Histidine-Mediated Intramolecular Electrostatic Repulsion for Controlling pH-Dependent Protein-Protein Interaction.

Protein-protein interactions that can be controlled by environmental triggers have immense potential in various biological and industrial applications. In the current study, we aimed to engineer a pH-dependent protein-protein interaction that employs intramolecular electrostatic repulsion through a structure-guided histidine substitution approach. We implemented this strategy on Streptococcal protein G, an affinity ligand for immunoglobulin G, and showed that even a single point mutation effectively improved the pH sensitivity of the binding interactions without adversely affecting its structural stability or its innate binding function. Depending on the pH of the environment, the protein-protein interaction was disrupted by the electrostatic repulsion between the substituted histidine and its neighboring positively charged residues. Structurally, the substituted histidine residue was located adjacent to a lysine residue that could form hydrogen bonds with immunoglobulin G. Thermodynamically, the introduced electrostatic repulsion was reflected in the significant loss of the exothermic heat of the binding under acidic conditions, whereas accompanying enthalpy-entropy compensation partly suppressed the improvement of the pH sensitivity. Thus, the engineered pH-sensitive protein G could enable antibody purification under mildly acidic conditions. This intramolecular design can be combined with conventional protein-protein interface design. Moreover, the method proposed here provides us with additional design criteria for optimization of pH-dependent molecular interactions.

Backbone Circularization Coupled with Optimization of Connecting Segment in Effectively Improving the Stability of Granulocyte-Colony Stimulating Factor.

Backbone circularization of protein is a powerful method to improve its structural stability. In this paper, we presumed that a tight connection leads to much higher stability. Therefore, we designed circularized variants of a granulocyte-colony stimulating factor (G-CSF) with a structurally optimized terminal connection. To estimate the appropriate length of the connection, we surveyed the Protein Data Bank to find local structures as a model for the connecting segment. We set the library of local structures composed of "helix-loop-helix," subsequently selected entries similar to the G-CSF terminus, and finally sorted the hit structures according to the loop length. Two, five, or nine loop residues were frequently observed; thus, three circularized variants (C163, C166, and C170) were constructed, prepared, and evaluated. All circularized variants demonstrated a higher thermal stability than linear G-CSF (L175). In particular, C166 that retained five connecting residues demonstrated apparent Tm values of 69.4 °C, which is 8.7 °C higher than that of the circularized variant with no truncation (C177), indicating that the optimization of the connecting segment is effective for enhancing the overall structural stability. C166 also showed higher proteolytic stability against both endoprotease and exopeptidase than L175. We anticipate that the present study will contribute to the improvement in the general design of circularized protein and development of G-CSF biobetters.

Development of systemic in vitro evolution and its application to generation of peptide-aptamer-based inhibitors of cathepsin E.

Proteases are involved in various biological functions. Thus, inhibition of their activities is scientifically interesting and medically important. However, there is no systematic method established to date to generate endopeptidase inhibitory peptides. Here, we report a general system to identify endopeptidase inhibitory peptides based on the use of in vitro evolution. Using this system, we generated peptides that inhibit cathepsin E (CE) specifically at a submicromolar IC(50). This system generates protease inhibitor peptides utilizing techniques of cDNA display, selection-by-function, Y-ligation-based block shuffling, and others. We further demonstrated the importance and effectiveness of a secondary library for obtaining small-sized and active peptides. CE inhibitory peptides generated by this method were characterized by a small size (8 to 12 aa) and quite different sequences, suggesting that they bind to different sites on CE. Typical CE inhibitory peptide aptamers obtained here (P(i)101; SCGG IIII SCIA) have half an inhibition activity (K(i); 5 nM) of pepstatin A (potent CE inhibitor) without inhibiting cathepsin D (structurally similar to CE). The general applicability of this system suggests that it may be useful to identify inhibitory peptides for various kinds of proteases and that it may therefore contribute to protein science and drug discovery. The peptide binding to a protein is discussed in comparison with the antibody binding to an antigen.

Molecular design guided by a local map of sequence space: DNA aptamers that inhibit cathepsin E.

It has been strongly demanded by a number of people to elevate activities of molecules of a particular function. Currently, there is no general guide available for this purpose. Here we present a novel approach for this; a local sequence space map-directed method for exploring molecules of a higher activity. This approach exploits the knowledge of a local sequence space so far established and obtains the shape of sequence space (map) by intra- and extrapolating the known landscape, which was drawn through the principal coordinates analysis. In this method, we successfully obtained 16 DNA aptamers of cathepsin E (CE) inhibitory activity that have comparable or higher activities than the ancestral ones on which the designed molecules were based. Some of them had a 30% higher activity than the previously reported top one (SFR-6-3). This high efficiency in obtaining functional molecules (16 out of 21 newly designed ones) is by no means usual because most of molecules generated at random are known to have no function, showing the effectiveness of the map-based approach. The selected molecules were confirmed to have the i-motif structure, consistent to the fact that they have a C-rich sequence and their CE-inhibitory activities were measured at an acidic pH, both of which are favorable for the i-motif. This structure of CE-inhibitory aptamers was inferred to contribute to the structural stability but not to the function itself directly.

GFPs of insertion mutation generated by molecular size-altering block shuffling.

Insertion and deletion analyses of a protein have been less common than point mutation analyses, partly due to the lack in effective methods. This is the case with the green fluorescent protein (GFP), which is so widely applied in molecular biology and other fields. In this paper we first introduce a systematic approach for generating insertion/deletion mutants of GFP. A new technology of Y-ligation-based block shuffling (YLBS) was successfully applied to produce size-altered GFPs, providing insertion-containing GFPs of fluorescence, though no deletion type of fluorescence was obtained so far as examined. The analysis of these proteins suggested that size alteration (deletion/insertion) is acceptable so far as some type of rearrangement in a local structure can accommodate it. This paper demonstrates that YLBS can generate insertion and deletion mutant libraries systematically, which are beneficial in the study of structure-function relationship.