Fluoroaromatics stacking direct protein assembly

24 04 2012

Stacked Fluoroaromatics as Supramolecular Synthons for Programming Protein Dimerization Specificity

Christopher J. Pace, Hong Zheng, Ruben Mylvaganam, Diane Kim, &  Jianmin Gao*

Angew. Chem. Int. Ed. 2011, 51, 103. DOI: 10.1002/anie.201105857

By Ana Victoria Morales

Because little is known about aromatic interactions for protein design, Gao and colleagues decided to study them. Previous work by this group showed that a stacked phenyl and perfluorophenyl pair controls the dimerization specificity of a protein. That led them to the studies done in this article that involved the analysis of the aromatic stacking energetics by incorporating several stacked aromatic pairs into the protein α2D.

Fluorinated analogues of phenylalanine in the residues Phe10 and Phe29 were incorporated into α2D. All mutant proteins were able to fold into homodimeric complexes. A van’t Hoff analysis was used to study the thermodynamic parameters of the dimerization. The melting temperatures for the α2D homodimers varied from 29-78 ºC, a difference of almost 50 ºC between the wild type and the (Z,Z) which is indicative of the greater hydrophobicity of fluorocarbon compounds. The folding free energies (∆Gf) ranged from -5.9 to -12.8 kcal mol-1. The plot of the folding free energy against LogP of the fluoroaromatic side chains showed a poor correlation as well as the plot of the folding free energy against the surface area. Because of this, other factors than hydrophobicity must be contributing to the stability of the homodimers. Interestingly, the folding stability agreed with the magnitude of the dipole moments, this is indicative that dipole-dipole coupling may be stabilizing the homodimers. For example, (F345F345) showed the most favorable folding free energy between all variants and it had the greatest dipole moment of all the amino acids. Then, another plot was done of the free energy against the combination of the LogP and the dipole moment. It had a much better correlation (R2=93), indicating the contribution of both hydrophobicity and dipole moments of the aromatic rings.

Dipole-induced-dipole interactions were studied on α2D single mutants. The stability is supposed to reveal the best fluorinated phenylalanine analogues for targeting a native phenylalanine. Surprisingly, the (F,Zo) mutant gave the most stable homodimer. This was surprising because Z is more hydrophobic and it was expected to have a more favorable quadrupole with the phenylalanine. This is indicative of the combination of the hydrophobicity and dipole moment for strong stacking interactions with the native aromatic residues. It was then hypothesized that aromatic interactions can direct orthogonal molecular assembly or self-sorting behavior of peptides. Thermodynamic equilibrium through a disulfide cross-linking experiment of a three component system was studied with mutants (F, F), (F345F, F345F), and (Z, Z). Theoretically speaking, random dimerization should have given six species. In the LC-MS experiment only two significant peaks were observed, those of the heterodimer of mutants (F,F) and (Z, Z) which is stabilized by quadrupole interaction and the other (F345F, F345F) homodimer stabilized by the dipole-dipole coupling.

I enjoyed reading this article because I learned a lot, and assume that everybody learns something because as the authors say that this one of the first investigation of the energetics of aromatic stacking in proteins. The article put into perspective how much fluorine could change the energetics of the aromatic residues. There are small but very significant details to be considered such as the amount of fluorines incorporated and their position. Fluorinated amino acids are useful because of the benefit of fluorination in NMR analysis, PET imaging and protein stabilization. The information obtained is very useful because it provides insights for the energetic considerations of incorporating fluorinated aromatic amino acids into target proteins. It also highlights the importance, not only of the hydrophobic contributions, but the dipole contributions to aromatic stacking. Zo was the most efficient in targeting native Phe residues through aromatic stacking, this finding helps in the design of enzyme inhibitors. It was also proven that self-sorting does occur with these peptides by the use of stacked aromatics as supramolecular synthons. Nevertheless, for this last study I would have liked to see more experiments with several combinations of the fluorine analogues to see if the amount of fluorines in the analogue and their position affect the self-sorting. It would have been nice to see if there were other types of assemblies, but that may need another article. Overall, it is a nice article that has to be read carefully to understand the next step and why the experiments were done.




