Abstract
Human acidic fibroblast growth factor (hFGF1) is an all beta-sheet protein that is involved in the regulation of key cellular processes including cell proliferation and wound healing. hFGF1 is known to aggregate when subjected to thermal unfolding. In this study, we investigate the equilibrium unfolding of hFGF1 using a wide array of biophysical and biochemical techniques. Systematic analyses of the thermal and chemical denaturation data on hFGF1 variants (Q54P, K126N, R136E, K126N/R136E, Q54P/K126N, Q54P/R136E, and Q54P/K126N/R136E) indicate that nullification of charges in the heparin-binding pocket can significantly increase the stability of wtFGF1. Triple variant (Q54P/K126N/R136E) was found to be the most stable of all the hFGF1 variants studied. With the exception of triple variant, thermal unfolding of wtFGF1 and the other variants is irreversible. Thermally unfolded triple variant refolds completely to its biologically native conformation. Microsecond-level molecular dynamic simulations reveal that a network of hydrogen bonds and salt bridges linked to Q54P, K126N, and R136E mutations, are responsible for the high stability and reversibility of thermal unfolding of the triple variant. In our opinion, the findings of the study provide valuable clues for the rational design of a stable hFGF1 variant that exhibits potent wound healing properties.
Highlights
Human acidic fibroblast growth factor is an all beta-sheet protein that is involved in the regulation of key cellular processes including cell proliferation and wound healing. hFGF1 is known to aggregate when subjected to thermal unfolding
We recently demonstrated that a charge reversal mutation (R136E) in the heparin-binding pocket (HBP) marginally decreases the heparinbinding affinity but enhances the cell proliferation activity of h FGF119. hFGF1 is known to be inherently unstable molecule at temperatures just above the physiological temperature[20]
Introduction of Q54P, K126N, and R136E plausibly aid in minimizing the exposure of hydrophobic regions to the surface and restricts the conformational fluctuations occurring in the heparin-binding region (HBR), thereby leading to refolding and higher stability of wtFGF1
Summary
Human acidic fibroblast growth factor (hFGF1) is an all beta-sheet protein that is involved in the regulation of key cellular processes including cell proliferation and wound healing. hFGF1 is known to aggregate when subjected to thermal unfolding. The propensity to aggregate is attributed to inter-chain interactions between solvent-exposed hydrophobic surfaces present in obligatory and non-obligatory partially structured intermediates that populate the kinetic/equilibrium folding/unfolding pathways of proteins[11]. In this context, understanding the structural determinants governing protein aggregation is critical for the rational design of drugs against the multitude of amyloid diseases[12]. It is believed that addition of low concentrations of the denaturant potentially destabilizes the stable obligatory intermediate that accumulates in the GdnHCl-induced equilibrium unfolding pathway of hFGF126. We believe that the findings of this study shed valuable insights into the interplay of structural forces that confer reversible unfolding behavior to the protein and provides useful clues for the rational design of a hFGF1-based therapeutics to manage chronic wounds
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call