Studying the salt dependence of the binding of sigma70 and sigma32 to core RNA polymerase using luminescence resonance energy transfer.

The study of protein-protein interactions is becoming increasingly important for understanding the regulation of many cellular processes. The ability to quantify the strength with which two binding partners interact is desirable but the accurate determination of equilibrium binding constants is a difficult process. The use of Luminescence Resonance Energy Transfer (LRET) provides a homogeneous binding assay that can be used for the detection of protein-protein interactions. Previously, we developed an LRET assay to screen for small molecule inhibitors of the interaction of sigma70 with thebeta' coiled-coil fragment (amino acids 100-309). Here we describe an LRET binding assay used to monitor the interaction of E. coli sigma70 and sigma32 with core RNA polymerase along with the controls to verify the system. This approach generates fluorescently labeled proteins through the random labeling of lysine residues which enables the use of the LRET assay for proteins for which the creation of single cysteine mutants is not feasible. With the LRET binding assay, we are able to show that the interaction of sigma70 with core RNAP is much more sensitive to NaCl than to potassium glutamate (KGlu), whereas the sigma32 interaction with core RNAP is insensitive to both salts even at concentrations >500 mM. We also find that the interaction of sigma32 with core RNAP is stronger than sigma70 with core RNAP, under all conditions tested. This work establishes a consistent set of conditions for the comparison of the binding affinities of the E.coli sigma factors with core RNA polymerase. The examination of the importance of salt conditions in the binding of these proteins could have implications in both in vitro assay conditions and in vivo function.

Scalable culture and cryopreservation of human embryonic stem cells on microcarriers.

As a result of their pluripotency and potential for unlimited self-renewal, human embryonic stem cells (hESCs) hold tremendous promise in regenerative medicine. An essential prerequisite for the widespread application of hESCs is the establishment of effective and efficient protocols for large-scale cell culture, storage, and distribution. At laboratory scales hESCs are cultured adherent to tissue culture plates; these culture techniques are labor-intensive and do not scale to high cell numbers. In an effort to facilitate larger scale hESC cultivation, we investigated the feasibility of culturing hESCs adherent to microcarriers. We modified the surface of Cytodex 3 microcarriers with either Matrigel or mouse embryonic fibroblasts (MEFs). hESC colonies were effectively expanded in a pluripotent, undifferentiated state on both Matrigel-coated microcarriers and microcarriers seeded with a MEF monolayer. While the hESC expansion rate on MEF-microcarriers was less than that on MEF-plates, the doubling time of hESCs on Matrigel-microcarriers was indistinguishable from that of hESCs expanded on Matrigel-coated tissue culture plates. Standard hESC cryopreservation methodologies are plagued by poor viability and high differentiation rates upon thawing. Here, we demonstrate that cryopreservation of hESCs adherent to microcarriers in cryovials provides a higher recovery of undifferentiated cells than cryopreservation of cells in suspension. Together, these results suggest that microcarrier-based stabilization and culture may facilitate hESC expansion and storage for research and therapeutic applications.

Optical mapping discerns genome wide DNA methylation profiles.

BACKGROUND: Methylation of CpG dinucleotides is a fundamental mechanism of epigenetic regulation in eukaryotic genomes. Development of methods for rapid genome wide methylation profiling will greatly facilitate both hypothesis and discovery driven research in the field of epigenetics. In this regard, a single molecule approach to methylation profiling offers several unique advantages that include elimination of chemical DNA modification steps and PCR amplification. RESULTS: A single molecule approach is presented for the discernment of methylation profiles, based on optical mapping. We report results from a series of pilot studies demonstrating the capabilities of optical mapping as a platform for methylation profiling of whole genomes. Optical mapping was used to discern the methylation profile from both an engineered and wild type Escherichia coli. Furthermore, the methylation status of selected loci within the genome of human embryonic stem cells was profiled using optical mapping. CONCLUSION: The optical mapping platform effectively detects DNA methylation patterns. Due to single molecule detection, optical mapping offers significant advantages over other technologies. This advantage stems from obviation of DNA modification steps, such as bisulfite treatment, and the ability of the platform to assay repeat dense regions within mammalian genomes inaccessible to techniques using array-hybridization technologies.

LRET-based HTS of a small-compound library for inhibitors of bacterial RNA polymerase.

