Professor Richard Berry D. Phil.

Abstract:

Over the past 50 years, protein complexes have been studied with techniques such as X-ray crystallography and electron microscopy, generating images which although detailed are static and homogeneous. More recently, limited application of in vivo fluorescence and other techniques has revealed that many complexes previously thought stable and compositionally uniform are dynamically variable, continually exchanging components with a freely circulating pool of “spares.” Here, we consider the purpose and prevalence of protein exchange, first reviewing the ongoing story of exchange in the bacterial flagella motor, before surveying reports of exchange in complexes across all domains of life, together highlighting great diversity in timescales and functions. Finally, we put this in the context of high-throughput proteomic studies which hint that exchange might be the norm, rather than an exception.

Abstract:

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents. Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol. Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo 3 -oxidase and F 1 F o ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to > 10 microns, as well as ATP production by this chain.