Subunits In Bacteria Research Articles

19] Assays for studying functional properties of in vitro translated Gsα subunit

This chapter discusses different procedure designed to study the functional properties of mutated in vitro translated G s α subunits. The relationship between the structure and function of a protein has been widely explored by in vitro mutagenesis of the cDNA encoding the protein and expression of the mutated protein in an eukaryotic cell. If the synthesis of the endogenous protein can be abolished, this approach becomes more fruitful because the functional properties of the mutated protein can be directly assayed on the transfected cell. Isolation of the cyc- variant of the mouse lymphoma S49 cell line, which does not synthesize the α subunit of the GTP-binding protein G s has provided this ideal situation for studying the functional domains of Gs α subunits. An alternative approach, based on the synthesis of large amounts of α subunit in bacteria and its use for reconstitution of cyc- membranes, is more attractive, but it faces the problem of posttranslational modifications which are lacking in the bacterially expressed proteins.

FT-IR studies on the triplet state of P680 in the photosystem II reaction center: triplet equilibrium within a chlorophyll dimer.

The structure and molecular interactions of the primary donor (P680) in the reaction center (D1-D2-cytochrome b-559 complex) of photosystem II (PS II) have been investigated by detecting light-induced FT-IR difference spectra upon the formation of its triplet state (3P680). The 3P680/P680 spectrum obtained was analyzed by comparing it with difference spectra between the ground and lowest triplet states of purified chlorophyll a (Chl) in organic solvents. The negative peaks at 1669 and 1707 cm-1 accompanied by the positive peaks at 1627 and 1659 cm-1 in the 3P680/P680 spectrum were assigned to the keto C = O stretching mode, and the appearance of these two pairs of bands indicated that P680 has a dimeric structure analogous to that of the bacterial primary donor. From the band positions of the keto and carbomethoxy C = O stretches, the hydrogen-bonding properties of these two Chl molecules were found to be asymmetrical; in one Chl molecule both the keto and carbomethoxy C = O groups form hydrogen bonds, while in the other Chl molecule the keto C = O is not hydrogen-bonded whereas the carbomethoxy C = O probably is hydrogen-bonded. The temperature dependence of the intensity ratios of the keto C = O bands revealed that the triplet state is equilibrated between the two Chl molecules with an energy gap of 8.4 +/- 0.7 meV. Most of the triplet population was found to be localized on one Chl molecule (86% at 80 K), in which both of the two C = O groups are hydrogen-bonded, that is probably attached to the D1 subunit. Considering the structure of the bacterial reaction center determined by X-ray crystallography and the sequence homology between the D1 and D2 subunits of PS II and the L and M subunits of bacteria, a model of the P680 structure and its interactions with apoproteins has been proposed.

Expression of mouse rod photoreceptor cGMP phosphodiesterase γ subunit in bacteria

We expressed the γ subunit of mouse rod photoreceptor cGMP phosphodiesterase (PDE) in the bacterial pGEX-2TK expression vector which produces a cleavable 40 kDa fusion protein. The fusion protein can be isolated in a one step procedure by affinity chromatography on glutathione beads. The yield of purified fusion protein is approximately 10 mg from 1 liter of bacterial culture, or about 3 mg of PDEγ, equivalent to the PDEγ content of approximately 200,000 mouse retinas. Both the fusion protein and the cleaved PDEγ, to which a short kinase domain remains attached, are biologically active, inhibiting activated PDE in a manner comparable to native PDEγ. Immobilized PDEγ binds transducin α subunit charged with GTP, PDE α and β subunits, and, unexpectedly, arrestin (S-antigen).

31] Expression and mutagenesis of the regulatory subunit of cAMP-dependent protein kinase in Escherichia coli

Publisher Summary The advancement of technology has provided new dimensions for asking questions about protein structure and function, about the role that any given protein plays, and about the regulation of its expression. Questions of expression now can be addressed directly. Of fundamental importance will be how the expression of R and C subunits is coordinated. With at least one R subunit gene cloned, it is possible to selectively mutate various regions of the regulatory subunit to better understand those specific structural features that convey functional properties to the protein. Following isolation of the cloned gene, the next step in this process is the construction of an expression vector. In addition to being a step on the pathway toward mutagenesis, the expression of R subunit in bacteria also can provide a mechanism for rapidly producing large amounts of protein. This chapter describes the construction of such expression vector. Because the R subunit is not thought to be subject to major posttranslational modification, such as glycosylation, the host of choice is bacteria, and a wide variety of vectors are available for designing constructs that will lead to the expression of a given protein.

Depression of colony formation by human thymus-derived lymphocytes with rifampin and other antimicrobial agents.

Colonies of human lymphocytes with thymus-derived (T) cell characteristics can be induced in phytohemagglutinin-P to grow in a semisolid medium. To expand the data base on the effects of antimicrobial agents on cell-mediated immunity, the effect of 30 antimicrobial agents on T-lymphocyte cloning was studied. All of the drugs were added to the cultures in concentrations ranging from 10(-5) to 10(-14) M, and the results were compared with those in cultures without the drug. Drugs that inhibit protein synthesis at the 50S ribosomal subunit in bacteria--in particular, chloramphenicol, clindamycin, erythromycin, and troleandomycin--suppressed colony formation. However, the most significantly immunosuppressive agent was rifampin; it suppressed colony formation at concentrations of up to 2.5 x 10(-9) M, a value significantly lower than that found in previous in vitro testing and well below therapeutic levels. Screening of drugs by lymphocyte cloning techniques for possible suppression of cell-mediated immunity appears to be a rapid, sensitive, and inexpensive procedure.