Insights into the oligomeric structure of the HIV-1 Vpu protein

The HIV-1-encoded protein Vpu forms an oligomeric ion channel/pore in membranes and interacts with host proteins to support the virus lifecycle. However, Vpu molecular mechanisms are currently not well understood. Here, we report on the Vpu oligomeric organization under membrane and aqueous conditions and provide insights into how the Vpu environment affects the oligomer formation. For these studies, we designed a maltose-binding protein (MBP)-Vpu chimera protein and produced it in E. coli in soluble form. We analyzed this protein using analytical size-exclusion chromatography (SEC), negative staining electron microscopy (nsEM), and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, we found that MBP-Vpu formed stable oligomers in solution, seemingly driven by Vpu transmembrane domain self-association. A coarse modeling of nsEM data as well as SEC and EPR data suggests that these oligomers most likely are pentamers, similar to what was reported regarding membrane-bound Vpu. We also noticed reduced MBP-Vpu oligomer stability upon reconstitution of the protein in β-DDM detergent and mixtures of lyso-PC/PG or DHPC/DHPG. In these cases, we observed greater oligomer heterogeneity, with MBP-Vpu oligomeric order generally lower than in solution; however, larger oligomers were also present. Notably, we found that in lyso-PC/PG, above a certain protein concentration, MBP-Vpu assembles into extended structures, which had not been reported for Vpu. Therefore, we captured various Vpu oligomeric forms, which can shed light on Vpu quaternary organization. Our findings could be useful in understanding Vpu organization and function in cellular membranes and could provide information regarding the biophysical properties of single-pass transmembrane proteins.     

The PurR binding site in the glyA prometer region of Escherichia coli

Site-directed mutagenesis was used to change the PurR binding site in the control region of a glyA-lac gene fusion. Mutations that changed the PurR binding sequence away from the consensus sequence reduced PurR binding, which correlated with reduced purine-mediated repression. Mutations that changed the binding sequence toward the consensus sequence had no significant effect on either PurR binding or purine-mediated repression. Hypoxanthine and guanine, co-repressors for PurR-mediated regulation of the pur regulon, increased binding of PurR to glyA operator DNA.     

The Almost-one Parameter Hypothesis Applied to Pharmacokinetics of Ethanol Infusion

They say that the pharmacokinetics of constant-rate intravenous infusion of ethanol obeys the Henri-Michaelis-Menten (H-M-M) model. In analysing the original data from the viewpoint of the almost-one parameter hypothesis (see box 1), we found that the whole process should be divided into four phases and the H-M-M model was valid only in the last phase.     

Nutrient limitation and nutrient‐driven shifts in plant species composition in a species‐rich fen meadow

Question: We studied the development and persistence of the effects of nutrient pulses on biomass production and species composition in a fen meadow. Location: Nature reserve, central Netherlands, 5 m a.s.l. Methods: Single pulse fertilization with N and P in a factorial design on an undrained central and a drained margin site in a species‐rich fen meadow (Cirsio dissecti‐Molinietum). Biomass production and species composition were monitored during four years. Results: At the central site, N addition boosted biomass production, but only during one year. The species composition was not changed. P fertilization increased the biomass production and changed the species composition from a vegetation dominated by Carex panicea to a grassland community with abundant Holcus lanatus, but not before the second year. At the margin site, P fertilization changed the species composition in a similar way, but biomass production was not increased. N fertilization had no effect. At both sites the P induced shift in species composition persisted for four years although the P effect declined during the experiment. Conclusions: The biomass responses show that N was limiting in the central site. Another nutrient, besides N and P (probably K) must have been limiting in the marginal site. The fast decline of the N effect on biomass is ascribed to increased denitrification and biomass removal. The delay in the P effect on biomass and species composition and the persistence of the P effect on species composition are ascribed to fast immobilisation and subsequent slow release of fertilizer P in the peat soil. Recurrence of the P pulses is expected to cause permanent changes in species composition.     

Cryo-EM Structure of the Full-length hnRNPA1 Amyloid Fibril

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a multifunctional RNA-binding protein that is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis and multisystem proteinopathy. In this study, we have used cryo-electron microscopy to investigate the three-dimensional structure of amyloid fibrils from full-length hnRNPA1 protein. We find that the fibril core is formed by a 45-residue segment of the prion-like low-complexity domain of the protein, whereas the remaining parts of the protein (275 residues) form a fuzzy coat around the fibril core. The fibril consists of two fibril protein stacks that are arranged into a pseudo-2₁ screw symmetry. The ordered core harbors several of the positions that are known to be affected by disease-associated mutations, but does not encompass the most aggregation-prone segments of the protein. These data indicate that the structures of amyloid fibrils from full-length proteins may be more complex than anticipated by current theories on protein misfolding.     

Activation volumes for horseradish peroxidase compound ii reactions

The activation volumes for the reactions of horseradish peroxidase compound II with L-tyrosine. 3-iodo-L-tyrosine. p-aminobenzoic acid and ferrocyanide were determined by using a high-pressure stopped-flow technique at 25°C and pH 7. For the tyrosines, the solvent electrostriction accompanying substrate ionization and H⁺ transfer from the substituted phenol to a basic group of the enzyme can account for the observed negative activation volumes. For p-aminobenzoic acid a simple electron transfer without H⁺ transfer appears to occur. The positive activation volume for ferrocyanide may be explained in terms of electron transfer associated with a large change in electrostriction of the inorganic redox couple.     

A theoretical approach to G-protein modulation of cellular responsiveness

 Structure and function of cells often depend critically on molecular signals arriving at their surface. There are universal mechanisms of signal transduction and signal processing across cell membranes. In this paper the mechanisms involving guanine-nucleotide regulatory proteins (“G-proteins”) and certain receptor-kinases are considered. On the basis of recent findings in molecular biology a mathematical model is developed taking into account all essential components in the biochemical network between first and second messenger. There are two coupled feedback loops inherent in this process. The model finally consists of three nonlinear equations, which are obtained from a system of originally ten equations by using conservation laws and quasi-steady state conditions. The second part of the paper contains a mathematical analysis of the model. Solutions describing the temporal development of the involved biochemical species are shown to be bounded, more specifically to remain, independent of the size of the input signal, in a bounded domain of the state space. For the situation of stationary input signals existence, uniqueness and asymptotic stability of steady states are derived. We also demonstrate biologically relevant stimulus-response properties like monotonicity and saturation effects. For temporally non-constant input signals we show numerically that the model is able to produce phenomena of hypersensitivity and desensitization which are important characteristics of cellular responsiveness. Received 18 March 1996; received in revised form 15 April 1996