Non-covalent binding of membrane lipids to membrane proteins

Polar lipids and membrane proteins are major components of biological membranes, both cell membranes and membranes of enveloped viruses. How these two classes of membrane components interact with each other to influence the function of biological membranes is a fundamental question that has attracted intense interest since the origins of the field of membrane studies. One of the most powerful ideas that driven the field is the likelihood that lipids bind to membrane proteins at specific sites, modulating protein structure and function. However only relatively recently has high resolution structure determination of membrane proteins progressed to the point of providing atomic level structure of lipid binding sites on membrane proteins. Analysis of X-ray diffraction, electron crystallography and NMR data over 100 specific lipid binding sites on membrane proteins. These data demonstrate tight lipid binding of both phospholipids and cholesterol to membrane proteins. Membrane lipids bind to membrane proteins by their headgroups, or by their acyl chains, or binding is mediated by the entire lipid molecule. When headgroups bind, binding is stabilized by polar interactions between lipid headgroups and the protein. When acyl chains bind, van der Waals effects dominate as the acyl chains adopt conformations that complement particular sites on the rough protein surface. No generally applicable motifs for binding have yet emerged. Previously published biochemical and biophysical data link this binding with function. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Reversal of coenzyme specificity of 2,3‐butanediol dehydrogenase from Saccharomyces cerevisae and in vivo functional analysis

Saccharomyces cerevisiae NAD(H)‐dependent 2,3‐butanediol dehydrogenase (Bdh1), a medium chain dehydrogenase/reductase is the main enzyme catalyzing the reduction of acetoin to 2,3‐butanediol. In this work we focused on altering the coenzyme specificity of Bdh1 from NAD(H) to NADP(H). Based on homology studies and the crystal structure of the NADP(H)‐dependent yeast alcohol dehydrogenase Adh6, three adjacent residues (Glu²²¹, Ile²²², and Ala²²³) were predicted to be involved in the coenzyme specificity of Bdh1 and were altered by site‐directed mutagenesis. Coenzyme reversal of Bdh1 was obtained with double Glu221Ser/Ile222Arg and triple Glu221Ser/Ile222Arg/Ala223Ser mutants. The performance of the triple mutant for NADPH was close to that of native Bdh1 for NADH. The three engineered mutants were able to restore the growth of a phosphoglucose isomerase deficient strain (pgi), which cannot grow on glucose unless an alternative NADPH oxidizing system is provided, thus demonstrating their in vivo functionality. These mutants are interesting tools to reduce the excess of acetoin produced by engineered brewing or wine yeasts overproducing glycerol. In addition, they represent promising tools for the manipulation of the NADP(H) metabolism and for the development of a powerful catalyst in biotransformations requiring NADPH regeneration. Biotechnol. Bioeng. 2009; 104: 381–389 © 2009 Wiley Periodicals, Inc.     

Steady-state,Pre-steady-state,and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, k_off). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (k_off). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (<1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E>S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.     

Correlation between the compact regions predicted by the Average Distance Map (ADM) and the biologically active sites in atrial natriuretic peptide

The prediction of the short-range compact regions of human atrial natriuretic peptide (a-hANP), one of the biologically active peptides, has been made by means of the Average Distance Map(ADM). We found out that the location of the predicted short-range compact regions is consistent with the structural units determined by the NMR analysis (Kobayashiet al., 1988). Furthermore, the short-range compact regions correspond well to the biologically active areas of atriopeptin (103–125)-amide (which is homologous peptide toa-hANP), detected by the glycine substitution technique (Konishiet al., 1987). The results suggest that a predicted short-range compact region can be regarded as a possible active site in a biologically active peptide.     