13 responses

26 04 2012

This article presents the first systematic study of the effects of substituting phenylalanine with a synthetic derivative containing fluorine in a synthetic protein. The enhanced hydrophobicity of fluorocarbons is not a novel discovery. The most important result arises from the fact that factors other than the hydrophobicity of the phenylalanine derivative influence the overall stabilization of the proteins. The same factors can also result in self-sorting of otherwise similar proteins, an impressive effect. I find that the manuscript is of adequate length for its contents. The authors don’t dwell on any experiments more than they should.

The authors measured the thermodynamics of the melting of the protein dimers. The control was the “wild-type” containing unsubstituted phenylalanines. The conclusions are supported by the results, as shown by several plots of varying correlation. The peptides were synthesized using solid-state synthesis. The purity of the synthetic peptides was of 95% or better.

The narrative of the article is engaging. It is easy to follow, keeps you in touch with the overall purpose of their experiments and their interpretation of the results.

The synopsis does not have noticeable grammatical errors. What I noticed is faulty sentence construction, which can be improved by practice and appropriate proofreading. I think this article is beyond a “general knowledge of chemistry” as it focuses on a specific type of intermolecular interaction not covered in your average general chemistry class. The blogger did not follow the guidelines but that was to be expected, she did not have them. The picture is not informative; it doesn’t give any notion of what the paper is about. She should have added the fluorine derivatives somewhere and made it more schematic. Ana did not use a single hyperlink in her synopsis, but this can be spared since it is a novel requirement. To improve her future synopses, if any, Ana should double check her sentences to see if they have appropriate syntax and make a picture that tells the story of the article one way or another.

26 04 2012
Luis Negrón

In this article Gao and coworkers “modify” or “modulate” the folding of a protein called α2D by using a “supramolecular synthon” when they mutate the residues Phe 10 and Phe 29 with different fluoroaromatic derivatives. They found that this group provides stability to the system due to the internal stacking that they describe as “unique”. They measure that stability with experimental thermodynamic data of Tm, dH, dG and ddG. They found that better stability in the supramolecular synthons of (Z0Z0)>(ZmZm)>(ZpZp).

I can accept that the “supramolecular synthon” can provide an additional stability to the system, but I don’t think that with the presented thermodynamic data they can attribute on aromatic interactions the general role in stability. Hydrophobicity is complex concept that needs to be taken in consideration better and not only as weak Van der Waals interaction. This simple 35-residue polypeptide has different side chains that make it more complex because those side chains provide additional and specific interactions (H-bond, salt-bridge, etc) that will increase the stability of the system. For this reason, I don’t think that the desire “specificity” is achieved as they said.

One interesting aspect of the article was that this approach can be used for self-sorting of this dimers. This concept of self-sorting can be achieved with this system, but the question is, we need high specificity or how specific has to be this system? Because according with this property, we can obtain different types of self-sorting (narcissistic or social).

About the article, I think that the message is simple, so have to be a short-article. They did a lot of effort not only with the thermodynamic experiments that were performed by thermal denaturation monitored by CD. It will be interesting how the thermodynamic information varies by measuring thermodynamic parameters by DSC.

About the synopsis, it was well written, but was a little complex to understand because for this simple and short article, there was a lot of important information but how is organized or explained is a little complex or challenging. The picture was interesting and helps for the understanding of the article, but a better association with the thermodynamic data or some illustration of the supramolecular synthon could help to understand what they want to address.

26 04 2012
Diana S.

