title: Negative Charge Neutralization in the Loops and Turns of Outer Membrane Phospholipase A Impacts Folding Hysteresis at Neutral pH.
abstract: Hysteresis in equilibrium protein folding titrations is an experimental barrier that must be overcome to extract meaningful thermodynamic quantities. Traditional approaches to solving this problem involve testing a spectrum of solution conditions to find ones that achieve path independence. Through this procedure, a specific pH of 3.8 was required to achieve path independence for the water-to-bilayer equilibrium folding of outer membrane protein OmpLA. We hypothesized that the neutralization of negatively charged side chains (Asp and Glu) at pH 3.8 could be the physical basis for path-independent folding at this pH. To test this idea, we engineered variants of OmpLA with Asp → Asn and Glu → Gln mutations to neutralize the negative charges within various regions of the protein and tested for reversible folding at neutral pH. Although not fully resolved, our results show that these mutations in the periplasmic turns and extracellular loops are responsible for 60% of the hysteresis in wild-type folding. Overall, our study suggests that negative charges impact the folding hysteresis in outer membrane proteins and their neutralization may aid in protein engineering applications.
authors.affiliation: T. C. Jenkins Department of Biophysics, Johns Hopkins University , 3400 North Charles Street, Baltimore, Maryland 21218, United States.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: McDonald
authors.affiliation: T. C. Jenkins Department of Biophysics, Johns Hopkins University , 3400 North Charles Street, Baltimore, Maryland 21218, United States.
authors.forename: Karen G
authors.initials: KG
authors.lastname: Fleming
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D054466:Phospholipases A1 / Q000737:chemistry*; D017510:Protein Folding
chemical_list: D001425:Bacterial Outer Membrane Proteins; D054466:Phospholipases A1; C412876:outer membrane phospholipase A