Monitoring the pH-Driven Transition of Anthrax Toxin Using Biolayer

Interferometry and Electron Microscopy

Jon F Tally

Department of Biochemistry and Molecular Biology

University of Kansas Medical Center

Outline

E) Prepore to Pore Transition Kinetic profiles ndash BLI endosomal transitions

F) Release of Protein for EM

D) Catching the Anthrax toxin prepore to pore translocon structure

B) Biolayer Interferometry Instrument

C) BLI tip activation and protein preparation

A) Anthrax and its toxin translocation

Protective Lethal EdemaAntigen Factor Factor

(produced in response to elevated C02 levels)

httpwwwproteinloungecomAnimationMechanism Of AnthraxToxins

Anthrax lethality Arises from the Production of Protein Toxins

Outline

E) Prepore to Pore Transition Kinetic profiles ndash BLI endosomal transitions

F) Release of Protein for EM

D) Catching the Anthrax toxin prepore to pore translocon structure

B) Biolayer Interferometry Instrument

C) BLI tip activation and protein preparation

A) Anthrax and its toxin translocation

Protective Lethal EdemaAntigen Factor Factor

(produced in response to elevated C02 levels)

httpwwwproteinloungecomAnimationMechanism Of AnthraxToxins

Anthrax lethality Arises from the Production of Protein Toxins

Protective Lethal EdemaAntigen Factor Factor

(produced in response to elevated C02 levels)

httpwwwproteinloungecomAnimationMechanism Of AnthraxToxins

Anthrax lethality Arises from the Production of Protein Toxins

Anthrax structure

BLI Instrument

ForteBIO httpwwwblitzmenowcomabouthtml

BLITZ

400-750nm

Biolayer Interferometry Methodology

fore

BIO

htt

p

ww

wf

ort

ebio

co

mi

nte

ract

ion

sSp

rin

g_2

01

2p

age5

htm

l

Biolayer Interferometry Methodology

fore

BIO

htt

p

ww

wf

ort

ebio

co

mi

nte

ract

ion

sSp

rin

g_2

01

2p

age5

htm

l

How Do We Create a BioreactiveSensor Tip to Display Kinetics

Anthrax Transition ---- Dnm shift Sensogram

Coupling Chemistry

LFn or Anthrax Prepore can be immobilized onto BLI or SPR surface via Thiol coupling

Thiol coupling an active disulfide moiety can be introduced either on the dextranmatrix or on the ligand molecule 2-(2-pyrdinyldithio) ethaneamine (PDEA) can be used to introduce the di-sulfide group for attachment exchange

C

OH

O C

O

O C

NH

OEDCNHS

NO O

SHPDEA

C

NH

O

S

S

S

S

NE126C LFn

Alternatively a SH containing protein --the prepore itself can be immobilizedon the tip

SH

E126C LFn Attachment to Matrix

C

OH

O C

O

O C

NH

OEDCNHS

NO O

SHPDEA

C

NH

O

S

S

S

S

NE126C LFn

SuccinimideCarboimide

Pyridinyl dithio ethane amide

Prepore attachment to E126C LFn

50 mM L-cysteineBlock

E126C LFn

Prepore

L-cysteine blocks remaining SH Groups

PA Binds with Kd = 1nM

LFN binding sites

Section of Domain-2 unfold and refoldsInto extendedstructure

Domain-2

Prepore to Pore transitions (overlay)Slideshow ndashclick for animation

BLI Transition Protocol

pH Induced Transitions

Kinetic Data of Anthrax pH-Induced Transitions

pH SPR BLI

(t12) sec k1 (sminus1

) k2 (sminus1

) k3 (sminus1

)

