A mechanism for the evolution of phosphorylation sites.

Protein phosphorylation provides a mechanism for the rapid, reversible control of protein function. Phosphorylation adds negative charge to amino acid side chains, and negatively charged amino acids (Asp/Glu) can sometimes mimic the phosphorylated state of a protein. Using a comparative genomics approach, we show that nature also employs this trick in reverse by evolving serine, threonine, and tyrosine phosphorylation sites from Asp/Glu residues. Structures of three proteins where phosphosites evolved from acidic residues (DNA topoisomerase II, enolase, and C-Raf) show that the relevant acidic residues are present in salt bridges with conserved basic residues, and that phosphorylation has the potential to conditionally restore the salt bridges. The evolution of phosphorylation sites from glutamate and aspartate provides a rationale for why phosphorylation sometimes activates proteins, and helps explain the origins of this important and complex process.

Systematic investigation of CRISPR-Cas9 configurations for flexible and efficient genome editing in Corynebacterium glutamicum NRRL-B11474.

This study details a reliable and efficient method for CRISPR-Cas9 genome engineering in the high amino acid-producing strain of Corynebacterium glutamicum, NRRL-B11474. Our investigation demonstrates that a plasmid-encoded single-guide RNA paired with different edit-encoding fragments is sufficient to generate edits without the addition of an exogenous recombinase. This approach leverages a genome-integrated copy of the cas9 gene for reduced toxicity, in combination with a single plasmid carrying the targeting guide RNA and matching edit fragment. Our study systematically investigated the impact of homology arm length on editing efficiency and demonstrates genome editing with homology arm lengths as small as 25 bp for single-nucleotide polymorphisms and 75 bp for 100 bp sequence swaps. These homology arm lengths are smaller than previously reported for other strains of C. glutamicum. Our study finds that C. glutamicum NRRL-B11474 is not amenable to efficient transformation with plasmids containing the BL1, NG2, or CC1 origins of replication. This finding differs from all previously reported approaches to plasmid-based CRISPR-Cas9 or Cpf1 editing in other strains of C. glutamicum. Two alternative origins of replication (CG1 and CASE1) can be used to successfully introduce genome edits; furthermore, our data demonstrate improved editing efficiency when guide RNAs and edit fragments are encoded on plasmids carrying the CASE1 origin of replication (compared to plasmids carrying CG1). In addition, this study demonstrates that efficient editing can be done using an integrated Cas9 without the need for a recombinase. We demonstrate that the specifics of CRISPR-Cas9 editing configurations may need to be tailored to enable different edit types in a particular strain background. Refining configuration parameters such as edit type, homology arm length, and plasmid origin of replication enables robust, flexible, and efficient CRISPR-Cas9 editing in differing genetic strain contexts.

A C-terminal inhibitory domain controls the activity of p63 by an intramolecular mechanism.

The human genome is far smaller than originally estimated, and one explanation is that alternative splicing creates greater proteomic complexity than a simple count of open reading frames would suggest. The p53 homologue p63, for example, is a tetrameric transcription factor implicated in epithelial development and expressed as at least six isoforms with widely differing transactivation potential. In particular, p63alpha isoforms contain a 27-kDa C-terminal region that drastically reduces their activity and is of clear biological importance, since patients with deletions in this C terminus have phenotypes very similar to patients with mutations in the DNA-binding domain. We have identified a novel domain within this C terminus that is necessary and sufficient for transcriptional inhibition and which acts by binding to a region in the N-terminal transactivation domain of p63 homologous to the MDM2 binding site in p53. Based on this mechanism, we provide a model that explains the transactivation potential of homo- and heterotetramers composed of different p63 isoforms and their effect on p53.

In-cell NMR spectroscopy.

