Localization of functional polypeptides in bacterial inclusion bodies.
(25/86)
Bacterial inclusion bodies, while showing intriguing amyloid-like features, such as a beta-sheet-based intermolecular organization, binding to amyloid-tropic dyes, and origin in a sequence-selective deposition process, hold an important amount of native-like secondary structure and significant amounts of functional polypeptides. The aggregation mechanics supporting the occurrence of both misfolded and properly folded protein is controversial. Single polypeptide chains might contain both misfolded stretches driving aggregation and properly folded protein domains that, if embracing the active site, would account for the biological activities displayed by inclusion bodies. Alternatively, soluble, functional polypeptides could be surface adsorbed by interactions weaker than those driving the formation of the intermolecular beta-sheet architecture. To explore whether the fraction of properly folded active protein is a natural component or rather a mere contaminant of these aggregates, we have explored their localization by image analysis of inclusion bodies formed by green fluorescent protein. Since the fluorescence distribution is not homogeneous and the core of inclusion bodies is particularly rich in active protein forms, such protein species cannot be passively trapped components and their occurrence might be linked to the reconstruction dynamics steadily endured in vivo by such bacterial aggregates. Intriguingly, even functional protein species in inclusion bodies are not excluded from the interface with the solvent, probably because of the porous structure of these particular protein aggregates. (+info)
19F-NMR detection of lacZ gene expression via the enzymic hydrolysis of 2-fluoro-4-nitrophenyl beta-D-galactopyranoside in vivo in PC3 prostate tumor xenografts in the mouse.
(26/86)
Gene therapy shows promise for treating prostate cancer and has been evaluated in several clinical trials. A major challenge that remains is to establish a method for verifying transgene activity in situ. The lacZ gene encoding beta-galactosidase historically has been the most popular reporter gene for molecular biology. We have designed a 19F NMR approach to reveal lacZ gene expression by assessing beta-galactosidase (beta-gal) activity in vivo. The substrate 2-fluoro-4-nitrophenyl beta-D-galactopyranoside (OFPNPG) is readily hydrolyzed by beta-gal with a corresponding decrease in the 19F-NMR signal from OFPNPG and the appearance of a new signal shifted 4-6 ppm upfield from the aglycone 2-fluoro-4-nitrophenol (OFPNP). We report proof of principle in cultures of PC3 prostate cancer cells using 19F NMR spectroscopy and 19F chemical shift imaging. More importantly, we demonstrate for the first time the ability to differentiate wild-type and lacZ-expressing prostate tumor xenografts in mice using this approach. (+info)
Structural analysis of lac repressor bound to allosteric effectors.
(27/86)
The lac operon is a model system for understanding how effector molecules regulate transcription and are necessary for allosteric transitions. The crystal structures of the lac repressor bound to inducer and anti-inducer molecules provide a model for how these small molecules can modulate repressor function. The structures of the apo repressor and the repressor bound to effector molecules are compared in atomic detail. All effectors examined here bind to the repressor in the same location and are anchored to the repressor through hydrogen bonds to several hydroxyl groups of the sugar ring. Inducer molecules form a more extensive hydrogen-bonding network compared to anti-inducers and neutral effector molecules. The structures of these effector molecules suggest that the O6 hydroxyl on the galactoside is essential for establishing a water-mediated hydrogen bonding network that bridges the N-terminal and C-terminal sub-domains. The altered hydrogen bonding can account in part for the different structural conformations of the repressor, and is vital for the allosteric transition. (+info)
The cost of expression of Escherichia coli lac operon proteins is in the process, not in the products.
(28/86)
Differential expression of ccrA in methicillin-resistant Staphylococcus aureus strains carrying staphylococcal cassette chromosome mec type II and IVa elements.
(31/86)
Determination of the roles of Glu-461 in beta-galactosidase (Escherichia coli) using site-specific mutagenesis.
(32/86)
Site-directed substitutions (Asp, Gly, Gln, His, and Lys) were made for Glu-461 of beta-galactosidase (Escherichia coli). All substitutions resulted in loss of most activity. Substrates and a substrate analog inhibitor were bound better by the Asp-substituted enzyme than by the normal enzyme, about the same for enzyme substituted with Gly, but only poorly when Gln, His, or Lys was substituted. This shows that Glu-461 is involved in substrate binding. Binding of the positively charged transition state analog 2-aminogalactose was very much reduced with Gly, Gln, His, and Lys, whereas the Asp-substituted enzyme bound this inhibitor even better than did the wild-type enzyme. Since Asp, like Glu, is negatively charged, this strongly supports the proposal that one role of Glu-461 is to electrostatically interact with a positively charged galactosyl transition state intermediate. The substitutions also affected the ability of the enzyme to bind L-ribose, a planar analog of D-galactose that strongly inhibits beta-galactosidase activity. This indicates that the binding of a planar "galactose-like" compound is somehow mediated through Glu-461. The data indicated that the presence of Glu-461 is highly important for the acid catalytic component of kappa 2 (glycosylic bond cleavage or "galactosylation"), and therefore Glu-461 must be involved in a concerted acid catalytic reaction, presumably by stabilizing a developing carbonium ion. The kappa 2 values with o- and p-nitrophenyl-beta-D-galactopyranoside as substrates varied more or less as did the K8 values, indicating that most of the glycolytic bond breaking activity found for the enzymes from the mutants with these substrates was probably a result of strain or other such effects. The kappa 3 values (hydrolysis or "degalactosylation") of the substituted enzymes were also low, indicating that Glu-461 is important for that part of the catalysis. The enzyme with His substituted for Glu-461 had the highest kappa 3 value. This is probably a result of the formation of a covalent bond between His and the galactosyl part of the substrate. (+info)