Location of the receptor-interaction site on CheB, the methylesterase response regulator of bacterial chemotaxis.

Sensory adaptation in bacterial chemotaxis is mediated by covalent modification of chemoreceptors, specifically methylation and demethylation of glutamates catalyzed by methyltransferase CheR and methylesterase CheB. The methylesterase is a two-domain response regulator in which phosphorylation of the regulatory domain enhances activity of the catalytic domain. In Escherichia coli and Salmonella typhimurium, a crucial determinant of efficient methylation and demethylation is a specific pentapeptide sequence at the chemoreceptor carboxyl terminus, a position distant from sites of enzymatic action. Each enzyme binds pentapeptide, but the site of binding has been located only for CheR. Here we locate the pentapeptide-binding site on CheB by assessing catalytic activity and pentapeptide binding of CheB fragments, protection of CheB from proteolysis by pentapeptide, and interference with pentapeptide-CheB interaction by a CheB segment. The results place the binding site near the hinge between regulatory and catalytic domains, in a segment spanning the carboxyl-terminal end of the regulatory domain and the beginning of the linker that stretches to the catalytic domain. This location is quite different from the catalytic domain location of the pentapeptide-binding site on CheR and is likely to reflect the rather different ways in which pentapeptide binding enhances enzymatic action for the methyltransferase and the methylesterase.

Efficient adaptational demethylation of chemoreceptors requires the same enzyme-docking site as efficient methylation.

The mechanistic basis of sensory adaptation and gradient sensing in bacterial chemotaxis is reversible covalent modification of transmembrane chemoreceptors, methylation, and demethylation at specific glutamyl residues in their cytoplasmic domains. These reactions are catalyzed by a dedicated methyltransferase CheR and a dedicated methylesterase CheB. The esterase is also a deamidase that creates certain methyl-accepting glutamyls by hydrolysis of glutamine side chains. We investigated the action of CheB and its activated form, phospho-CheB, on a truncated form of the aspartate receptor of Escherichia coli that was missing the last 5 aa of the intact receptor. The deleted pentapeptide is conserved in several chemoreceptors in enteric and related bacteria. The truncated receptor was much less efficiently demethylated and deamidated than intact receptor, but essentially was unperturbed for kinase activation or transmembrane signaling. CheB bound specifically to an affinity column carrying the isolated pentapeptide, implying that in the intact receptor the pentapeptide serves as a docking site for the methylesterase/deamidase and that the truncated receptor was inefficiently modified because the enzyme could not dock. It is striking that the same pentapeptide serves as an activity-enhancing docking site for the methyltransferase CheR, the other enzyme involved in adaptational covalent modification of chemoreceptors. A shared docking site raises the tantalizing possibility that relative rates of methylation and demethylation could be influenced by competition between the two enzymes at that site.

Comparison in vitro of a high- and a low-abundance chemoreceptor of Escherichia coli: similar kinase activation but different methyl-accepting activities.

In Escherichia coli, high-abundance chemoreceptors are present in cellular amounts approximately 10-fold greater than low-abundance chemoreceptors. Cells containing only low-abundance receptors exhibit abnormally low tumble frequencies and do not migrate effectively in spatial gradients. These defects reflect an inherent activity difference between the two receptor classes. We used in vitro assays to investigate this difference. The low-abundance receptor Trg mediated an approximately 100-fold activation of the kinase CheA, only twofold less than activation by the high-abundance receptor Tar. In contrast, Trg was less than 1/20 as active as Tar for in vitro methylation. As observed for high-abundance receptors, kinase activation by Trg varied with the extend of modification at methyl-accepting sites; low methylation corresponded to low kinase activation. Thus, in Trg-only cells, low receptor methylation would result in low kinase activation, correspondingly low content of phospho-CheY, and a decreased dynamic range over which attractant binding could modulate kinase activity. These features could account for the low tumble frequency and inefficient taxis exhibited by Trg-only cells. Thus, the crucial functional difference between the receptor classes is likely to be methyl-accepting activity. We investigated the structural basis for this functional difference by introducing onto the carboxy terminus of Trg a CheR-binding pentapeptide, usually found only at the carboxy termini of high-abundance receptors. This addition enhanced the in vitro methyl-accepting activity of Trg 10-fold.

Sequential treatment by phosphotungstic acid and uranyl acetate enhances the adherence of lipid membranes and membrane proteins to hydrophobic EM grids.

A simple procedure for screening by electron microscopic observations of conditions for the reconstitution of membrane proteins into lipid bilayers is described. This procedure consists of a 5-10 s treatment of electron microscopic grids, to which the sample has already been applied, with 1% phosphotungstic acid before proceeding with final staining in uranyl acetate. The method substantially enhances the adherence of lipid membranes and membrane protein particles to hydrophobic collodion/carbon grids.

Studies of the structural organization of a bacterial chemoreceptor by electron microscopy.

