Improved data visualization techniques for analyzing macromolecule structural changes

Abstract

The empirical phase diagram (EPD) is a colored representation of overall structural integrity and conformational stability of macromolecules in response to various environmental perturbations. Numerous proteins and macromolecular complexes have been analyzed by EPDs to summarize results from large data sets from multiple biophysical techniques. The current EPD method suffers from a number of deficiencies including lack of a meaningful relationship between color and actual molecular features, difficulties in identifying contributions from individual techniques, and a limited ability to be interpreted by color-blind individuals. In this work, three improved data visualization approaches are proposed as techniques complementary to the EPD. The secondary, tertiary, and quaternary structural changes of multiple proteins as a function of environmental stress were first measured using circular dichroism, intrinsic fluorescence spectroscopy, and static light scattering, respectively. Data sets were then visualized as (1) RGB colors using three-index EPDs, (2) equiangular polygons using radar charts, and (3) human facial features using Chernoff face diagrams. Data as a function of temperature and pH for bovine serum albumin, aldolase, and chymotrypsin as well as candidate protein vaccine antigens including a serine threonine kinase protein (SP1732) and surface antigen A (SP1650) from S. pneumoniae and hemagglutinin from an H1N1 influenza virus are used to illustrate the advantages and disadvantages of each type of data visualization technique.

Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application

“…28 Major histocompatibility complex 1 (MHC-1) binding predictions were made using the IEDB analysis resource Consensus tool. 29 The average predicted affinity for a given non-apeptide centered at the site of mutation was calculated for all MHC alleles. The predicted aggregation potential for WT FGF-1 and the C16S/A66C/C117A/P134A mutant were calculated from the X-ray structures (WT FGF-1 PDB accession 1JQZ) using the AggreScan 3D server.…”

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio‐layer interferometry

“…The three-index empirical phase diagrams (EPDs) were constructed as described previously 18,48 to summarize and facilitate the analysis of the biophysical stability data sets from different instruments (CD signal at 222 nm, ANS fluorescence intensity, and OD 360 ). Calculations were performed and data visualization phase diagrams were prepared using in-house laboratory software (Middaugh Suite) as described in detail elsewhere.…”

“…Calculations were performed and data visualization phase diagrams were prepared using in-house laboratory software (Middaugh Suite) as described in detail elsewhere. 18 GFP refolding assay. GFP refolding was used to test the GroEL partitioning and refolding efficiencies.…”

“…Using these biophysical stability data sets, we constructed two EPDs for the wild-type and biotinylated GroEL proteins [ Fig. 1(I-J)], as described in detail elsewhere, 18 to summarize and visualize their physical stability profile. The two EPDs appeared similar in terms of the pH and temperature conditions demonstrating that biotinylation does not significantly affect the physical stability profile of GroEL.…”

Accelerated Formulation Development of Monoclonal Antibodies (mAbs) and mAb-Based Modalities: Review of Methods and Tools

“…Presentation of the data can be done using empirical phase diagrams, which depict the changes in biophysical properties by color. This approach was used for the analysis of highly concentrated antibody solutions 115,116 and in other studies of antibody structure and stability. 117,118 …”