Chloroplast-encoded small subunits of the three multiprotein complexes in photosynthetic electron transport

The photosystem I, photosystem II, and cytochromeb ₆ f complexes that are involved in electron transport of oxygenic photosynthesis consist of a number of subunits encoded by either the chloroplast or nuclear genomes. In addition to the major subunits that carry redox components or photosynthetic pigments, these complexes contain several to more than ten subunits with molecular masses of less than 10 kDa. Directed mutagenesis has served as a powerful tool for investigation of the roles of these small subunits in the organization or function of the complexes. Various chloroplast transformants of the green algaChlamydomonas reinhardtii and mutants of cyanobacteria in which a gene encoding a small subunit was deleted or altered have been constructed. Evidence has accumulated suggesting that these small subunits function in the assembly, stabilization, or protection from photoinhibition of the complexes or in the modulation or regulation of electron transport. This article presents an overview of the properties and functions of the chloroplast-encoded small subunits of the three multiprotein complexes of photosynthetic electron transport that have been mainly analyzed with chloroplast transformants ofC. reinhardtii and the corresponding cyanobacterial transformants. Recipient of the Botanical Society Award for Young Scientists, 1995.     

Delineation of in vivo assembled multiprotein complexes via biomolecular interaction analysis mass spectrometry

Biomolecular interaction analysis mass spectrometry (BIA-MS) is a multiplexed bioanalytical approach used in analysis of proteins from complex biological mixtures. It utilizes surface-immobilized ligands for protein affinity retrieval, surface plasmon resonance for monitoring the ligand-protein interaction and matrix-assisted laser desorption/ionization-time of flight mass spectrometry for revealing the masses of the biomolecules retrieved by the ligand. In order to explore the utility of BIA-MS in delineation of multiprotein complexes, an in vivo assembled protein complex comprised of retinol binding protein (RBP) and transthyretin (TTR) was investigated. Antibodies to RBP and TTR were utilized as ligands in the analysis of the protein complex present in human plasma. The RBP-TTR complex was retrieved by the anti-RBP antibody as indicated by the presence of both RBP and TTR signals in the mass spectra. RBP signals were not observed in the mass spectra of the material retained on the anti-TTR derivatized surface. In addition, the mass-specific detection in BIA-MS allowed detection of RBP and TTR analyte variants.     

Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics

β-Barrel proteins, or outer membrane proteins (OMPs), perform many essential functions in Gram-negative bacteria, but questions remain about the mechanism by which they are assembled into the outer membrane (OM). In Escherichia coli, β-barrels are escorted across the periplasm by chaperones, most notably SurA and Skp. However, the contributions of these two chaperones to the assembly of the OM proteome remained unclear. We used differential proteomics to determine how the elimination of Skp and SurA affects the assembly of many OMPs. We have shown that removal of Skp has no impact on the levels of the 63 identified OM proteins. However, depletion of SurA in the skp strain has a marked impact on the OM proteome, diminishing the levels of almost all β-barrel proteins. Our results are consistent with a model in which SurA plays a primary chaperone role in E. coli. Furthermore, they suggest that while no OMPs prefer the Skp chaperone pathway in wild-type cells, most can use Skp efficiently when SurA is absent. Our data, which provide a unique glimpse into the protein content of the nonviable surA skp mutant, clarify the roles of the periplasmic chaperones in E. coli.     

Three-dimensional structures of protein-protein complexes in the E. coli PTS

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) includes a collection of proteins that accomplish phosphoryl transfer from phosphoenolpyruvate (PEP) to a sugar in the course of transport. The soluble proteins of the glucose transport pathway also function as regulators of diverse systems. The mechanism of interaction of the phosphoryl carrier proteins with each other as well as with their regulation targets has been amenable to study by nuclear magnetic resonance (NMR) spectroscopy. The three-dimensional solution structures of the complexes between the N-terminal domain of enzyme I and HPr and between HPr and enzyme IIA(Glc) have been elucidated. An analysis of the binding interfaces of HPr with enzyme I, IIA(Glc) and glycogen phosphorylase revealed that a common surface on HPr is involved in all these interactions. Similarly, a common surface on IIA(Glc) interacts with HPr, IIB(Glc) and glycerol kinase. Thus, there is a common motif for the protein-protein interactions characteristic of the PTS.     

