Elena Pazos

Highly Sensitive SERS Quantification of the Oncogenic Protein c‑Jun in Cellular Extracts

A surface-enhanced Raman scattering (SERS)-based sensor was developed for the detection of the oncoprotein c-Jun at nanomolar levels. c-Jun is a member of the bZIP (basic zipper) family of dimeric transcriptional activators, and its overexpression has been associated with carcinogenic mechanisms in several human cancers. For our sensing purpose, we exploited the ability of c-Jun to heterodimerize with its native protein partner, c-Fos, and therefore designed a c-Fos peptide receptor chemically modified to incorporate a thiophenol (TP) group at the N-terminal site. The TP functionality anchors the c-Fos protein onto the metal substrate and works as an effective SERS probe to sense the structural rearrangements associated with the c-Fos/c-Jun heterodimerization.

DNA Recognition by Synthetic Constructs

The interaction of transcription factors with specific DNA sites is key for the regulation of gene expression. Despite the availability of a large body of structural data on protein–DNA complexes, we are still far from fully understanding the molecular and biophysical bases underlying such interactions. Therefore, the development of non‐natural agents that can reproduce the DNA‐recognition properties of natural transcription factors remains a major and challenging goal in chemical biology. In this review we summarize the basics of double‐stranded DNA recognition by transcription factors, and describe recent developments in the design and preparation of synthetic DNA binders. We mainly focus on synthetic peptides that have been designed by following the DNA interaction of natural proteins, and we discuss how the tools of organic synthesis can be used to make artificial constructs equipped with functionalities that introduce additional properties to the recognition process, such as sensing and controllability.

Cyclin A Probes by Means of Intermolecular Sensitization of Terbium-Chelating Peptides

Intermolecular sensitization of lanthanide ions was effectively implemented in the development of fluorescent sensors targeting cyclin A. A chelating unit has been conjugated to peptides containing a known cyclin A binding motif (CBM). Upon interaction of the modified terbium-chelating peptides with the cyclin A substrate recruitment groove, the Tb3+ ion is placed in the vicinity of the Trp217 side chain, which results in efficient intermolecular terbium sensitization and specific long wavelength fluorescent emission from the metal center.

Temporary Electrostatic Impairment of DNA Recognition: Light-Driven DNA Binding of Peptide Dimers†

Gene expression relies on a myriad of carefully orchestrated interactions between specialized proteins called transcription factors (TFs) and regulatory DNA sequences. In general, such interactions are subtly regulated in time and space, so that many TFs remain inactive until receiving an appropriate activation signal. It is well-established that the DNA readout by TFs largely relies on interactions between amino acid side chains and the DNA bases and phosphates. Among these contacts, those involving positively charged basic amino acids are critical for the thermodynamic stability of their DNA complexes. We reasoned that the temporary electrostatic deactivation of such contacts might provide for the development of TF-based systems where DNA binding activity could be externally controllable, for instance by light. These systems could be useful methods for transcriptional control, or for probing spatiotemporal patterns of gene expression in living organisms. It is curious that despite the well-established use of light-activated compounds in chemical biology, examples of photocontrolled DNA binding are certainly scarce. These include reversible switches based on the photostationary equilibrium of azo-modified DNA binders, special chromophores with poor DNA binding affinity and/or specificity, and single-use caging strategies for triggering minor groove binding or intercalation. Thus, inspired by the use of negatively charged elements to modulate cell internalization, we sought to develop a general strategy for photocontrolling the sequencespecific DNA binding of TF peptide mimics. Herein we demonstrate that tethering polyanionic tails to basic DNA-binding bZIP peptides through a light-sensitive linker suppresses the DNA interaction. Upon irradiation, the negatively charged appendages are released, and the DNAbinding activity is thus restored. As reference system for implementing the strategy we chose the GCN4 transcription factor, an archetypical bZIP TF that specifically binds to ATF/CREB (5’-ATGA(c/g)TCAT-3’) or AP1 (5’-ATGA(c)TCAT-3’) sites as a leucine zipper-mediated dimer of uninterrupted a helices. The N-terminal basic regions feature many positively charged amino acids that are key for the DNA recognition (10 Lys or Arg out of 31 residues in the basic region; Figure 1, GCN4br). It has been shown that the leucine zipper itself can be substituted by a number of dimerizing units without significant loss in the DNA binding properties. Therefore, in our first iteration for the design of the electrostatically impaired DNA-binding peptides, we selected the minimum sequence of the bZIP basic region (br) that it is known to retain the DNA binding ability when engineered as a disulfide dimer. This minimal peptide was extended at the N-terminus by adding acidic extensions with four or eight Glu residues linked to the core br sequence