675 15 0190 plusmn 0005 0029 plusmn 0001 0005 plusmn 00009

65 15 0813 plusmn 0016 009 plusmn 0005 0006 plusmn 00004

6 lt1 0872 plusmn 0021 0082 plusmn 0004 0010 plusmn 00005

55 lt1 1143 plusmn 0024 0076 plusmn 0024 0008 plusmn 00004

5 lt1 1095 plusmn 0018 0025 plusmn 0004 0010 plusmn 00041

BLI Instrument

ForteBIO httpwwwblitzmenowcomabouthtml

BLITZ

400-750nm

Biolayer Interferometry Methodology

fore

BIO

htt

p

ww

wf

ort

ebio

co

mi

nte

ract

ion

sSp

rin

g_2

01

2p

age5

htm

l

Biolayer Interferometry Methodology

fore

BIO

htt

p

ww

wf

ort

ebio

co

mi

nte

ract

ion

sSp

rin

g_2

01

2p

age5

htm

l

How Do We Create a BioreactiveSensor Tip to Display Kinetics

Anthrax Transition ---- Dnm shift Sensogram

Coupling Chemistry

LFn or Anthrax Prepore can be immobilized onto BLI or SPR surface via Thiol coupling

Thiol coupling an active disulfide moiety can be introduced either on the dextranmatrix or on the ligand molecule 2-(2-pyrdinyldithio) ethaneamine (PDEA) can be used to introduce the di-sulfide group for attachment exchange

C

OH

O C

O

O C

NH

OEDCNHS

NO O

SHPDEA

C

NH

O

S

S

S

S

NE126C LFn

Alternatively a SH containing protein --the prepore itself can be immobilizedon the tip

SH

E126C LFn Attachment to Matrix

C

OH

O C

O

O C

NH

OEDCNHS

NO O

SHPDEA

C

NH

O

S

S

S

S

NE126C LFn

SuccinimideCarboimide

Pyridinyl dithio ethane amide

Prepore attachment to E126C LFn

50 mM L-cysteineBlock

E126C LFn

Prepore

L-cysteine blocks remaining SH Groups

PA Binds with Kd = 1nM

LFN binding sites

Section of Domain-2 unfold and refoldsInto extendedstructure

Domain-2

Prepore to Pore transitions (overlay)Slideshow ndashclick for animation

BLI Transition Protocol

pH Induced Transitions

Kinetic Data of Anthrax pH-Induced Transitions

pH SPR BLI

(t12) sec k1 (sminus1

) k2 (sminus1

) k3 (sminus1

)

675 15 0190 plusmn 0005 0029 plusmn 0001 0005 plusmn 00009

65 15 0813 plusmn 0016 009 plusmn 0005 0006 plusmn 00004

6 lt1 0872 plusmn 0021 0082 plusmn 0004 0010 plusmn 00005

55 lt1 1143 plusmn 0024 0076 plusmn 0024 0008 plusmn 00004

5 lt1 1095 plusmn 0018 0025 plusmn 0004 0010 plusmn 00041

Biolayer Interferometry Methodology

fore

BIO

htt

p

ww

wf

ort

ebio

co

mi

nte

ract

ion

sSp

rin

g_2

01

2p

age5

htm

l

Biolayer Interferometry Methodology

fore

BIO

htt

p

ww

wf

ort

ebio

co

mi

nte

ract

ion

sSp

rin

g_2

01

2p

age5

htm

l

How Do We Create a BioreactiveSensor Tip to Display Kinetics

Anthrax Transition ---- Dnm shift Sensogram

Coupling Chemistry

LFn or Anthrax Prepore can be immobilized onto BLI or SPR surface via Thiol coupling

Thiol coupling an active disulfide moiety can be introduced either on the dextranmatrix or on the ligand molecule 2-(2-pyrdinyldithio) ethaneamine (PDEA) can be used to introduce the di-sulfide group for attachment exchange

C

OH

O C

O

O C

NH

OEDCNHS

NO O

SHPDEA

C

NH

O

S

S

S

S

NE126C LFn

Alternatively a SH containing protein --the prepore itself can be immobilizedon the tip

