An empirical phase diagram approach to investigate conformational stability of “second‐generation” functional mutants of acidic fibroblast growth factor‐1

Abstract

Acidic fibroblast growth factor-1 (FGF-1) is an angiogenic protein which requires binding to a polyanion such as heparin for its mitogenic activity and physicochemical stability. To evaluate the extent to which this heparin dependence on solution stability could be reduced or eliminated, the structural integrity and conformational stability of 10 selected FGF-1 mutants were examined as a function of solution pH and temperature by a series of spectroscopic methods including circular dichroism, intrinsic and extrinsic fluorescence spectroscopy and static light scattering. The biophysical data were summarized in the form of colored empirical phase diagrams (EPDs). FGF-1 mutants were identified with stability profiles in the absence of heparin comparable to that of wild-type FGF-1 in the presence of heparin while still retaining their biological activity. In addition, a revised version of the EPD methodology was found to provide an information rich, high throughput approach to compare the effects of mutations on the overall conformational stability of proteins in terms of their response to environmental stresses such as pH and temperature.

Improved data visualization techniques for analyzing macromolecule structural changes

“…no reversibility is implied), but rather simple, empirical representations of the physical behavior of the macromolecule. [1][2][3] As shown in Fig-ure 5(B), a small pink-colored region between the native and unfolded state is also observed in the three-index EPD. The calculated structural indices used for the construction of the three-index EPD for the protein antigen SP1732 are shown in Figure 6(G-I; see Methods section).…”

“…In addition, differences between EPDs can be directly compared (e.g., between mutants of the same protein) by processing all the data together. 3 By making measurements as a function of variables such as temperature, pH, concentration, ionic strength, and mechanical stress, the colored EPD diagram represents an information rich type of ''stress/response'' diagram that has been useful in a wide variety of applications including stabilization and formulation of protein therapeutics and a variety of macromolecular vaccines.…”

High-Throughput Biophysical Analysis and Data Visualization of Conformational Stability of an IgG1 Monoclonal Antibody After Deglycosylation

“…], followed by the application of multidimensional mathematical analysis techniques, to produce a colored diagram representing structural changes as a function of environmental stress. Furthermore, the ability of EPDs to detect major conformational stability changes in a series of site‐directed mutants of acidic fibroblast growth factor (FGF‐1) was recently demonstrated . Recent work has also established additional data visualization methods such as radar charts, Chernoff faces, and comparative signature diagrams to better compare protein samples …”

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

“…FGF-1 was provided by Dr. Michael Blaber, Florida State University School of Medicine, and was prepared as described previously. 30 Polyclonal human antibody preparation and green fluorescent protein (GFP) were purchased from MP biochemicals (Santa Ana, CA) and Calbiochem (Billerica, MA), respectively. The polyclonal antibody was further purified in our labs as described below.…”