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An empirical phase diagram approach to investigate conformational stability of “second‐generation” functional mutants of acidic fibroblast growth factor‐1
Author(s) -
Alsenaidy Mohammad A.,
Wang Tingting,
Kim Jae Hyun,
Joshi Sangeeta B.,
Lee Jihun,
Blaber Michael,
Volkin David B.,
Middaugh C. Russell
Publication year - 2012
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2008
Subject(s) - circular dichroism , chemistry , mutant , biophysics , heparin , dynamic light scattering , fibroblast growth factor , fibroblast growth factor receptor , phase diagram , phase (matter) , crystallography , biochemistry , materials science , receptor , biology , organic chemistry , nanotechnology , gene , nanoparticle
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.