Statistical potentials from the Gaussian scaling behaviour of chain fragments buried within protein globules

Knowledge-based approaches use the statistics collected from protein data-bank structures to estimate effective interaction potentials between amino acid pairs. Empirical relations are typically employed that are based on the crucial choice of a reference state associated to the null interaction case. Despite their significant effectiveness, the physical interpretation of knowledge-based potentials has been repeatedly questioned, with no consensus on the choice of the reference state. Here we use the fact that the Flory theorem, originally derived for chains in a dense polymer melt, holds also for chain fragments within the core of globular proteins, if the average over buried fragments collected from different non-redundant native structures is considered. After verifying that the ensuing Gaussian statistics, a hallmark of effectively non-interacting polymer chains, holds for a wide range of fragment lengths, although with significant deviations at short spatial scales, we use it to define a 'bona fide' reference state. Notably, despite the latter does depend on fragment length, deviations from it do not. This allows to estimate an effective interaction potential which is not biased by the presence of correlations due to the connectivity of the protein chain. We show how different sequence-independent effective statistical potentials can be derived using this approach by coarse-graining the protein representation at varying levels. The possibility of defining sequence-dependent potentials is explored.

Nature-inspired Circular-economy Recycling (NaCRe) for Proteins

Simone Giaveri

Since a few decades our planet has been loaded with billion tons of synthetic polymer-based materials, commonly named plastics. The large scale of plastic production, associated with its limited recyclability, are the driving force for the accumulation of such materials into the environment. Garbage patches, i.e. islands of plastics in the ocean, are striking evidences of this issue. A sustainable handling, that is production and disposal, of synthetic polymer-based materials is one of the greatest challenges that humanity has to face.Proteins and nucleic acids are natural polymers. Arguably, Nature produces an amount of such polymers that is higher with respect to man-made polymers. However, these materials do not accumulate into the environment, that is the approach used by Nature to handle these polymers is sustainable. The secret for its sustainaibility lies on the circularity in the materialsâ use. Indeed, proteins and nucleic acids are sequence-defined polymers that undergo depolymerization to monomers, and recycling into new materials by reassembling the so obtained monomers into arbitrarily different sequences. Organisms digest proteins into amino acids. These monomers are in turn polymerized to produce the protein of need, at the time of the protein synthesis.In this thesis we show that this process is achievable outside living organisms. Indeed, we depolymerized structurally different short peptides, and peptides mixtures into their constitutive amino acids, and recycled such monomers into biotechnologically relevant proteins (green, and red fluorescent proteins), by using an amino acid-free cell-free transcription-translation system. We further applied our methodology to recycle proteins with high relevance in materials engineering such as Î²-lactoglobulin films, used for water filtration, or silk fibroin solutions into green fluorescent protein. We were also successful in recycling mixtures composed of short peptides, and technologically relevant proteins into fluorescent proteins, as well as into bioactive enzymes (catechol 2,3-dioxygenase). Finally, we achieved multiple cycles of recycling (two cycles), and we demonstrated that the strategy can be expanded beyond the set of the twenty proteinogenic amino acids.Presented herein is a nature-inspired approach to recycling of soft materials, where unknown mixtures of polymers are recycled into the polymer of need, by the local community, at the time of recycling. The materials are costantly transformed into different ones, without any external feed, and there is no way to distinguish a new polymer from a recycled one. The strength of this method lies in its compatibility with the principles of circular economy.

EPFL2021

Structural Studies and Protein Engineering of Human O6-Alkylguanine-DNA Alkyltransferase

Birgit Mollwitz

The specific labeling of proteins with synthetic probes is a powerful approach to study protein function and protein tags have been widely used for this purpose. A well-established example for a self-labeling protein tag is SNAP-tag. It specifically reacts with a wide variety of O6-benzylguanine derivatives (BG-derivatives) and was derived from the human O6-alkylguanine-DNA alkyltransferase (hAGT) by protein engineering. Relative to hAGT, SNAP-tag possesses a 52-fold higher reactivity towards BG-derivatives, does not bind to DNA and expresses well in cells as on cell surfaces. It is known that alkylation of hAGT results in protein unfolding and degradation. However, an increased degradation of SNAP-tag fusion proteins after labeling has not been observed. The first part of this work focused on the structural basis underlying the differences in protein stability between SNAP-tag and hAGT. A detailed biochemical and structural analysis was performed to determine (i) the interaction of SNAP-tag with its substrate, (ii) the factors responsible for its increased reactivity and (iii) how the labeling affected the stability of the protein. Besides an increased reactivity with BG-derivatives the superior stability of SNAP-tag compared to the parent protein hAGT could be confirmed. Whereas wild-type hAGT was rapidly degraded in cells after alkyl transfer, benzylated SNAP-tag showed a higher stability against proteolytic degradation. Moreover, the combination of our crystallographic and computational data provided further insight into the structural basis for the improved properties. The data indicated that the intrinsic stability of a key alpha helix was an important factor in triggering the unfolding and degradation of wild-type hAGT and provided new insights into the structure-function relationship of this DNA repair protein. The second part was aiming for the generation of a new SNAP-tag-based inhibitor complex. It was envisaged that this complex would interact with the target protein via amino acid loops and decrease its function only upon labeling with BG-inhibitor molecules. Therefore, SNAP-tag was modified by the insertion of stretches of randomized amino acids and the generated protein libraries were screened for binding affinity. The utilization of two yeast-based systems, the yeast three-hybrid and two hybrid technologies, allowed for the differentiation of small-molecule dependent and independent binding interactions. It could be demonstrated that a specific protein-loop interaction can be generated by this approach. It could further be shown that inhibition of the catalytic activity of the target protein E.coli dihydrofolate reductase by a SNAP-loop mutant was possible. In summary this work revealed new insights into the stability of hAGT and SNAP-tag and the structure-function relationship of AGTs in general. Further, SNAP-tag engineering generated a new protein-binder whose affinity towards the target protein was leading to protein inhibition.

EPFL2012

On the use of direct-coupling analysis with a reduced alphabet of amino acids combined with super-secondary structure motifs for protein fold prediction

Jaume Bonet Martinez

Direct-coupling analysis (DCA) for studying the coevolution of residues in proteins has been widely used to predict the three-dimensional structure of a protein from its sequence. We present RADI/raDIMod, a variation of the original DCA algorithm that groups chemically equivalent residues combined with super-secondary structure motifs to model protein structures. Interestingly, the simplification produced by grouping amino acids into only two groups (polar and non-polar) is still representative of the physicochemical nature that characterizes the protein structure and it is in line with the role of hydrophobic forces in protein-folding funneling. As a result of a compressed alphabet, the number of sequences required for the multiple sequence alignment is reduced. The number of long-range contacts predicted is limited; therefore, our approach requires the use of neighboring sequence-positions. We use the prediction of secondary structure and motifs of super-secondary structures to predict local contacts. We use RADI and raDIMod, a fragment-based protein structure modelling, achieving near native conformations when the number of super-secondary motifs covers >30-50% of the sequence. Interestingly, although different contacts are predicted with different alphabets, they produce similar structures.