Month: May 2018

María’s comment on: Artificially Engineered Protein Hydrogels Adapted from the Nucleoporin Nsp1 for Selective Biomolecular Transport

Maria Luisa Adrover

 

MLAC blog image 18-05

The main goal of the article by the Olsen group is to present a new protein hydrogel composed of nucleoporin like polypeptides that allow a selective transport of biomolecules into the formed hydrogel. The engineering of this system was inspired by the selectivity of the protein matrix in the nuclear pore complex. The nuclear pore complex is composed of nucleoporins like Nsp1 (16 repeats of a 19 amino acid sequence). These are proteins containing Phe-Gly (FG) repeats that contribute to specific binding of the nuclear transport receptors. The authors took advantage of the finding that individual nucleoporins can form hydrogels in vitro. They designed three different nucleoporin-like polypeptides (NLP) using pentameric (P) coiled-coil domains in the C and N terminus of the polypeptide: P-1NLP-P, P-2NLP-P and P-cNsp1-P (the original nucleoporin).

In their experiments, they conducted rheology studies (using oscillations as a type stress) to evaluate the flow properties of the protein hydrogel. They found that the hydrogel in behaves in some cases as a viscous liquid (when G“ > G`) while in other cases it behaves as an elastic solid (when G` > G“). Also, they noticed by adding hexanediol, the FG repeats were weakened making the hydrogel less elastic. Another study was done using Raman spectroscopy too see any changes in the peaks of the spectra. They saw that by adding hexanediol there was a reduction the Phe vibrational mode of 486 cm-1 meaning that the FG repeats where indeed being disrupted. Lastly, using capillary experiments they studied the selective transport of the protein hydrogels. They studied the selectivity of the biomolecules measuring the fluorescence intensity of: Importin β, IBB-MBP-EGFP (Importin β binding with maltose binding protein and enhanced green fluorescent protein) and MBP-mCherry. Fluorescence studies showed that Importin β was diffused inside P-cNsp1-P hydrogel with the highest intensity, followed by P-2NLP-P and lastly P-1NLP-P. Interestingly, they showed that just by changing one amino acid (Serine to Aspartic Acid) the selectivity for Importin β changed. This suggested that small changes to the nucleoporin like polypeptides affects the selectivity. Adding 10% hexanediol to P-2NLP-P barely showed fluorescence mainly due to the FG disruptions mentioned before. Finally, a successful engineering of hydrogels was achieved showing a high selectivity just like the natural nucleoporin system. A relatively simple polypeptide chain just like the cNsp1 allow the design of nucleoporin-like polypeptides as selective as the protein matrix found inside the nuclear pore complex.

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Maxier’s comment on: Macromolecular Crowding Modifies the Impact of Specific Hofmeister Ions on the Coil–Globule Transition of PNIPAM

By Maxier Acosta Santiago

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While the recently discussed Cremer paper described efforts towards understanding the effect on the LCST of PNIPAM by salts of the Hofmeister’s series, Sakota’s article went somewhat deeper into the study of this phenomenon by taking in consideration molecular crowding. Sakota’s group decided to use a PEG polymer as a crowding agent. Crowding may affect the biding of anions from the Hofmeister’s series and the PNIPAM resulting in a change in the LCST. First, they studied the presence of PEG in a PNIPAM solution showing that the crowding agent reduces the LCST. Kosmotropic anions, that decrease the LCST, but chaotropes increase the LCST. For the Hofemeister series effect on LCST we can go back to Luis Prieto’s blog post and Cremer’s paper which explains this effect better.

When the article begins to look at the presence of PEG at different salt concentrations, they see a close correlation between the LCST and the Hofmeister series. Yet, for the chaotropes ClO4⁻ and SCN⁻, the presence of PEG lead to a larger increase in the LCST. From here they decide to apply different theories to explain the results. Within the examined theories they discuss around thermodynamics of the system. Their explanation evolves as follows, even if the organization via LCST of PNIPAM is not thermodynamically favorable, the overlapping excluded volumes of PEG and the PNIPAM particles increase the translational entropy of water molecules in solution, which makes the formation of the system possible.

Although this paper brings something new to the table to discuss (molecular crowding and LCST), I do have some concerns. When taking into consideration so many different factors like molecular crowding, salt, and the responsive system itself, we should look deeper into the behavior. To limit certain factors, they maintained certain constant concentrations throughout the paper. Yet, in the discussion, I feel they lacked additional experimental results or computational studies (using molecular and/or coarse-grained simulations) to support their theoretical thermodynamics analysis.