Observation and quantitation of metal-binding sites in the sex steroid-binding protein of human and rabbit sera using the luminescent probe terbium

The first direct evidence for specific metal-binding sites in pure human and pure rabbit sex steroid-binding protein (SBP) is obtained using the luminescent lanthanide terbium. Terbium, a probe for calcium sites in proteins, provided protection of the SBP steroid-binding activity in diluted human serum samples equivalent to that provided by calcium. Pure SBP, first treated with ethylenediaminetetraacetate, was dialyzed against buffer containing TbCl₃. After gel filtration to remove nonspecifically bound terbium, the protein was denatured in urea. The amount of protein-bound terbium was determined by luminescence enhancement of the lanthanide using the chelator dipicolinate, yielding four metal-binding sites per mole of dimer protein from both species.     

Strategies for rapid assessment of fungal diversity

The problems of estimating fungal diversity are considerable, due especially to the large numbers of species even in small study sites, only a small proportion of which are adequately characterized, and the lack of trained staff and appropriate identification manuals. These difficulties are multiplied in the tropics. Strategies for providing preliminary assessments of fungal diversity which can be carried out within short time-frames are discussed, which will allow initial site valuations to be made for conservation purposes. The necessity of identifying all species discovered is considered, and the need emphasized for strictly defined sampling protocols which allow proper comparison between sites. The advantages and disadvantages of preliminary diversity analyses involving different taxonomic and ecological groups, fungus/plant associations, spatial sampling, soil and litter analysis, molecular methods and indirect assessment are considered.     

Modeling of substrate and inhibitor binding to phospholipase A2.

Molecular graphics and molecular mechanics techniques have been used to study the mode of ligand binding and mechanism of action of the enzyme phospholipase A2. A substrate-enzyme complex was constructed based on the crystal structure of the apoenzyme. The complex was minimized to relieve initial strain, and the structural and energetic features of the resultant complex analyzed in detail, at the molecular and residue level. The minimized complex was then used as a basis for examining the action of the enzyme on modified substrates, binding of inhibitors to the enzyme, and possible reaction intermediate complexes. The model is compatible with the suggested mechanism of hydrolysis and with experimental data about stereoselectivity, efficiency of hydrolysis of modified substrates, and inhibitor potency. In conclusion, the model can be used as a tool in evaluating new ligands as possible substrates and in the rational design of inhibitors, for the therapeutic treatment of diseases such as rheumatoid arthritis, atherosclerosis, and asthma.     

Landscape structure influences tree density patterns in fragmented woodlands in semi‐arid eastern Australia

Landscape and local‐scale influences are important drivers of plant community structure. However, their relative contribution and the degree to which they interact remain unclear. We quantified the extent to which landscape structure, within‐patch habitat and their confounding effects determine post‐clearing tree densities and composition in agricultural landscapes in eastern subtropical Australia. Landscape structure (incorporating habitat fragmentation and loss) and within‐patch (site) features were quantified for 60 remnant patches of Eucalyptus populnea (Myrtaceae) woodland. Tree density and species for three ecological maturity classes (regeneration, early maturity, late maturity) and local site features were assessed in one 100 × 10 m plot per patch. All but one landscape characteristic was determined within a 1.3‐km radius of plots; Euclidean nearest neighbour distance was measured inside a 5‐km radius. Variation in tree density and composition for each maturity class was partitioned into independent landscape, independent site and joint effects of landscape and site features using redundancy analysis. Independent site effects explained more variation in regeneration density and composition than pure landscape effects; significant predictors were the proportion of early and late maturity trees at a site, rainfall and the associated interaction. Conversely, landscape structure explained greater variation in early and late maturity tree density and composition than site predictors. Area of remnant native vegetation within a landscape and patch characteristics (area, shape, edge contrast) were significant predictors of early maturity tree density. However, 31% of the explained variation in early mature tree differences represented confounding influences of landscape and local variables. We suggest that within‐patch characteristics are important in influencing semi‐arid woodland tree regeneration. However, independent and confounding effects of landscape structure resulting from previous vegetation clearing may have exerted a greater historical influence on older cohorts and should be accounted for when examining woodland dynamics across a broader range of environments.