Abstract
Flavodoxin is an essential protein for Helicobacter pylori, a pathogen living in the very acidic environment of the gastric tract and responsible for several diseases. We report the conformational stability of the protein in neutral and acidic pH. The apoprotein remains native between pH 12 and 5 and adopts a monomeric molten globule conformation at more acidic pH values. The equilibrium unfolding in urea appears two-state for either conformation, but the native one coexists with a hidden equilibrium intermediate of very similar properties. The stability of H. pylori apoflavodoxin is higher than that of the Anabaena homologue throughout the entire pH interval, which may be related to better charge compensation. H. pylori apoflavodoxin is strongly stabilized by its FMN cofactor. A global analysis of apo- and holoflavodoxin equilibrium unfolding, with and without excess FMN, indicates that the cofactor only binds to the native state. Some physical-chemical properties of the protein may represent an adaptation to the acidic environment. Unlike the apoflavodoxin from Anabaena, which becomes highly insoluble at pH 5.0, that from H. pylori remains soluble to at least 40 microm. This fact, together with the high stability of the apoprotein at this low pH that can arise in the bacteria cytoplasm, seems useful to allow newly synthesized apoflavodoxin molecules to fold and remain soluble to accomplish cofactor binding, which in turn increases the stability. Also, whenever the cytoplasmic pH drops to 5, preexisting flavodoxin molecules will remain folded and soluble and will retain the FMN cofactor, thus remaining functional.
Highlights
Flavodoxins are bacterial electron-transfer proteins that bear a noncovalently bound redox cofactor (FMN) that allows them to participate in a variety of reactions [1]
H pylori flavodoxin lacks a fairly conserved tryptophan residue involved in FMN binding in many flavodoxins and bears instead an alanine residue, which creates an exposed pocket near the redox cofactor [12] that is being used to target small inhibitory molecules [13]
We report here the conformational stability of H. pylori flavodoxin in a wide pH interval to try to understand how the protein can remain functional at moderately acidic pH values where flavodoxin is expected to be either aggregated or highly insoluble [3], and as a contribution to understanding the somewhat controversial equilibrium and binding behavior of the flavodoxin family
Summary
We report here the conformational stability of H. pylori flavodoxin in a wide pH interval to try to understand how the protein can remain functional at moderately acidic pH values where flavodoxin is expected to be either aggregated or highly insoluble [3], and as a contribution to understanding the somewhat controversial equilibrium and binding behavior of the flavodoxin family. A global analysis of the unfolding of the apo- and holo-forms, with and without excess FMN, shows that only the native state can interact with the cofactor The apoflavodoxin from H. pylori remains folded, soluble. and in an FMN-competent state at the fairly low pH values that can arise in the cytoplasm [27] of the pathogen, and the holoprotein is highly stable
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