SH

E126C LFn Attachment to Matrix

C

OH

O C

O

O C

NH

OEDCNHS

NO O

SHPDEA

C

NH

O

S

S

S

S

NE126C LFn

SuccinimideCarboimide

Pyridinyl dithio ethane amide

Prepore attachment to E126C LFn

50 mM L-cysteineBlock

E126C LFn

Prepore

L-cysteine blocks remaining SH Groups

PA Binds with Kd = 1nM

LFN binding sites

Section of Domain-2 unfold and refoldsInto extendedstructure

Domain-2

Prepore to Pore transitions (overlay)Slideshow ndashclick for animation

BLI Transition Protocol

pH Induced Transitions

Kinetic Data of Anthrax pH-Induced Transitions

pH SPR BLI

(t12) sec k1 (sminus1

) k2 (sminus1

) k3 (sminus1

)

675 15 0190 plusmn 0005 0029 plusmn 0001 0005 plusmn 00009

65 15 0813 plusmn 0016 009 plusmn 0005 0006 plusmn 00004

6 lt1 0872 plusmn 0021 0082 plusmn 0004 0010 plusmn 00005

55 lt1 1143 plusmn 0024 0076 plusmn 0024 0008 plusmn 00004

5 lt1 1095 plusmn 0018 0025 plusmn 0004 0010 plusmn 00041

Biolayer Interferometry Methodology

fore

BIO

htt

p

ww

wf

ort

ebio

co

mi

nte

ract

ion

sSp

rin

g_2

01

2p

age5

htm

l

How Do We Create a BioreactiveSensor Tip to Display Kinetics

Anthrax Transition ---- Dnm shift Sensogram

Coupling Chemistry

LFn or Anthrax Prepore can be immobilized onto BLI or SPR surface via Thiol coupling

Thiol coupling an active disulfide moiety can be introduced either on the dextranmatrix or on the ligand molecule 2-(2-pyrdinyldithio) ethaneamine (PDEA) can be used to introduce the di-sulfide group for attachment exchange

C

OH

O C

O

O C

NH

OEDCNHS

NO O

SHPDEA

C

NH

O

S

S

S

S

NE126C LFn

Alternatively a SH containing protein --the prepore itself can be immobilizedon the tip

SH

E126C LFn Attachment to Matrix

C

OH

O C

O

O C

NH

OEDCNHS

NO O

SHPDEA

C

NH

O

S

S

S

S

NE126C LFn

SuccinimideCarboimide

Pyridinyl dithio ethane amide

Prepore attachment to E126C LFn

50 mM L-cysteineBlock

E126C LFn

Prepore

L-cysteine blocks remaining SH Groups

PA Binds with Kd = 1nM

LFN binding sites

Section of Domain-2 unfold and refoldsInto extendedstructure

Domain-2

Prepore to Pore transitions (overlay)Slideshow ndashclick for animation

BLI Transition Protocol

pH Induced Transitions

Kinetic Data of Anthrax pH-Induced Transitions

pH SPR BLI

(t12) sec k1 (sminus1

) k2 (sminus1

) k3 (sminus1

)

675 15 0190 plusmn 0005 0029 plusmn 0001 0005 plusmn 00009

65 15 0813 plusmn 0016 009 plusmn 0005 0006 plusmn 00004

6 lt1 0872 plusmn 0021 0082 plusmn 0004 0010 plusmn 00005

55 lt1 1143 plusmn 0024 0076 plusmn 0024 0008 plusmn 00004

5 lt1 1095 plusmn 0018 0025 plusmn 0004 0010 plusmn 00041

How Do We Create a BioreactiveSensor Tip to Display Kinetics

Anthrax Transition ---- Dnm shift Sensogram

Coupling Chemistry

LFn or Anthrax Prepore can be immobilized onto BLI or SPR surface via Thiol coupling

Thiol coupling an active disulfide moiety can be introduced either on the dextranmatrix or on the ligand molecule 2-(2-pyrdinyldithio) ethaneamine (PDEA) can be used to introduce the di-sulfide group for attachment exchange

C

OH

O C

O

O C

NH

OEDCNHS

NO O

SHPDEA

C

NH

O

S

S

S

S

NE126C LFn

Alternatively a SH containing protein --the prepore itself can be immobilizedon the tip

