The flavodoxin from Helicobacter pylori: structural determinants of thermostability and FMN cofactor binding.


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. Study holds ProTherm entries: 23067, 23068, 23069, 23070, 23071, 23072, 23073, 23074, 23075, 23076, 23077, 23078, 23079, 23080, 23081, 23082, 23083, 23084, 23085, 23086, 23087, 23088, 23089, 23090, 23091, 23092, 23093, 23094, 23095, 23096, 23097, 23098, 23099 Extra Details: 1st transition Flavodoxin, Helicobacter pylori, thermostability, structural characteristics

Submission Details

ID: iYo5VWYk3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:54 p.m.

Version: 1

Publication Details
Cremades N;Velazquez-Campoy A;Freire E;Sancho J,Biochemistry (2008) The flavodoxin from Helicobacter pylori: structural determinants of thermostability and FMN cofactor binding. PMID:18095659
Additional Information

Study Summary

Number of data points 72
Proteins Flavodoxin ; Flavodoxin
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dHcal buffers:MOPS: 50 mM, prot_conc:20 microM ; Experimental Assay: Tm buffers:MOPS: 50 mM, prot_conc:20 microM ; Experimental Assay: dHcal prot_conc:20 microM, buffers:EPPS: 50 mM ; Experimental Assay: dHcal buffers:EPPS: 50 mM, prot_conc:40 microM ; Experimental Assay: dHcal buffers:MOPS: 50 mM, prot_conc:20 microM, pH:7.0 ; Experimental Assay: Tm buffers:MOPS: 50 mM, prot_conc:20 microM, pH:7.0 ; Experimental Assay: dCp prot_conc:40 microM ; Experimental Assay: Tm prot_conc:20 microM, buffers:EPPS: 50 mM ; Experimental Assay: dHvH prot_conc:20 microM ; Experimental Assay: dHcal buffers:MOPS: 50 mM, prot_conc:2-20 microM, pH:7.0 ; Experimental Assay: Tm buffers:MOPS: 50 mM, prot_conc:2-20 microM, pH:7.0 ; Experimental Assay: dHcal prot_conc:2-20 microM, buffers:EPPS: 50 mM ; Experimental Assay: dCp prot_conc:50 microM ; Experimental Assay: dHcal prot_conc:50 microM, buffers:EPPS: 50 mM ; Experimental Assay: Tm prot_conc:50 microM, buffers:EPPS: 50 mM ; Experimental Assay: dHvH prot_conc:50 microM ; Experimental Assay: Tm buffers:EPPS: 50 mM, ionic:NaCl: 45 mM, prot_conc:20-40 microM ; Experimental Assay: dHvH ionic:NaCl: 45 mM, prot_conc:20-40 microM ; Experimental Assay: Tm prot_conc:20 microM, buffers:EPPS: 50 mM, ionic:NaCl: 45 mM ; Experimental Assay: dHvH prot_conc:20 microM, ionic:NaCl: 45 mM ; Experimental Assay: Tm prot_conc:2-20 microM, buffers:EPPS: 50 mM ; Experimental Assay: dHvH prot_conc:2-20 microM ; Experimental Assay: Tm buffers:EPPS: 50 mM, prot_conc:40 microM ; Experimental Assay: dHvH prot_conc:40 microM
Libraries Mutations for sequence MGKIGIFFGTDSGNAEAIAEKISKAIGNAEVVDVAKASKEQFNSFTKVILVAPTAGAGDLQTDWEDFLGTLEASDFANKTIGLVGLGDQDTYSETFAEGIFHIYEKAKAGKVVGQTSTDGYHFEASKAVEGGKFVGLVIDEDNQDDLTDERISKWVEQVKGSFA

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2BMV 2006-06-22 2.11 Apoflavodoxin from Helicobacter pylori
1FUE 2002-02-06 2.4 FLAVODOXIN FROM HELICOBACTER PYLORI
2W5U 2009-12-22 2.62 Flavodoxin from Helicobacter pylori in complex with the C3 inhibitor

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
97.6 Flavodoxin Q9ZK53 FLAV_HELPJ
100.0 Flavodoxin O25776 FLAV_HELPY