Sota Yagi

Addition of negatively charged residues can reverse the decrease in the solubility of an acidic protein caused by an artificially introduced non-polar surface patch

A non-polar patch on the surface of a protein can cause a reduction in the solubility and stability of the protein, and thereby induce aggregation. However, a non-polar patch may be required so that the protein can bind to another molecule. The mutant 6L-derived from the acidic, dimeric α-helical protein sulerythrin and containing six additional leucines arranged to form a non-polar patch on its surface when properly folded-has a substantially reduced solubility in comparison with that of wild-type sulerythrin. This reduced solubility appears to cause 6L to aggregate. To reverse this aggregation, we mutated 6L so that it contained three to six additional glutamates or aspartates that we predicted would surround the non-polar leucine patch on natively folded 6L. Although the introduction of three glutamates or aspartates increased solubility, the mutants still aggregate and have a reduced α-helical content. Conversely, mutants with six additional glutamates or aspartates appear to exist mostly as dimers and to have the same α-helical content as that of wild-type sulerythrin. Notably, the introduction of five lysines or five arginines at the positions held by the glutamates or aspartates did not recover solubility as effectively as did the negatively charged residues. These results demonstrate that negatively charged residues, but not positively charged ones, surrounding a non-polar patch on an acidic protein can completely reverse the decrease in its solubility caused by the patch of non-polar surface residues.

De novo design of protein-protein interactions through modification of inter-molecular helix-helix interface residues.

For de novo design of protein-protein interactions (PPIs), information on the shape and chemical complementarity of their interfaces is generally required. Recent advances in computational PPI design have allowed for de novo design of protein complexes, and several successful examples have been reported. In addition, a simple and easy-to-use approach has also been reported that arranges leucines on a solvent-accessible region of an α-helix and places charged residues around the leucine patch to induce interactions between the two helical peptides. For this study, we adopted this approach to de novo design a new PPI between the helical bundle proteins sulerythrin and LARFH. A non-polar patch was created on an α-helix of LARFH around which arginine residues were introduced to retain its solubility. The strongest interaction found was for the LARFH variant cysLARFH-IV-3L3R and the sulerythrin mutant 6L6D (KD=0.16 μM). This artificial protein complex is maintained by hydrophobic and ionic interactions formed by the inter-molecular helical bundle structure. Therefore, by the simple and easy-to-use approach to create de novo interfaces on the α-helices, we successfully generated an artificial PPI. We also created a second LARFH variant with the non-polar patch surrounded by positively charged residues at each end. Upon mixing this LARFH variant with 6L6D, mesh-like fibrous nanostructures were observed by atomic force microscopy. Our method may, therefore, also be applicable to the de novo design of protein nanostructures.

Creation of artificial protein–protein interactions using α-helices as interfaces

Designing novel protein–protein interactions (PPIs) with high affinity is a challenging task. Directed evolution, a combination of randomization of the gene for the protein of interest and selection using a display technique, is one of the most powerful tools for producing a protein binder. However, the selected proteins often bind to the target protein at an undesired surface. More problematically, some selected proteins bind to their targets even though they are unfolded. Current state-of-the-art computational design methods have successfully created novel protein binders. These computational methods have optimized the non-covalent interactions at interfaces and thus produced artificial protein complexes. However, to date there are only a limited number of successful examples of computationally designed de novo PPIs. De novo design of coiled-coil proteins has been extensively performed and, therefore, a large amount of knowledge of the sequence–structure relationship of coiled-coil proteins has been accumulated. Taking advantage of this knowledge, de novo design of inter-helical interactions has been used to produce artificial PPIs. Here, we review recent progress in the in silico design and rational design of de novo PPIs and the use of α-helices as interfaces.

Characterization of the low-temperature activity of Sulfolobus tokodaii glucose-1-dehydrogenase mutants

Thermophilic enzymes are potentially useful for industrial processes because they are generally more stable than are mesophilic or psychrophilic enzymes. However, a crucial drawback for their use in such processes is that most thermophilic enzymes are nearly inactive at moderate and low temperatures. We have previously proposed that modulation of the coenzyme-binding pocket of thermophilic dehydrogenases can produce mutated proteins with enhanced low-temperature activities. In the current study, we produced and characterized mutants of an NADP-dependent glucose-1-dehydrogenase from the hyperthermophile Sulfolobus tokodaii in which a predicted coenzyme-binding, non-polar residue was replaced by another non-polar residue. Detailed analyses of the kinetic properties of the wild-type enzyme and its mutants showed that one of the mutants (V254I) had improved kcat and kcat/Km values at both 25°C and 80°C. Temperature-induced unfolding experiments showed that the thermal stability of the mutant enzyme was comparable to that of the wild-type enzyme. Calculation of the energetic contribution of the V254I mutation for the dehydrogenase reaction revealed that the mutation destabilizes the enzyme-NADP(+)-glucose ternary complex and reduces the transition-state energy, thus enhancing catalysis.

Coarse-Grained Molecular Dynamics Simulation of Sulerythrin and LARFH for Producing Protein Nanofibers

Artificial creation of fibers utilizing proteins has been a target of bionanotechnology. Yagi et al. succeeded in designing artificial protein fibers using two types of proteins: LARFH and sulerythrin. Binding interfaces were designed for sulerythrin and LARFH by introducing mutations, and the fibrous structures were confirmed by atomic force microscopy. However, branching was observed in the fibrous structure, possibly because of non-specific interactions between the proteins. In this study, we analyzed the behavior and binding sites of sulerythrin mutants and LARFH mutants using coarse-grained molecular dynamics (MD) simulation. Binding simulations were performed for a system of one sulerythrin and one LARFH, and also of two sulerythrin molecules and four LARFH molecules. These results suggested that glutamic acids originally possessed by sulerythrin contribute to non-specific binding at sites other than the designed interfaces.

Selection of a platinum-binding sequence in a loop of a four-helix bundle protein

Protein-metal hybrids are functional materials with various industrial applications. For example, a redox enzyme immobilized on a platinum electrode is a key component of some biofuel cells and biosensors. To create these hybrid materials, protein molecules are bound to metal surfaces. Here, we report the selection of a novel platinum-binding sequence in a loop of a four-helix bundle protein, the Lac repressor four-helix protein (LARFH), an artificial protein in which four identical α-helices are connected via three identical loops. We created a genetic library in which the Ser-Gly-Gln-Gly-Gly-Ser sequence within the first inter-helical loop of LARFH was semi-randomly mutated. The library was then subjected to selection for platinum-binding affinity by using the T7 phage display method. The majority of the selected variants contained the Tyr-Lys-Arg-Gly-Tyr-Lys (YKRGYK) sequence in their randomized segment. We characterized the platinum-binding properties of mutant LARFH by using quartz crystal microbalance analysis. Mutant LARFH seemed to interact with platinum through its loop containing the YKRGYK sequence, as judged by the estimated exclusive area occupied by a single molecule. Furthermore, a 10-residue peptide containing the YKRGYK sequence bound to platinum with reasonably high affinity and basic side chains in the peptide were crucial in mediating this interaction. In conclusion, we have identified an amino acid sequence, YKRGYK, in the loop of a helix-loop-helix motif that shows high platinum-binding affinity. This sequence could be grafted into loops of other polypeptides as an approach to immobilize proteins on platinum electrodes for use as biosensors among other applications.