SH

E126C LFn Attachment to Matrix

C

OH

O C

O

O C

NH

OEDCNHS

NO O

SHPDEA

C

NH

O

S

S

S

S

NE126C LFn

SuccinimideCarboimide

Pyridinyl dithio ethane amide

Prepore attachment to E126C LFn

50 mM L-cysteineBlock

E126C LFn

Prepore

L-cysteine blocks remaining SH Groups

PA Binds with Kd = 1nM

LFN binding sites

Section of Domain-2 unfold and refoldsInto extendedstructure

Domain-2

Prepore to Pore transitions (overlay)Slideshow ndashclick for animation

BLI Transition Protocol

pH Induced Transitions

Kinetic Data of Anthrax pH-Induced Transitions

pH SPR BLI

(t12) sec k1 (sminus1

) k2 (sminus1

) k3 (sminus1

)

675 15 0190 plusmn 0005 0029 plusmn 0001 0005 plusmn 00009

65 15 0813 plusmn 0016 009 plusmn 0005 0006 plusmn 00004

6 lt1 0872 plusmn 0021 0082 plusmn 0004 0010 plusmn 00005

55 lt1 1143 plusmn 0024 0076 plusmn 0024 0008 plusmn 00004

5 lt1 1095 plusmn 0018 0025 plusmn 0004 0010 plusmn 00041

Coupling Chemistry

LFn or Anthrax Prepore can be immobilized onto BLI or SPR surface via Thiol coupling

Thiol coupling an active disulfide moiety can be introduced either on the dextranmatrix or on the ligand molecule 2-(2-pyrdinyldithio) ethaneamine (PDEA) can be used to introduce the di-sulfide group for attachment exchange

C

OH

O C

O

O C

NH

OEDCNHS

NO O

SHPDEA

C

NH

O

S

S

S

S

NE126C LFn

Alternatively a SH containing protein --the prepore itself can be immobilizedon the tip

SH

E126C LFn Attachment to Matrix

C

OH

O C

O

O C

NH

OEDCNHS

NO O

SHPDEA

C

NH

O

S

S

S

S

NE126C LFn

SuccinimideCarboimide

Pyridinyl dithio ethane amide

Prepore attachment to E126C LFn

50 mM L-cysteineBlock

E126C LFn

Prepore

L-cysteine blocks remaining SH Groups

PA Binds with Kd = 1nM

LFN binding sites

Section of Domain-2 unfold and refoldsInto extendedstructure

Domain-2

Prepore to Pore transitions (overlay)Slideshow ndashclick for animation

BLI Transition Protocol

pH Induced Transitions

Kinetic Data of Anthrax pH-Induced Transitions

pH SPR BLI

(t12) sec k1 (sminus1

) k2 (sminus1

) k3 (sminus1

)

675 15 0190 plusmn 0005 0029 plusmn 0001 0005 plusmn 00009

65 15 0813 plusmn 0016 009 plusmn 0005 0006 plusmn 00004

6 lt1 0872 plusmn 0021 0082 plusmn 0004 0010 plusmn 00005

55 lt1 1143 plusmn 0024 0076 plusmn 0024 0008 plusmn 00004

5 lt1 1095 plusmn 0018 0025 plusmn 0004 0010 plusmn 00041

LFn or Anthrax Prepore can be immobilized onto BLI or SPR surface via Thiol coupling

Thiol coupling an active disulfide moiety can be introduced either on the dextranmatrix or on the ligand molecule 2-(2-pyrdinyldithio) ethaneamine (PDEA) can be used to introduce the di-sulfide group for attachment exchange

C

OH

O C

O

O C

NH

OEDCNHS

NO O

SHPDEA

C

NH

O

S

S

S

S

NE126C LFn

Alternatively a SH containing protein --the prepore itself can be immobilizedon the tip

SH

E126C LFn Attachment to Matrix

C

OH

O C

O

O C

NH

OEDCNHS

NO O

SHPDEA

C

NH

O

S

S

S

S

NE126C LFn

SuccinimideCarboimide

Pyridinyl dithio ethane amide

Prepore attachment to E126C LFn

50 mM L-cysteineBlock

E126C LFn

Prepore

L-cysteine blocks remaining SH Groups

PA Binds with Kd = 1nM

LFN binding sites

Section of Domain-2 unfold and refoldsInto extendedstructure

Domain-2

Prepore to Pore transitions (overlay)Slideshow ndashclick for animation

BLI Transition Protocol

pH Induced Transitions

Kinetic Data of Anthrax pH-Induced Transitions

pH SPR BLI

(t12) sec k1 (sminus1

) k2 (sminus1

) k3 (sminus1

)

