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. The results indicate that Skp modulates the observed rate of PagP folding in a manner that is dependent on the composition of the membrane and the ionic strength of the buffer used. These data suggest that electrostatic interactions play an important role in Skp-assisted substrate delivery to the membrane. In contrast, SurA showed no effect on the observed folding rates of PagP, consistent with the view that these chaperones act by distinct mechanisms in partially redundant parallel chaperone pathways that facilitate OMP assembly. In addition to delivery of the substrate protein to the membrane, the ability of Skp to prevent OMP aggregation was investigated. The results show that folding and membrane insertion of PagP can be restored, in part, by Skp in conditions that strongly favour PagP aggregation. These results illustrate the utility of in vitro systems for dissecting the complex folding environment encountered by OMPs in the periplasm and demonstrate the key role of Skp in holding aggregation-prone OMPs prior to their direct or indirect delivery to the membrane.

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. Here the role played by individual residues in the native helix III sequence in locking Im7 into a stable native structure is further examined. This work commenced with an Im7 sequence trapped in the partially folded state by substitution of the six residues in helix III with a polyglycine sequence. Biophysical analysis of variants in which individual residues from the native helix III sequence, and combinations of these residues, were introduced into this background demonstrated a critical requirement for three residues, Leu 53, Ile 54 and Tyr 55, to lock Im7 into its unique native structure. The results demonstrate a stringent constraint on the evolution of the Im7 helix III sequence rationalizing its high-sequence identity in the fold family. Thus, Leu 53 and Ile 54 provide crucial stabilizing interactions in the hydrophobic core of native Im7, while Tyr 55 is required for both stability and function. In contrast, Tyr 56 is critical for colicin binding and has no role in maintaining a stable native fold.

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). Glycans were introduced at seven solvent exposed sites within the Im7 sequence and the kinetic and thermodynamic consequences of N-linked glycosylation analyzed. The ΔΔG° values for glycan incorporation were found to range from +5.2 to -3.8 kJ·mol(-1). In several cases, glycosylation influences folding by modulating the local conformational preferences of the glycosylated sequence. These locally mediated effects are most prominent in the center of α-helices where glycosylation negatively effects folding and in compact turn motifs between segments of ordered secondary structure where glycosylation promotes folding and enhances the overall stability of the native protein. The studies also provide insight into why glycosylation is commonly identified at the transition between different types of secondary structure and when glycosylation may be used to elaborate protein structure to protect disordered sequences from proteolysis or immune system recognition.

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. This small, four-helix protein has previously been shown to fold via a compact three-helical intermediate state. Here, overpacking substitutions, in which residue side-chain size is increased, were used to examine the specificity and malleability of core packing in the folding intermediate and rate-limiting transition state. In parallel, polar groups were introduced into the Im7 hydrophobic core via Val-->Thr or Phe-->Tyr substitutions and used to determine the solvation status of core residues at different stages of folding. Over 30 Im7 variants were created allowing both series of substitutions to cover all regions of the protein structure. Phi-value analysis demonstrated that the major changes in Im7 core solvation occur prior to the population of the folding intermediate, with key regions involved in docking of the short helix III remaining solvent-exposed until after the rate-limiting transition state has been traversed. In contrast, overpacking core residues revealed that some regions of the native Im7 core are remarkably malleable to increases in side-chain volume. Overpacking residues in other regions of the Im7 core result in substantial (>2.5 kJ mol(-1)) destabilisation of the native structure or even prevents efficient folding to the native state. This study provides new insights into Im7 folding; demonstrating that whilst desolvation occurs early during folding, adoption of a specifically packed core is achieved only at the very last step in the folding mechanism.

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.