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Minelise’s comment on: Synthesis and Direct Observation of Thermoresponsive DNA Copolymers

By Minelise E. Rivera

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In this paper, Li and Schroeder use single molecule techniques to have a direct observation on DNA-PNIPAM copolymers. First, they synthetized DNA-PNIPAM copolymers using a two-step strategy based on polymerase chain reaction (PCR) for generating linear DNA backbones containing dibenzocyclooctyne-dUTP, then grafted thermoresponsive side branches (PNIPAM) onto DNA backbones using copper-free click chemistry. Subsequent single molecule fluorescence spectroscopy studies unveiled more clearly the molecular heterogeneity association with the stretching and relaxing of the polymer above and below their LCST. Their results showed that intramolecular conformational dynamics of DNA-PNIPAM copolymers are affected by properties of the branches like molecular weight, density, leading to a change in transition temperatures. In other words, the single molecule technique provided a better understanding in a molecular perspective of chemically heterogeneous and stimuli-response polymers.

As I read this paper and looked for information to better understand it, I was amazed by the details with which they worked with throughout their study. I would have thought of working better with a bunch of them instead of just single molecules. It didn’t cross my mind that someone was going to, not only synthesized the molecule, but also study its characteristics. It was very interesting to learn about the methods that they used for characterization and synthesis. It got me wondering if those methods were the only ones that would work in this case and why. But, what I think that was very useful for me is that I got to understand better the importance of the LCST and the role that it played in their system. It reminded me of our project in which the SGQ self-assembles into the SHS and how it is to study it and understand its influence in the SHS as it was important for the copolymers with which it was worked with in the paper.

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Kiara’s comment on: A 2,7-diamino-1,4,8-triazanaphthalene derivative selectively binds to cytosine bulge DNA only at a weakly acidic pH

By Kiara N. Villa Del Valle

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This paper focuses reports how through a change in the pH, their compound gets protonated and through hydrogen bonds, the compound and the base binds. Lastly, the better-known DNA structure the double helix, but there are other varieties such as hairpin loops, interior loops, multi-branched loops and bulge loops. Bulge loops, are when an extra base winds up on one side of the DNA strand. This can happen when the helix missing a base or an extra one was inserted during the DNA copying process. This anomaly can cause cancer or triplet diseases. They first performed experiments to determine the pH dependence if the azaDANP. They changed the pH from 1.0 to 9.0 and it shows a hypochromic and hypsochromic shifts as the pH was changing. They also plotted the absorption in terms of the pH and determined a pKaH (pKa of the protonated form) of 4.3, a lower pKaH of 6.8 for a previously described compound. This decrease was due to the decreased basicity of the ring nitrogen by the substitution at C4-N. To get information about the azaDANP binding to the C-bulge, they simulated possible complexes between azaDANP and cytosine, it showed that the protonation likely occurs at N1 for the complex formation. They performed more experiments to measure the absorption spectra of azaDANP in presence of T, G, A and C bulged DNA, at pH 7.0 and 5.5 at room temperature. We want to focus in this, at 5.5 pH there is a new absorption band at 407 nm that in neutral pH is not observed and therefore suggesting that the formation is pH sensitive. The thermal stability of the azaDANP-C-bulge was investigated with absorption with variable temperature. We can see that the peak observed at 407 nm at 2 °C decreases as the temperature is increased to 80 °C. This shows the equilibrium of azaDANP and GCG/CC DNA to form a binary complex and, therefore, appearing to be temperature dependent.

 

Aikawa, 2017. A 2,7-diamino-1,4,8-triazanaphthalene derivative selectively binds to cytosine bulge DNA only at a weakly acidic pH

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José’s comment on: Intelligent, Biodegradable, and Self-Healing Hydrogels Utilizing DNA Quadruplexes

By José M. Martinez

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This paper is about making of a water based gel made from the supramolecular interactions of guanosine rich oligonucleotides. They attached these oligonucleotides symmetrically into the ends of PEG chains of variable lengths and conformations. This was made through High Efficiency Liquid Phase (HELP) synthesis. This synthetic strategy, as its name says, occurs in a liquid phase instead of the usual solid phase synthesis. The purpose of this is to allow them to obtain greater quantities of product more easily without risking the effectiveness. These DNA-PEG-DNA monomers have the ability, when diluted and presented with a cation (K+ or Na+), to self-assemble its DNA moieties through non-covalent interactions to for G-quadruplexes. Those strong interactions change the physical properties of the solution by forming a hydrogel. In the study, it was proven that the concentration of the cation required to trigger the self-assembly is relatively small. It is even compared to the concentration of salts found in body-related fluids like sweat, saliva, and tears. The hydrogel also has the capability to heal itself and interact by diffusion with another of the same kind. Finally, it was found that by adding a few extra nucleotides to the DNA moiety of the monomer and a matching strand for that part, the G-quadruplexes could be disassembled, returning the gel back to its liquid phase. All the previously stated properties indicate potential uses of this hydrogel for biomedical purposes.
Regarding myself, I learned mostly about the molecular aspect of gels and how they are formed. It was very interesting seeing the role that the supramolecular interactions played in the formation of this particular gel and the experiments made to investigate further into its properties.

