9 research outputs found
Extension of a de novo TIM barrel with a rationally designed secondary structure element
The ability to construct novel enzymes is a major aim in de novo protein design. A popular enzyme fold for design attempts is the TIM barrel. This fold is a common topology for enzymes and can harbor many diverse reactions. The recent de novo design of a four-fold symmetric TIM barrel provides a well understood minimal scaffold for potential enzyme designs. Here we explore opportunities to extend and diversify this scaffold by adding a short de novo helix on top of the barrel. Due to the size of the protein, we developed a design pipeline based on computational ab initio folding that solves a less complex sub-problem focused around the helix and its vicinity and adapt it to the entire protein. We provide biochemical characterization and a high-resolution X-ray structure for one variant and compare it to our design model. The successful extension of this robust TIM-barrel scaffold opens opportunities to diversify it towards more pocket like arrangements and as such can be considered a building block for future design of binding or catalytic sites
Fine-tuning spermidine binding modes in the putrescine binding protein PotF
A profound understanding of the molecular interactions between receptors and ligands is important throughout diverse research, such as protein design, drug discovery, or neuroscience. What determines specificity and how do proteins discriminate against similar ligands? In this study, we analyzed factors that determine binding in two homologs belonging to the well-known superfamily of periplasmic binding proteins, PotF and PotD. Building on a previously designed construct, modes of polyamine binding were swapped. This change of specificity was approached by analyzing local differences in the binding pocket as well as overall conformational changes in the protein. Throughout the study, protein variants were generated and characterized structurally and thermodynamically, leading to a specificity swap and improvement in affinity. This dataset not only enriches our knowledge applicable to rational protein design but also our results can further lay groundwork for engineering of specific biosensors as well as help to explain the adaptability of pathogenic bacteria
Extension of a de novo TIM barrel with a rationally designed secondary structure element
Change in protein-ligand specificity through binding pocket grafting
Recognition and discrimination of small molecules are crucial for biological processes in living systems. Understanding the mechanisms that underlie binding specificity is of particular interest to synthetic biology, e.g. the engineering of biosensors with de novo ligand affinities. Promising scaffolds for such biosensors are the periplasmic binding proteins (PBPs) due to their ligand-mediated structural change that can be translated into a physically measurable signal. In this study we focused on the two homologous polyamine binding proteins PotF and PotD. Despite their structural similarity, PotF and PotD have different binding specificities for the polyamines putrescine and spermidine. To elucidate how specificity is determined, we grafted the binding site of PotD onto PotF. The introduction of 7 mutations in the first shell of the binding pocket leads to a swap in the binding profile as confirmed by isothermal titration calorimetry. Furthermore, the 1.7Ă
crystal structure of the new variant complexed with spermidine reveals the interactions of the specificity determining residues including a defined water network. Altogether our study shows that specificity is encoded in the first shell residues of the PotF binding pocket and that transplantation of these residues allows the swap of the binding specificity
Potential of Fragment Recombination for Rational Design of Proteins
ABSTRACT: It is hypothesized that protein domains evolved from smaller intrinsically stable subunits via combinatorial assembly. Illegitimate recombination of fragments that encode protein subunits could have quickly led to diversification of protein folds and their functionality. This evolutionary concept presents an attractive strategy to protein engineering, e.g., to create new scaffolds for enzyme design. We previously combined structurally similar parts from two ancient protein folds, the (ÎČα)8-barrel and the flavodoxin-like fold. The resulting âhopeful monster â differed significantly from the intended (ÎČα)8-barrel fold by an extra ÎČ-strand in the core. In this study, we ask what modifications are necessary to form the intended structure and what potential this approach has for the rational design of functional proteins. Guided b
Potential of Fragment Recombination for Rational Design of Proteins
It is hypothesized that protein domains evolved from
smaller intrinsically
stable subunits via combinatorial assembly. Illegitimate recombination
of fragments that encode protein subunits could have quickly led to
diversification of protein folds and their functionality. This evolutionary
concept presents an attractive strategy to protein engineering, e.g.,
to create new scaffolds for enzyme design. We previously combined
structurally similar parts from two ancient protein folds, the (ÎČα)<sub>8</sub>-barrel and the flavodoxin-like fold. The resulting âhopeful
monsterâ differed significantly from the intended (ÎČα)<sub>8</sub>-barrel fold by an extra ÎČ-strand in the core. In this
study, we ask what modifications are necessary to form the intended
structure and what potential this approach has for the rational design
of functional proteins. Guided by computational design, we optimized
the interface between the fragments with five targeted mutations yielding
a stable, monomeric protein whose predicted structure was verified
experimentally. We further tested binding of a phosphorylated compound
and detected that some affinity was already present due to an intact
phosphate-binding site provided by one fragment. The affinity could
be improved quickly to the level of natural proteins by introducing
two additional mutations. The study illustrates the potential of recombining
protein fragments with unique properties to design new and functional
proteins, offering both a possible pathway of protein evolution and
a protocol to rapidly engineer proteins for new applications
De novo designed peptides for cellular delivery and subcellular localisation
Increasingly, it is possible to design peptide and protein assemblies de novo from first principles or computationally. This approach provides new routes to functional synthetic polypeptides, including designs to target and bind proteins of interest. Much of this work has been developed in vitro. Therefore, a challenge is to deliver de novo polypeptides efficiently to sites of action within cells. Here we describe the design, characterisation, intracellular delivery, and subcellular localisation of a de novo synthetic peptide system. This system comprises a dual-function basic peptide, programmed both for cell penetration and target binding, and a complementary acidic peptide that can be fused to proteins of interest and introduced into cells using synthetic DNA. The designs are characterised in vitro using biophysical methods and X-ray crystallography. The utility of the system for delivery into mammalian cells and subcellular targeting is demonstrated by marking organelles and actively engaging functional protein complexes