675 15 0190 plusmn 0005 0029 plusmn 0001 0005 plusmn 00009

65 15 0813 plusmn 0016 009 plusmn 0005 0006 plusmn 00004

6 lt1 0872 plusmn 0021 0082 plusmn 0004 0010 plusmn 00005

55 lt1 1143 plusmn 0024 0076 plusmn 0024 0008 plusmn 00004

5 lt1 1095 plusmn 0018 0025 plusmn 0004 0010 plusmn 00041

E126C LFn Attachment to Matrix

C

OH

O C

O

O C

NH

OEDCNHS

NO O

SHPDEA

C

NH

O

S

S

S

S

NE126C LFn

SuccinimideCarboimide

Pyridinyl dithio ethane amide

Prepore attachment to E126C LFn

50 mM L-cysteineBlock

E126C LFn

Prepore

L-cysteine blocks remaining SH Groups

PA Binds with Kd = 1nM

LFN binding sites

Section of Domain-2 unfold and refoldsInto extendedstructure

Domain-2

Prepore to Pore transitions (overlay)Slideshow ndashclick for animation

BLI Transition Protocol

pH Induced Transitions

Kinetic Data of Anthrax pH-Induced Transitions

pH SPR BLI

(t12) sec k1 (sminus1

) k2 (sminus1

) k3 (sminus1

)

675 15 0190 plusmn 0005 0029 plusmn 0001 0005 plusmn 00009

65 15 0813 plusmn 0016 009 plusmn 0005 0006 plusmn 00004

6 lt1 0872 plusmn 0021 0082 plusmn 0004 0010 plusmn 00005

55 lt1 1143 plusmn 0024 0076 plusmn 0024 0008 plusmn 00004

5 lt1 1095 plusmn 0018 0025 plusmn 0004 0010 plusmn 00041

Prepore attachment to E126C LFn

50 mM L-cysteineBlock

E126C LFn

Prepore

L-cysteine blocks remaining SH Groups

PA Binds with Kd = 1nM

LFN binding sites

Section of Domain-2 unfold and refoldsInto extendedstructure

Domain-2

Prepore to Pore transitions (overlay)Slideshow ndashclick for animation

BLI Transition Protocol

pH Induced Transitions

Kinetic Data of Anthrax pH-Induced Transitions

pH SPR BLI

(t12) sec k1 (sminus1

) k2 (sminus1

) k3 (sminus1

)

675 15 0190 plusmn 0005 0029 plusmn 0001 0005 plusmn 00009

65 15 0813 plusmn 0016 009 plusmn 0005 0006 plusmn 00004

6 lt1 0872 plusmn 0021 0082 plusmn 0004 0010 plusmn 00005

55 lt1 1143 plusmn 0024 0076 plusmn 0024 0008 plusmn 00004

5 lt1 1095 plusmn 0018 0025 plusmn 0004 0010 plusmn 00041

LFN binding sites

Section of Domain-2 unfold and refoldsInto extendedstructure

Domain-2

Prepore to Pore transitions (overlay)Slideshow ndashclick for animation

BLI Transition Protocol

pH Induced Transitions

Kinetic Data of Anthrax pH-Induced Transitions

pH SPR BLI

(t12) sec k1 (sminus1

) k2 (sminus1

) k3 (sminus1

)

675 15 0190 plusmn 0005 0029 plusmn 0001 0005 plusmn 00009

65 15 0813 plusmn 0016 009 plusmn 0005 0006 plusmn 00004

6 lt1 0872 plusmn 0021 0082 plusmn 0004 0010 plusmn 00005

55 lt1 1143 plusmn 0024 0076 plusmn 0024 0008 plusmn 00004

5 lt1 1095 plusmn 0018 0025 plusmn 0004 0010 plusmn 00041

BLI Transition Protocol

pH Induced Transitions

Kinetic Data of Anthrax pH-Induced Transitions

pH SPR BLI

(t12) sec k1 (sminus1

) k2 (sminus1

) k3 (sminus1

)

