Effect of carboxylate-binding mode on metal binding/selectivity and function in proteins

We delineate the factors governing the carboxylate-binding mode (monodentate vs bidentate) in metalloproteins. We reveal how the carboxylate-binding mode affects the binding affinity and selectivity of a metal ion as well as the function of a metalloprotein using Ca2+-binding proteins and enzymes (ribonuclease H1, phosphoserine phosphatase, and ribonucleotide reductase) as examples. The collected data indicate that a carboxylate monodentate left arrow over right arrow bidentate switch, in addition to other structural factors, could be used to fine tune the metal-binding site affinity and/or selectivity, thus modifying the function/properties of the metalloprotein.     

Fatty acid binding proteins and mammary-derived growth inhibitor

The multigene superfamily of intracellular lipid binding proteins in mammals comprises up to now 13 different types of 14–15 kDa proteins, whose foremost high-affinity ligands are long-chain fatty acids, retinoids and bile acids, respectively. The phylogenetically related proteins of highly conserved tertiary structure are encoded by genes, that are canonically structured into four exons and three introns. In addition, these genes are characterized by promoter regions with responsive elements common to many genes encoding lipid-metabolizing enzymes, that interact with nuclear receptors activated by peroxisome proliferators or fatty acids. On the basis of interactions of fatty acids – actually macronutrients – with the ligand activated nuclear receptors and with the fatty acid binding proteins (FABPs) the regulation of FABP-expression and the functions of the various FABP-types in cellular lipid homeostasis, signal transduction and growth regulation will be reviewed.     

Multiplicity of VLLE equations: Case studies

Phase equilibrium calculation involving VLLE is often encountered in design, simulation and optimisation of many chemical processes. Practically, VLLE is numerically solved by a set of equations of chemical potential equilibrium and mass balances, which in theory may lead to multiple solutions due to the high nonlinearity of equations. Here, the multiplicity of VLLE is analysed by continuation method with pressure and composition as parameters. The detailed features of the output multiplicity of VLLE, e.g. the multiple liquid compositions and relative holdups, multiple liquid-liquid boundaries as well as the mixture composition region resulting in multiplicity, are presented. The influence of VLLE on process multiplicity is illustrated through the flash process. Moreover, the effect of the activity model is investigated, which shows that multiplicity of VLLE may change significantly and even disappear using different activity models. The multiplicity study of the VLLE problem is very helpful for evaluating the activity models and parameters and for providing proper initial values for simulation of system with VLLE multiplicity.     

The prediction and characterization of metal binding sites in proteins

The rational engineering of novel functions into proteins canonly be attempted when the underlying structural scaffold onwhich the new function is displayed and the structure of thetarget protein are both well understood. To introduce functionsmediated by metals it is therefore necessary to identify theprincipal liganding residues for the chosen metal, the requiredarchitecture of the metal-ligand complex and sites within thetarget protein that could accommodate such sites. Here we presenta method that applies structural information from the proteindata bank to the ab initio design and characterization of novelmetal binding sites. The prediction method has been tested on28 metalloprotein structures from the Brookhaven Protein DataBank. It successfully identified >90% of the metal bindingsites. In addition, we have used the method to design and characterizezinc binding sites in two antibody structures. Metal bindingstudies on one of these putative metalloantibodies showed metalbinding, confirming the predictive power of the method.     

Acyl-CoA:cholesterol acyltransferase activity in the rat mammary gland: Variation during pregnancy and lactation