Our article this week performs a systematic study on the effect of hydrogen to fluorine mutations on the interactions between stacked aromatic groups. I guess the aspect that would call most the readers’ attention in this article, as the authors point out, is the large difference brought about by the relatively small permutations performed.
They chose an accessible system: and artificial protein-like peptide, with the advantage that it can be readily prepared through solid phase synthesis, also allowing the incorporation of non-natural amino acids relatively easily. The system is not only synthetically accessible, but is also has reversible folding, allowing the melting profiles of the samples to be obtained without destroying the sample. Some synthesis is also presented, though minor attention goes to it because the emphasis is, as usual, to the supramolecular stuff… but kudos for incorporating the use of a different new chiral auxiliary.
I think that using such a small protein-mimic allowed them to see the effect of the mutations more clearly, since it lacks many other interactions to dilute the effect of the fluorine permutations.
Self-sorting illustrates well just how thermodynamically different each of the attractions are. By allowing enough equilibration time to “trap” each of the dimers with their minimum-energy partners, they could use disulfide chemistry to covalently bind each of the pairs.
The narrative is well enough… the R^2 values they discuss for linearity are scary, but they’re saved by the 0.93 value they get for the linear combination of dipole-dipole + calculated logP values.
The synopsis was far to complicated to understand without having actually read the paper: too many details, too much specialized vocabulary, not really engaging in the narrative, and probably longer than what the paper merited. The picture illustrates one of the pictorial aspects of the paper,: the self-sorting (both narcissistic and social) of the peptides prepared. I’m assuming that the pic comes from the pdb file. It was nice…

26 04 2012

This weeks article presented by Gao et al dragged my attention in the interest of the article as it is reported to be the first investigation on the energetics of aromatic stacking in proteins. The authors examined the aromatic stacking energetics by introducing various stacked aromatic pairs into model protein α2D, which is a denovo designed 35 residue polypeptide that has the ability to fold into dimeric helix with reversible folding behavior.
As a known fact that amino acids are building blocks of proteins and the amino acids have polar hydrophilic and hydrophobic aliphatic and aromatic side chains which dictate their chemical properties in biological systems. The experiment has its own significance as the authors made use of aromatic pairs in the design of peptides and proteins in contrast to much of the study using hydrophilic and polar groups. α2D mutants were synthesized through solid phase peptide synthesis with the replacement of phenylalanine residues at 10 and 29 positions with fluorine analogues bearing varied number of fluorine atoms which were able to fold into homodimeric complexes
The central theme of the experiment appears very interesting as it depicts the involvement of quadrupole interactions and thermodynamics. These thermodynamic parameters were obtained through vant Hoff analysis of thermal melting curves at varied concentrations which indicated the involvement of other factors such as dipole-dipole coupling other than hydrophobicity, to the stability of the homodimers. These interactions are very important as they dictate the stability and integrity of the peptides in addition to maintaining the secondary and tertiary structure of the proteins.
These types of article of course, help us to rethink the possibility of involvement of all the aspects within the range.
Overall Ana could manage to gather all the points in her synopsis. The picture is too simple and would have been much more artistic and informative involving fluorine derivatives.

26 04 2012

The authors reported an experimental assessment on the influence of dipoles on aromatic stacking by means of fluor additions on Phe residues that stabilize a dimeric protein thorough Phe stacking interactions. The author’s selection of the α2D dimeric protein for this type of assessment is suitable and well supported in the narrative. I found interesting that the Phe stacking interactions interlocked the dimer.

The aromatic stacking on wild-type α2D has the aromatic rings located in opposite directions therefore in the experiments with double mutants the self-complementarity of F345F combine with its net dipole being the biggest of all is what makes up for the best ΔΔG. However on experiments with single mutants is Zo the one with the most favorable/best ΔΔG even though it has a net dipole value very similar to that of F345F. What is different on Zo is that it also has an additional Fluor atom, which potentially enhance the complementarity between the quadrupole moments of the two-aryl rings doing the stacking (F and Zo). Considering the difference in hydrophobicity and complementarity of Zo or F345 with native Phe, I would have recommend the authors another self-sorting experiment mixing (F,F), (Zo,Zo), and (Z,Z) to evaluate what type of dimerization occurs from the thermodynamic equilibrium of these three-component system. The results from this experiment combine with their additional data might strengthen their statement suggesting Zo as a potential “warhead” to target native Phe residues through aromatic stacking. It could have also expanded the scope of their strategy since they propose the use of their system towards the design of enzyme inhibitors for binding pockets having aromatic residues.