Advances in lanthanide-based luminescent peptide probes for monitoring the activity of kinase and phosphatase

Signaling pathways based on protein phosphorylation and dephosphorylation play critical roles in the orchestration of complex biochemical events and form the core of most signaling pathways in cells (i.e. cell cycle regulation, cell motility, apoptosis, etc.). The understanding of these complex signaling networks is based largely on the biochemical study of their components, i.e. kinases and phosphatases. The development of luminescent sensors for monitoring kinase and phosphatase activity is therefore an active field of research. Examples in the literature usually rely on the modulation of the fluorescence emission of organic fluorophores. However, given the exceptional photophysical properties of lanthanide ions, there is an increased interest in their application as emissive species for monitoring kinase and phosphatase activity. This review summarizes the advances in the development of lanthanide‐based luminescent peptide sensors as tools for the study of kinases and phosphatases and provides a critical description of current examples and synthetic approaches to understand these lanthanide‐based luminescent peptide sensors.

Ultrasensitive multiplex optical quantification of bacteria in large samples of biofluids

Efficient treatments in bacterial infections require the fast and accurate recognition of pathogens, with concentrations as low as one per milliliter in the case of septicemia. Detecting and quantifying bacteria in such low concentrations is challenging and typically demands cultures of large samples of blood (~1 milliliter) extending over 24–72 hours. This delay seriously compromises the health of patients. Here we demonstrate a fast microorganism optical detection system for the exhaustive identification and quantification of pathogens in volumes of biofluids with clinical relevance (~1 milliliter) in minutes. We drive each type of bacteria to accumulate antibody functionalized SERS-labelled silver nanoparticles. Particle aggregation on the bacteria membranes renders dense arrays of inter-particle gaps in which the Raman signal is exponentially amplified by several orders of magnitude relative to the dispersed particles. This enables a multiplex identification of the microorganisms through the molecule-specific spectral fingerprints.

A Folding-Based Approach for the Luminescent Detection of a Short RNA Hairpin

We report the rational design of a 20-mer basic peptide, derived from the transcriptional antitermination protein N of bacteriophage P22, equipped with a luminescent DOTA[Tb(3+)] macrocyclic complex and a sensitizing tryptophan antenna. Folding of this peptide into an α helical conformation, which occurs upon binding to its target boxB RNA hairpin, results in a large increase in luminescence emission. Therefore, the peptide construct works as a highly sensitive and selective probe for this RNA hairpin.

Rational design of a cyclin A fluorescent peptide sensor

We report the design and development of a fluorescent sensor specifically designed to target cyclin A, a protein that plays a key role in the regulation of the cell cycle. Computational studies provide a molecular picture that explains the observed emission increase, suggesting that the 4-DMAP fluorophore in the peptide is protected from the bulk solvent when inserted into the hydrophobic binding groove of cyclin A.

Sensing coiled-coil proteins through conformational modulation of energy transfer processes – selective detection of the oncogenic transcription factor c-Jun

Designed terbium-chelating peptides were used to monitor the formation of leucine zipper associations. The sensing strategy is based on the modulation of the antenna effect from a Trp residue donor and the luminescence increase arising from the folding that takes place upon the formation of the coiled-coil structure. An application in the specific sensing of the oncogenic c-Jun transcription factor is demonstrated.

Identification of Cyclin A Binders with a Fluorescent Peptide Sensor

A peptide sensor that integrates the 4-dimethylaminophthalimide (4-DMAP) fluorophore in a short cyclin A binding sequence displays a large fluorescence emission increase upon interacting with the cyclin A Binding Groove (CBG). Competitive displacement assays of this probe allow the straightforward identification of peptides that interact with the CBG, which could potentially block the recognition of CDK/cyclin A kinase substrates.