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When nanoparticles and nanoplastics enter biological fluids, their surfaces are rapidly coated with proteins, forming a corona that governs biological responses. However, understanding protein- surface interaction energetics remains a significant challenge. Here, we examine how protein charge distribution affects adsorption to polystyrene nanoparticles (PSNPs) by generating a series of lysine-to-alanine variants of the GB3 protein. Using isothermal titration calorimetry (ITC), we found that the K19A variant binds most strongly to both non-functionalized and carboxylate- functionalized PSNPs. ITC thermograms indicate that K19A forms a stable monolayer, while other variants exhibit multilayer adsorption. We hypothesize that removing lysine at position 19 creates a flatter, more neutral interaction surface that promotes efficient initial binding. Fluorescence denaturation experiments show that PSNPs destabilize GB3 protein variants, and binding correlates strongly with protein unfolding (r = 0.82, p < 0.01 for COOH-PSNPs and r = 0.76, p < 0.03 for non-functionalized PSNPs). These results reveal how protein stability and charge distribution shape adsorption thermodynamics, offering a framework for predicting protein-surface interactions.
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Characterization of the structural forces governing the reversibility of the thermal unfolding of the human acidic fibroblast growth factor
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.
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- Award ID(s):
- 1945465
- PAR ID:
- 10282938
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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