ABSTRACT
For the successful generative engineering of functional artificial cells, a convenient and controllable means of delivering membrane proteins into membrane lipid bilayers is necessary. Here we report a delivery system that achieves this by employing membrane protein-carrying nanodiscs and the calcium-dependent fusion of phosphatidylserine lipid membranes. We show that lipid nanodiscs can fuse a transported lipid bilayer with the lipid bilayers of small unilamellar vesicles (SUVs) or giant unilamellar vesicles (GUVs) while avoiding recipient vesicles aggregation. This is triggered by a simple, transient increase in calcium concentration, which results in efficient and rapid fusion in a one-pot reaction. Furthermore, nanodiscs can be loaded with membrane proteins that can be delivered into target SUV or GUV membranes in a detergent-independent fashion while retaining their functionality. Nanodiscs have a proven ability to carry a wide range of membrane proteins, control their oligomeric state, and are highly adaptable. Given this, our approach may be the basis for the development of useful tools that will allow bespoke delivery of membrane proteins to protocells, equipping them with the cell-like ability to exchange material across outer/subcellular membranes.
ABSTRACT
We report the identification and characterization of a bacteriophage λ-encoded protein, NinH. Sequence homology suggests similarity between NinH and Fis, a bacterial nucleoid-associated protein (NAP) involved in numerous DNA topology manipulations, including chromosome condensation, transcriptional regulation and phage site-specific recombination. We find that NinH functions as a homodimer and is able to bind and bend double-stranded DNA in vitro. Furthermore, NinH shows a preference for a 15â bp signature sequence related to the degenerate consensus favored by Fis. Structural studies reinforced the proposed similarity to Fis and supported the identification of residues involved in DNA binding which were demonstrated experimentally. Overexpression of NinH proved toxic and this correlated with its capacity to associate with DNA. NinH is the first example of a phage-encoded Fis-like NAP that likely influences phage excision-integration reactions or bacterial gene expression.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacteriophage lambda/genetics , Bacteriophage lambda/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Bacterial Proteins/chemistry , Base Sequence , Binding Sites , Computer Simulation , DNA/metabolism , DNA, Viral/metabolism , DNA-Binding Proteins/chemistry , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Factor For Inversion Stimulation Protein/chemistry , Factor For Inversion Stimulation Protein/genetics , Gene Expression , Mutant Proteins/metabolism , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Multimerization/genetics , Viral Proteins/chemistryABSTRACT
DNA nanotechnology research has long-held promise as a means of developing functional molecules capable of delivery to cells. Recent advances in DNA origami have begun to realize this potential but is still at the earliest stage and a number of hurdles remain. This review focuses on progress in addressing these hurdles and considers some of the challenges still outstanding. These include stability of such structures necessary to reach target cells after administration; methods of cell targeting and uptake; strategies to avoid or escape endosomes and techniques for achieving specific subcellular localization. Finally, the functionality that can be expected once DNA origami structures reach their final intracellular targets will be considered.