We used analysis by electron microscopy to obtain structural information about a transmembrane receptor that mediates chemotaxis in Escherichia coli. Two-dimensional arrays of regularly packed particles of the receptor Trg were obtained by reconstitution of purified, detergent-solubilized protein into lipid bilayers. Preliminary image processing of negatively stained arrays revealed an almost square 8.8 x 8.8-nm unit cell and resolved the particles into four peaks of density around a central depression. In certain conditions, reconstituted, Trg-containing bilayers associated into membrane stacks. The regular spacing of the stacks provided a value of 15 nm for the dimension of the receptor normal to the membrane. Using these dimensions, the estimated occupied volume of the structure would be sufficient to contain four monomers of Trg. This tetramer form may be a dimer of two antiparallel or parallel homodimers. Our analysis indicates that a receptor monomer is approximately 4.4 nm at the widest point and 15 nm long. Given the dimensions of the periplasmic domain of the closely related receptor Tars, determined by X-ray crystallography, and a minimum bilayer thickness of 3 nm, the cytoplasmic domain would be approximately 5.0 by 4.4 nm. Higher resolution analysis should reveal additional information about receptor structure.

[Electron microscopic study of ATP synthetase from bovine heart mitochondria]. / Elektronno-mikroskopicheskoe issledovanie ATP-sintetazy iz mitokhondrii serdtsa byka.

Two-dimensional crystals of the mitochondrial ATP synthase up to 0.4 microns in size were obtained from the detergent-lipid-protein micelles by detergent dialysis. A projected map of the negatively stained crystal was calculated from electron microscopical images by the Fourier-filtering procedure at ca. 2.8 nm resolution. The unit cell (with not more than two ATP synthase molecules) has the following parameters: a 13.0 nm, b 25.6 nm and gamma 86 degrees. In line with this conclusion, two alternative models for the crystal structural organization are plausible, viz., with one or two protein molecules per unit cell. The first model suggests an asymmetric incorporation of ATP synthase molecules into the lipid bilayer: extramembranous portions F1 are located on one side of the crystal membrane plane. According to the second model, the incorporation occurs on each side of the lipid bilayer, the unit cell containing the two oppositely oriented protein molecules. Based on the absence of another type of the projected crystal images (a rear view of the membrane), unique to the first model, preference is given to the second model.

Electron microscopy of two-dimensional crystals of mitochondrial ATP synthase.

Two-dimensional crystals of the mitochondrial ATP synthase up to 0.4 microns in size were obtained from the detergent-lipid-protein micelles by detergent dialysis. A projected map of the negatively stained crystal was calculated from electron microscopical images by the Fourier-filtering procedure at about 2.8 nm resolution. The unit cell (with not more than two ATP synthase molecules) has the following parameters: a = 13.0 nm, b = 25.6 nm and gamma = 86 degrees. Two alternative models for the crystal structural organization were suggested, viz. with one or two protein molecules per unit cell.

Two-dimensional crystallization of reaction centers from Chloroflexus aurantiacus.

Two-dimensional crystals of photosynthetic reaction centers from Chloroflexus aurantiacus were obtained from protein-lipid-detergent micelles by detergent dialysis. The size of crystals was up to 2 microns. Some of them were multilayered crystals. However, other crystal forms were also observed. Preliminary image processing analysis showed that crystals of one crystal form referred to two-sided plane group p2 and had the following unit cell parameters: a = 17.6 nm, b = 18.0 nm, gamma = 84 degrees. The contour map of the crystal stain-excluding region was calculated by the Fourier-filtering procedure at about 2 nm resolution.

Three-dimensional structure of (Na+ + K+)-ATPase revealed by electron microscopy of two-dimensional crystals.

Prolonged incubation of membrane fragments containing homogeneous (Na+ + K+)-ATPase with Mg2+, K+ and VO-3 at 4 degrees C resulted in formation of two-dimensional crystals of this enzyme with unit cell parameters: a = 66 A, b = 118 A, gamma = 108 degrees. The crystals correspond to the two-sided plane group p21. By combining tilted electron microscopic views of the crystals, a three-dimensional structure of (Na+ + K+)- ATPase was calculated at approximately 20 A resolution. The unit cell is formed by two (alpha beta)-promoters which are in contact in their central parts. The structure was compared with chemical modification and immunochemical data; the arrangement of intra- and extramembrane domains was proposed.

[Device for the high-speed cryofixation of biological matter]. / Ustroistvo dlia vysokoskorostnoi kriofiksatsii biologicheskikh ob"ektov.

A device of simple construction is proposed for rapid cryofixation of biological objects for freeze--fracture studies. The rapid freezing of the object is achieved by it immersion into the fast coolant stream, which is created in a centrifugal rotor of special construction. The freezing rate of the test-object in liquid freon-22 stream is 10 times greater compared with the ordinary cryofixation procedure using the same coolant.