Project description:Until recently, actin was thought to act merely as a passive track for its motility partner, myosin, during actomyosin interactions. Yet a recent report having observed dynamical conformational changes in labeled skeletal muscle alpha-actin suggests that actin has a more active role. Because the labeling technique was still immature, however, conclusions regarding the significance of the different conformations are difficult to make. Here, we describe the preparation of fully active alpha-actin obtained from a baculovirus expression system. We developed alpha-actin recombinants, of which subdomains 1 and 2 have specific sites for fluorescent probes. This specific labeling technique offers to significantly expand the information acquired from actin studies.
Project description:Fluorescent tagging is a powerful tool for imaging proteins in living cells. However, the steric effects imposed by fluorescent tags impair the behavior of many proteins. Here, we report a novel technique, Instant with DTT, EDT, And Low temperature (IDEAL)-labeling, for rapid and specific FlAsH-labeling of tetracysteine-tagged cell surface proteins by using prion protein (PrP) and amyloid precursor protein (APP) as models. In prion-infected cells, FlAsH-labeled tetracysteine-tagged PrP converted from the normal isoform (PrPsen) to the disease-associated isoform (PrPres), suggesting minimal steric effects of the tag. Pulse-chase analysis of PrP and APP by fluorescent gel imaging demonstrated the utility of IDEAL labeling in investigating protein metabolism by identifying an as-yet-unrecognized C-terminal fragment (C3) of PrPsen and by characterizing the kinetics of PrPres and APP metabolism. C3 generation and N-terminal truncation of PrPres were inhibited by the anti-prion compound E64, a cysteine protease inhibitor. Surprisingly, E64 did not inhibit the synthesis of new PrPres, providing insight into the mechanism by which E64 reduces steady-state PrPres levels in prion-infected cells. To expand the versatility of tetracysteine tagging, we created new Alexa Fluor- and biotin-conjugated tetracysteine-binding molecules that were applied to imaging PrP endocytosis and ultrastructural localization. IDEAL-labeling extends the use of biarsenical derivatives to extracellular proteins and beyond microscopic imaging.
Project description:Chemokines control cell migration in many contexts including development, homeostasis, immune surveillance and inflammation. They are also involved in a wide range of pathological conditions ranging from inflammatory diseases and cancer, to HIV. Chemokines function by interacting with two types of receptors: G protein-coupled receptors on the responding cells, which transduce signaling pathways associated with cell migration and activation, and glycosaminoglycans on cell surfaces and the extracellular matrix which organize and present some chemokines on immobilized surface gradients. To probe these interactions, imaging methods and fluorescence-based assays are becoming increasingly desired. Herein, a method for site-specific fluorescence labeling of recombinant chemokines is described. It capitalizes on previously reported 11-12 amino acid tags and phosphopantetheinyl transferase enzymes to install a fluorophore of choice onto a specific serine within the tag through a coenzyme A-fluorophore conjugate. The generality of the method is suggested by our success in labeling several chemokines (CXCL12, CCL2, CCL21 and mutants thereof) and visualizing them bound to chemokine receptors and glycosaminoglycans. CXCL12 and CCL2 showed the expected co-localization on the surface of cells with their respective receptors CXCR4 and CCR2 at 4 °C, and co-internalization with their receptors at 37 °C. By contrast, CCL21 showed the presence of large discrete puncta that were dependent on the presence of both CCR7 and glycosaminoglycans as co-receptors. These data demonstrate the utility of this labeling approach for the detection of chemokine interactions with GAGs and receptors, which can vary in a chemokine-specific manner as shown here. For some applications, the small size of the fluorescent adduct may prove advantageous compared to other methods (e.g. antibody labeling, GFP fusion) by minimally perturbing native interactions. Other advantages of the method are the ease of bacterial expression, the versatility of labeling with any maleimide-fluorophore conjugate of interest, and the covalent nature of the fluorescent adduct.
Project description:DNA-encapsulated silver clusters are readily conjugated to proteins and serve as alternatives to organic dyes and semiconductor quantum dots. Stable and bright on the bulk and single molecule levels, Ag nanocluster fluorescence is readily observed when staining live cell surfaces. Being significantly brighter and more photostable than organics and much smaller than quantum dots with a single point of attachment, these nanomaterials offer promising new approaches for bulk and single molecule biolabeling.
