| Autor: |
Stuut Balsam S; Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire 03755, United States., Zhong F; Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755, United States., Pence N; Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755, United States., Levintov L; Department of Chemical Engineering and Bioengineering, University of New Hampshire, Durham, New Hampshire 03824, United States., Andhare D; Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755, United States., Hammond JH; Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire 03755, United States., Ragusa MJ; Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755, United States., Vashisth H; Department of Chemical Engineering and Bioengineering, University of New Hampshire, Durham, New Hampshire 03824, United States.; Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824, United States.; Integrated Applied Mathematics Program, University of New Hampshire, Durham, New Hampshire 03824, United States.; Molecular and Cellular Biotechnology Program, University of New Hampshire, Durham, New Hampshire 03824, United States., Hogan DA; Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire 03755, United States., Pletneva EV; Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755, United States. |
| Abstrakt: |
Many bacteria have hemerythrin (Hr) proteins that bind O 2 , including Pseudomonas aeruginosa , in which microoxia-induced Hr (Mhr) provide fitness advantages under microoxic conditions. Mhr has a 23 amino-acid extension at its C -terminus relative to a well-characterized Hr from Methylococcus capsulatus , and similar extensions are also found in Hrs from other bacteria. The last 11 amino acids of this extended, C -terminal tail are highly conserved in gammaproteobacteria and predicted to form a helix with positively charged and hydrophobic faces. In cellular fractionation assays, wild-type (WT) Mhr was found in both membrane and cytosolic fractions, while a Mhr W143* variant lacking the last 11 residues was largely in the cytosol and did not complement Mhr function in competition assays. Mhr L112Y , a variant that has a much longer-lived O 2 -bound form, was fully functional and had a similar localization pattern to that of WT Mhr. Both Mhr W143* and Mhr L112Y had secondary structures, stabilities, and O 2 -binding kinetics similar to those of WT Mhr. Fluorescence studies revealed that the C -terminal tail, and particularly the fragment corresponding to its last 11 residues, was sufficient and necessary for association with lipid vesicles. Molecular dynamics simulations and subsequent cellular analysis of Mhr variants have demonstrated that conserved, positively charged residues in the tail are important for Mhr interactions with negatively charged membranes and the contribution of this protein to competitive fitness. Together, these data suggest that peripheral interactions of Mhr with membranes are guided by the C-terminal tail and are independent of O 2 -binding. |
