Dissecting the effects of periplasmic chaperones on the in vitro folding of the outer membrane protein PagP.

Although many periplasmic folding factors have been identified, the mechanisms by which they interact with unfolded outer membrane proteins (OMPs) to promote correct folding and membrane insertion remain poorly understood. Here, we have investigated the effect of two chaperones, Skp and SurA, on the folding kinetics of the OMP, PagP. Folding kinetics of PagP into both zwitterionic diC12:0PC (1,2-dilauroyl-sn-glycero-3-phosphocholine) liposomes and negatively charged 80:20 diC12:0PC:diC12:0PG [1,2-dilauroyl-sn-glycero-3-phospho-(1'-rac-glycerol)] liposomes were investigated using a combination of spectroscopic and SDS-PAGE assays.

2-O-alkylated para-benzamide α-helix mimetics: the role of scaffold curvature.

The design and synthesis of a new 2-O-alklyated benzamide α-helix mimetic is described. Comparison with regioisomeric 3-O-alkylated benzamides permits a preliminary evaluation of the role that mimetic curvature has in determining molecular recognition properties.

Dissecting key residues in folding and stability of the bacterial immunity protein 7.

The small four-helix immunity protein, Im7, has previously been shown to fold via a compact intermediate containing three of the four native helices. The short, six-residue helix III only docks onto the developing Im7 structure after the rate-limiting second transition state has been traversed. Previous work demonstrated that mutation of the helix III sequence can be used to trap the protein in the on-pathway intermediate ensemble at equilibrium.

Perturbing the folding energy landscape of the bacterial immunity protein Im7 by site-specific N-linked glycosylation.

N-linked glycosylation modulates protein folding and stability through a variety of mechanisms. As such there is considerable interest in the development of general rules to predict the structural consequences of site-specific glycosylation and to understand how these effects can be exploited in the design and development of modified proteins with advantageous properties. In this study, expressed protein ligation is used to create site-specifically glycosylated variants of the bacterial immunity protein Im7 modified with the chitobiose disaccharide (GlcNAc-GlcNAc).

Desolvation and development of specific hydrophobic core packing during Im7 folding.

Development of a tightly packed hydrophobic core drives the folding of water-soluble globular proteins and is a key determinant of protein stability. Despite this, there remains much to be learnt about how and when the hydrophobic core becomes desolvated and tightly packed during protein folding. We have used the bacterial immunity protein Im7 to examine the specificity of hydrophobic core packing during folding.

An expanding arsenal of experimental methods yields an explosion of insights into protein folding mechanisms.

In recent years, improvements in experimental techniques and enhancements in computing power have revolutionized our understanding of the mechanisms of protein folding. By combining insights gained from theory, experiment and simulation we are moving toward an atomistic view of folding landscapes. Future challenges involve exploiting the knowledge gained and methods developed to enable us to elucidate a molecular description of folding dynamics in the complex environment of the cell.