Reversible Thermal Denaturation of Human FGF-1 Induced by Low Concentrations of Guanidine Hydrochloride

Blaber, Sachiko I.; Culajay, Juan F.; Khurana, Archana; Blaber, Michael

doi:10.1016/s0006-3495(99)76904-3

Cited by 67 publications

(142 citation statements)

References 44 publications

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“…Addition of low concentrations of GdmHCl (0.6 -1.0 M) eliminated the formation of protein aggregates and resulted in quasireversible behavior of the sample (Fig. 3) as previously reported for the human fibroblast growth factor-1 protein (24). For these samples, ϳ20 -50% of the protein contributed to a well developed unfolding endotherm on successive scans indicating that at least a fraction of the protein was able to refold reversibly under these conditions.…”

Section: Differential Scanning Calorimetrysupporting

confidence: 77%

Calorimetric Studies on the Tight Binding Metal Interactions of Escherichia coli Manganese Superoxide Dismutase

Mizuno

Whittaker

Bächinger

et al. 2004

Journal of Biological Chemistry

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Escherichia coli apomanganese superoxide dismutase, prepared by removing the native metal ion under denaturing conditions, exhibits thermally triggered metal uptake behavior previously observed for thermophilic and hyperthermophilic superoxide dismutases but over a lower temperature range. Superoxide dismutases (SODs) 1 (E.C. 1.15.1.1) are important antioxidant metalloenzymes protecting cells against oxidative stress arising from reactive oxygen species (1-4). SODs are ubiquitous, and multiple forms often exist within a single organism or cell. Four major forms of SODs (Mn, Fe, Cu/Zn, and Ni) have been identified, distinguished by the protein fold and by the nature of the catalytic metal ion (5-8). Mn-and Fe-SODs share extensive homology in protein structure, whereas the Ni and Cu/Zn enzymes are structurally distinct. Despite their similar structures (9), the Mn-and Fe-SODs exhibit a strict specificity for the catalytic metal ion (manganese or iron). Much lower selectivity is generally observed for metal binding, and the dimeric Mn-SOD from Escherichia coli is typically isolated as a mixture of Mn 2 -, (Mn,Fe)-, Fe 2 -, and half-apoforms (10, 11). Both the manganese-and iron-replete forms may be enriched by supplementing the culture medium with either manganese or iron salts (12). Apo-Mn-SOD binds metals nonselectively in the presence of guanidinium denaturants, and this method has been used to prepare manganese-, iron-, and cobalt-reconstituted forms (13-17).Each subunit of the E. coli Mn-SOD homodimer is composed of two domains, a predominantly ␣-helical N-terminal domain and a mixed ␣/␤ C-terminal domain (18). The metal-binding site lies on the interface between these two domains buried in the interior of the protein. The mononuclear metal ion is coordinated by four amino acid side chains (Fig. 1), two arising from the N-terminal domain (His-26 and His-81) and two from the C-terminal domain (Asp-167 and His-171), resulting in a metal ion cross-link between the two domains. A buried solvent molecule serves as a fifth ligand forming a hydrogen bond in the outer sphere of the metal complex with the amido head group of Glu-146 (E. coli Mn-SOD sequence numbering). The metal ion appears to be very tightly bound, being released only under denaturing conditions and resisting extraction by metal chelators like EDTA (13).Earlier work has shown that metal-free apo-SOD is produced during expression of recombinant thermophilic SODs in the mesophilic host, E. coli. The purified apoprotein has been shown to efficiently take up metal ion in vitro at elevated temperatures, and the binding is essentially irreversible under these conditions. At lower temperatures, metal uptake is not detected (19,20). The sigmoidal temperature profile for metalation of the recombinant thermophilic apo-SODs suggests that a two-state activated process is involved. The requirements for denaturing conditions to remove the metal and thermally triggered metal uptake imply the existence of large activation barriers for changes in the metalation state ...

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Section: Differential Scanning Calorimetrysupporting

confidence: 77%

Calorimetric Studies on the Tight Binding Metal Interactions of Escherichia coli Manganese Superoxide Dismutase

Mizuno

Whittaker

Bächinger

et al. 2004

Journal of Biological Chemistry

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“…2, upper panel) supporting the two-state denaturation assumption. 25 A plot of the derived u-values (Fig. 2, lower panel) indicates that FGF-1 has a highly polarized transition state, with the vast majority of evaluated positions having u-values clustered around 1.0 or 0.0 (and are therefore either fully native-like, or denatured-like, in the folding transition state, respectively).…”

Section: Resultsmentioning

confidence: 99%

“…FGF-1 is a weak mesophile as regards thermostability (DG unfolding ¼ 21.1 kJ/ mol), 25,28 and certain turn mutations can destabilize the protein by a magnitude approaching the overall DG unfolding ; thus, stabilizing background mutations were necessary for a subset of mutations. Of the 44 positions evaluated, 28 yielded |DDG| !…”

