this issue range from evolution on the billion year scale to the colour of roses, from Darwin to Rachel Carson; all as seen through the lens of cytochrome P450.     

Bacterial reduction of hexavalent molybdenum to molybdenum blue

A bacterium that was able to tolerate and reduce as high as 50 mM of sodium molybdate to molybdenum blue has been isolated from a metal recycling ground. The isolate was tentatively identified as Serratia sp. strain Dr.Y8 based on the carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny. ANOVA analysis showed that isolate Dr.Y8 produced significantly higher (P < 0.05) amount of Mo-blue with 3, 5.1 and 11.3 times more molybdenum blue than previously isolated molybdenum reducers such as Serratia marcescens strain Dr.Y6, E. coli K12 and E. cloacae strain 48, respectively. Its molybdate reduction characteristics were studied in this work. Electron donor sources such as sucrose, mannitol, fructose, glucose and starch supported molybdate reduction. The optimum phosphate, pH and temperature that supported molybdate reduction were 5 mM, pH 6.0 and 37°C, respectively. The molybdenum blue produced from cellular reduction exhibited a unique absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. Metal ions such as chromium, silver, copper and mercury resulted in approximately 61, 57, 80, and 69% inhibition of the molybdenum-reducing activity at 1 mM, respectively. The reduction characteristics of strain Dr.Y8 suggest that it would be useful in future molybdenum bioremediation.     

Kinetic and proteolytic identification of heat-induced conformational changes in the urea herbicide binding site of isolated Phaseolus vulgaris chloroplast thylakoids

Kinetic parameters of 3-(3, 4-dichlorophenyl)-1, 1-dimethyl urea (DCMU)-induced inhibition of electron transport in chloroplast thylakoids isolated from Phaseolus vulgaris L. cv. Oregon 1604 were determined from analysis of a convergent, parallel electrical circuit. Through this analogue, the apparent affinity of the purported binding site for DCMU (K₁) and the relative amount of DCMU-insensitive electron transport (v^max₁/v_o) were obtained using a reiterative non-linear least squares curve-fitting procedure. Exposure of thylakoids to heat caused a gradual increase in K₁ (or decrease in the affinity of the thylakoid for DCMU) with an apparent activation energy of 134 kJ mol⁻¹. Tryptic susceptibility of a protein region regulating K₁ also decreased gradually with exposure to 45°C, suggesting that the heat-induced increase in K₁ might be due to a protein conformational change. On the other hand, thylakoid exposure to 45°C resulted in a rapid (<5 min) irreversible increase in v^max_I/v_o, which was also the apparent result of a conformational change in a region of the protein which regulates this function. These results are suggestive of the existence of differential thermal sensitivities of proteins within the thylakoids and, perhaps, of different regions within a single membrane protein.     

The effect of antibiotics on the photocycle and protoncycle of purple membrane suspensions

The interrelation was studied between the phototransient absorbing maximally at 412 nm (M₄₁₂) and light-induced proton release under steady-state conditions in aqueous suspensions of ‘purple membrane’ derived from Halobacterium halobium. The decay of M₄₁₂ was slowed down by the simultaneous application of the ionophoric antibiotics valinomycin and beauvericin. The former had only slight activity alone and the latter was effective only in conjunction with valinomycin. The steady-state concentration of M₄₁₂ which was formed on illumination was a direct function of the concentration of valinomycin. Maximum stabilization of M₄₁₂ was obtained when the valinomycin was approximately equimolar with the bacteriorhodopsin. Addition of salts to the medium increased the number of protons released per molecule of M₄₁₂ without affecting the level of M₄₁₂ which was produced by continuous illumination. The effectiveness of the salts in this respect depended on the nature of the cation. Ca²⁺ and their antagonists La³⁺ and ruthenium red were found to have especially high affinity for the system. The extent of light-induced acidification could not be enhanced by increasing the pH of the medium from 6.5 to 7.8. The possible mechanism of action of the ionophores and of the cations on the photocycle and on the proton cycle is discussed.     

Critical Role of Lipid Scramblase TMEM16F in Phosphatidylserine Exposure and Repair of Plasma Membrane after Pore Formation

1. Download : Download high-res image (253KB)
2. Download : Download full-size image

     

Effects of detergents on catalytic activity of human endometase/matrilysin 2, a putative cancer biomarker

Matrix metalloproteinases (MMPs) are a family of hydrolytic enzymes that play significant roles in development, morphogenesis, inflammation, and cancer invasion. Endometase (matrilysin 2 or MMP-26) is a putative early biomarker for human carcinomas. The effects of the ionic and nonionic detergents on catalytic activity of endometase were investigated. The hydrolytic activity of endometase was detergent concentration dependent, exhibiting a bell-shaped curve with its maximum activity near the critical micelle concentration (CMC) of nonionic detergents tested. The effect of Brij-35 on human gelatinase B (MMP-9), matrilysin (MMP-7), and membrane-type 1 MMP (MT1-MMP) was further explored. Their maximum catalysis was observed near the CMC of Brij-35 (∼ 90 μM). Their IC₅₀ values were above the CMC. The inhibition mechanism of MMP-7, MMP-9, and MT1-MMP by Brij-35 was a mixed type as determined by Dixon’s plot; however, the inhibition mechanism of endometase was noncompetitive with a K_i value of 240 μM. The catalytic activities of MMPs are influenced by detergents. Monomer of detergents may activate and stabilize MMPs to enhance catalysis, but micelle of detergents may sequester enzyme and block the substrate binding site to impede catalysis. Under physiological conditions, a lipid or membrane microenvironment may regulate enzymatic activity.