675 15 0190 plusmn 0005 0029 plusmn 0001 0005 plusmn 00009

65 15 0813 plusmn 0016 009 plusmn 0005 0006 plusmn 00004

6 lt1 0872 plusmn 0021 0082 plusmn 0004 0010 plusmn 00005

55 lt1 1143 plusmn 0024 0076 plusmn 0024 0008 plusmn 00004

5 lt1 1095 plusmn 0018 0025 plusmn 0004 0010 plusmn 00041

pH Induced Transitions

Kinetic Data of Anthrax pH-Induced Transitions

pH SPR BLI

(t12) sec k1 (sminus1

) k2 (sminus1

) k3 (sminus1

)

675 15 0190 plusmn 0005 0029 plusmn 0001 0005 plusmn 00009

65 15 0813 plusmn 0016 009 plusmn 0005 0006 plusmn 00004

6 lt1 0872 plusmn 0021 0082 plusmn 0004 0010 plusmn 00005

55 lt1 1143 plusmn 0024 0076 plusmn 0024 0008 plusmn 00004

5 lt1 1095 plusmn 0018 0025 plusmn 0004 0010 plusmn 00041

Kinetic Data of Anthrax pH-Induced Transitions

pH SPR BLI

(t12) sec k1 (sminus1

) k2 (sminus1

) k3 (sminus1

)

675 15 0190 plusmn 0005 0029 plusmn 0001 0005 plusmn 00009

65 15 0813 plusmn 0016 009 plusmn 0005 0006 plusmn 00004

6 lt1 0872 plusmn 0021 0082 plusmn 0004 0010 plusmn 00005

55 lt1 1143 plusmn 0024 0076 plusmn 0024 0008 plusmn 00004

5 lt1 1095 plusmn 0018 0025 plusmn 0004 0010 plusmn 00041

Generating Image of Macromolecule

Problem - The pore aggregates in solution

Crystal structure of the prepore

Low pHdetergent1M urea +37˚C

DetergentLiposomes

Standard approaches canrsquot prevent aggregation

X

Hydrophobic Residues

Idea ndash capture pore using Chaperonin as stabilizer (binds hydrophobic tip)

Chaperonin protein (prevents Protein aggregation LargeHydrophobic binding site)

Toxin Coupled with GroELBlocks Hydrophobic Pore Elements

Toxin Coupled with GroELViewed by EM

(Katayama et al NSMB July 2008)

Method to release the immobilized biological molecules from biosensor tip and visualized them by EM (A way of validating inhibitors ndash Secondary screens)

Removing Biosensor tip Dip into lt2microl buffer+decoupling reagent

Prepare EM grid

EM Grids may be negative stained or transferred to blot apparatus for CryoEM

Untransitioned Prepore Visualized

Early Experiments Show Hydrophobic Attraction Nanodisk Formation Blocks Attraction

Unprotected NanoDiskProtected

Lipid Nanodisks

Scaffold protein organizes phospholipidsforming a variable diameter disk

Lipids Membrane Scaffold Protein

Transitioned Pore ProteinNanodisk Protected

PA Transitioned Pore

Nanodisk

Removal from solid surface (BLItz tip) indicating that

insertion of PA in to lipid nanodisc confirmed by TEM

Negative Stain EM Field

Additional Applications

Tetanus Toxin

Nanodisk

TeNTAvg n=64

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry

Naik S1 Kumru OS Cullom M Telikepalli SN Lindboe E Roop TLJoshi SB Amin D Gao P Middaugh CR Volkin DB Fisher MT Protein Sci 2014 Oct23(10)1461-78

Attached Biotinylated GroEL to strepavidin coated BLI tip

Collected binding data upon mAb denaturation ndash exposing hydrophobic sites on mAb

Imaged GroEL and GroEL Conjugates when released from tip

Established a proof of concept for a tracking assay to detect structurally alteredandor aggregated species of pharmaceutically relevant proteins

