A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell-free protein synthesis systems.

Cell-free protein synthesis (CFPS) systems allow for robust protein expression with easy manipulation of conditions to improve protein yield and folding. Recent technological developments have significantly increased the productivity and reduced the operating costs of CFPS systems, such that they can compete with conventional in vivo protein production platforms, while also offering new routes for the discovery and production of biotherapeutics. As cell-free systems have evolved, productivity increases have commonly been obtained by addition of components to previously designed reaction mixtures without careful re-examination of the essentiality of reagents from previous generations.

Confocal single-molecule FRET for protein conformational dynamics.

Single-molecule Fӧrster-type resonance energy transfer (smFRET) is a unique technique capable of following conformational motions of individual protein molecules. The direct observation of individual proteins provides rich information that would be washed away in ensemble measurements, hence opening up new avenues for establishing the protein structure-function relationships through dynamics. Retrieving dynamics information of biomolecular motions via smFRET, though, requires careful experiment design and rigorous treatment of single-molecule statistics.

Structural distributions from single-molecule measurements as a tool for molecular mechanics.

A mechanical view provides an attractive alternative for predicting the behavior of complex systems since it circumvents the resource-intensive requirements of atomistic models; however, it remains extremely challenging to characterize the mechanical responses of a system at the molecular level. Here, the structural distribution is proposed to be an effective means to extracting the molecular mechanical properties. End-to-end distance distributions for a series of short poly-L-proline peptides with the sequence P(n)CG(3)K-biotin (n = 8, 12, 15 and 24) were used to experimentally illustrate this new approach.

Engagement of arginine finger to ATP triggers large conformational changes in NtrC1 AAA+ ATPase for remodeling bacterial RNA polymerase.

The NtrC-like AAA+ ATPases control virulence and other important bacterial activities through delivering mechanical work to σ54-RNA polymerase to activate transcription from σ54-dependent genes. We report the first crystal structure for such an ATPase, NtrC1 of Aquifex aeolicus, in which the catalytic arginine engages the γ-phosphate of ATP. Comparing the new structure with those previously known for apo and ADP-bound states supports a rigid-body displacement model that is consistent with large-scale conformational changes observed by low-resolution methods.

Dynamic active-site protection by the M. tuberculosis protein tyrosine phosphatase PtpB lid domain.

The Mycobacterium tuberculosis protein tyrosine phosphatase PtpB shows resistance to the oxidative conditions that prevail within an infected host macrophage, but the mechanism of this molecular adaptation is unknown. Crystal structures of PtpB revealed previously that a closed, two-helix lid covers the active site. By measuring single-molecule Forster-type resonance energy transfer to probe the dynamics of two helices that constitute the lid, we obtained direct evidence for large, spontaneous opening transitions of PtpB with the closed form of both helices favored approximately 3:1.

Direct Mg(2+) binding activates adenylate kinase from Escherichia coli.

We report evidence that adenylate kinase (AK) from Escherichia coli can be activated by the direct binding of a magnesium ion to the enzyme, in addition to ATP-complexed Mg(2+). By systematically varying the concentrations of AMP, ATP, and magnesium in kinetic experiments, we found that the apparent substrate inhibition of AK, formerly attributed to AMP, was suppressed at low magnesium concentrations and enhanced at high magnesium concentrations. This previously unreported magnesium dependence can be accounted for by a modified random bi-bi model in which Mg(2+) can bind to AK directly prior to AMP binding.

Quantitation of steroid hormones in thin fresh frozen tissue sections.

As analytical technologies in proteomics and metabolomics continue to mature, there is an increasing need to apply these to clinically relevant biologic samples. In this study, a liquid chromatography-tandem mass spectrometry method that utilizes selected reaction monitoring was used to measure the absolute quantity of estrogens and estrogen metabolites and testosterone in 8-microm tissue sections obtained from a fresh frozen lymph node tumor infiltrated by metastatic breast carcinoma. Total (conjugated plus unconjugated) and unconjugated levels of these steroid hormones were measured using two cohorts, each containing five adjacent serial sections cut from this tumor.

Illuminating the mechanistic roles of enzyme conformational dynamics.

Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzyme's lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AK's domain movements on its catalytic time scale.

Gene promoter methylation in prostate tumor-associated stromal cells.

Background: Gene expression can be silenced through the methylation of specific sites in the promoter region. This mechanism of gene silencing has an important role in the carcinogenesis of prostate and other cancers. Although tumor-associated stromal cells also exhibit changes in gene expression, promoter methylation has not been described in these cells.

Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations--implications for the study of ribosome dynamics.

Protein biosynthesis requires numerous conformational rearrangements within the ribosome. The structural core of the ribosome is composed of RNA and is therefore dependent on counterions such as magnesium ions for function. Many steps of translation can be compromised or inhibited if the concentration of Mg(2+) is too low or too high.