共表达甘油脱氢酶和二羟丙酮激酶对大肠杆菌生长及甘油代谢的影响

考察共表达甘油脱氢酶(GldA)和二羟丙酮激酶(DhaKLM)对大肠杆菌生长及甘油代谢的影响。结果表明:在好氧条件下,共表达甘油脱氢酶及二羟丙酮激酶可以提高大肠杆菌利用甘油合成菌体的效率,利用等量的甘油,重组菌最高菌密度比对照菌提高了70%,细胞干质量为3.54 g(以每升发酵液计)。在厌氧条件下,仅共表达甘油脱氢酶并不能促进大肠杆菌的甘油代谢,而同时共表达甘油脱氢酶和二羟丙酮激酶可以明显提高大肠杆菌代谢甘油的能力,每克菌体消耗的甘油量提高了42%,每克干细胞中达11.08 g,代谢产物组成也发生显著变化,乙酸成为主要产物。这说明共表达gldA及dhaKLM基因能有效促进大肠杆菌好氧利用甘油生长及厌氧甘油代谢的能力。     

Relief of polarity in E. coli depleted of 30S ribosomal subunits.

Summary Escherichia coli was depleted of ribosomes by a thermal shock at 47° C which quantitatively destroyed the 30S ribosomal subunits. During recovery in minimal medium at 30° C RNA is synthesized while protein synthesis resumes only after about 90 min. It is shown that lac mRNA is synthesized in the complete absence of ribosomal activity and hence RNA synthesis is not coupled to protein synthesis. Lac mRNA from a series of lac nonsense mutants was examined in both heated and untreated cells. It was found that the polar effect of nonsense mutation is relieved in the absence of ribosomes and that this relief is due to the synthesis of larger mRNA molecules. Since Rho remained active in thermally treated cells, premature termination at secondary signals within the lac operon must also depend on the presence of active ribosomes.Abbreviations used SSC 0.15 M Nacl, 0.015 M sodium citrate (pH 7.0) - mRNA messenger ribonucleic acid - IPTG isopropyl--D-thiogalactopyranoside - cAMP adenosine 3: 5-cyclic monophosphoric acid - LDS lithium dodecyl sulfate - TCA trichloroacetic acid The paper forms part of the first author's M.Sc. thesis     

Quantitative study of interaction of deacylated tRNA with Escherichia coli ribosomes. Role of 50 S subunits in formation of the E site

The 30 S subunit contains 2 sites for tRNA binding (Phe-tRNA, AcPhe-tRNA, tRNAPheOH) with the functional properties of D and A sites of the 70 S ribosome after attachment of 50 S subunit. The third (E) site specific for deacylated tRNA is introduced into 70 S ribosome by its 50 S subunit. The E-site binding of tRNAPheOH is not sensitive to either tetracycline and edeine, and practically codon-independent. The affinity constant of tRNAPheOH for the E site is 2-3 orders of magnitude lower than that for the D site.     

Disassembly of intact multiprotein complexes in the gas phase

The observation of multiprotein complexes by mass spectrometry formerly relied upon chemical cross-linking to maintain interactions. Recent technological developments have enabled the observation of intact macromolecular complexes without modification. These assemblies, with masses far in excess of those measured previously, can be examined through controlled dissociation in the mass spectrometer, revealing information about their subunit interactions and topology.     

The integration of signaling by multiprotein complexes containing Raf kinases

In vivo, eukaryotic cells are subjected simultaneously to a broad array of signals ranging from mitogens and inflammatory inputs to environmental stresses and developmental cues. The combinatorial nature of cellular signaling necessitates that a cell integrate its signal transduction pathways so as to implement rapidly and efficiently an appropriate suite of responses. Emerging evidence indicates that, over the course of evolution, cells have developed multiprotein signaling complexes, or "signalosomes" that mediate the coordinate regulation of different signaling pathways. Such molecular signal integration contrasts with the classical notion of signaling complexes assembled by scaffold proteins-entities that function to segregate specific pathways from one another. This review will focus on two signal integrating multiprotein complexes that involve Raf family kinases: the MLK3-B-Raf-Raf-1 complex and the Raf-1-Mst-2 complex.     

Neural network optimization for E. coli promoter prediction.

Methods for optimizing the prediction of Escherichia coli RNA polymerase promoter sequences by neural networks are presented. A neural network was trained on a set of 80 known promoter sequences combined with different numbers of random sequences. The conserved -10 region and -35 region of the promoter sequences and a combination of these regions were used in three independent training sets. The prediction accuracy of the resulting weight matrix was tested against a separate set of 30 known promoter sequences and 1500 random sequences. The effects of the network's topology, the extent of training, the number of random sequences in the training set and the effects of different data representations were examined and optimized. Accuracies of 100% on the promoter test set and 98.4% on the random test set were achieved with the optimal parameters.