Cholesterol esterification by acyl-CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) has been studied in microsomes isolated from the mammary glands of rats in late pregnancy, in early and mid-lactation, and following premature weaning. The mammary glands were freeze-clamped and the microsomes prepared in the presence of phosphatase inhibitors to preserve the phosphorylation status of the enzyme. Optimal conditions were established for the assay of enzyme activity in the presence of endogenous cholesterol. Supplementation of the microsomes with exogenous cholesterol as a dispersion in Triton WR-1339 was shown to lead to an increase in enzyme activity. Incubation of microsomes with MgATP led to an increase in ACAT activity which could be reversed by treatment of the microsomes with a phosphoprotein phosphatase preparation from rat liver. The results suggested that ACAT activity in the mammary gland was activated by phosphorylation in a similar way to that observed for the hepatic enzyme. The mammary glands from pregnant animals contained a higher level of ACAT activity than did the glands of the lactating animals and this correlated with the higher cholesteryl ester content of the pregnant glands. The highest level of ACAT activity was found in the weaned animals but the cholesteryl ester content of the microsomes was lower than expected. The influence of progesterone levels and changes in feeding patterns during gestation were considered as factors in these variations in ACAT activity.     

Expression of fatty acyl-CoA binding proteins in colon cells: response to butyrate and transformation

Fatty acyl-CoA affect many cellular functions as well as serving as cellular building blocks. Several families of cytosolic fatty acyl-CoA binding proteins may modulate the activities of fatty acyl-CoA. Intestinal enterocytes contain at least three unique families of cytosolic proteins that bind fatty acyl-CoA: acyl-CoA binding protein (ACBP), fatty acid binding proteins (including the liver, L-FABP and intestinal, I-FABP), and sterol carrier protein-2 (SCP-2). Immortalized rat colon epithelial cell lines expressed only ACBP and SCP-2 at levels of 0.75±0.13 and 0.42±0.02 ng/μg protein. Ras and src transformation increased colon cell density and differentially altered ACBP and SCP-2 expression without affecting I-FABP or L-FABP levels. ACBP levels were 1.8-fold and 1.5-fold increased in ras- and src-transformed cells, respectively. In contrast, SCP-2 expression was significantly decreased 55 and 67% in ras- and src-transformed cells, respectively. Butyrate treatment of ras- and src-transformed cells decreased cell proliferation up to 60–85% as compared to 25–30% in control cells. Butyrate treatment decreased ACBP expression in all cell lines but had no effect on the levels of SCP-2, I-FABP, or L-FABP. These studies suggest that the differential expression of ACBP and SCP-2 in rat colonic cell lines, as well as their modulation by butyrate, may be altered by cell transformation.     

Diatoms and Plants Acyl-CoA:lysophosphatidylcholine Acyltransferases (LPCATs) Exhibit Diverse Substrate Specificity and Biochemical Properties

The search of the Phaeodactylum tricornutum genome database revealed the existence of six genes potentially encoding lysophospholipid acyltransferases. One of these genes, Phatr3_J20460, after introduction to yeast ale1 mutant disrupted in the LPCAT gene, produced a very active acyl-CoA:lysophosphatidylcholine (LPCAT) enzyme. Using in vitro assays applying different radioactive and non-radioactive substrates and microsomal fractions from such yeast, we have characterized the biochemical properties and substrate specificities of this PtLPCAT1. We have found that the substrate specificity of this enzyme indicates that it can completely supply phosphatidylcholine (PC) with all fatty acids connected with a biosynthetic pathway of very long-chain polyunsaturated fatty acids (VLC-PUFAs) used further for the desaturation process. Additionally, we have shown that biochemical properties of the PtLPCAT1 in comparison to plant LPCATs are in some cases similar (such as the dependency of its activity on pH value), differ moderately (such as in response to temperature changes), or express completely different properties (such as in reaction to calcium and magnesium ions or toward some acyl-CoA with 20C polyunsaturated fatty acids). Moreover, the obtained results suggest that cloned “Phatr3_J20460” gene can be useful in oilseeds plant engineering toward efficient production of VLC-PUFA as LPCAT it encodes can (contrary to plant LPCATs) introduce 20:4-CoA (n-3) to PC for further desaturation to 20:5 (EPA, eicosapentaenoic acid).     

