Description of project to engineer fluorophore bacterial periplasmic binding proteins for use as biosensors in liposomes

1. Lab Meeting: “Turning Houseflies into Fireflies” Szostak Lab Howard Hughes Medical Institute, and Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114

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12. Binding Proteins of Interest 34.470 kD 28.460 kD 34.473 kD 40.775 kD 26.141 kD 17.822 kD 31.000 kD 24.988 kD 29.714 kD 57.536 kD 33.196 kD Protein Size L65C sbp Sulfate A234C rbp Ribose phoS malE hisJ mglB gltI/ybeJ glnH fhuD dppA araF Gene F126C Glutamate-Aspartate S164C (S197C) Phosphate D95C Maltose V163C Histidine H152C Glucose-Galactose Y163C Glutamine Iron D450C Dipeptide K301C, C64A Arabinose Mutations Binding Protein

13. Binding Proteins of Interest 37.696 kD 28.474 kD 35.832 kD 40.928 kD 59.239 kD 33.334 kD 31.000 kD 24.988 kD 29.714 kD 66.8982 kD 33.196 kD Protein Size L65C sbp Sulfate Functional A234C rbp Ribose Functional phoS malE hisJ mglB gltI/ybeJ glnH fhuD dppA araF Gene Functional Functional Functional Functional Expressed Cloned WT Cloned WT Purified 1 st of double mutant Status F126C Glutamate-Aspartate S164C (A197C) Phosphate D95C Maltose V163C Histidine H152C Glucose-Galactose Y163C Glutamine E203C Iron D450C Dipeptide K301C, C64A Arabinose Mutations Binding Protein

23. And the Others? More of the same… Sbp Rbp HisJ MglB MalE

25. SDS-PAGE

26. Still More Proteins… DppA: Dipeptide Binding Protein (D450C) Digest Confirmation Gel Sequenced & Expressed ↓

28. Still More Proteins… MalE: Maltose Binding Protein (D95C) Digest Confirmation Gel Sequenced & Expressed ↓ ↓

29. Still More Proteins… MglB: Glucose-Galactose Binding Protein (H152C) Digest Confirmation Gel Sequenced & Expressed ↓ ↓

30. Still More Proteins… YbeJ (GltI): Glutamate-Aspartate Binding Protein (H152C) Digest Confirmation Gel Sequenced & Expressed ↓

31. Still More Proteins… GlnH: Glutamine Binding Protein (Y163C) FhuD: Fe 3+ Binding Protein

34. ??Questions??