Correctly folded membrane proteins underlie a plethora of cellular processes, but little is known about how they fold. Knowledge of folding mechanisms centres on reversible folding of chemically denatured membrane proteins. However, this cannot replicate the unidirectional elongation of the protein chain during co-translational folding in the cell, where insertion is assisted by translocase apparatus. We show that a lipid membrane (devoid of translocase components) is sufficient for successful co-translational folding of two bacterial α-helical membrane proteins, DsbB and GlpG. Folding is spontaneous, thermodynamically driven, and the yield depends on lipid composition. Time-resolving structure formation during co-translational folding revealed different secondary and tertiary structure folding pathways for GlpG and DsbB that correlated with membrane interfacial and biological transmembrane amino acid hydrophobicity scales. Attempts to refold DsbB and GlpG from chemically denatured states into lipid membranes resulted in extensive aggregation. Co-translational insertion and folding is thus spontaneous and minimises aggregation whilst maximising correct folding.

There is a limited understanding of the folding of multidomain membrane proteins. Lactose permease (LacY) of Escherichia coli is an archetypal member of the major facilitator superfamily of membrane transport proteins, which contain two domains of six transmembrane helices each. We exploit chemical denaturation to determine the unfolding free energy of LacY and employ Trp residues as site specific thermodynamic probes. Single Trp LacY mutants are created with the individual Trps situated at mirror image positions on the two LacY domains. The changes in Trp fluorescence induced by urea denaturation are used to construct denaturation curves from which unfolding free energies can be determined. The majority of the single Trp tracers report the same stability and an unfolding free energy of ~ +2 kcal.mol-1. There is one exception; the fluorescence of W33 at the cytoplasmic end of helix I on the N domain is unaffected by urea. In contrast, the equivalent position on the first helix, VII, of the C terminal domain exhibits wild type stability, with the single Trp tracer at position 243 on helix VII reporting an unfolding free energy of +2 kcal.mol-1. This indicates that the region of the N domain of LacY at position 33 on helix I has enhanced stability to urea, when compared the corresponding location at the start of the C domain. We also find evidence for a potential network of stabilising interactions across the domain interface, which reduces accessibility to the hydrophilic substrate binding pocket between the two domains. 1 Relative domain folding and stability of a membrane transport proteinHarris et al. Response to referees' commentsWe thank the referees for their helpful comments and have made changes to the manuscript in accordance with the issues raised by the editor and referees as detailed below. Additionally to improve clarity we have made colour version of the figures. EditorWe have re-written the abstract and expanded the explanation of the single Trp mutants, p2&5.It is hard to comment on the absolute magnitude of the free energy value determined for LacY and GalP, in the absence of thermodynamic measurements on other membrane proteins as well as studies using different methods to assess thermodynamic stability in lipid bilayers and kinetic stability. The free energy is small implying that the inherent protein structure has relatively low thermodynamic stability. It could be that the bilayer increases the kinetic stability, or that the absolute magnitude is partly dependent on the renaturing solvent since it is hard to separate the folding systems from the solvent surroundings in membrane protein unfolding studies. Referee 1Technical concerns: 1. DDM CMC in ureaThe CMC of DDM in different concentrations of urea was measured and showed that micelles are present with a DDM concentration of 1mM even at the highest urea concentration of 8M (as previously used for GalP). We have clarified this in the methods (p15) and added a figure S8 to the supplementary information showing the increase in DDM CM...

Transmembrane transporters are responsible for maintaining a correct internal cellular environment. The inherent flexibility of transporters together with their hydrophobic environment means that they are challenging to study in vitro, but recently significant progress been made. This review will focus on in vitro stability and folding studies of transmembrane alpha helical transporters, including reversible folding systems and thermal denaturation. The successful re-assembly of a small number of ATP binding cassette transporters is also described as this is a significant step forward in terms of understanding the folding and assembly of these more complex, multi-subunit proteins. The studies on transporters discussed here represent substantial advances for membrane protein studies as well as for research into protein folding. The work demonstrates that large flexible hydrophobic proteins are within reach of in vitro folding studies, thus holding promise for furthering knowledge on the structure, function and biogenesis of ubiquitous membrane transporter families. This article is part of a Special Issue entitled: Protein Folding in Membranes.

Nature encapsulates reactions within membrane-bound compartments, affording sequential and spatial control over biochemical reactions. Droplet Interface Bilayers are evolving into a valuable platform to mimic this key biological feature in artificial systems. A major issue is manipulating flow across synthetic bilayers. Droplet Interface Bilayers must be functionalised, with seminal work using membrane-inserting toxins, ion channels and pumps illustrating the potential. Specific transport of biomolecules, and notably transport against a concentration gradient, across these bilayers has yet to be demonstrated. Here, we successfully incorporate the archetypal Major Facilitator Superfamily transporter, lactose permease, into Droplet Interface Bilayers and demonstrate both passive and active, uphill transport. This paves the way for controllable transport of sugars, metabolites and other essential biomolecular substrates of this ubiquitous transporter superfamily in DIB networks. Furthermore, cell-free synthesis of lactose permease during DIB formation also results in active transport across the interface bilayer. This adds a specific disaccharide transporter to the small list of integral membrane proteins that can be synthesised via in vitro transcription/translation for applications of DIB-based artificial cell systems. The introduction of a means to promote specific transport of molecules across Droplet Interface Bilayers against a concentration gradient gives a new facet to droplet networks.

Membrane proteins must be inserted into a membrane and folded into their correct structure to function correctly. This insertion occurs during translation and synthesis by the ribosome for most α-helical membrane proteins. Precisely how this co-translational insertion and folding occurs, and the role played by the surrounding lipids, is still not understood. Most of the work on the influence of the lipid environment on folding and insertion has focussed on denatured, fully translated proteins, and thus does not replicate folding during unidirectional elongation of nascent chains that occurs in the cell. This review aims to highlight recent advances in elucidating lipid composition and bilayer properties optimal for insertion and folding of nascent chains in the membrane and in the assembly of oligomeric proteins.