Abstract:
:Acidic fibroblast growth factors (FGF1s) are heparin binding proteins that regulate a wide array of key cellular processes and are also candidates for promising biomedical applications. FGF1-based therapeutic applications are currently limited due to their inherent thermal instability and susceptibility to proteases. Using a wide range of biophysical and biochemical techniques, we demonstrate that reversal of charge on a well-conserved positively charged amino acid, R136, in the heparin binding pocket drastically increases the resistance to proteases, thermal stability, and cell proliferation activity of the human acidic fibroblast growth factor (hFGF1). Two-dimensional NMR data suggest that the single point mutations at position-136 (R136G, R136L, R136Q, R136K, and R136E) did not perturb the backbone folding of hFGF1. Results of the differential scanning calorimetry experiments show that of all the designed R136 mutations only the charge reversal mutation, R136E, significantly increases (ΔTm = 7 °C) the thermal stability of the protein. Limited trypsin and thrombin digestion results reveal that the R136E mutation drastically increases the resistance of hFGF1 to the action of the serine proteases. Isothermal titration calorimetry data show that the R136E mutation markedly decreases the heparin binding affinity of hFGF1. Interestingly, despite lower heparin binding affinity, the cell proliferation activity of the R136E variant is more than double of that exhibited by either the wild type or the other R136 variants. The R136E variant due to its increased thermal stability, resistance to proteases, and enhanced cell proliferation activity are expected to provide valuable clues for the development of hFGF1- based therapeutics for the management of chronic diabetic wounds.
journal_name
Biochem Biophys Res Communjournal_title
Biochemical and biophysical research communicationsauthors
Kerr R,Agrawal S,Maity S,Koppolu B,Jayanthi S,Suresh Kumar G,Gundampati RK,McNabb DS,Zaharoff DA,Kumar TKSdoi
10.1016/j.bbrc.2019.08.029subject
Has Abstractpub_date
2019-10-15 00:00:00pages
191-196issue
2eissn
0006-291Xissn
1090-2104pii
S0006-291X(19)31540-2journal_volume
518pub_type
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