Stoichiometry and absolute quantification of proteins with mass spectrometry using fluorescent and isotope-labeled concatenated peptide standards

We have explored a general approach for the determination of absolute amounts and the relative stoichiometry of proteins in a mixture using fluorescence and mass spectrometry. We engineered a gene to express green fluorescent protein (GFP) with a synthetic fusion protein (GAB-GFP) in Escherichia coli to function as a spectroscopic standard for the quantification of an analogous stable isotope-labeled, non-fluorescent fusion protein (GAB*) and for the quantification and stoichiometric analysis of purified transducin, a heterotrimeric G-protein complex. Both GAB-GFP and GAB* contain concatenated sequences of specific proteotypic peptides that are derived from the alpha, beta, and gamma protein subunits of transducin and that are each flanked by spacer regions that maintain the native proteolytic properties for these peptide fragments. Spectroscopic quantification of GAB-GFP provided a molar scale for mass spectrometric ratios from tryptic peptides of GAB* and defined molar responses for mass spectrometric signal intensities from a purified transducin complex. The stoichiometry of transducin subunits alpha, beta, and gamma was measured to be 1:1.1:1.15 over a 5-fold range of labeled internal standard with a relative standard deviation of 9%. Fusing a unique genetically coded spectroscopic signal element with concatenated proteotypic peptides provides a powerful method to accurately quantify and determine the relative stoichiometry of multiple proteins present in complexes or mixtures that cannot be readily assessed using classical gravimetric, enzymatic, or antibody-based technologies.     

Extended polypeptide linkers establish the spatial architecture of a pyruvate dehydrogenase multienzyme complex

Icosahedral pyruvate dehydrogenase (PDH) enzyme complexes are molecular machines consisting of a central E2 core decorated by a shell of peripheral enzymes (E1 and E3) found localized at a distance of approximately 75-90 A from the core. Using a combination of biochemical, biophysical, and cryo-electron microscopic techniques, we show here that the gap between the E2 core and the shell of peripheral enzymes is maintained by the flexible but extended conformation adopted by 60 linker polypeptides that radiate outwards from the inner E2 core, irrespective of the E1 or E3 occupancy. The constancy of the gap is thus not due to protein-protein interactions in the outer protein shell. The extended nature of the E2 inner-linker regions thereby creates the restricted annular space in which the lipoyl domains of E2 that carry catalytic intermediates shuttle between E1, E2, and E3 active sites, while their conformational flexibility facilitates productive encounters.     

Three-dimensional imaging of the highly bent architecture of Bdellovibrio bacteriovorus by using cryo-electron tomography

Bdellovibrio bacteriovorus cells are small deltaproteobacterial cells that feed on other gram-negative bacteria, including human pathogens. Using cryo-electron tomography, we demonstrated that B. bacteriovorus cells are capable of substantial flexibility and local deformation of the outer and inner membranes without loss of cell integrity. These shape changes can occur in less than 2 min, and analysis of the internal architecture of highly bent cells showed that the overall distribution of molecular machines and the nucleoid is similar to that in moderately bent cells. B. bacteriovorus cells appear to contain an extensive internal network of short and long filamentous structures. We propose that rearrangements of these structures, in combination with the unique properties of the cell envelope, may underlie the remarkable ability of B. bacteriovorus cells to find and enter bacterial prey.     

Chemoreceptors in Caulobacter crescentus: trimers of receptor dimers in a partially ordered hexagonally packed array

Chemoreceptor arrays are macromolecular complexes that form extended assemblies primarily at the poles of bacterial cells and mediate chemotaxis signal transduction, ultimately controlling cellular motility. We have used cryo-electron tomography to determine the spatial distribution and molecular architecture of signaling molecules that comprise chemoreceptor arrays in wild-type Caulobacter crescentus cells. We demonstrate that chemoreceptors are organized as trimers of receptor dimers, forming partially ordered hexagonally packed arrays of signaling complexes in the cytoplasmic membrane. This novel organization at the threshold between order and disorder suggests how chemoreceptors and associated molecules are arranged in signaling assemblies to respond dynamically in the activation and adaptation steps of bacterial chemotaxis.     

Over-expression of proteins using a modified pBAD24 vector in <Emphasis Type="Italic">E. coli</Emphasis> expression system

A modified pBAD24 vector (pBAD24M) was constructed with the araBAD promoter of the arabinose operon along with T7g10 sequence elements and a modified Shine–Dalgarno sequence. While both green fluorescent protein and granulocyte colony stimulating factor showed negligible expression under the original pBAD24 vector, they were expressed at >35% of total cellular protein with the modified vector. Similar results were obtained for staphylokinase wherein the pBAD24-SAK construct yielded 8 ng/10⁶ c.f.u. of E. coli induced cells while the pBAD24M-SAK vector showed nearly 55 ng/10⁶ c.f.u. induced bacterial cells as tested by ELISA. Interestingly, the expression levels using modified pBAD24 vector matched that achieved with T7 promoter based vector system. The modified pBAD24 vector therefore represents a simple and a useful prokaryotic expression system for efficient repression, modulation and elevated protein expression levels. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A microwave-assisted facile regioselective Fischer indole synthesis and antitubercular evaluation of novel 2-aryl-3,4-dihydro-2H-thieno[3,2-b]indoles