The role of a protein inside a cell is determined by both its location and its conformational state. Although fluorescence techniques are widely used to determine the cellular localization of proteins in vivo, these approaches cannot provide detailed information about a protein's three-dimensional state. This gap, however, can be filled by NMR spectroscopy, which can be used to investigate both the conformation as well as the dynamics of proteins inside living cells. In this chapter we describe technical aspects of these "in-cell NMR" experiments. In particular, we show that in the case of (15)N-labeling schemes the background caused by labeling all cellular components is negligible, while (13)C-based experiments suffer from high background levels and require selective labeling schemes. A correlation between the signal-to-noise ratio of in-cell NMR experiments with the overexpression level of the protein shows that the current detection limit is 150-200 muM (intracellular concentration). We also discuss experiments that demonstrate that the intracellular viscosity is not a limiting factor since the intracellular rotational correlation time is only approximately two times longer than the correlation time in water. Furthermore, we describe applications of the technique and discuss its limitations.

Low-Cost, High-Throughput Sequencing of DNA Assemblies Using a Highly Multiplexed Nextera Process.

In recent years, next-generation sequencing (NGS) technology has greatly reduced the cost of sequencing whole genomes, whereas the cost of sequence verification of plasmids via Sanger sequencing has remained high. Consequently, industrial-scale strain engineers either limit the number of designs or take short cuts in quality control. Here, we show that over 4000 plasmids can be completely sequenced in one Illumina MiSeq run for less than $3 each (15× coverage), which is a 20-fold reduction over using Sanger sequencing (2× coverage). We reduced the volume of the Nextera tagmentation reaction by 100-fold and developed an automated workflow to prepare thousands of samples for sequencing. We also developed software to track the samples and associated sequence data and to rapidly identify correctly assembled constructs having the fewest defects. As DNA synthesis and assembly become a centralized commodity, this NGS quality control (QC) process will be essential to groups operating high-throughput pipelines for DNA construction.

Rapid and reliable DNA assembly via ligase cycling reaction.

Assembly of DNA parts into DNA constructs is a foundational technology in the emerging field of synthetic biology. An efficient DNA assembly method is particularly important for high-throughput, automated DNA assembly in biofabrication facilities and therefore we investigated one-step, scarless DNA assembly via ligase cycling reaction (LCR). LCR assembly uses single-stranded bridging oligos complementary to the ends of neighboring DNA parts, a thermostable ligase to join DNA backbones, and multiple denaturation-annealing-ligation temperature cycles to assemble complex DNA constructs. The efficiency of LCR assembly was improved ca. 4-fold using designed optimization experiments and response surface methodology. Under these optimized conditions, LCR enabled one-step assembly of up to 20 DNA parts and up to 20 kb DNA constructs with very few single-nucleotide polymorphisms (<1 per 25 kb) and insertions/deletions (<1 per 50 kb). Experimental comparison of various sequence-independent DNA assembly methods showed that circular polymerase extension cloning (CPEC) and Gibson isothermal assembly did not enable assembly of more than four DNA parts with more than 50% of clones being correct. Yeast homologous recombination and LCR both enabled reliable assembly of up to 12 DNA parts with 60-100% of individual clones being correct, but LCR assembly provides a much faster and easier workflow than yeast homologous recombination. LCR combines reliable assembly of many DNA parts via a cheap, rapid, and convenient workflow and thereby outperforms existing DNA assembly methods. LCR assembly is expected to become the method of choice for both manual and automated high-throughput assembly of DNA parts into DNA constructs.

Tuning the activation threshold of a kinase network by nested feedback loops.

Determining proper responsiveness to incoming signals is fundamental to all biological systems. We demonstrate that intracellular signaling nodes can tune a signaling network's response threshold away from the basal median effective concentration established by ligand-receptor interactions. Focusing on the bistable kinase network that governs progesterone-induced meiotic entry in Xenopus oocytes, we characterized glycogen synthase kinase-3beta (GSK-3beta) as a dampener of progesterone responsiveness. GSK-3beta engages the meiotic kinase network through a double-negative feedback loop; this specific feedback architecture raises the progesterone threshold in correspondence with the strength of double-negative signaling. We also identified a marker of nutritional status, l-leucine, which lowers the progesterone threshold, indicating that oocytes integrate additional signals into their cell-fate decisions by modulating progesterone responsiveness.

