The synthesis of an EDTA-like chelating peptidomimetic building block suitable for solid-phase peptide synthesis.

A novo trifunctional EDTA-like peptidomimetic amino acid is described. This unique building block, which is prepared in a straightforward manner from commercialized starting materials, contains three moieties: a hexadentate chelating unit similar to that present in EDTA, and amino and carboxylic groups, which facilitate its introduction into the backbone of peptides using conventional SPPS. As a proof of concept, this building block is introduced into a cyclic peptide inspired from the family of Gratisin analogues. The designed peptide contains the amino acid analogue in one of the turns, and chelates Ca2+ with nanomolar affinity at physiological pH.

ADP-ribose-derived nuclear ATP synthesis by NUDIX5 is required for chromatin remodeling.

Key nuclear processes in eukaryotes, including DNA replication, repair, and gene regulation, require extensive chromatin remodeling catalyzed by energy-consuming enzymes. It remains unclear how the ATP demands of such processes are met in response to rapid stimuli. We analyzed this question in the context of the massive gene regulation changes induced by progestins in breast cancer cells and found that ATP is generated in the cell nucleus via the hydrolysis of poly(ADP-ribose) to ADP-ribose. In the presence of pyrophosphate, ADP-ribose is used by the pyrophosphatase NUDIX5 to generate nuclear ATP. The nuclear source of ATP is essential for hormone-induced chromatin remodeling, transcriptional regulation, and cell proliferation.

Structural Analysis of the Pin1-CPEB1 interaction and its potential role in CPEB1 degradation.

The Cytoplasmic Polyadenylation Element Binding proteins are RNA binding proteins involved in the translational regulation of mRNA. During cell cycle progression, CPEB1 is labeled for degradation by phosphorylation-dependent ubiquitination by the SCF(ß-TrCP) ligase. The peptidyl-prolyl isomerase Pin1 plays a key role in CPEB1 degradation. Conditioned by the cell cycle stage, CPEB1 and Pin1 interactions occur in a phosphorylation-independent or -dependent manner. CPEB1 contains six potential phosphorylatable Pin1 binding sites. Using a set of biophysical techniques, we discovered that the pS210 site is unique, since it displays binding activity not only to the WW domain but also to the prolyl-isomerase domain of Pin1. The NMR structure of the Pin1 WW-CPEB1 pS210 (PDB ID: 2n1o) reveals that the pSerPro motif is bound in trans configuration through contacts with amino acids located in the first turn of the WW domain and the conserved tryptophan in the ß3-strand. NMR relaxation analyses of Pin1 suggest that inter-domain flexibility is conferred by the modulation of the interaction with peptides containing the pS210 site, which is essential for degradation.

RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains.

Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1-4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1-RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins' ability to interact with RNA as demonstrated by ion mobility-mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1-RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the ß-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1-RRM2-CPE complex that illustrates the experimental data.

Rapid spectroscopic velocity quantification using periodically oscillating gradients.

In this work two spectroscopic methods are described which allow rapid flow velocity quantification in the presence of a parabolic velocity distribution. This method requires only a single excitation and is based on flow encoding by periodically oscillating gradients. In the shown spin echo variant additional refocusing pulses correct for field inhomogeneities. A theoretical model is introduced, which describes the course of the derived spectra even in high flow region, where a significant part of the encoded spins leaves the sensitive area of the coil during data acquisition (outflow-effect). It was demonstrated that both methods can quantify flow velocities within the velocity range of 1mm/s up to 36 cm/s in the presence of a parabolic flow velocity distribution. The maximum velocity of the parabolic distribution is indicated in this method by a peak in the acquired spectrum from which the velocity could be quantified. Flow velocity quantification by periodically oscillating gradients seems a reasonable and fast alternative to established imaging techniques.