A Tunable and Reversible Platform for the Intracellular Formation of Genetically Engineered Protein Microdomains

Citations Over TimeTop 19% of 2012 papers

Abstract

From mitochondria to the nuclear envelope, the controlled assembly of micro- and nanostructures is essential for life; however, the level at which we can deliberately engineer the assembly of microstructures within intracellular environments remains primitive. To overcome this obstacle, we present a platform to reversibly assemble genetically engineered protein microdomains (GEPMs) on the time scale of minutes within living cells. Biologically inspired from the human protein tropoelastin, these protein polymers form a secondary aqueous phase above a tunable transition temperature. This assembly process is easily manipulated to occur at or near physiological temperature by adjusting molecular weight and hydrophobicity. We fused protein polymers to green fluorescent protein (GFP) to visualize their behavior within the cytoplasm. While soluble, these polymers have a similar intracellular diffusion constant as cytosolic proteins at 7.4 μm(2)/s; however, above their phase transition temperature, the proteins form distinct microdomains (0.1-2 μm) with a reduced diffusion coefficient of 1.1 μm(2)/s. Microdomain assembly and disassembly are both rapid processes with half-lives of 3.8 and 1.0 min, respectively. Via selection of the protein polymer, the assembly temperature is tunable between 20 and 40 °C. This approach may be useful to control intracellular formation of genetically engineered proteins and protein complexes into concentrated microdomains.

Related Papers

• → Distinct Steps of Cross-linking, Self-association, and Maturation of Tropoelastin Are Necessary for Elastic Fiber Formation(2007)77 cited
• → Gene Expression of Tropoelastin Is Enhanced in the Aorta Proximal to the Coarctation in Rabbits(2002)12 cited
• → The Role of the Carboxy Terminus of Tropoelastin in its Assembly into the Elastic Fiber(1999)31 cited
• → Tropoelastin Inhibits Vascular Calcification via 67-kDa Elastin Binding Protein in Cultured Bovine Aortic Smooth Muscle Cells(2004)29 cited
• → Kinetics of the Incorporation of Tropoelastin into Elastic Fibers in Embryonic Chick Aortad(1982)9 cited