DNA damage in oocytes induces a switch of the quality control factor TAp63α from dimer to tetramer.

TAp63α, a homolog of the p53 tumor suppressor, is a quality control factor in the female germline. Remarkably, already undamaged oocytes express high levels of the protein, suggesting that TAp63α's activity is under tight control of an inhibitory mechanism. Biochemical studies have proposed that inhibition requires the C-terminal transactivation inhibitory domain. However, the structural mechanism of TAp63α inhibition remains unknown. Here, we show that TAp63α is kept in an inactive dimeric state. We reveal that relief of inhibition leads to tetramer formation with ∼20-fold higher DNA affinity. In vivo, phosphorylation-triggered tetramerization of TAp63α is not reversible by dephosphorylation. Furthermore, we show that a helix in the oligomerization domain of p63 is crucial for tetramer stabilization and competes with the transactivation domain for the same binding site. Our results demonstrate how TAp63α is inhibited by complex domain-domain interactions that provide the basis for regulating quality control in oocytes.

Transmembrane segment enhanced labeling as a tool for the backbone assignment of alpha-helical membrane proteins.

Recent advances in cell-free expression protocols have opened a new avenue toward high-resolution structural investigations of membrane proteins by x-ray crystallography and NMR spectroscopy. One of the biggest challenges for liquid-state NMR-based structural investigations of membrane proteins is the significant peak overlap in the spectra caused by large line widths and limited chemical shift dispersion of alpha-helical proteins. Contributing to the limited chemical shift dispersion is the fact that approximately 60% of the amino acids in transmembrane regions consist of only six different amino acid types. This principle disadvantage, however, can be exploited to aid in the assignment of the backbone resonances of membrane proteins; by (15)N/(13)C-double-labeling of these six amino acid types, sequential connectivities can be obtained for large stretches of the transmembrane segments where number and length of stretches consisting exclusively of these six amino acid types are enhanced compared with the remainder of the protein. We show by experiment as well as by statistical analysis that this labeling scheme provides a large number of sequential connectivities in transmembrane regions and thus constitutes a tool for the efficient assignment of membrane protein backbone resonances.

The better tag remains unseen.

One of the most crucial steps in protein structure determination by nuclear magnetic resonance (NMR) spectroscopy is the preparation of highly concentrated and well behaving protein samples. Here we present a system of modular tags which allows for high level expression, sophisticated purification of full-length protein, and solubility enhancement while keeping the amount of additional resonances low. This system consists of two different expression constructs and utilizes the tight binding of human calmodulin (hCaM) to the calmodulin binding peptide (CBP), which has already been used as a purification tag.

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.

Strategies for the cell-free expression of membrane proteins.

Cell-free expression offers an interesting alternative method to produce membrane proteins in high amounts. Elimination of toxicity problems, reduced proteolytic degradation and a nearly unrestricted option to supply potentially beneficial compounds like cofactors, ligands or chaperones into the reaction are general advantages of cell-free expression systems. Furthermore, the membrane proteins may be translated directly into appropriate hydrophobic and membrane-mimetic surrogates, which might offer significant benefits for the functional folding of the synthesized proteins. Cell-free expression is a rapidly developing and highly versatile technique and several systems of both, prokaryotic and eukaryotic origins, have been established. We provide protocols for the cell-free expression of membrane proteins in different modes including their expression as precipitate as well as their direct synthesis into detergent micelles or lipid bilayers.

Membrane protein expression in cell-free systems.

Cell-free expression has emerged as a promising tool for the fast and efficient production of membrane proteins. The rapidly growing number of successfully produced targets in combination with the continuous development of new applications significantly promotes the distribution of this technology. Membrane protein synthesis by cell-free expression does not appear to be restricted by origin, size or topology of the target, and its global application is therefore a highly valuable characteristic. The technology is relatively fast to establish in standard biochemical labs, and it does not require expensive equipment. Moreover, it enables the production of membrane proteins in completely new modes, like the direct translation into detergent micelles, which is not possible with any other expression system. In this protocol, we focus on the currently most efficient cell-free expression system for membrane proteins based on Escherichia coli extracts.

E-cadherin but not beta-catenin expression is decreased in laryngeal biopsies from patients with laryngopharyngeal reflux.

Abnormal exposure of acid refluxate on the esophageal mucosa has been shown to decrease the epithelial barrier function through an alteration in the intercellular junctional complex. However, only few studies have examined the molecular effects caused by abnormal exposure of gastric refluxate on the laryngeal epithelium. E-cadherin and beta-catenin are cell membrane-associated proteins playing a major role in the maintenance of cell-cell adhesion in epithelial tissues. In this study we tried to analyse the molecular effect of laryngopharyngeal reflux (LPR) on the cellular expression of these proteins. Therefore, we compared the expression of E-caherin and beta-catenin in laryngeal biopsies from patients with and without pH-documented laryngopharyngeal reflux. Paraffin-embedded archival laryngeal biopsies taken from 21 patients, who had undergone rigid laryngoscopy under general anaesthesia and postoperative 24-h pH monitoring, were evaluated immunohistochemically with antibodies to E-cadherin and beta-catenin. The membrane expression of the two proteins was categorized in no expression, mild, moderate and strong (grade 0-3). In LPR patients (n=14) the mean grade of E-cadherin and beta-catenin expression was 1.57 and 1.21, while in specimens of patients without pH-documented LPR it was 2.57 and 1.29. The difference in E-cadherin expression was statistically significant (P=0.011). From our findings we conclude that LPR can cause a decrease in the laryngeal expression of E-cadherin but not of beta-catenin. The reduction of E-cadherin-mediated adhesion could contribute to the development of laryngeal neoplasms. E-cadherin immunostaining of laryngeal biopsies could be a further diagnostic tool to confirm the diagnosis in patients with suspected LPR.

Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems.

Cell-free expression is emerging as a prime method for the rapid production of preparative quantities of high-quality membrane protein samples. The technology facilitates easy access to large numbers of proteins that have been extremely difficult to obtain. Most frequently used are cell-free systems based on extracts of Escherichia coli cells, and the reaction procedures are reliable and efficient. This protocol describes the preparation of all essential reaction components such as the E. coli cell extract, T7 RNA polymerase, DNA templates as well as the individual stock solutions. The setups of expression reactions in analytical and preparative scales, including a variety of reaction designs, are illustrated. We provide detailed reaction schemes that allow the preparation of milligram amounts of functionally folded membrane proteins of prokaryotic and eukaryotic origin in less than 24 h.