Project description:The impact of the Drosophila experimental system on studies of modern biology cannot be understated. The ability to tag endogenously expressed proteins is essential to maximize the use of this model organism. Here, we describe a method for labeling endogenous proteins with self-complementing split fluorescent proteins (split FPs) in a cell-type-specific manner in Drosophila A short fragment of an FP coding sequence is inserted into a specific genomic locus while the remainder of the FP is expressed using an available GAL4 driver line. In consequence, complementation fluorescence allows examination of protein localization in particular cells. Besides, when inserting tandem repeats of the short FP fragment at the same genomic locus, we can substantially enhance the fluorescence signal. The enhanced signal is of great value in live-cell imaging at the subcellular level. We can also accomplish a multicolor labeling system with orthogonal split FPs. However, other orthogonal split FPs do not function for in vivo imaging besides split GFP. Through protein engineering and in vivo functional studies, we report a red split FP that we can use for duplexed visualization of endogenous proteins in intricate Drosophila tissues. Using the two orthogonal split FP systems, we have simultaneously imaged proteins that reside in distinct subsynaptic compartments. Our approach allows us to study the proximity between and localization of multiple proteins endogenously expressed in essentially any cell type in Drosophila.
Project description:As newly synthesized proteins emerge from the ribosome, they interact with a variety of cotranslational cellular machineries that facilitate their proper folding, maturation, and localization. These interactions are essential for proper function of the cell, and the ability to study these events is crucial to understanding cellular protein biogenesis. To this end, we have developed a highly efficient method to generate ribosome-nascent chain complexes (RNCs) site-specifically labeled with a fluorescent dye on the nascent polypeptide. The fluorescent RNC provides real-time, quantitative information on its cotranslational interaction with the signal recognition particle and will be a valuable tool in elucidating the role of the translating ribosome in numerous biochemical pathways.
Project description:Glycosylation is a common modification found on numerous proteins and lipids. However, direct detection of glycans on these intact biomolecules has been challenge. Here, utilizing enzymatic incorporation of fluorophore-conjugated sialic acids, dubbed as direct fluorescent glycan labeling, we report the labeling and detection of N- and O-glycans on glycoproteins. The method allows detection of specific glycans without the laborious gel blotting and chemiluminescence reactions used in Western blotting. The method can also be used with a variety of fluorescent dyes.
Project description:The tendency for tetramerization is the main disadvantage in the green fluorescent protein homologues from Anthozoa species. We report a universal method called hetero-oligomeric tagging, which diminishes troublesome consequences of tetramerization of Anthozoa-derived fluorescent proteins (FP) in intracellular protein labelling. This approach is based on the co-expression of the FP-tagged protein of interest together with an excess of free non-fluorescent FP mutant. The resulting FP heterotetramers contain only a single target polypeptide and, therefore, can be considered pseudo-monomeric. Feasibility of the method has been demonstrated with a red FP fused with cytoplasmic beta-actin or tubulin-binding protein Tau34. In addition, heterotetramers appeared to be a unique model for biophysical characterization of Anthozoa FPs in pseudo-monomeric state.
Project description:Rapid, one-pot, concerted, site-specific labeling of proteins at genetically encoded unnatural amino acids with distinct small molecules at physiological pH, temperature, and pressure is an important challenge. Current approaches require sequential labeling, low pH, and typically days to reach completion, limiting their utility. We report the efficient, genetically encoded incorporation of alkyne- and cyclopropene-containing amino acids at distinct sites in a protein using an optimized orthogonal translation system in E. coli. and quantitative, site-specific, one-pot, concerted protein labeling with fluorophores bearing azide and tetrazine groups, respectively. Protein double labeling in aqueous buffer at physiological pH, temperature, and pressure is quantitative in 30 min.
Project description:The development of novel methods for highly efficient protein purification remains a research focus in the biotechnology field because conventional purification approaches, including affinity purification, gel filtration, and ion-exchange chromatography, require complex manipulation steps and are costly. Here, we describe a simple and rapid protein purification strategy in which the SUMO tag and Ulp1 protease are surface-displayed separately on Escherichia coli cells. After protein induction, the cells are harvested, resuspended in cleavage buffer, and incubated together for cleavage. In this approach, the surface-displayed Ulp1 cleaves the membrane-anchored SUMO fusion protein, resulting in the release of the target protein from the C-terminal of SUMO into the solution. The bacterial cells harboring SUMO and Ulp1 on their surfaces can be easily removed by centrifugation. To evaluate the purification method, we used red fluorescent protein (mCherry). Purified mCherry protein (7.72 ± 1.05 mg from 1 L of bacterial culture) was obtained after only 30 min of incubation. The protein purity was higher than 80%, and could be further improved (> 90%) by simple ultrafiltration. This study offers a promising and simple strategy for the purification of recombinant protein in its native form that requires only cleavage and centrifugation steps.