Protein function microarrays based on self-immobilizing and self-labeling fusion proteins

Protein microarrays are an attractive approach for the high-throughput analysis of protein function, but their impact on proteomics has been limited by the technical difficulties associated with their generation. Here we demonstrate that fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT) can be used for the simple and reliable generation of protein microarrays for the analysis of protein function. Important features of the approach are the selectivity of the covalent immobilization; this allows for direct immobilization of proteins out of cell extracts, and the option both to label and to immobilize AGT fusion proteins, which allows for direct screening for protein-protein interactions between different AGT fusion proteins. In addition to the identification of protein-protein interactions, AGT-based protein microarrays can be used for the characterization of small molecule-protein interactions or post-translational modifications. The potential of the approach was demonstrated by investigating the post-translational modification of acyl carrier protein (ACP) from E. coli by different phosphopantetheine transferases (PPTases), yielding insights into the role of selected ACP amino acids in the ACP-PPTase interaction.     

Selection of single domain binding proteins by covalent DNA display

Selection technologies such as phage and ribosome display, which provide a physical linkage between genetic information and encoded polypeptide, are important tools for the engineering of proteins for diagnostic and therapeutic applications. We have recently described a selection strategy called covalent DNA display, in which individual proteins are covalently linked to the cognate encoding DNA template in separate droplets of a water-in-oil emulsion. We here report on the optimization of several experimental steps in covalent DNA display technology, such as the elution conditions and the PCR strategy used for the amplification of selected DNA templates. A PCR assembly strategy was developed, which allows the amplification of the DNA templates over repeated rounds of selection. In addition, we could demonstrate that approximately 50% of the DNA templates form a covalent adduct with the corresponding proteins in the compartments of a water-in-oil emulsion. In model selection experiments, differences in recovery efficiency <100 000 per round of selection could be observed when comparing a specific binding polypeptide with a binder of irrelevant specificity. Furthermore, the optimized protocol was successfully applied for the selection of single domain proteins, capable of specific binding to mouse serum albumin (MSA). A mutant derived from the SH3 domain of the Fyn kinase, with millimolar affinity to MSA, was affinity matured using covalent DNA display and yielded several MSA binding FynSH3 variants with dissociation constants in the 100 nM range.     

Epidermal growth factor receptor downregulation by small heterodimeric binding proteins

No single engineered protein has been shown previously to robustly downregulate epidermal growth factor receptor (EGFR), a validated cancer target. A panel of fibronectin-based domains was engineered to bind with picomolar to nanomolar affinity to multiple epitopes of EGFR. Monovalent and homo- and hetero-bivalent dimers of these domains were tested for EGFR downregulation. Selected orientations of non-competitive heterodimers decrease EGFR levels by up to 80% in multiple cell types, without activating receptor signaling. These heterodimers inhibit autophosphorylation, proliferation and migration, and are synergistic with the monoclonal antibody cetuximab in these activities. These small (25 kDa) heterodimers represent a novel modality for modulating surface receptor levels.     

Reversible binding of heme to proteins in cellular signal transduction

Heme plays critical roles in numerous biological phenomena. Recent evidence has uncovered a new role of heme in cellular signal transduction, and its mechanism involves reversible binding of heme to proteins. This Account highlights the novel function of heme as an intracellular messenger in the regulation of gene expression and ion channel function.     

Multiplicity and stability of premixed laminar flames: an application of bifurcation theory

Numerical bifurcation techniques are used to describe multiple steady states for a premixed, laminar flame stabilized on a flat flame burner. The flame is assumed to be adiabatic, and the kinetic mechanism is approximated by a single reaction. The numerical methods make it possible to determine all steady states and eliminate computational difficulties near singular points. The possibility of defining solutions near singularities is particularly important in flame modeling for it is near such points that ignition and burn-out may occur. Three steady states are identified: a stable upper state corresponding to a flame burning at or near the adiabatic flame temperature, a lower solution representing an extinguished stable state, and an unstable intermediate state. Sensitivity of the solutions to changes in kinetic parameters is enhanced near burn-out. It is expected that the ability to predict flame behavior near such singular points will be particularly useful in the determination of flame kinetics.     

