Role of methyl groups in dynamics and evolution of biomolecules

Recent studies have discovered strong differences between the dynamics of nucleic acids (RNA and DNA) and proteins, especially at low hydration and low temperatures. This difference is caused primarily by dynamics of methyl groups that are abundant in proteins, but are absent or very rare in RNA and DNA. In this paper, we present a hypothesis regarding the role of methyl groups as intrinsic plasticizers in proteins and their evolutionary selection to facilitate protein dynamics and activity. We demonstrate the profound effect methyl groups have on protein dynamics relative to nucleic acid dynamics, and note the apparent correlation of methyl group content in protein classes and their need for molecular flexibility. Moreover, we note the fastest methyl groups of some enzymes appear around dynamical centers such as hinges or active sites. Methyl groups are also of tremendous importance from a hydrophobicity/folding/entropy perspective. These significant roles, however, complement our hypothesis rather than preclude the recognition of methyl groups in the dynamics and evolution of biomolecules.     

Phosphatidylinositol 4-kinase from Saccharomyces cerevisiae. Kinetic analysis using Triton X-100/phosphatidylinositol-mixed micelles.

Phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified from Saccharomyces cerevisiae by an improved procedure over that previously reported (Belunis, C.J., Bae-Lee, M., Kelley, M.J., and Carman, G.M. (1988) J. Biol. Chem. 263, 18897-18903) for the enzyme. The molecular mass of the enzyme was 45 kDa. The 35-kDa protein previously identified as PI 4-kinase was a proteolysis product of the 45-kDa protein. A detailed kinetic analysis of the purified enzyme was performed with Triton X-100/phosphatidylinositol-mixed micelles according to the "surface dilution" (Deems, R.A., Eaton, B.R., and Dennis, E.A. (1975) J. Biol. Chem. 250, 9013-9020) and "dual phospholipid" (Hendrickson, H.S., and Dennis, E.A. (1984) J. Biol. Chem. 259, 5734-5739) kinetic models. Phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol at concentrations of phosphatidylinositol below 0.1 mM. Above 0.1 mM activity was only dependent on the surface concentration of phosphatidylinositol. The enzyme more closely followed the dual phospholipid model where the enzyme associated with Triton X-100 micelles when phosphatidylinositol was present. The interfacial Michaelis constant (KmB) for phosphatidylinositol was 0.0036 mol fraction and the dissociation constant (KsA) for phosphatidylinositol in the micelle surface was 0.26 mM. The results of glycerol gradient centrifugation studies showed that the enzyme was physically associated with Triton X-100/phosphatidylinositol micelles.     

Cofactor requirements and reconstitution of microcin B17 synthetase: a multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic microcin B17

In the maturation of the Escherichia coli antibiotic Microcin B17 (MccB17), the McbA prepro-antibiotic is modified post-translationally by the multimeric microcin synthetase complex (composed of the McbB, -C, and -D proteins), which cyclizes four cysteines and four serines to thiazoles and oxazoles, respectively. Herein, we report the purification of individual subunits of MccB17 synthetase as fusions to maltose binding protein (MBP), and the in vitro reconstitution of heterocyclization activity. Preliminary characterization of each subunit reveals McbB to be a zinc-containing protein that may catalyze the initial cyclodehydration step, and McbC to contain flavin, consistent with an anticipated role for a dehydrogenase. We have previously demonstrated that McbD is a regulated ATPase/GTPase that may function as a conformational switch. Photolabeling experiments with the McbA propeptide now identify McbD as the initial site of substrate recognition. Heterocyclization activity was reconstituted only by combining all three subunits, demonstrating that each protein is required for heterocycle formation. Titration assays indicate that the subunits bind to each other with at least micromolar affinities, although McbD affords activity only after the MBP tag is proteolytically removed. Subunit competition assays with an McbDD147A mutant, which yields a catalytically deficient synthetase in vivo, show it to be defective in complex formation, whereas the McbBC181A/C184A double mutant, which is also inactive, competitively inhibits reconstitution by native McbB. Addition of the HtpG chaperone (originally shown to copurify with MccB17 synthetase), does not stimulate synthetase reconstitution or heterocyclization activity in vitro. A model for synthetase activity is proposed.     

Atomic force microscopy and light scattering of small unilamellar actin-containing liposomes

Three-dimensional networks of filamentous actin (F-actin) encapsulated inside phosphatidylcholine liposomes are currently being used in an effort to model the cytoskeleton and plasma membrane of eukaryotic cells. In this article, unilamellar lipid vesicles consisting of egg yolk-derived phosphatidylcholine encapsulating monomeric actin (G-actin) were made via extrusion in low ionic strength buffer (G-buffer). Vesicle shape and structure in these dispersions was studied using a combination of fluid-tapping atomic force microscopy, and multiangle static light scattering. After subjecting the liposome dispersion to high ionic strength polymerization buffer (F-buffer) containing K(+) ions, atomic force microscopy imaging and light scattering of these liposomes indicated the formation of specialized structures, including an overall liposome structure transformation from spherical to torus, disk-shaped geometries and tubular assemblies. Several atomic force microscopy control measurements were made to ascertain that the specialized structures formed were not due to free G-actin and F-actin self-assembling on the sample surface, plain liposomes exposed to G- and F-buffer, or liposomes encapsulating G-actin. Liposomes encapsulating G-actin assumed mostly thin disk shapes and some large irregularly shaped aggregates. In contrast, liposomes encapsulating polymerized actin assumed mostly torus or disk shapes along with some high aspect ratio tubular structures.     

Validation of signature polarlipid fatty acid biomarkers for alkane-utilizing bacteria in soils and subsurface aquifer materials

Abstract Extractable cell membrane-derived polarlipid ester-linked fatty acids (PLFA) obtained from aerated soils gassed with methane or propane and from methane- and propane-oxidizing bacteria isolated from the soils were analyzed by capillary gas chromatography/mass spectrometry. Exposure of aerated soils to methane resulted in the formation of a high proportion of an unusual 18-carbon mono-unsaturated PLFA, 18:lw8c. High proportions of this fatty acid biomarker are found in monocultures from this soil grown in minimal media with methane. This PLFA has been previously established as associated with authentic type II methane-oxidizing bacteria. The microbiota in aerated soils exposed to hydrocarbons containing propane, formed a suite of PLFA characterized by high proportions of a 16-carbon mono-unsaturated acid, 16:lw6c, and an 18-carbon saturated fatty acid with an additional methyl branch at the 10 position, 10 Me 18:0. This PLFA pattern has been detected in several monocultures enriched from the soil with propane-amended minimal media. The correspondence of high proportions of these unusual mono-unsaturated PLFA in the isolated monocultures and in situ in the soils after stimulation with the appropriate hydrocarbon is a strong validation of the utility of these biomarkers in defining the community structure of the surface soil microbial community.