In general the narrative was ok with good descriptions from the authors about their findings even though I still think I have not fully get all the details on the nomenclature used by the authors to described the derivatives analyzed. Ana’s synopsis incorporates the main findings in a somewhat too much-detailed way, which might not be necessary to describe the overall story of the article. It was good to see that she suggest to do more experiments with several combinations of the fluorine analogues but I think they cover the basic permutations of single fluorine additions preserving the peptide isosteric. Her figure was good because it highlights two very important findings in an engaging way.

26 04 2012

In this article Gao and coworker present their studies in the energetics involved in the aromatic stacking of phenylalanine fluorinated derivatives in the context of proteins. In these studies, they observed that the fluorinated derivatives possessed a higher stability than the wild type. This was expected because the fluorinated derivatives are more hydrophobic. Other studies suggested other factors beside hydrophobicity must be contributing to the stability of the overall protein. They found out that such factor were dipole-dipole, dipole-induced dipole which contributed to the aromatic stacking.

I think that the narrative from this article was engaging. They performed enough experiment to support their hypothesis and they explain the data very well. I also think that they had the proper control groups and their conclusions could be correlated with their data. Overal the paper was easy to follow and understand.

As for Ana’s synopsis, it did have the main idea of the paper, and se mentioned the most important data. However I did find it a little hard to read sometimes, maybe because of the way the sentences are written. Maybe if she uses more transitional words to connect her ideas, this could improve her writing. I don’t think that a person with only a general knowledge of chemistry can fully get the main idea of the paper, it’s too specialized. The picture was kind of helpful in understanding some aspects of the article.

26 04 2012
Rocio Roque

This other week’s article by Gao and colleagues studies the energetics of stacked aromatic pairs incorporated into the protein α2D. This, done by mutating Phe10 and Phe29 residues with fluorine derivatives and incorporating them into the latter protein. Their main findings suggest that other important aspects besides hidrophobicity affect the stabilization of the protein. This, might as well be the “first systematic investigation of the energetics of aromatic stacking in the context of proteins”, as the authors readily state.

About the article, its narrative was good and engaging. For me, it was easier to follow than our other paper of the week. I think, they did the experiments that were needed and they presented their data in a clear way. Overall it was a good article.

The synopsis was well written, but Ana doesn’t do smooth transitions between sentences (not engaging enough?!). Overall it was ok. Her figure was good and stated the main findings of the paper.

26 04 2012

In this article Gao and colleagues present a systematic analysis of the aromatic stacking energetics of several stacked aromatic pairs into the protein α2D. To accomplish this they use a set of fluorinated analogues of phenylalanine in the Phe10 and Phe29 positions of α2D. As expected all the mutated proteins were able to fold into homodimeric complexes, this complexes were use to study the thermodynamic parameters of the dimerization. The wide range of melting temperatures for the α2D homodimers were used as an indicative of the hydrophobicity of fluorocarbon compounds. The fact that the folding free energy of the fluoroaromatic side chains show a poor correlation just as the plot of the folding free energy against the surface area, make them get to the conclution that other factors than hydrophobicity are contributing to the stability of the homodimers. On the contraire the folding stability agreed with the dipole moment of the fluoroaromatic side chains, indicating that dipole-dipole interactions play a role in the stability of the homodimers. By re-analyzing they identified that the contribution in stability is do to both, hydrophobicity and dipole moments of the aromatic rings. To further explore the contribution of dipole-induced-dipole interactions they study single mutants of α2D. As a result they identified that the F,Zo mutant was the most stable homodimer. To study the possible self-sorting behavior of peptides, thermodynamic equilibrium studies were performed using a disulfide cross-linking experiment. This methodology were use to study three mutant systems. LC-MS experiments show that only two significant species were observed, heterodimer F,F and Z, Z. This two systems are stabilized by pi-pi interaction and the other homodimer, F345F, F345F, by the dipole-dipole interactions.

