Biochimica et biophysica acta. Biomembranes最新文献

This work correlates the effects of benzohydroxamate (BH) and nitrobenzohydroxamate (NBH) anions in two membrane models which may be used for anti-tuberculosis (anti-TB) spectroscopic studies and/or computational studies. Firstly, the BH and NBH influence in the physico-chemical properties of soy asolectin (ASO)-based large multilamellar vesicles (MLVs) were evaluated by spectroscopic and calorimetric studies. In parallel, the BH and NBH interaction with a Mycobacterium tuberculosis (Mtb) inner membrane model, composed of phosphatidyl-myo-inositol-dimannoside (PIM₂), was investigated by molecular dynamics (MD) simulations. Spectroscopic data showed a localization of BH close to the lipid phosphate group, while NBH was found close to the choline region. The BH ordered the ASO choline, phosphate and carbonyl regions and disrupted the acyl methylenes, reducing the membrane packing of the lipid hydrophobic region. On the other hand, NBH showed an ordering effect in all the lipid groups (polar, interface and hydrophobic ones). By MD studies, it was found that NBH enhanced the stability of the PIM₂ membrane more than BH, while also being positioned closer to its mannosyl oxygens. As in ASO MLVs, BH was localized close to the PIM₂ phosphate group and disrupted its acyl chains. However, higher values of lateral diffusion were observed for NBH than BH. Despite this, BH and NBH increased the membrane thickness by 35 %, which suggests a global ordering effect of both drugs. Findings of this work reinforce the accordance and complementarity between MLVs based on ASO and the PIM₂ MD model results to study the drug effects in Mtb membrane properties.

Apolipoprotein A-I (apoA-I), the primary protein component of plasma high-density lipoproteins (HDL), is comprised of two structural regions, an N-terminal amphipathic α-helix bundle domain (residues 1–184) and a hydrophobic C-terminal domain (residues 185–243). When a recombinant fusion protein construct [bacterial pelB leader sequence – human apoA-I (1–243)] was expressed in Escherichia coli shaker flask cultures, apoA-I was recovered in the cell lysate. By contrast, when the C-terminal domain was deleted from the construct, large amounts of the truncated protein, apoA-I (1–184), were recovered in the culture medium. Consequently, following pelB leader sequence cleavage in the E. coli periplasmic space, apoA-I (1–184) was secreted from the bacteria. When the pelB-apoA-I (1–184) fusion construct was expressed in a 5 L bioreactor, substantial foam production (~30 L) occurred. Upon foam collection and collapse into a liquid foamate, SDS-PAGE revealed that apoA-I (1–184) was the sole major protein present. Incubation of apoA-I (1–184) with phospholipid vesicles yielded reconstituted HDL (rHDL) particles that were similar in size and cholesterol efflux capacity to those generated with full-length apoA-I. Mass spectrometry analysis confirmed that pelB leader sequence cleavage occurred and that foam fractionation did not result in unwanted protein modifications. The facile nature and scalability of bioreactor-based apolipoprotein foam fractionation provide a novel means to generate a versatile rHDL scaffold protein.

Lateral heterogeneity, or mosaicity, is a fundamental property inherent to cell membranes that is crucial for their functioning. While microscopic inhomogeneities (e.g. rafts) are easily detected experimentally, lipid domains with nanoscale dimensions (nanoclusters of nanodomains, NDs) resist reliable characterization by instrumental methods. In such a case, important insight can be gained via computer modeling. Here, NDs composed of lipid's head groups in the mixed zwitterionic dioleoylphosphatidylcholine (DOPC) and negatively charged dioleoylphosphatidylserine (DOPS) bilayers were studied by molecular dynamics. A new algorithm has been developed to identify NDs. Unlike most similar methods, it implicitly considers the heterogeneous distribution of lipid head atomic density and does not require subjectively chosen parameters. In DOPS-rich membranes, lipids form more compact and stable NDs due to strong interlipid interactions. In DOPC-rich systems, NDs arise due to the “packing” effect of weakly bound lipid heads. The clustering picture is related to the physical properties of the bilayer surface: DOPS-rich systems show more pronounced surface heterogeneity of hydrophilic/hydrophobic regions compared to DOPC-rich ones. The results obtained are important for the effective quantitative characterization of the “dynamic molecular portrait” of a membrane surface – its “fingerprint” characterizing dynamical distribution of its physicochemical properties.

KCNQ1, also known as Kv7.1, is a voltage gated potassium channel that associates with the KCNE protein family. Mutations in this protein has been found to cause a variety of diseases including Long QT syndrome, a type of cardiac arrhythmia where the QT interval observed on an electrocardiogram is longer than normal. This condition is often aggravated during strenuous exercise and can cause fainting spells or sudden death. KCNE1 is an ancillary protein that interacts with KCNQ1 in the membrane at varying molar ratios. This interaction allows for the flow of potassium ions to be modulated to facilitate repolarization of the heart. The interaction between these two proteins has been studied previously with cysteine crosslinking and electrophysiology. In this study, electron paramagnetic resonance (EPR) spectroscopy line shape analysis in tandem with site directed spin labeling (SDSL) was used to observe changes in side chain dynamics as KCNE1 interacts with KCNQ1. KCNE1 was labeled at different sites that were found to interact with KCNQ1 based on previous literature, along with sites outside of that range as a control. Once labeled KCNE1 was incorporated into vesicles, KCNQ1 (helices S1-S6) was titrated into the vesicles. The line shape differences observed upon addition of KCNQ1 are indicative of an interaction between the two proteins. This method provides a first look at the interactions between KCNE1 and KCNQ1 from a dynamics perspective using the full transmembrane portion of KCNQ1.