JMM: Tanaka, 2017. Intelligent, Biodegradable, and Self-Healing Hydrogels Utilizing DNA Quadruplexes

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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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Valeria’s comment on: Reversible Regulation of Thermoresponsive Property of Dithiomaleimide-Containing Copolymers via Sequential Thiol Exchange Reactions

By Valeria Burgos Caldero

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The main purpose of this article was to synthesize multi-responsive polymers that could be reversibly modified to adjust their LCST. Indeed, these researchers were able to develop a system in which multiple thiol exchanges were made, and in turn, they could determine how the thiols affected the transition temperature of the polymer. They used a copolymer containing P(TEGA) and DMMA. By performing transmission measurements at various temperatures, they concluded that as the thiol changed, the transition temperature of polymer varied depending on the resulting hydrophobicity. More polar functionalities increased the transition temperature and less polar ones decreased it. They were able to demonstrate the reversibility of the modifications since they managed to return to their original functionality after various thiol exchanges. Finally, they implemented a fluorescence signal to monitor the reaction progress. They found that thioglucose quenches the polymer’s fluorescence while making the compound soluble throughout the range of temperatures. With these findings, a wide range of possibilities were opened, since now, if you want a polymer for a specific type of function where a specific temperature response is needed, it is easily accessible by adding the corresponding thiol to the polymer solution. The mechanism of turning off the fluorescence may give access to reversible systems in aqueous conditions.
In general, I found it much simpler to prepare for this article than for the first one I presented. I feel that by doing these exercises of presenting scientific articles I have been acquiring maturity in the analysis process since it was difficult for me to understand articles in the beginning. Something that I found missing in the article is that they never explained the experimental procedure on how they achieved reversibility after adding different thiols to the same sample. I liked that they used common thiols, some of which we use in our research and others that maybe we could apply. In general, the article relates a lot to the research I’m doing with Diana. It could be useful to try to see the stimuli-responsive variations in the compounds that we are synthesizing. Maybe because it is related to variations in functionalities with thiols, similar to my own research, I found it more enjoyable to prepare the discussion and understand the material in the article.

Tang, 2016. Reversible Regulation of Thermoresponsive Property of Dithiomaleimide-Containing Copolymers via Sequential Thiol Exchange Reactions

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

By Luxene Belfleur

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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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Luis’ comment on: Guanidinium can both Cause and Prevent the Hydrophobic Collapse of Biomacromolecules

By Luis A. Prieto

LAPC-Blog-GM1805

Heyda, Jungwirth and Cremer collaborated in a study of guanidinium (Gnd+) salts and their effect in lower critical solution temperature (LCST). They studied molecular details of the cause of these transitions by IR-ATR and molecular dynamics simulation where they wanted to understand how Gnd+ salts interacted with the backbone of the Elastin-like peptides (ELP). In previous studies ELPs showed a change in LCST that followed the Hofmeister series in sodium salts but using Gnd+ salts proved to be different, especially Guanidinium thiocyanate (GndSCN) that at low concentrations the LCST decreases, but at high concentrations the LCST increases. They studied particular phenomenon using ATR-IR where they found GndSCN binds strongly with ELPs and resulted in an interesting behavior when the concentration of salt is increased. At low concentrations the polymer collapsed (salting-out) because of cross-linking of the peptide and at high concentrations resolubilization occurred (salting-in). Other salts followed typical behavior of salting-in (guanidinium chloride, weak binding) or salting-out (guanidinium sulfate, poor binding). Coarse-grain and all atom simulations corroborated this finding where they found particular detail of the interaction of the carbonyl groups of the peptide backbone with Gnd+, most likely through H-bonds.

The thermodynamics of this paper I found particularly interesting since it reminded me of everything that I have to re-learn.  An attractive experiment was that they used a melting point apparatus to measure the LCST, meaning the use of small amount of sample to gather fundamental information of the system which is also the case with ATR-IR. An elegant work and also inspirational since our lab works with responsive systems and we will definitively see if we can do the LCST measurement with a melting point instrument. I got to say that I particularly like the all atom simulations and Figure 5, where we can see in molecular detail the interactions of the salts with the peptide where thiocyanate and Gnd+ interact strongly with the hydrophobic parts (V, G) and hydrophilic part (peptide bond), respectively.

LAPC: Heyda, 2017. Guanidinium can both Cause and Prevent the Hydrophobic Collapse of Biomacromolecules

 

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