A series of novel 2-aryl-3,4-dihydro-2H-thieno[3,2-b]indoles has been synthesised regioselectively in good yields from the reaction of 5-aryldihydro-3(2H)-thiophenones and arylhydrazine hydrochloride. This reaction is found to be assisted by microwaves. The thieno[3,2-b]indoles were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv (MTB) and multi-drug resistant M. tuberculosis (MDR-TB). Among 22 compounds screened, [2-(2,4-dichlorophenyl)-7-fluoro-3,4-dihydro-2H-thieno[3,2-b]indole] (6t) was found to the most active compound with MIC of 0.4 μg/mL against MTB and MDR-TB.     

5-Nitrofuran-2-yl derivatives: Synthesis and inhibitory activities against growing and dormant mycobacterium species

Eighteen 5-nitrofuran-2-yl derivatives were prepared by reacting 5-nitro-2-furfural with various (sub)phenyl/pyridyl thiosemicarbazide using microwave irradiation. The compounds were tested for their in vitro activity against tubercular and various non-tubercular mycobacterium species in log-phase and 6-week-starved cultures. Compound N-(3,5-dibromopyridin-2-yl)-2-((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide (4r) was found to be the most potent compound (MIC: 0.22 μM) and was 3 times more active than standard isoniazid (INH) and equally active as rifampicin (RIF) in log-phase culture of Mycobacterium tuberculosis H37Rv. In starved M. tuberculosis H37Rv, 4r inhibited with MIC of 13.9 μM and was found to be 50 times more active than INH and slightly more active than RIF.     

Minocycline attenuates microglial activation but fails to mitigate striatal dopaminergic neurotoxicity: role of tumor necrosis factor-alpha

Activated microglia are implicated in the pathogenesis of disease-, trauma- and toxicant-induced damage to the CNS, and strategies to modulate microglial activation are gaining impetus. A novel action of the tetracycline derivative minocycline is the ability to inhibit inflammation and free radical formation, factors that influence microglial activation. Minocycline is therefore being tested as a neuroprotective agent to alleviate CNS damage, although findings so far have yielded mixed results. Here, we showed that administration of a single low dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or methamphetamine (METH), a paradigm that causes selective degeneration of striatal dopaminergic nerve terminals without affecting the cell body in substantia nigra, increased the expression of mRNAs encoding microglia-associated factors F4/80, interleukin (IL)-1alpha, IL-6, monocyte chemoattractant protein-1 (MCP-1, CCL2) and tumor necrosis factor (TNF)-alpha. Minocycline treatment attenuated MPTP- or METH-mediated microglial activation, but failed to afford neuroprotection. Lack of neuroprotection was shown to be due to the inability of minocycline to abolish the induction of TNF-alpha and its receptors, thereby failing to modulate TNF signaling. Thus, TNF-alpha appeared to be an obligatory component of dopaminergic neurotoxicity. To address this possibility, we examined the effects of MPTP or METH in mice lacking genes encoding IL-6, CCL2 or TNF receptor (TNFR)1/2. Deficiency of either IL-6 or CCL2 did not alter MPTP neurotoxicity. However, deficiency of both TNFRs protected against the dopaminergic neurotoxicity of MPTP. Taken together, our findings suggest that attenuation of microglial activation is insufficient to modulate neurotoxicity as transient activation of microglia may suffice to initiate neurodegeneration. These findings support the hypothesis that TNF-alpha may play a role in the selective vulnerability of the nigrostriatal pathway associated with dopaminergic neurotoxicity and perhaps Parkinson's disease.     

Design and characterisation of piperazine-benzofuran integrated dinitrobenzenesulfonamide as Mycobacterium tuberculosis H37Rv strain inhibitors

Molecular hybridisation of four bioactive fragments piperazine, substituted-benzofuran, amino acids, and 2,4-dinitrobenzenesulfonamide as single molecular architecture was designed. A series of new hybrids were synthesised and subjected to evaluation for their inhibitory activity against Mycobacterium tuberculosis (Mtb) H37Rv. 4d–f and 4o found to exhibit MIC as 1.56 µg/mL, equally active as ethambutol whereas 4a, 4c, 4j displayed MIC 0.78 µg/mL were superior to ethambutol. Tested compounds demonstrated an excellent safety profile with very low toxicity, good selectivity index, and antioxidant properties. All the newly synthesised compounds were thoroughly characterised by analytical methods. The result was further supported by molecular modelling studies on the crystal structure of Mycobacterium tuberculosis enoyl reductase.