Beyond Molecular Beacons: Optical Sensors Based on the Binding-Induced Folding of Proteins and Polypeptides (original) (raw)
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Construction of a fluorescent biosensor family
Protein Science, 2009
Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share a common physical signal transduction functionality and detect many different analytes. We demonstrate the facility of designing optical biosensors based on fluorophore conjugates using 8 environmentally sensitive fluorophores and 11 bPBPs specific for diverse ligands, including sugars, amino acids, anions, cations, and dipeptides. Construction of reagentless fluorescent biosensors relies on identification of sites that undergo a local conformational change in concert with the global, ligand-mediated hinge-bending motion. Construction of cysteine mutations at these locations then permits site-specific coupling of environmentally sensitive fluorophores that report ligand binding as changes in fluorescence intensity. For 10 of the bPBPs presented in this study, the three-dimensional receptor structure was used to predict the location of reporter sites. In one case, a bPBP sensor specific for glutamic and aspartic acid was designed starting from genome sequence information and illustrates the potential for discovering novel binding functions in the microbial genosphere using bioinformatics.
Angewandte Chemie International Edition, 2010
Protein or peptide nanowires can occur either naturally in, for example, amyloid proteins, or be genetically fabricated by gene fusion or phage display technology. One of the major advantages of protein nanowires is that they can self-assemble into predetermined patterns with functionalized surfaces. Because of their unique properties, nanowires have great potential in the development of next-generation circuits, new functional materials, nanostructures, ordered liquid crystalline systems, high powered batteries, and diagnostic tools. The self-assembly of biological structures has been shown to be advantageous in the study of biosynthesis and nanobiotechnology.
Array-Based Sensing of Proteins Using Conjugated Polymers
Journal of the American Chemical Society, 2007
Convenient, precise, and rapid protein sensing methods are of great importance in medical diagnostics and proteomics. 1 Widely used specific interaction-based sensing protocols (e.g., ELISA) require protein receptors of high affinity and specificity requiring the generation of pertinent protein receptors/ligands for multiprotein detection. 1 In this regard, sensor array approaches are attractive, using differential binding interactions that are selective rather than specific. 2 This "electronic nose/tongue" strategy provides highly versatile sensors. 3,4 Recently, this principle has been used for protein detection through either fluorescence quenching 5 or indicator displacement. 6 While these sensors have been effective, they feature high limits of detection, and only relatively small sets (4-5 proteins) were studied. Effective protein sensing requires efficient protein receptors and competent signal transducers. Water-soluble conjugated polymers with pendant-charged residues provide an excellent scaffold for sensor design. 7,8 These materials can bind protein surfaces through multivalent interactions. Moreover, their optical properties are sensitive to minor conformational or environmental changes, 7,9 enabling efficient signal transduction of the binding events. In this work, we use six functionalized poly(p-phenyleneethynylene)s (PPEs) 10 to build a protein sensor array (Figure 1). These highly fluorescent polymers possess various charge characteristics and molecular scales. Such structural features provide tremendous binding diversity upon interaction with protein analytes, generating distinct fluorescence response patterns for protein discrimination. We have chosen 17 proteins as the sensing targets (Table 1). These proteins possess diverse structural characteristics including metal/nonmetal-containing, molecular weight (MW), isoelectric point (pI), and UV absorbencies. Notably, many protein targets have comparable MW and pI values, thereby providing excellent objects for examining the differentiation ability of the PPE-based sensor array.
Coiled-Coil Peptide Beacon: A Tunable Conformational Switch for Protein Detection
The understanding of protein folding and assembly is of central importance for the design of proteins and enzymes with novel or improved functions.Minimalistic model systems, such as coiled-coils,p rovidea ne xcellent platform to improve this understanding and to construct novel molecular devices. Along those lines,wedesigned aconformational switch that is composed of two coiled-coil forming peptides and ac entral binding epitope.Inthe absence of abinding partner,this switch adopts ah airpin-like conformation that opens upon receptor binding.V ariation of the coiled-coil length modulates the strength of the intramolecular constraint. The two conformational states of this switch have been linked with characteristic fluorescent properties,w hiche nables the detection of the receptor in real-time.
A novel fluorescent probe for protein binding and folding studies:p-cyano-phenylalanine
Biopolymers, 2006
Recently, it is has been shown that the C¼ ¼N stretching vibration of a non-natural amino acid, p-cyano-phenylalanine (Phe CN ), could be used as an infrared reporter of local environment. Here, we further showed that the fluorescence emission of Phe CN is also sensitive to solvent and, therefore, could be used as a novel optical probe for protein binding and folding studies. Moreover, we found that the fluorescence quantum yield of Phe CN is nearly five times larger than that of phenylalanine and, more importantly, can be selectively excited even when other aromatic amino acids are present, thus making it a more versatile fluorophore. To test the feasibility of using Phe CN as a practical fluorescent probe, we studied the binding of calmodulin (CaM) to a peptide derived from the CaM-binding domain of skeletal muscle myosin light chain kinase (MLCK). The peptide (MLCK 3CN ) contains a single Phe CN residue and has been shown to bind to CaM with high affinity. As expected, addition of CaM into a MLCK 3CN solution resulted in quenching of the Phe CN fluorescence. A series of stochiometric titrations further allowed us to determine the binding affinity (K d ) of this peptide to CaM. Taken together, these results indicated that the Phe CN fluorescence is sensitive to environment and could be applicable to a wide variety of biological problems.
Protein recognition by a pattern-generating fluorescent molecular probe
Nature nanotechnology, 2017
Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this app...
Coiled-coil Peptide Beacon: A Tuneable Conformational Switch for Protein Detection
Angewandte Chemie International Edition
The understanding of protein folding and assembly is of central importance for the design of proteins and enzymes with novel or improved functions.Minimalistic model systems, such as coiled-coils,p rovidea ne xcellent platform to improve this understanding and to construct novel molecular devices. Along those lines,wedesigned aconformational switch that is composed of two coiled-coil forming peptides and ac entral binding epitope.Inthe absence of abinding partner,this switch adopts ah airpin-like conformation that opens upon receptor binding.V ariation of the coiled-coil length modulates the strength of the intramolecular constraint. The two conformational states of this switch have been linked with characteristic fluorescent properties,w hiche nables the detection of the receptor in real-time.
Fluorescent Dynamic Covalent Polymers for DNA Complexation and Templated Assembly
Molecules
Dynamic covalent polymers (DCPs) offer opportunities as adaptive materials of particular interest for targeting, sensing and delivery of biological molecules. In this view, combining cationic units and fluorescent units along DCP chains is attractive for achieving optical probes for the recognition and delivery of nucleic acids. Here, we report on the design of acylhydrazone-based DCPs combining cationic arginine units with π-conjugated fluorescent moieties based on thiophene-ethynyl-fluorene cores. Two types of fluorescent building blocks bearing neutral or cationic side groups on the fluorene moiety are considered in order to assess the role of the number of cationic units on complexation with DNA. The (chir)optical properties of the building blocks, the DCPs, and their complexes with several types of DNA are explored, providing details on the formation of supramolecular complexes and on their stability in aqueous solutions. The DNA-templated formation of DCPs is demonstrated, whi...