Abstract
Investigations into protein folding are largely dominated by studies on monomeric proteins. However, the transmembrane domain of an important group of membrane proteins is only formed upon multimerization. Here, we use in vitro translation-coupled folding and insertion into artificial liposomes to investigate kinetic steps in the assembly of one such protein, the outer membrane secretin PulD of the bacterial type II secretion system. Analysis of the folding kinetics, measured by the acquisition of distinct determinants of the native state, provides unprecedented evidence for a sequential multistep process initiated by membrane-driven oligomerization. The effects of varying the lipid composition of the liposomes indicate that PulD first forms a "prepore" structure that attains the native state via a conformational switch.
Highlights
PulD forms the dodecameric outer membrane portal in the type II secretion system and can self-assemble
Multimers obtained in this way are indistinguishable by all available criteria from those isolated from the outer membrane [28, 29, 36]
By analyzing features of the PulD native state in model membranes, we show that PulD can form an alternative, probably loosely packed multimeric intermediate that is membrane-embedded (Fig. 4, stage ii) before a conformational switch triggers formation of the native state
Summary
PulD forms the dodecameric outer membrane portal in the type II secretion system and can self-assemble. Membrane protein folding is a rapidly expanding field of research in which experiment and computation on model systems deliver important insights into how membrane proteins are stabilized and interact with lipids [1,2,3,4,5,6,7,8] and how they assemble in vivo (9 –11) In many such analyses, the protein under study is monomeric or is maintained in a monomeric form, often to reduce the complexity of the system [8, 12,13,14]. Several membrane proteins reach their native state in such in vitro systems [27], including secretins from type II secretion systems [28, 29] Such tractability could allow a detailed analysis of the assembly of multimeric membrane proteins, if ways can be found to trap assembly intermediates. We further characterize a membrane-associated intermediate state and speculate that a conformational switch occurs at the same time as native pore formation
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