The article was well written and easy to follow, the figures were appropriate for the delivery of the data. I also appreciate the SI it was very well prepared. As Ana mention, I enjoy this article because I learn a lot from it. Ana’s synopsis was very helpful as well as the picture.

27 04 2012

In this article is presente a library of pi-pi interacting aromatic rings with fluorine substituents in the α2D protein. Library consisted on changing the position of the F in the ring and increasing the amount of F. First they took the folding free energy and F345F (referring to the position of the F), Z(ortho), Z(meta), Z(para) [referring to the remaining H position) dimers seemed to be the most stable in comparison with the F (no F ring). Later they combined (F,X) [X= a monomer of the most stable dimers]. It seemed to have the best stability with [F,Z(ortho)]. They correlate the stability with hydrophobicity and dipole-dipole interaction. As far as they know they are the first to do energetics of aromatics stacking in proteins via fluorinated aromatic rings for the possible use of fluoronation in NMR and PET.

The paper is well organized, engaging and very straightforward. What I think they did not took in consideration was the possible H-bonding. I know we are talking about aromatic systems and the main interaction here are pi-pi interactions and as they mention the dipole-dipole moment of each molecule. I also have in mind that H-bonds are mainly strong dipole-dipole interactions. Yet we should not let them go under the radar, lets not forget that F can interact in H-bonding. H-bond is pushed down our throats almost the whole B.A. its kind of hard to ignore them lol. Something that caught my eye also was the position of the H in the Z compounds. The closer they get to the main chain the more stable the folding is.

About Ana’s synopsis it was overall good explaining the significance of the article and their results. Maybe it went too specific for my taste when it came to review the folding free energy tendencies, could get confusing if you get too deep in data. The picture is ok.

27 04 2012

In this article, Gao et al. introduce phenylalanine analogues with varying degrees of fluorine substitution into α2D. The followed by studying the thermodynamic stability of these. I guess one of the most significant things they find is that all of these variants have the capability to form homodimeric complexes. They also observed that these fluorinated complexes are more stable (according to their thermodynamic data) than the wild type (control group). They attributed this increase in stability to an increase of hydrophobicity in the fluorocarbons. They also mention dipole-dipole coupling is another contributor to the enhanced stability of the fluorinated compounds, and also that dipole-induced dipole type interactions help contribute to aromatic stacking.

I think the paper was pretty decent, I think they used the correct control groups, as well as the pertinent experiments and the length of it was in accordance with the contents. I think the narrative was pretty easy to follow and easy to understand. However, I don’t think this would be a paper for someone that hasn’t taken more advanced courses in chemistry, since the material is somewhat more specific.

As for the synopsis, I thought it was ok, but kind of confusing in the way that some of the sentences were written, although it does well in summarizing the important aspects of the article. The picture, however, I just don’t think that the relation with the article is obvious enough.

27 04 2012

didn’t understand the synopsis cartoon when I first see it, a legend would have been of great help to understand it better or maybe for substitute this nomenclature for one easier to follow, at least for the cartoon purpose. Good synopsis, it helps me to understand the article. A very challenging article, I am still confused with the nomenclature. How the two types (heterodimer, homodimmer) of mutants are defined in terms of their components? This is the case of mainly fluorophobic effect and in the case of (F345F, F345F) other interactions may lead to the increased stability. I am wandering how our last week speaker, Dr. Wheeler, explain this last result?

27 04 2012

Sorry for the typo at the beginning of my comment. The first sentence should start with an “I”.

14 11 2012
Michel Repasky

DL-Phenylalanine (DLPA) is marketed as a nutritional supplement for its supposed analgesic and antidepressant activities. DL-Phenylalanine is a mixture of D-phenylalanine and L-phenylalanine.”

Consider our own internet page as well

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s

%d bloggers like this: