Bacterial Periplasmic Binding Proteins as Biosensors in Liposomes

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Information about Bacterial Periplasmic Binding Proteins as Biosensors in Liposomes

Published on June 1, 2008

Author: Water_Xta1

Source: slideshare.net

Description

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

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

Problem: How can one track the locations and relative concentrations of molecules (esp. those of prebiotic interest) in liposomes? http://bioweb.wku.edu/courses/biol22000/2Bonds/ http://bioweb.wku.edu/pix/Pix.htm

How can one track the locations and relative concentrations of molecules (esp. those of prebiotic interest) in liposomes?

Background: Biosensors Indicate the presence of molecules Specificity to targets & signal transduction: ligand binding  detectable physical changes Should be reagentless: do not change composition as a consequence of making the measurement Ligand-Specific Periplasmic Binding Proteins Superfamily of macromolecules that facilitate the active transport of ions, sugars and amino acids Can be engineered to sense these substrates

Indicate the presence of molecules

Specificity to targets & signal transduction: ligand binding  detectable physical changes

Should be reagentless: do not change composition as a consequence of making the measurement

Ligand-Specific Periplasmic Binding Proteins

Superfamily of macromolecules that facilitate the active transport of ions, sugars and amino acids

Can be engineered to sense these substrates

Background: E. coli bPBPs b acterial P eriplasmic B inding P roteins 2 domains linked by a hinge region Ligand-binding site is located at the interface between them. 2 conformations that interconvert via hinge bending Ligand-free open Liganded closed Conformational change attributed to reporter functions Fluorophore (Acrylodan) conjugates of bPBPs specific for various ligands Felder, C.B., et al. (1999) AAPS PharmSci. 1 (2). 1-20. www.botgard.ucla.edu /. ../b0567tx.html

b acterial P eriplasmic B inding P roteins

2 domains linked by a hinge region

Ligand-binding site is located at the interface between them.

2 conformations that interconvert via hinge bending

Ligand-free open

Liganded closed

Conformational change attributed to reporter functions

Fluorophore (Acrylodan) conjugates of bPBPs specific for various ligands

Background: E. coli bPBPs Changes in fluorescence intensity indicate ligand binding Environmentally-sensitive fluorophores are placed in locations that undergo conformational change (local  global) Cys mutations are placed at these sites so that changes in fluorescence may be directly linked to ligand binding. Sites identified by De Lorimier et al. Protein Science (2002), 11:2655–2675. Closed structure examination, open vs. closed & structural/sequence homology Hinge Mutated Residue Bound Ligand De Lorimier et al. Protein Science. 11:2655–2675.

Changes in fluorescence intensity indicate ligand binding

Environmentally-sensitive fluorophores are placed in locations that undergo conformational change (local  global)

Cys mutations are placed at these sites so that changes in fluorescence may be directly linked to ligand binding.

Sites identified by De Lorimier et al.

Protein Science (2002), 11:2655–2675.

Closed structure examination, open vs. closed & structural/sequence homology

ABC Transport System: A TP B inding C assette Periplasmic Protein-Dependent Transport System (G-) Binding Lipoprotein-Dependent Transport System (G+) bPBPs are initial receptors in active transport across cellular membranes &/or chemotaxis Each binds a specific substrate (e.g. sugar, amino acid, or ion) with high affinity. 3 Types of Constituents: Genes typically found in an operon 2 integral membrane proteins (permeases) each having 6- transmembrane segment 2 peripheral membrane proteins that bind & hydrolyze ATP (most evolutionarily conserved) A periplasmic (or lipoprotein) substrate-binding protein (the part we seek to exploit in our assays)

A TP B inding C assette

Periplasmic Protein-Dependent Transport System (G-)

Binding Lipoprotein-Dependent Transport System (G+)

bPBPs are initial receptors in active transport across cellular membranes &/or chemotaxis

Each binds a specific substrate (e.g. sugar, amino acid, or ion) with high affinity.

3 Types of Constituents: Genes typically found in an operon

2 integral membrane proteins (permeases) each having 6- transmembrane segment

2 peripheral membrane proteins that bind & hydrolyze ATP (most evolutionarily conserved)

A periplasmic (or lipoprotein) substrate-binding protein (the part we seek to exploit in our assays)

    

 

Labeling with Acrylodan 6-acryloyl-2-dimethylaminonaphthalene Molecular Formula: C 15 H 15 NO Molecular Weight: 225.29 Labeling Rxn: 36.4 μM protein stock (0.014 g/ml) for 300 μL protein Add 2.16 μl of 50 mM TCEP (50 mM TCEP; 10 mM dye) Add 10.8 μl of 10 mM dye (0.001g/0.5ml) Leave mixture in fridge overnight. Emission: 500 nm Excitation: 390 nm

6-acryloyl-2-dimethylaminonaphthalene

Molecular Formula: C 15 H 15 NO

Molecular Weight: 225.29

Labeling Rxn:

36.4 μM protein stock (0.014 g/ml) for 300 μL protein

Add 2.16 μl of 50 mM TCEP (50 mM TCEP; 10 mM dye)

Add 10.8 μl of 10 mM dye (0.001g/0.5ml)

Leave mixture in fridge overnight.

Background: Biosensors Objective: Synthesize a collection of binding proteins that exploits a common signal transduction mechanism Fluorophore reports binding of specific molecular targets in liposomes Amino acids, cations, anions, dipeptides & sugars

Objective: Synthesize a collection of binding proteins that exploits a common signal transduction mechanism

Fluorophore reports binding of specific molecular targets in liposomes

Amino acids, cations, anions, dipeptides & sugars

Background: Biosensors Objective: Synthesize a collection of binding proteins that exploits a common signal transduction mechanism Fluorophore reports binding of specific molecular targets in liposomes Amino acids, cations, anions, dipeptides & sugars http://www.darnellworks.com/a52/nr0007.htm

Objective: Synthesize a collection of binding proteins that exploits a common signal transduction mechanism

Fluorophore reports binding of specific molecular targets in liposomes

Amino acids, cations, anions, dipeptides & sugars

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

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

phoS : Cloning with pET100/TOPO Champion TM pET Directional TOPO® Expression Kit (Invitrogen) Digest Confirmation Gel Sequenced & Expressed ↓ PhoS: S164C (A197C)

Champion TM pET Directional TOPO® Expression Kit (Invitrogen)

PhoS: Mass Spectrophotometry PhoS = 35.832 kD His-Tag = 3 kD 38,832 D 

PhoS = 35.832 kD

His-Tag = 3 kD

hisJ : Cloning with pET100/TOPO HisJ : V163C ↓ Digest Confirmation Gel Sequenced & Expressed

HisJ : V163C

HisJ Purification Process 1L culture Ni-NTA column Dialysis: 50 mM HEPES, 50 mM NaCl, pH = 7.4 M.W. = 26 kD Labeled with Acrylodan 50 kD

1L culture

Ni-NTA column

Dialysis: 50 mM HEPES, 50 mM NaCl, pH = 7.4

M.W. = 26 kD

Labeled with Acrylodan

Affinity of HisJ: Titrations 20 mM MOPS, 100 mM NaCl pH 6.9 20 mM NaH 2 PO 4 , 100 mM NaCl pH = 6.9

20 mM MOPS, 100 mM NaCl pH 6.9

20 mM NaH 2 PO 4 , 100 mM NaCl pH = 6.9

sbp : Cloning with TOPO/pET100 Sbp: L65C Digest Confirmation Gel Sequenced & Expressed ↓

Sbp: L65C

Sbp Purification Process 1L culture Ni-NTA column Dialysis: 20 mM Tris-HCl, pH = 8.0, cholestryramine (Dowex 1X2-100) resin M.W. = 35 kD Labeled with Acrylodan

1L culture

Ni-NTA column

Dialysis: 20 mM Tris-HCl, pH = 8.0, cholestryramine (Dowex 1X2-100) resin

M.W. = 35 kD

Labeled with Acrylodan

rbp : Cloning with TOPO/pET100 Rbp: A234C Digest Confirmation Gel Sequenced & Expressed ↓

Rbp: A234C

Rbp Purification Process Expressed Ni-NTA column M.W. = 28 kD Found in pellet? Run through column with urea May need refolding… 

Expressed

Ni-NTA column

M.W. = 28 kD

Found in pellet?

Run through column with urea

May need refolding…

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

What can be eliminated: Buffer pH is not near pI Storage (never frozen, tested immediately & still dysfunctional; also tried 5 & 30% glycerol when frozen) Sequences (Mutation clearly sequenced & no other errors) Mass (Mass Spec/SDS-PAGE) Column contamination (each protein has it’s own Ni-NTA column)

Buffer pH is not near pI

Storage (never frozen, tested immediately & still dysfunctional; also tried 5 & 30% glycerol when frozen)

Sequences (Mutation clearly sequenced & no other errors)

Mass (Mass Spec/SDS-PAGE)

Column contamination (each protein has it’s own Ni-NTA column)

SDS-PAGE

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

DppA: D450C Mutation Very difficult to sequence mutagenized segment (N’s); 3 different sequencing primers! Expressed to test functionality, SDS-PAGE & M.S.

Very difficult to sequence mutagenized segment (N’s); 3 different sequencing primers!

Expressed to test functionality, SDS-PAGE & M.S.

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

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

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

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

Future Plans High Priority: Sbp, Rbp, MglB, MalE & HisJ: Repair defective binding measurement Investigate effect of salts, pH (unlikely) Possible contamination? DppA: Test kinetics (M.S.) PhoS: Keep working or buy? Lower Priority: AraF: C64A (K301C done) YbeJ: F126C GlnH: Y163C FhuD: E203C Devise a new assay system? Concerns: - Cost to produce “in-house” Stability Ease of use/time to produce System limitations

High Priority:

Sbp, Rbp, MglB, MalE & HisJ: Repair defective binding measurement

Investigate effect of salts, pH (unlikely)

Possible contamination?

DppA: Test kinetics (M.S.)

PhoS: Keep working or buy?

Lower Priority:

AraF: C64A (K301C done)

YbeJ: F126C

GlnH: Y163C

FhuD: E203C

Devise a new assay system?

Concerns:

- Cost to produce “in-house”

Stability

Ease of use/time to produce

System limitations

Acknowledgments Jack Sheref Mark Raphael Yollette Florian

Jack

Sheref

Mark

Raphael

Yollette

Florian

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