The function of tryptophan residues in membrane proteins

Membrane proteins have a significantly higher Trp content thando soluble proteins. This is especially true for the M and Lsubunits of the photosynthetic reaction center from purple bacteria.The Trp residues are not uniformly distributed through the membranebut are concentrated at the periplasmic side of the complex.In addition, Trp residues are not randomly aligned. Within theprotein subunits, many form hydrogen bonds with carbonyl oxygensof the main chain, thereby stabilizing the protein. On the surfaceof the molecule, they are correctly positioned to form hydrogenbonds with the lipid head groups while their hydrophobic ringsare immersed in the lipid part of the bilayer. These observationssuggest that Trp residues are involved in the translocationof protein through the membrane and that following translocation,Trp residues serve as anchors on the periplasmic side of themembrane. Received August 13, 1991; accepted February 11, 1992.     

Enantioselective proteins: selection, binding studies and molecular modeling of antibodies with affinity towards hydrophobic BINOL derivatives

In this paper, the initial steps towards the design of novel artificial metalloenzymes that exploit proteins as a second coordination sphere for traditional metal-ligand catalysis are described. Phage display was employed to select and study antibody fragments capable of recognizing hydrophobic BINOL derivatives designed to mimic BINAP, a widely used ligand in asymmetric metal-catalyzed reactions. The binding affinities of the selected antibodies towards a series of haptens were evaluated by using ELISA assays. A homology model of one of the most selective antibodies was constructed, and a computer-assisted ligand-docking study was carried out to elucidate the binding of the hapten. It was shown that, due to the hydrophobic nature of the haptens, a higher level of theoretical treatment was required to identify the correct binding modes. A small selection of the antibodies was found to discriminate between enantiomers and small structural modifications of the BINOL derivatives. The selectivities arise from hydrophobic interactions, and we propose that the identified set of antibodies provides a foundation for a novel route to artificial metalloenzymes.     

Nonspecific lipid transfer proteins as probes of membrane structure and function

A protein that accelerates transfer of phospholipids of varying head group and fatty acid composition has been purified from bovine liver. As previously found for other phospholipid transfer proteins, “nonspecific lipid transfer protein” stimulates a kinetically biphasic transfer of radioactively labeled phospholipid from small unilamellar vesicles to unlabeled multilamellar vesicles. The kinetics are consistent with rapid transfer of phospholipid from the outer monalyer and slow transfer of that localized in the inner monolayer (half-times greater than 3 days for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol). Protein catalyzed transfer is inhibited by high ionic strength and has an activation energy of 35 kJ/mol. The broad lipid specificity and ease of large-scale purification make these proteins candidates for membrane phospholipid compositional modification. The compositions of rat liver mitochondrial and microsomal membranes and Morris hepatoma 7288c mitochondrial membranes were altered by incubation with lipid vesicles and nonspecific lipid transfer protein. Incubation with phosphatidylcholine vesicles led to increased levels of phosphatidylcholine and decreased levels of other transferrable lipids (phosphatidylethanolamine, phosphatidylinositol, and cholesterol) unless the latter were included in the vesicles. When vesicles containing dipalmitoylphosphatidylcholine were incubated with microsomal membranes, a large increase in disaturated phosphatidylcholine was also observed. These changes in composition were correlated with activities of membrane enzymes. It appears that microsomal glucose-6-phosphatase is inhibited by increased phosphatidylcholine saturation. Moreover, this enzyme is also inhibited by decreases in the phosphatidylethanolamine/phosphatidylcholine ratio whereas NADPH cytochrome c reductase is not. Likewise, decreased cholesterol to phospholipid ratios did not greatly affect the abnormally low levels of hepatoma succinate cytochrome c reductase activity. This paper was presented at the 73rd AOCS annual meeting, Toronto, Canada, May 1982.