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Amphiphilic Peptoids as General Tool for Membrane Proteins Stabilization and Crystallization

Periodic Reporting for period 1 - Pro-Membrane (Amphiphilic Peptoids as General Tool for Membrane Proteins Stabilization and Crystallization)

Reporting period: 2015-05-01 to 2017-04-30

"- What is the problem/issue being addressed?

This action aimed to face the still unsolved challenge of stabilizing and crystallizing Membrane-Proteins (MPs). The innovative tool to reach this result is a particular class of amphiphilic peptoids. The intrinsically modular nature and the cell membrane permeability of peptoids, combined with a better understanding of their physical properties can establish the base of a general method for structural determination of MPs.

- Why is it important for society?

Membrane proteins (MPs) represent almost 60% of pharmaceutical targets. However, despite their fundamental role, only 2% of the protein of known structure are that of MPs, and, unfortunately, this lack of knowledge seriously affects structure-based drug design. While difficulties lay on overexpression, purification, stability and crystallization of MPs, the major bottleneck for their structural and functional study arises from their instability outside a lipid bilayer environment, where specific hydrophobic interactions keep the protein in its native and active conformational state. Therefore detergents that mimic the physical properties of the original bilayer are needed. Despite some success, currently available reagents did’nt open up the field, and a pressing need for new amphiphiles remains.

- What are the overall objectives?

A reiterative and comparative study of differently designed APOs libraries has been realized. In order to understand the macroscopic effects due to molecular changes, thermodynamic studies on the micellization process, and stabilization/crystallization screenings on model proteins was done and it's still an ongoing research through a strict collaboration between the partners and the two planned secondments of the researcher. Peptoids represent an endless source to “tune” the physical properties of micelles. These studies still need further investigations to reach the main objective, which is a a general and fast method to tune the aphipathic properties of the molecules ""on demand""."
"The synthetic part of the project, to produce and characterize, amphiphilic peptoids was performed at the institute of chemistry of Clermont-Ferrand, the thermodynamic studies in the first secondment at the institute of Biotechnology of Vilnius, and the study of interactions with membrane proteins for their stabilization and characterization, at the Institute of Structural Biology in Grenoble, during the second secondments.
Partial divulgation of the results was done in international conferences, in Berkeley-California at the Peptoid summit through oral presentation, and to the conference of the Biophysics Society in Lithuania with poster presentation. I could could give lectures on my research at the ""Institut de Biologie Structurale"" of Grenoble (France), the Institute of Biotechnology in Vilnius (Lithuania), and at the Department of Chemistry of the University of Copenaghen (Denmark). In each of these events I could acknowledge the contribution of European Commission and IEF-MC-H2020 fellowship on my research.
The Individual Fellowship for the Marie Curie project ""Pro-Membrane"" had to be ended earlier (December 2016) in respect to the final date.
Indeed, one of the objective of the IEF-MC is to promote professional maturity and career of young researchers, through mobility in Europe, realizing scientific projects of their own design. In that respect, the fellowship worked really well, as I was recently appointed Assistant Professor at the University of Pisa (Italy)."
Ideally, detergents for MPs manipulation should be amphiphilic molecules, able to self-assemble in well defined micelles, allowing the solubilisation of the protein at first, and through specific interactions, stabilize the active conformation of the MP. The so-formed Protein-Detergent complex (PDC) should then form well-ordered lattices suitable for crystallographic diffraction or be stable enough for NMR studies in solution. Examples of new types of detergents are hemifluorinated detergents, tripod detergents, cholesterol based molecules, amphiphilic polymers, peptide surfactants etc. Despite some success, the number of Membrane Proteins of resolved crystal structure is still really low. In conclusion, at the state of the art, despite all these efforts, the way to cut this Gordian Knot is still missing.
The new family of amphiphilic peptoids could contribute to solve this problem, because for the intrinsically modular nature of these molecules is possible to tune their self-assembly and their amphipatic properties. The results obtained needs further studies and analysis. However, even though the project had to be ended earlier, the Supervisor, the 2 secondments collaborators and myself, will meet at the university of Pisa on April the 18th to discuss the continuation of the project and the publication strategy.
On the occasion of this meeting, on April 19th, we will present in a conference our results, open to the public, at the department of chemistry of the university of Pisa. The event will be filmed and uploaded in the social networks of the university, for a broader divulgation. European commission and IEF-MC-H2020 will be properly aknowledged in this event and in the future publications.
In conclusion, I feel really grateful for having had the chance to work on this European Project. Not only because was personally the boost to continue my work, but because it created a european network of researchers and institutions that will continue to work together on a challenging problem, like the structure of membrane proteins.
Formation of Protein-Detergent complex for Membrane Proteins crystallization