Resistance mechanisms against whole classes of antibiotics are currently developing faster than research generates new structurally different biologically active agents. The demand for new antimicrobial drugs has not been matched by the speed of discovery. The interface between sigma and core of bacterial RNA polymerase offers an attractive target for drug discovery, and we have previously described the development of a very robust high-throughput assay for this target based on luminescence resonance energy transfer. Here we describe a semiautomated screen of a commercially available library (Chembridge, San Diego, CA) that led to the identification of four small molecules, two of which have activity in preventing in vitro transcription and growth of Escherichia coli.

Identification and characterization of the gene encoding the Acidobacterium capsulatum major sigma factor.

Acidobacterium capsulatum is an acid-tolerant, encapsulated, Gram-negative member of the ubiquitous, but poorly understood Acidobacteria phylum. Little is known about the genetics and regulatory mechanisms of A. capsulatum. To begin to address this gap, we identified the gene encoding the A. capsulatum major sigma factor, rpoD, which encodes a 597-amino acid protein with a predicted sequence highly similar to the major sigma factors of Solibacter usitatus Ellin6076 and Geobacter sulfurreducens PCA. Purified hexahistidine-tagged RpoD migrates at approximately 70 kDa under SDS-PAGE conditions, which is consistent with the predicted MW of 69.2 kDa, and the gene product is immunoreactive with monoclonal antibodies specific for either bacterial RpoD proteins or the N-terminal histidine tag. A. capsulatum RpoD restored normal growth to E. coli strain CAG20153 under conditions that prevent expression of the endogenous rpoD. These results indicate we have cloned the gene encoding the A. capsulatum major sigma factor and the gene product is active in E. coli.

Homogeneous fluorescent assay for RNA polymerase.

A new method for determination of RNA polymerase (RNAP) activity is presented. The method uses nucleoside tri- and tetraphosphate derivatives carrying 4-methylumbelliferone residue at the terminal phosphate. Incorporation of such compounds in RNA by RNA polymerase is accompanied by release of di- and triphosphate derivatives of 4-methylumbelliferone. Subsequent treatment by alkaline phosphatase produces free 4-methylumbelliferone that is highly fluorescent and can be easily detected. The sensitivity of the method is higher than that reported in previous studies. The validity of the assay has been demonstrated by retrieving the RNAP inhibitors from a collection of 16,000 compounds.

A cross-reactive polyol-responsive monoclonal antibody useful for isolation of core RNA polymerase from many bacterial species.

The use of antibodies for protein purification is a powerful technique but the release of the target protein in its active form is often difficult. So called "polyol-responsive" monoclonal antibodies (PR-MAbs) have a feature that allows elution of the antigen under very gentle conditions, so that even multi-subunit proteins can be released in their active form. In this work a PR-MAb, 8RB13, was isolated that can purify RNA polymerase (RNAP) from many different bacterial species. High specificity towards RNAP with a broad species cross-reactivity was achieved by immunization with RNAP from Escherichia coli and screening with Bacillus subtilis RNA polymerase. The isolated MAb could detect the beta-subunit of RNA polymerase from 10 out of 12 species tested on a Western blot indicating its potential for purification of core RNAP from these organisms. Representatively, four of these species E. coli, B. subtilis, Pseudomonas aeruginosa, and Streptomyces coelicolor were subjected to immunoaffinity purification yielding RNA polymerases that were active in in vitro transcription and seemed to be primarily core polymerase, lacking sigma-subunits.

A fast Western blot procedure improved for quantitative analysis by direct fluorescence labeling of primary antibodies.

The procedures for Western blots have been around for a long time and recent developments have increased the sensitivity for luminescent techniques so that the need for radioactive probes has been limited to only a few applications. Nevertheless, most protocols require more than 6 h and are often performed over more than a day. The majority of techniques require a secondary antibody conjugated to an enzyme that catalyzes a color reaction in order to amplify a detectable signal. However, both processes, the binding of a secondary antibody and the catalyzed reaction with the dye, are sources for errors and the latter is disadvantageous for a signal that is linear over a larger range of detected antigen. In order to improve the procedure most commonly used for quantitative analysis and convenience, we investigated the use of fluorescence labeling of primary monoclonal antibodies against Escherichia coli RNA polymerase subunits (beta', sigmaE and sigmaFecI) and their use in Western blots. We achieved a sensitivity (<1 ng detectable protein) comparable to most luminescent techniques. Additionally, we reduced the procedure time significantly to less than 1 h after SDS-PAGE and transfer to a membrane. Above all, we obtained a linear signal over the range of 30 ng to 1 microg of protein (dependent on protein size) making quantitative analysis of Western blots easier and more reliable.

Luminescence resonance energy transfer-based high-throughput screening assay for inhibitors of essential protein-protein interactions in bacterial RNA polymerase.

The binding of sigma factors to core RNA polymerase is essential for the specific initiation of transcription in eubacteria and is thus critical for cell growth. Since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic RNA polymerases, sigma factor binding is a promising target for drug discovery. A homogeneous assay for sigma binding to RNA polymerase (Escherichia coli) based on luminescence resonance energy transfer (LRET) was developed by using a europium-labeled sigma70 and an IC5-labeled fragment of the beta' subunit of RNA polymerase (amino acid residues 100 through 309). Inhibition of sigma binding was measured by the loss of LRET through a decrease in IC5 emission. The technical advances offered by LRET resulted in a very robust assay suitable for high-throughput screening, and LRET was successfully used to screen a crude natural-product library. We illustrate this method as a powerful tool to investigate any essential protein-protein interaction for basic research and drug discovery.

On-column tris(2-carboxyethyl)phosphine reduction and IC5-maleimide labeling during purification of a RpoC fragment on a nickel-nitrilotriacetic acid Column.

Fluorescence labeling of proteins has become increasingly important since fluorescent techniques like FRET and fluorescence polarization are now commonly used in protein binding studies, proteomics, and for high-throughput screening in drug discovery. In our efforts to study the binding of the beta(')-subunit from Escherichia coli RNA polymerase (RNAP) to sigma70, we synthesized a fluorescent-labeled beta(')-fragment (residues 100-309) in a very convenient way, that could be used as a general protocol for hexahistidine-tagged proteins. By performing all the following steps, purification, reduction, derivatization with IC5-maleimide, and free dye removal while the protein was bound to the column, we were able to reduce the procedure time significantly and at the same time achieve better labeling efficiency and quality. The beta(')-fragment with a N-terminal His(6)-tag was purified from inclusion bodies and could be refolded prior to or after binding to a Ni-NTA affinity column. Reduction prior to labeling was achieved with TCEP that does not interfere with Ni-NTA chemistry. The labeled beta(')-fragment was tested with sigma70 that was labeled with an europium-based fluorophore for binding in a electrophoretic mobility-shift assay. The sigma-to-core protein interaction in bacterial RNA polymerase offers a potentially specific target for drug discovery, since it is highly conserved among the eubacteria, but differs significantly from eukaryotes.

Mutational analysis of the C-domain in nonribosomal peptide synthesis.

The initial condensation event in the nonribosomal biosynthesis of the peptide antibiotics gramicidin S and tyrocidine A takes place between a phenylalanine activating racemase GrsA/TycA and the first proline-activating module of GrsB/TycB. Recently we established a minimal in vitro model system for NRPS with recombinant His6-tagged GrsA (GrsAPhe-ATE; 127 kDa) and TycB1 (TycB1Pro-CAT; 120 kDa) and demonstrated the catalytic function of the C-domain in TycB1Pro-CAT to form a peptide bond between phenylalanine and proline during diketopiperazine formation (DKP). In this work we took advantage of this system to identify catalytically important residues in the C-domain of TycB1Pro-CAT using site-directed mutagenesis and peptide mapping. Mutations in TycB1Pro-CAT of 10 strictly conserved residues among 80 other C-domains with potential catalytic function, revealed that only R62A, H147R and D151N are impaired in peptide-bond formation. All other mutations led to either unaffected (Q19A, C154A/S, Y166F/W and R284A) or insoluble proteins (H146A, R67A and W202L). Although 100 nm of the serine protease inhibitors N-alpha-tosyl-l-phenylalanylchloromethane or phenylmethanesulfonyl fluoride completely abolished DKP synthesis, no covalently bound inhibitor derivatives in the C-domain could be identified by peptide mapping using HPLC-MS. Though the results do not reveal a particular mechanism for the C-domain, they exhibit a possible way of catalysis analogous to the functionally related enzymes chloramphenicol acetyltransferase and dihydrolipoyl transacetylase. Based on this, we propose a mechanism in which one catalytic residue (H147) and two other structural residues (R62 and D151) are involved in amino-acid condensation.