Folding Of Water-soluble Proteins Research Articles

New Insights into the Molecular Mechanism of Beta Barrel Outer Membrane Protein Folding

Inspired by the seminal work of Anfinsen, investigations of the folding of small, water-soluble proteins have culminated in detailed insights into how these molecules attain and stabilise their native folds. In contrast, despite their overwhelming importance in biology, progress in understanding the folding and stability of membrane proteins remains relatively limited. Focusing on the β-barrel outer membrane protein, PagP, we have been using mutational analysis to determine how this protein folds from its urea denatured state into lipid vesicles and how this process is facilitated by molecular chaperones. In this lecture I will describe our recent experiments that have investigated the initial interactions of PagP with a bilayer, its mechanism of insertion into lipid, and how this process is facilitated by the molecular chaperones skp and surA. The work is at an early stage compared with the plethora of knowledge about the folding of water soluble proteins and how this is assisted by chaperones. Nonetheless the folding of this membrane protein is revealing new insights, new challenges and fascinating synergies with the folding mechanisms of water soluble counterparts.ReferencesHuysmans, G., Radford, S.E., Brockwell, D.J. & Baldwin, S.A. (2007) J. Mol. Biol. 373, 529-540Huysmans, G., Baldwin, S.A., Brockwell, D.J. & Radford, S.E. (2010) PNAS 107, 4099-4104Huysmans, G., Radford, S.E., Baldwin, S.A., Brockwell, D.J. (2012) J. Mol. Biol. 416, 453-464

Design of single-layer beta-sheets without a hydrophobic core.

The hydrophobic effect is the main thermodynamic driving force in the folding of water-soluble proteins. Exclusion of nonpolar moieties from aqueous solvent results in the formation of a hydrophobic core in a protein, which has been generally considered essential for specifying and stabilizing the folded structures of proteins. Outer surface protein A (OspA) from Borrelia burgdorferi contains a three-stranded beta-sheet segment which connects two globular domains. Although this single-layer beta-sheet segment is exposed to solvent on both faces and thus does not contain a hydrophobic core, the segment has a high conformational stability. Here we report the engineering of OspA variants that contain larger single-layer beta-sheets (comprising five and seven beta-strands) by duplicating a beta-hairpin unit within the beta-sheet. Nuclear magnetic resonance and small-angle X-ray scattering analyses reveal that these extended single-layer beta-sheets are formed as designed, and amide hydrogen-deuterium exchange and chemical denaturation show that they are stable. Thus, interactions within the beta-hairpin unit and those between adjacent units, which do not involve the formation of a hydrophobic core, are sufficient to specify and stabilize the single-layer beta-sheet structure. Our results provide an expanded view of protein folding, misfolding and design.