PDBsum entry 3osr

Fluorescent protein, transport protein

PDB id: 3osr

Contents

Protein chains

604 a.a.

Ligands

GLC-GLC ×2

Waters ×559

PDB id:

3osr

Name:

Fluorescent protein, transport protein

Title:

Maltose-bound maltose sensor engineered by insertion of circularly permuted green fluorescent protein into e. Coli maltose binding protein at position 311

Structure:

Maltose-binding periplasmic protein,green fluorescent protein. Chain: a, b. Fragment: gfp p42212 residues 2-146, 147-238, mbp p0aex9 residues 27- 199, 201-396. Synonym: mbp,mmbp,maltodextrin-binding protein. Engineered: yes

Source:

Escherichia coli o157:h7, aequorea victoria. Jellyfish. Organism_taxid: 83334, 6100. Gene: male, z5632, ecs5017, gfp. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

2.00Å R-factor: 0.185 R-free: 0.226

Authors:

I.M.Echevarria,J.S.Marvin,L.L.Looger,E.R.Schreiter

Key ref:

J.S.Marvin et al. (2011). A genetically encoded, high-signal-to-noise maltose sensor. Proteins, 79, 3025-3036. PubMed id: 21989929

Date:

09-Sep-10 Release date: 26-Oct-11

Protein chains

P0AEY0  (MALE_ECO57) -  Maltose/maltodextrin-binding periplasmic protein from Escherichia coli O157:H7

Seq: 396 a.a.

Struc: 604 a.a.*

Protein chains

P42212  (GFP_AEQVI) -  Green fluorescent protein from Aequorea victoria

Seq: 238 a.a.

Struc: 604 a.a.*

* PDB and UniProt seqs differ at 121 residue positions (black crosses)

Proteins 79:3025-3036 (2011)

PubMed id: 21989929

A genetically encoded, high-signal-to-noise maltose sensor.

J.S.Marvin, E.R.Schreiter, I.M.Echevarría, L.L.Looger.

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.