title: A genetically encoded, high-signal-to-noise maltose sensor.
abstract: We describe the generation of a family of high-signal-to-noise single-wavelength genetically encoded indicators for maltose. This was achieved by insertion of circularly permuted fluorescent proteins into a bacterial periplasmic binding protein (PBP), Escherichia coli maltodextrin-binding protein, resulting in a four-color family of maltose indicators. The sensors were iteratively optimized to have sufficient brightness and maltose-dependent fluorescence increases for imaging, under both one- and two-photon illumination. We demonstrate that maltose affinity of the sensors can be tuned in a fashion largely independent of the fluorescent readout mechanism. Using literature mutations, the binding specificity could be altered to moderate sucrose preference, but with a significant loss of affinity. We use the soluble sensors in individual E. coli bacteria to observe rapid maltose transport across the plasma membrane, and membrane fusion versions of the sensors on mammalian cells to visualize the addition of maltose to extracellular media. The PBP superfamily includes scaffolds specific for a number of analytes whose visualization would be critical to the reverse engineering of complex systems such as neural networks, biosynthetic pathways, and signal transduction cascades. We expect the methodology outlined here to be useful in the development of indicators for many such analytes.
authors: Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA. marvinj@janelia.hhmi.org
authors: Jonathan S
authors: JS
authors: Marvin
authors: Eric R
authors: ER
authors: Schreiter
authors: Ileabett M
authors: IM
authors: Echevarría
authors: Loren L
authors: LL
authors: Looger
mesh_terms: D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000648:ultrastructure*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D029968:Escherichia coli Proteins; D008024:Ligands; D058619:Maltose-Binding Proteins; D033902:Periplasmic Binding Proteins; C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins