No abstract

Real-time imaging of molecular events in living cells is important for understanding the basis of physiological processes and diseases. [1][2][3] Genetically encoded sensors that use fluorescence resonance energy transfer (FRET) [4] between two fluorescent proteins are attractive in this respect because they do not require cell-invasive procedures, can be targeted to different locations in the cell, and are easily adapted through mutagenesis and directed evolution approaches. [5][6][7] Following the pioneering work of Roger Tsien and others, on genetically encoded protease and calcium sensors, FRET-based imaging probes have been developed for many other small molecules and cell signaling events. [8][9][10][11][12][13] In these probes, conformational changes in a sensor domain are translated into a change in energy-transfer efficiency between donor and acceptor fluorescent domains, which is detected by a change in the ratio of donor and acceptor emission. This ratiometric response is independent of the sensor concentration, which is an important advantage of FRET-based sensors. In practice, however, most FRET-based sensors display only a relatively small difference in emission ratio upon activation. Improvement of these ratiometric changes has been recognized as an important prerequisite for use of these sensor systems in high-throughput applications based on fluorescence plate readers and fluorescence assisted cell sorting (FACS). [14,15] Recently a pair of CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein) variants, CyPet and YPet, respectively, have been reported that were optimized for FRET through a process of directed evolution.[16] When incorporated in a protease sensor, a 20-fold change in emission ratio was observed upon cleavage of a flexible peptide that linked CyPet and YPet, compared to only a fourfold change for the same construct with enhanced CFP (ECFP) and enhanced YFP (EYFP) domains. However, the mechanism behind their remarkable FRET properties has remained unclear. A total of eighteen mutations were introduced in the course of their development, many of which were at the exterior of the protein, at a large distance from the fluorophore. Moreover, no large differences in quantum yield or extinction coefficient were reported; this suggests that the photophysical properties of the fluorescent proteins were not significantly altered. We therefore hypothesized that the increase in FRET observed for CyPet and YPet could be due to an enhanced tendency to interact when connected by a peptide linker. The parent green fluorescent protein (GFP) has a known tendency to dimerize, [17] and analysis of the mutations in YPet have identified two residues, S208F and V224L, that are present at the dimer interface, as shown by the X-ray structure of the GFP dimer. Here, we show that A C H T U N G T R E N N U N G introduction of just these two mutations in both fluorescent domains of ECFP-linker-EYFP constructs results in a fourfold increase in the EYFP-to-ECFP emission ratio, which yields a 16-fol...

Liposomes have become popular drug delivery vehicles and have more recently also been applied as contrast agents for molecular imaging. Most current methods for functionalization of liposomes with targeting proteins rely on reactions of amine or thiol groups at the protein exterior, which generally result in nonspecific conjugation at multiple sites on the protein. In this study, we present native chemical ligation (NCL) as a general method to covalently couple recombinant proteins in a highly specific and chemoselective way to liposomes containing cysteine-functionalized phospholipids. A cysteine-functionalized phospholipid (Cys-PEG-DSPE) was prepared and shown to readily react with the MESNA thioester of EYFP, which was used as a model protein. Characterization of the EYFP-liposomes using fluorescence spectroscopy showed full retention of the fluorescent properties of conjugated EYFP and provides a lower limit of 120 proteins per liposome. The general applicability of NCL was further tested using CNA35, a collagen-binding protein recently applied in fluorescent imaging of collagen. NCL of CNA35 thioester yielded liposomes containing approximately 100 copies of CNA35 per liposome. The CNA35-liposomes were shown to be fully functional and bind collagen with a 150-fold higher affinity compared to CNA35. Our results show that NCL is an attractive addition to existing conjugation methods that allows direct, covalent, and highly specific coupling of recombinant proteins to liposomes and other lipid-based assemblies.

Collagen is an important marker for the assessment of tissue remodeling, both in normal tissue maturation and in a variety of prevalent disease processes. Given the importance of multivalency in the natural interactions of collagen, multivalent ligands provide unique opportunities to target collagen architectures. Here, we explored the use of micelles as dynamic self-assembling multivalent scaffolds for the collagen binding protein CNA35. Despite the increased popularity of micelles as nanosized carriers in targeted drug delivery and molecular imaging, few studies have actually directly addressed the importance of multivalent interactions for micelle-based targeting. Native chemical ligation was used as a chemoselective and efficient method to prepare relatively well-defined and stable micelles with a tunable average protein content between 0 and 20 copies of CNA35 per micelle. The thermodynamics and kinetics of CNA35 micelle binding to collagen was studied using solid-phase and surface plasmon resonance assays. Multivalent interactions between the micelles and collagen had a remarkable effect on micellar stability, since no dissociation of collagen-bound micelles was observed even after extensive washing. In addition, an impressive enhancement of collagen affinity was observed both in vitro and ex vivo resulting from multivalent display of a so-called "nonbinding" variant of CNA35. This "restoration" of collagen affinity was subsequently also observed for liposomes displaying the same low-affinity CNA35 variant at a sufficient density. These results demonstrate the importance of multivalent interactions for micelle-based targeting and illustrate the strength of multiple weak interactions when targeting intrinsically multivalent extracellular matrix (ECM) proteins such as collagen.