CHalf: Folding Stability Made Simple

Abstract

The structure of a protein defines its function and integrity and correlates with the protein folding stability (PFS). Quantifying PFS allows researchers to assess differential stability of proteins in different disease or ligand binding states, providing insight into protein efficacy and potentially serving as a metric of protein quality. There are a number of mass spectrometry (MS)-based methods to assess PFS, such as Thermal Protein Profiling (TPP), Stability of Proteins from Rates of Oxidation (SPROX), and Iodination Protein Stability Assay (IPSA). Despite the critical value that PFS studies add to the understanding of mechanisms of disease and treatment development, proteomics research is still primarily dominated by concentration-based studies. We found that a major reason for the lack of PFS studies is the lack of a user-friendly data processing tool. Here we present the first user-friendly software, C_Half, with a graphical user interface for calculating PFS. Besides calculating site-specific PFS of a given protein from chemical denature folding stability assays, C_Half is also compatible with thermal denature folding stability assays. C_Half also includes a set of data visualization tools to help identify changes in PFS across protein sequences and in between different treatment conditions. We expect the introduction of C_Half to lower the barrier of entry for researchers to investigate PFS, promoting the usage of PFS in studies. In the long run, we expect this increase in PFS research to accelerate our understanding of the pathogenesis and pathophysiology of disease.

Keywords: IPSA; SPROX; TPP; graphical user interface; protein folding stability.

Figure 1

The workflow of C_Half. This figure maps the workflow of C_Half, shows the image of the GUI for each tool, and shows example visualization outputs. Each panel is a module in the program and leads to the next module. The blue text represents the input files required for the next module; the * represents the product output file from the referenced module; and the + represents the function that is excluded for thermal denature experiments. The full size of screenshot of the interface and output are included in Supporting Figure 2 for the details.

Figure 2

Experiment flow of protein folding stability (PFS) assay. (A) Chemical denature based PFS assay denature protein using denaturant like GdmCl or urea and then covalently modify the surface exposed amino acid residues. The labeled peptides serve as the probe for protein stability measurement. (B) Thermal denature based PFS assays denature protein using an increasing temperature gradient. The protein aggregates as the temperature increases. The soluble protein fraction serves as the probe for protein stability measurement.

Figure 3

C_Half graphic result from sample data set. (A–C) The graphic results of IPSA experiment. (D–F) The graphics results of SPROX experiment. For the IPSA and SPROX experiments, a combined site figure for a given label site of a protein is shown on the left column, and the combined residue mapper (the middle column) shows the site C_1/2 across a protein’s sequence. (G–I) The graphic results of a TPP experiment. Since the TPP experiment does not label specific site of a protein, the denature curves are used to show the treatment effect on the same peptide. The scatter plot of site C_1/2 or peptide T_m (the right column) is used to show the global PFS change due to ligand binding or drug treatment.