Section: Resultsmentioning

confidence: 99%

“…The good agreement between these DDG values supports the two-state denaturation model. 25 Bottom panel, foreground: DDG iso values plotted against u f -values for turn mutations. u f -values were calculated according to the equation u f ¼ 1 À u u , due to the greater accuracy with which unfolding kinetic parameters can be measured for FGF-1.…”

Section: Discussionmentioning

confidence: 99%

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Experimental support for the foldability–function tradeoff hypothesis: Segregation of the folding nucleus and functional regions in fibroblast growth factor‐1

2012

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The acquisition of function is often associated with destabilizing mutations, giving rise to the stability-function tradeoff hypothesis. To test whether function is also accommodated at the expense of foldability, fibroblast growth factor-1 (FGF-1) was subjected to a comprehensive u-value analysis at each of the 11 turn regions. FGF-1, a b-trefoil fold, represents an excellent model system with which to evaluate the influence of function on foldability: because of its threefold symmetric structure, analysis of FGF-1 allows for direct comparisons between symmetryrelated regions of the protein that are associated with function to those that are not; thus, a structural basis for regions of foldability can potentially be identified. The resulting u-value distribution of FGF-1 is highly polarized, with the majority of positions described as either foldedlike or denatured-like in the folding transition state. Regions important for folding are shown to be asymmetrically distributed within the protein architecture; furthermore, regions associated with function (i.e., heparin-binding affinity and receptor-binding affinity) are localized to regions of the protein that fold after barrier crossing (late in the folding pathway). These results provide experimental support for the foldability-function tradeoff hypothesis in the evolution of FGF-1. Notably, the results identify the potential for folding redundancy in symmetric protein architecture with important implications for protein evolution and design.

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“…In contrast, results of other studies suggest a protective role for heparin/heparan sulfate (12)(13)(14). Binding of hFGF to the glycosaminoglycans is shown to protect FGFs against proteolytic digestion and heat-and acid-induced unfolding (15)(16)(17).…”

Section: Human Acidic Fibroblast Growth Factor (Hfgf-1)mentioning

confidence: 99%

15N NMR Relaxation Studies of Free and Ligand-bound Human Acidic Fibroblast Growth Factor

Chi

Kumar

Chiu

et al. 2000

Journal of Biological Chemistry

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15N NMR relaxation data have been used to characterize the backbone dynamics of the human acidic fibroblast growth factor (hFGF-1) in its free and sucrose octasulfate (SOS)-bound states.15 N longitudinal (R 1 ), transverse (R 2 ) relaxation rates and { 1 H}-15 N steadystate nuclear Overhauser effects were obtained at 500 and 600 MHz (at 25°C) for all resolved backbone amide groups using 1 H-detected two-dimensional NMR experiments. Relaxation data were fit to the extended model free dynamics for each NH group. The overall correlation time ( m ) for the free and SOS-bound forms were estimated to be 10.4 ؎ 1.07 and 11.1 ؎ 1.35 ns, respectively. Titration experiments with SOS reveals that the ligand binds specifically to the C-terminal domain of the protein in a 1:1 ratio. Binding of SOS to hFGF-1 is found to induce a subtle conformational change in the protein. Significant conformational exchange (R ex ) is observed for several residues in the free form of the protein. However, in the SOS-bound form only three residues exhibit significant R ex values, suggesting that the dynamics on the micro-to millisecond time scale in the free form is coupled to the cis-trans-proline isomerization. hFGF-1 is a rigid molecule with an average generalized parameter (S 2 ) value of 0.89 ؎ 0.03. Upon binding to SOS, there is a marked decrease in the overall flexibility (S 2 ‫؍‬ 0.94 ؎ 0.02) of the hFGF-1 molecule. However, the segment comprising residues 103-111 shows increased flexibility in the presence of SOS. Significant correlation is found between residues that show high flexibility and the putative receptor binding sites on the protein.

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Reversible Thermal Denaturation of Human FGF-1 Induced by Low Concentrations of Guanidine Hydrochloride

Cited by 67 publications

References 44 publications

Calorimetric Studies on the Tight Binding Metal Interactions of Escherichia coli Manganese Superoxide Dismutase

Calorimetric Studies on the Tight Binding Metal Interactions of Escherichia coli Manganese Superoxide Dismutase

Experimental support for the foldability–function tradeoff hypothesis: Segregation of the folding nucleus and functional regions in fibroblast growth factor‐1

15N NMR Relaxation Studies of Free and Ligand-bound Human Acidic Fibroblast Growth Factor

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