CiteULike: jwm Benson

Converting a maltose receptor into a nascent binuclear copper oxygenase by computational design.

DE Benson — 2008-04-27T14:26:10-00:00

Biochemistry, Vol. 41, No. 9. (5 March 2002), pp. 3262-3269.

Computational protein design methods were used to identify mutations that are predicted to introduce a binuclear copper center coordinated by six histidines, replacing the maltose-binding site in Escherichia coli maltose-binding protein (MBP) with an oxygen-binding site. A small family of five candidate designs consisting of 9 to 10 mutations each was constructed by oligonucleotide-directed mutagenesis. These mutant proteins were expressed and purified, and their stability, copper- and cobalt-binding properties, and interactions of the resulting metalloprotein complexes with azide, hydrogen peroxide, and dioxygen were characterized. We identified one 10-fold mutant, MBP.Hc.E, that can form Cu(II)(2) and Co(II)(2) complexes that interact with H(2)O(2) and O(2). The Co(II)(2) protein reacts with H(2)O(2) to form a complex that is spectroscopically similar to a synthetic model that structurally mimics the oxy-hemocyanin core, whereas the Cu(II)(2) protein reacted with O(2) or H(2)O(2) does not. We postulate that the equilibrium between the open and closed conformations of MBP allows species with variable Cu-Cu distances to form, and that such species can bind ligands in geometries that are not observed in natural type III centers. Introduction of one additional mutation in the hinge region of MBP, I329F, known to favor formation of the closed state, results in a binuclear copper center that when reacted with low concentrations of H(2)O(2) mimics the spectroscopic signature of oxy-hemocyanin.

Rational design of nascent metalloenzymes.

DE Benson — 2008-04-27T14:25:38-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 97, No. 12. (6 June 2000), pp. 6292-6297.

Understanding the early genesis of new enzymatic functions is one of the challenges in protein design, mechanistic enzymology, and molecular evolution. We have experimentally mimicked starting points in this process by introducing primitive iron and oxygen binding sites at various locations in thioredoxin, a small protein lacking metal centers, by using computational design. These rudimentary active sites show emerging enzymatic activities that select to varying degrees between different oxygen chemistries. Even within these nascent enzymes, mechanisms by which different reactions are controlled can be discerned. These involve both stabilizing and destabilizing interactions imposed on the metal center by the surrounding protein matrix.

Regulation of sigma B levels and activity in Bacillus subtilis.

AK Benson — 2005-11-03T20:52:34-00:00

J Bacteriol, Vol. 175, No. 8. (April 1993), pp. 2347-2356.

The sigB operon of Bacillus subtilis encodes sigma B plus three additional proteins (RsbV, RsbW, and RsbX) that regulate sigma B activity. Using an anti-sigma B monoclonal antibody to monitor the levels of sigma B protein, PSPAC to control the expression of the sigB operon, and a ctc-lacZ reporter system to monitor sigma B activity, we observed that the rsbV and rsbW products control sigma B activity at the ctc promoter independently of their effects on sigma B levels. In contrast, RsbX was found to have no effect on expression of ctc when the sigB operon was controlled by PSPAC. The data are consistent with RsbV and RsbW being regulators of sigma B activity and RsbX acting primarily as a negative regulator of sigB operon expression. Evidence that stationary-phase induction of the sigma B-dependent ctc promoter is accomplished by a reduction in RsbW-dependent inhibition of sigma B activity is also presented. In addition, Western blot (immunoblot) analyses of sigB operon expression demonstrated that sigma B accumulation is coupled to the synthesis of its primary inhibitor (RsbW). This finding is consistent with RsbW and sigma B being present within the cell in equivalent amounts, a circumstance that would permit RsbW to directly influence sigma B activity by a direct protein-protein interaction.

Light-harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions.

J Kargul — 2005-10-03T11:21:48-00:00

FEBS J, Vol. 272, No. 18. (September 2005), pp. 4797-4806.

The State 1 to State 2 transition in the photosynthetic membranes of plants and green algae involves the functional coupling of phosphorylated light-harvesting complexes of photosystem II (LHCII) to photosystem I (PSI). We present evidence suggesting that in Chlamydomonas reinhardtii this coupling may be aided by a hyper-phosphorylated form of the LHCII-like CP29 protein (Lhcbm4). MS analysis of CP29 showed that Thr6, Thr16 and Thr32, and Ser102 are phosphorylated in State 2, whereas in State 1-exposed cells only phosphorylation of Thr6 and Thr32 could be detected. The LHCI-PSI supercomplex isolated from the alga in State 2 was found to contain strongly associated CP29 in phosphorylated form. Electron microscopy suggests that the binding site for this highly phosphorylated CP29 is close to the PsaH protein. It is therefore postulated that redox-dependent multiple phosphorylation of CP29 in green algae is an integral part of the State transition process in which the structural changes of CP29, induced by reversible phosphorylation, determine the affinity of LHCII for either of the two photosystems.

The sigma B-dependent promoter of the Bacillus subtilis sigB operon is induced by heat shock.

AK Benson — 2005-06-29T12:15:35-00:00

J Bacteriol., Vol. 175, No. 7. (1993), pp. 1929-1935.

Bacillus subtilis $σ$B is Regulated by a Binding Protein (RsbW) that Blocks its Association with Core RNA Polymerase

AK Benson — 2005-06-29T12:15:35-00:00

Proc. Natl. Acad. Sci. USA, Vol. 90, No. 6. (1993), pp. 2330-2334.