Communication pubs.acs.org/Biomac
A Tunable and Reversible Platform for the Intracellular Formation of Genetically Engineered Protein Microdomains Martha K. Pastuszka, Siti M. Janib, Isaac Weitzhandler, Curtis T. Okamoto, Sarah Hamm-Alvarez, and J. Andrew MacKay* Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue, Los Angeles, California 90033-9121, United States S Supporting Information *
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 μm2/s; however, above their phase transition temperature, the proteins form distinct microdomains (0.1−2 μm) with a reduced diffusion coefficient of 1.1 μm2/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.
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INTRODUCTION The formation of synthetic subcellular compartments has long been of interest to bioengineer cellular behavior.1,2 There are numerous approaches to generate microparticles outside of cells,3 which can be loaded with cargo and incorporated into cells where they act as intracellular microdomains.4,5 However, these synthetic microparticles require cellular uptake and disruption of the plasma membrane to reach the cytosol. Herein we characterize a method for reversible intracellular assembly of genetically encodable protein microdomains (GEPMs). This approach would allow virtually any protein to become temperature sensitive and assemble into structures, circumventing the need to perform large temperature mutant screens or the addition of chemical modulators.6,7 This triggered microdomain formation has potential applications including protein sequestration, scaffold construction, and modulation of intracellular trafficking. Additionally, the reversibility of this platform affords the ability to study the transitory effects of such domains in a biological environment. To direct GEPM assembly, protein polymers have been selected from the family of elastin-like polypeptides (ELPs).8,9 Inspired from human tropoelastin, ELPs are composed from the motif (VPGXG)n. As polypeptides, ELPs can be genetically encoded and expressed inside living cells.8,10 ELPs undergo © 2012 American Chemical Society
temperature-dependent phase separation that is rapid and reversible. Below a phase transition temperature (Tt), ELPs are highly soluble. Above Tt, ELPs assemble into an aqueous twophase system. Tt can be genetically controlled by modifying the identities of X and n.11 Hydrophobic guest residues, X, such as valine and isoleucine, have low Tt's, while hydrophilic guest residues, such as alanine or serine, have higher Tt's. Similarly, ELPs of higher molecular weight, n, result in lower Tt's. Importantly, these two orthogonal parameters can be used to modify Tt such that it occurs at physiologically relevant temperatures and concentrations following expression in the cytoplasm. Previously, recombinant DNA technology has produced stimuli-responsive polypeptides that form multimer associations, also referred to as microdomains, that self-assemble extracellularly based on a pH-dependent trigger.12 Here, we expand the means with which to form biopolymer microdomain assemblies by constructing a tunable and reversible selfassembling polypeptide comprised of an ELP that forms intracellular microdomains (Figure 1a). The potential of Received: July 13, 2012 Revised: October 21, 2012 Published: October 22, 2012 3439
dx.doi.org/10.1021/bm301090x | Biomacromolecules 2012, 13, 3439−3444
Biomacromolecules
Communication
Figure 1. Tunable assembly of GEPMs. (a) Schematic representation of triggered microdomain assembly inside living cells. Before heating, the GFPELPs are soluble throughout the cytosol. After heating, the protein polymers assemble microdomains enriched in the GFP fusion protein. Similar to ELP-mediated phase separation, this process is reversible. (b) Demonstration of polymer phase separation across a valine library of ELPs (Xaa = Val). (c) Correlation between transition temperatures for intracellular GFP-ELPs and ELPs in free PBS based on a valine library (MW 19, 24, 28, and 38 kD) (R2 = 0.78; p =
