Tag: article

Yanira’s comment on: Intracellular Guest Exchange between Dynamic Supramolecular Hosts

By Yanira Rodríguez Valdéz

YRV blog image 18-05

The article by Swaminathan et al. studies the Förster Resonance Energy Transfer (FRET) between two dyes encapsulated in self-assembled nanoparticles made from amphiphilic polymers. The encapsulation of hydrophobic chromophores such as borondipyrromethene (BODIPY) and anthracene inside the hydrophobic interior of these nanoparticles allows their study in aqueous media. In close proximity (inside the same host), BODIPY and anthracene undergo FRET (anthracene = donor, BODIPY = acceptor). The authors observed that mixing nanoparticles loaded with anthracene and nanoparticles loaded with BODIPY has the same effect as loading nanoparticles with both anthracene and BODIPY, which suggests guests being exchanged when nanoparticles are mixed and that they are being captured by the same nanoparticle(s). These studies are performed both in aqueous media and in vitro (HeLa cells), which confirms that the guests can also be exchanged intracellularly.

Two mechanisms for guest transfer between hosts are proposed: 1) guests escape a host and subsequently are captured by another or 2) collision between hosts leads to exchange of guests. They indicate that because of the poor aqueous solubility of the fluorophore guests, mechanism 2 is more likely, but the authors do not perform any experiments to confirm this. They also state that 10-fold dilution with PBS does not affect energy-transfer efficiency. This is not expected if mechanism 2 is at play. Dilution would lead to less frequent collisions between hosts, and therefore, to less guest exchange, and less energy-transfer. The energy-transfer observed could be explained by diffusion, which is more akin to mechanism 1. Poor solubility might not be an issue, because as long as a small amount is soluble, escapes the host and is further entrapped by a host carrying the opposite guest, energy-transfer can still occur.

The authors attempt to determine where the supramolecular assemblies are localized intracellularly by incubating HeLa cells with two sets of nanoparticles, loaded separately with anthracene and BODIPY, and either chlorpromazine or genistein, which inhibit either clathrin- or caveolae-mediated endocytosis, respectively. The results indicated that intracellular fluorescence was reduced to a greater extent by the addition of chlorpromazine, which suggests that the particles are predominantly internalized by clathrin-mediated endocytosis. However, the authors do not perform further experiments that show the particles localized in endosomes and/or lysosomes, which would result from clathrin-mediated endocytosis. They seem to be attempting to rule out any other methods of internalization, but given that chlorpromazine only reduced the intracellular fluorescence to 59%, other methods of internalization could still be taking place.

Swaminathan, 2014. Intracellular Guest Exchange between Dynamic Supramolecular Hosts

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Carla’s comment on: Active Targeting of the Nucleus Using Nonpeptidic Boronate Tags

By Carla M. Quiñones

CMQ blog image 18-05

The main objective of the research described in this article by Rotello and coworkers was to use a synthetic non-peptide targeting motif that accesses the nucleus of the cell through an active transport mechanism. Active and passive transport take place in the cellular membrane as well as in the nuclear membrane. Intracellular targeting is as important as cellular targeting due to their relevance in drug delivery and potential therapies. The two challenges faced by the research group was to deliver the nuclear-targeted protein into the cytosol and to comprehend the mode of nuclear entry. They delivered the protein successfully to the cytosol with the help of a previously synthesised nanoparticle stabilized particle, which encapsulated the proteins of interest and liberated them into the cytosol via membrane fusion. The most significant finding was the efficiency of targeting the nucleus when modifying each protein of interest with benzyl boronate tags (BB tags).
They performed experiments with different proteins modified with BB tags resulting in a successful high-efficiency delivery to the nucleus. To assess the role of boronic acid in the BB tags, they modified GFP with the benzyl tag alone and saw less fluorescence inside the nucleus. This suggests that the boronic acid is necessary in the BB tags for a successful nuclear targeting. Furthermore, they determined if the mechanism of transport into the nucleus was either active or passive. To accomplish this goal, they added Ivermectin, an inhibitor for the α/β importin pathway (active transport), to the cells and also depleted ATP (required for all active transport pathways) in another set of experiments. They delivered GFP with 3 BB tags in both cases and saw a lower nuclear efficiency. They concluded that these modified proteins targeted the nucleus effectively through the importin α/β pathway (active transport) rather than passive transport.
Through the last part of the paper, I was curious of how they acknowledged that this happened the way the proposed since they didn’t explain the mechanism behind the α/β importin pathway, nor the chemistry of how the BB tag contributes to the nuclear targeting. Intrigued by this, I searched more about this phenomenon, but found no relevant studies, probably because this is a novel research field. In general, this was a good article and useful to our lab since we work with supramolecular systems which, in the future, we could adapt this strategy to achieve nuclear targeting.

