title: The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces.
abstract: Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Hückel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution.
authors.affiliation: Medical Research Council Centre for Protein Engineering, Cambridge, United Kingdom.
authors.forename: Eyal
authors.initials: E
authors.lastname: Arbely
authors.forename: Hannes
authors.initials: H
authors.lastname: Neuweiler
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Sharpe
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Johnson
authors.forename: Alan R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D040681:Structural Homology, Protein; D013816:Thermodynamics
chemical_list: D011768:Pyruvate Dehydrogenase Complex