The flavodoxin from Helicobacter pylori

Structural determinants of thermostability and FMN cofactor binding

Nunilo Cremades, Adrián Velazquez-Campoy, Ernesto I Freire, Javier Sancho

Research output: Contribution to journalArticle

Abstract

Flavodoxin has been recently recognized as an essential protein for a number of pathogenic bacteria including Helicobacter pylori, where it has been proposed to constitute a target for antibacterial drug development. One way we are exploring to screen for novel inhibitory compounds is to perform thermal upshift assays, for which a detailed knowledge of protein thermostability and cofactor binding properties is of great help. However, very little is known on the stability and ligand binding properties of H. pylori flavodoxin, and its peculiar FMN binding site together with the variety of behaviors observed within the flavodoxin family preclude extrapolations. We have thus performed a detailed experimental and computational analysis of the thermostability and cofactor binding energetics of H. pylori flavodoxin, and we have found that the thermal unfolding equilibrium is more complex that any other previously described for flavodoxins as it involves the accumulation of two distinct equilibrium intermediates. Fortunately the entire stability and binding data can be satisfactorily fitted to a model, summarized in a simple phase diagram, where the cofactor only binds to the native state. On the other hand, we show how variability of thermal unfolding behavior within the flavodoxin family can be predicted using structure-energetics relationships implemented in the COREX algorithm. The different distribution and ranges of local stabilities of the Anabaena and H. pylori apoflavodoxins explain the essential experimental differences observed: much lower Tm1, greater resistance to global unfolding, and more pronounced cold denaturation in H. pylori. Finally, a new strategy is proposed to identify using COREX structural characteristics of equilibrium intermediate states populated during protein unfolding.

Original languageEnglish (US)
Pages (from-to)627-639
Number of pages13
JournalBiochemistry®
Volume47
Issue number2
DOIs
StatePublished - Jan 15 2008

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Flavodoxin
Flavin Mononucleotide
Helicobacter pylori
Hot Temperature
Anabaena
Protein Unfolding
Proteins
Denaturation
Extrapolation
Phase diagrams
Assays
Bacteria
Binding Sites
Ligands
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Biochemistry

Cite this

The flavodoxin from Helicobacter pylori : Structural determinants of thermostability and FMN cofactor binding. / Cremades, Nunilo; Velazquez-Campoy, Adrián; Freire, Ernesto I; Sancho, Javier.

In: Biochemistry®, Vol. 47, No. 2, 15.01.2008, p. 627-639.

Research output: Contribution to journalArticle

Cremades, Nunilo ; Velazquez-Campoy, Adrián ; Freire, Ernesto I ; Sancho, Javier. / The flavodoxin from Helicobacter pylori : Structural determinants of thermostability and FMN cofactor binding. In: Biochemistry®. 2008 ; Vol. 47, No. 2. pp. 627-639.
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