 

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Diana’s comment on: Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers

By Diana Silva Brenes

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Relating molecular structure to function is the first step and one of the greatest challenge to understand nature’s designs or to make novel “functional designs” of our own. This paper by the Chilkoti group begins with statistical analysis of some of the most relevant proteins displaying LCST and UCST behavior. By analyzing the peptide sequences, the authors identify as common motif for both behaviors a high glycine & proline content. Furthermore, for LCST abundance of aromatic residues seems to be a requirement whereas UCST peptides seem to be encoded by a pair of zwitterionic residues.
To test if these observations lead to LCST/UCST phenomena, over 80 model peptides were recombinantly synthesized and their thermoresponsive behavior was measured by UV absorbance while changing the temperature. Each peptide presented the predicted behavior, giving support to their observations. Furthermore, by comparing a few selected examples, they show how an increase in hydrophobicity leads to an increased UCST cloud point and how eliminating one of the residues from azwitterionic pair turns a UCST peptide to an LCST peptide.
The LCST and UCST behavior is, however, a complex phenomenon dependent on protein-protein versus protein-water interactions, which in turn are modulated by more factors aside from the sequence of the protein. The possible scenarios are limitless, and the authors give insight on the most significant: peptide length, concentration, and pH (charge state of protonable atoms).
The robustness of the behavior encoded in the rules they found can be seen by a hybrid peptide containing both an LCST portion and a UCST one. The resulting peptide displays both behaviors, albeit at different temperatures from the “parent” sequences.
Finally, the authors show that searching for the characteristics they determined as important for LCST/UCST behavior throughout the human proteome produces examples of proteins whose function could very well be related to a thermoresponsive behavior, highlighting the applicability of their observations to understand the phenomena that make life as we know it possible.

Quiroz, 2015. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers

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Luxene’s comment on: Selective Tuning of Elastin-like Polypeptide Properties via Methionine Oxidation

By Luxene Belfleur

Luxene blog image 18-05

Figure 1. Kekule structure of and synthesis of ELP 2 and 3. Cartoon representation of ELPs solution, the LCST behavior and measurement of ELPs

In this article, the authors reported the LCST modulation of Elastin-like polymers (ELP) by the modification towards oxidation reactions of the methionine thioester group of the ELP 1 to form sulfoxide and sulfone ELPs derivatives 2 and 3 respectively (Figure 1). They isolated ELP 1 from plasmid DNA of E.coli bacteria and purified it by SDS-Page. In acidic media, using 30% hydrogen peroxide and 1% of acetic acid or formic acid in water, they obtained ELP derivatives 2 and 3 respectively and the molecular weight and structural elucidation of ELP 1 and its derivatives 2 and 3 had been examined and confirmed by mass spectrometry and NMR respectively.
They performed turbidity experiments to determine the cloud points of those three ELPs. As expected, both ELP derivatives 2 and 3 had a higher LCST behavior (55 °C and 43 °C for 2 and 3 respectively) compared to ELP 1 which is 25 °C (Figure 1). This is due to the increasing of water solubility of the modified ELPs (2 and 3), which required more energy to break the interaction between water and them (ELP 2 and 3) in order to evacuate the hydration shell around these ELPs and favored the collapse of the letters. Moreover, it was expected that ELP 3 would have a higher cloud point than the ELP 2 counterpart, however, this was not the case because the large dipole moment of ELP 3 favored intramolecular and intermolecular interactions between them and the protein respectively which promoted a decrease of the water solubility. They evaluated the influence of the I¯ and NO3¯ anions on the phase transition of the three ELPs and found that those anions had no significant effect on the LCST point of the ELP 1 while I¯ increased the LCST of ELP 2 and NO3¯ decreased the solubility of ELP 3 in agreement with the Hofmeister series effect.
They reported an interesting and straightforward work by turning the LCST behavior of ELPs towards synthetic modification. This work has inspired and motivated my team for designing and synthesizing sulfoxides and sulfones containing guanosine derivatives in order to both, stabilize the thioester containing guanosine derivatives compounds that we synthesize, and modulate the LCST properties of the SGQs that will be made from oxidizing 8ArG derivatives product.

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Exploring the periodic table with supramolecular G-quadruplexes

20130815_mAGi-C+_TOC

 

Our latest article just came out on the web: Tuning supramolecular G-quadruplexes with mono- and divalent cations (Mariana Martín-Hidalgo, Marilyn García-Arriaga, Fernando González, José M. Rivera,  Supramolecular Chemistry, DOI: 10.1080/10610278.2014.924626) In it we describe a series of systematic studies of the effect of a series of cations on the structure and other properties of supramolecular G-quadruplexes. You can download a free (!) copy of the article here, but you better hurry as this is a limited time offer available only to the first 50 people to take advantage of it. If, however, you are not one of the “50 lucky winners”, don’t despair, you can still get a copy article by simply leaving a request in the comments. In the meantime, here’s the abstract:

“Supramolecular G-quadruplexes (SGQs) are formed via the cation promoted self-assembly of guanine derivatives into stacks of planar hydrogen-bonded tetramers. Here, we present results on the formation of SGQs made from the 8-(m-acetylphenyl)-2′-deoxyguanosine (mAGi) derivative in the presence of various mono- and divalent cations. NMR and HR ESI-MS data indicate that varying the cation can efficiently tune the molecularity, the fidelity and stability (thermal and kinetic) of the resulting SGQs. The results show that, parallel to the previously reported potassium-templated hexadecamer (mAGi16·3K+), Na+, Rb+and NH4+ also promote the formation of similar supramolecules with high fidelity and molecularity. In contrast, the divalent cations Pb2+, Sr2+ and Ba2+ template the formation of octamers (mAGi8), with the latter two inducing higher thermal stabilities. Molecular dynamics simulations for the hexadecamers containing monovalent cations enabled critical insights that help explain the experimental observations.”

 

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