Tuning bulk electrostatics to regulate protein function.

Cyclin-dependent kinase activation can prevent yeast cells from responding to mating pheromone. Strickfaden et al. (2007) now show that this block arises from the multisite phosphorylation of Ste5. This provides a beautiful example of how phosphorylation can produce decisive changes in protein function through bulk electrostatics, without the necessity of intricate conformational changes.

Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes.

We introduce a eukaryotic cellular system, the Xenopus laevis oocyte, for in-cell NMR analyses of biomolecules at high resolution and delineate the experimental reference conditions for successful implementations of in vivo NMR measurements in this cell type. This approach enables quantitative NMR experiments at defined intracellular concentrations of exogenous proteins, which is exemplified by the description of in-cell NMR properties of the protein G B1 domain (GB1). Additional experiments in Xenopus egg extracts and artificially crowded in vitro solutions suggest that for this biologically inert protein domain, intracellular viscosity and macromolecular crowding dictate its in vivo behavior. These contributions appear particularly pronounced for protein regions with high degrees of internal mobility in the pure state. We also evaluate the experimental limitations of this method and discuss potential applications toward the in situ structural characterization of eukaryotic cellular activities.

Investigating macromolecules inside cultured and injected cells by in-cell NMR spectroscopy.

The noninvasive character of NMR spectroscopy, combined with the sensitivity of the chemical shift, makes it ideally suited to investigate the conformation, binding events and dynamics of macromolecules inside living cells. These 'in-cell NMR' experiments involve labeling the macromolecule of interest with a nonradioactive but NMR-active isotope (15N or 13C). Cellular samples are prepared either by selectively overexpressing the protein in suitable cells (e.g., bacterial cells grown on isotopically labeled media), or by injecting isotopically labeled proteins directly into either cells or cell extracts. Here we provide detailed protocols for in-cell NMR experiments in the prokaryotic organism Escherichia coli, as well as eukaryotic cells and extracts employing Xenopus laevis oocytes or egg extracts. In-cell NMR samples with proteins overexpressed in E. coli can be produced within 13-14 h. Preparing Xenopus oocyte samples for in-cell NMR experiments takes 6-14 h depending on the oocyte preparation scheme and the injection method used.

Quantitative identification of the protonation state of histidines in vitro and in vivo.

The C[bond]N coupling constants centered at the C(epsilon 1) and C(delta 2) carbons in histidine residues depend on the protonation state and tautomeric form of the imidazole ring, making them excellent indicators of pH or pK(a), and the ratio of the tautomeric states. In this paper, we demonstrate that the intensity ratios for the C(epsilon 1)-H and C(delta 2)-H cross-peaks measured with a constant time HSQC experiment without and with J(C[bond]N) amplitude modulation are determined by the ratios of the protonated and deprotonated forms and tautomeric states. This allows one to investigate the tautomeric state of histidines as well as their pK(a) in situations where changing the pH value by titration is difficult, for example, for in-cell NMR experiments. We apply this technique to the investigation of the bacterial protein NmerA and determine that the intracellular pH in the Escherichia coli cytoplasm is 7.1 +/- 0.1.

Methyl groups as probes for proteins and complexes in in-cell NMR experiments.

Studying protein components of large intracellular complexes by in-cell NMR has so far been impossible because the backbone resonances are unobservable due to their slow tumbling rates. We describe a methodology that overcomes this difficulty through selective labeling of methyl groups, which possess more favorable relaxation behavior. Comparison of different in-cell labeling schemes with three different proteins, calmodulin, NmerA, and FKBP, shows that selective labeling with [(13)C]methyl groups on methionine and alanine provides excellent sensitivity with low background levels at very low costs.