PSS: An enabling QTY server for designing water-soluble α-helical transmembrane proteins

Abstract

Membrane proteins, especially the α-helical ones such as G-protein coupled receptors (GPCRs), are considered extremely important owing to their significant biological roles. However, their expression and purification pose difficulties because of their poor solubility in water, which seriously impedes research progress in this field. Recently, QTY method, a revolutionary code-based protein engineering approach, was developed for the purpose of producing soluble transmembrane proteins. Here we describe a web server built for QTY design and certain analyses related to it (pss.sjtu.edu.cn). Typically, the Simple Design model is expected to take only 2-4 min, and the Library Design 2-5 h, of computer time, depending on target protein size and the number of transmembrane helices. Further, we describe a protocol for using the server with both Simple and Library Design modules. Protocols for experiments based on QTY design are also included. In summary, utilization of the web server, and associated protocols, will enable QTY-based protein-engineering to be implemented in a convenient, fast, accurate, and rational manner.Although the QTY strategy is straightforward in comparison to previous methods, doing QTY design manually is still time-consuming and tedious. The library design, especially, is virtually impractical to complete without computing power. Moreover, QTY related bioinformatics analyses are extremely tedious, as 7 different software programs are involved and the resulting output needs to be integrated. Therefore, PSS, a web-based server, was developed to facilitate the QTY method by avoiding the tedious analyses involved. The web-based server was developed to provide a graphical and friendly interface to a broader group of users. Software programs for QTY-related analysis were incorporated into the server to prepare elaborate analysis reports for QTY design. Using PSS for typical QTY Simple Design, a standard analysis report can be completed within 4 minutes and only requires very simple operations such as copy and pasting. PSS is expected to be widely used by scientists endeavoring in the research about α-helical proteins. METHODS AND IMPLEMENTATIONThe workflow and architecture of PSS server are illustrated in Figure 1. The following subsections describe the main features of each step/part in detail. InputTwo types of information are needed for QTY design. These are the protein sequence and its corresponding TM region information (Fig. 2). By default, a user input must include a UniProt number, which is the unique ID of a protein record in the UniProt database. Then, the server will retrieve that protein sequence from the UniProt database (https://www.uniprot.org/), and use it for QTY substitutions. If multiple sequences are represented by a single UniProt number, only the canonical sequence will be used for the design. Alternatively, a user can also input a protein sequence by simply typing/pasting that protein sequence in the provided text field (Supplementary File 1).With regard to TM region information, t...