Beyond molecular beacons: optical sensors based on the binding-induced folding of proteins and polypeptides.
ABSTRACT Many polypeptides and small proteins can be readily engineered such that they only fold upon binding a specific target ligand. This approach couples target recognition with a considerable change in polymer structure and dynamics. Recent years have seen the development of a number of biosensors that couple these large changes to readily measurable optical (fluorescent) outputs. These sensors afford the detection of a wide variety of macromolecular targets including proteins, polypeptides, and nucleic acids. Here we describe the design of such biosensors, from the first iterations as protein engineering experiments, to the development of biosensors targeting a range of protein and nucleic acid targets.
- Methods in Enzymology 02/2004; 380:350-79. · 2.00 Impact Factor
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ABSTRACT: Existing strategies for creating biosensors mainly rely on large conformational changes to transduce a binding event to an output signal. Most molecules, however, do not exhibit large-scale structural changes upon substrate binding. Here, we present a general approach (alternate frame folding, or AFF) for engineering allosteric control into ligand binding proteins. AFF can in principle be applied to any protein to establish a binding-induced conformational change, even if none exists in the natural molecule. The AFF design duplicates a portion of the amino acid sequence, creating an additional "frame" of folding. One frame corresponds to the wild-type sequence, and folding produces the normal structure. Folding in the second frame yields a circularly permuted protein. Because the two native structures compete for a shared sequence, they fold in a mutually exclusive fashion. Binding energy is used to drive the conformational change from one fold to the other. We demonstrate the approach by converting the protein calbindin D(9k) into a molecular switch that senses Ca2+. The structures of Ca2+-free and Ca2+-bound calbindin are nearly identical. Nevertheless, the AFF mechanism engineers a robust conformational change that we detect using two covalently attached fluorescent groups. Biological fluorophores can also be employed to create a genetically encoded sensor. AFF should be broadly applicable to create sensors for a variety of small molecules.ACS Chemical Biology 11/2008; 3(11):723-32. · 5.44 Impact Factor
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ABSTRACT: We have characterised a series of C-terminal fragments of barnase by different biophysical techniques to find out when they acquire secondary and tertiary native-like structure. Fragments B96-110 (which comprises the last 15 residues of the intact protein) up to B37-110 (which involves most of the protein except the two first helices and a loop) were mainly disordered. Only fragment B23-110, which lacks alpha-helix1, showed native-like near and far-UV CD and fluorescence spectra. The intensities of these spectra were lower than those of the full-length protein, which suggests the absence of complete side-chain packing. Urea denaturation followed by fluorescence, far-UV CD and gel-filtration chromatography techniques indicated a co-operative transition only for B23-110. None of the fragments melted co-operatively with temperature. Thus, the formation of secondary and tertiary structure requires most of the polypeptide chain to be present, that is, secondary and tertiary structure are formed in parallel. This agrees with the proposed model for barnase folding, where the residual structure in small fragments is weak and flickering, and it is only consolidated when there are enough tertiary interactions. Thus, the development of structure in the series of C-terminal fragments follows a similar behaviour to that observed in the series of N-terminal fragments of barnase.Journal of Molecular Biology 04/1999; 287(2):421-32. · 3.91 Impact Factor