Problem - The pore aggregates in solution

Crystal structure of the prepore

Low pHdetergent1M urea +37˚C

DetergentLiposomes

Standard approaches canrsquot prevent aggregation

X

Hydrophobic Residues

Idea ndash capture pore using Chaperonin as stabilizer (binds hydrophobic tip)

Chaperonin protein (prevents Protein aggregation LargeHydrophobic binding site)

Toxin Coupled with GroELBlocks Hydrophobic Pore Elements

Toxin Coupled with GroELViewed by EM

(Katayama et al NSMB July 2008)

Method to release the immobilized biological molecules from biosensor tip and visualized them by EM (A way of validating inhibitors ndash Secondary screens)

Removing Biosensor tip Dip into lt2microl buffer+decoupling reagent

Prepare EM grid

EM Grids may be negative stained or transferred to blot apparatus for CryoEM

Untransitioned Prepore Visualized

Early Experiments Show Hydrophobic Attraction Nanodisk Formation Blocks Attraction

Unprotected NanoDiskProtected

Lipid Nanodisks

Scaffold protein organizes phospholipidsforming a variable diameter disk

Lipids Membrane Scaffold Protein

Transitioned Pore ProteinNanodisk Protected

PA Transitioned Pore

Nanodisk

Removal from solid surface (BLItz tip) indicating that

insertion of PA in to lipid nanodisc confirmed by TEM

Negative Stain EM Field

Additional Applications

Tetanus Toxin

Nanodisk

TeNTAvg n=64

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry

Naik S1 Kumru OS Cullom M Telikepalli SN Lindboe E Roop TLJoshi SB Amin D Gao P Middaugh CR Volkin DB Fisher MT Protein Sci 2014 Oct23(10)1461-78

Attached Biotinylated GroEL to strepavidin coated BLI tip

Collected binding data upon mAb denaturation ndash exposing hydrophobic sites on mAb

Imaged GroEL and GroEL Conjugates when released from tip

Established a proof of concept for a tracking assay to detect structurally alteredandor aggregated species of pharmaceutically relevant proteins

Idea ndash capture pore using Chaperonin as stabilizer (binds hydrophobic tip)

Chaperonin protein (prevents Protein aggregation LargeHydrophobic binding site)

Toxin Coupled with GroELBlocks Hydrophobic Pore Elements

Toxin Coupled with GroELViewed by EM

(Katayama et al NSMB July 2008)

Method to release the immobilized biological molecules from biosensor tip and visualized them by EM (A way of validating inhibitors ndash Secondary screens)

Removing Biosensor tip Dip into lt2microl buffer+decoupling reagent

Prepare EM grid

EM Grids may be negative stained or transferred to blot apparatus for CryoEM

Untransitioned Prepore Visualized

Early Experiments Show Hydrophobic Attraction Nanodisk Formation Blocks Attraction

Unprotected NanoDiskProtected

Lipid Nanodisks

Scaffold protein organizes phospholipidsforming a variable diameter disk

Lipids Membrane Scaffold Protein

Transitioned Pore ProteinNanodisk Protected

PA Transitioned Pore

Nanodisk

Removal from solid surface (BLItz tip) indicating that

insertion of PA in to lipid nanodisc confirmed by TEM

Negative Stain EM Field

Additional Applications

Tetanus Toxin

Nanodisk

TeNTAvg n=64

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry

Naik S1 Kumru OS Cullom M Telikepalli SN Lindboe E Roop TLJoshi SB Amin D Gao P Middaugh CR Volkin DB Fisher MT Protein Sci 2014 Oct23(10)1461-78

Attached Biotinylated GroEL to strepavidin coated BLI tip

Collected binding data upon mAb denaturation ndash exposing hydrophobic sites on mAb

Imaged GroEL and GroEL Conjugates when released from tip

Established a proof of concept for a tracking assay to detect structurally alteredandor aggregated species of pharmaceutically relevant proteins

Toxin Coupled with GroELViewed by EM

(Katayama et al NSMB July 2008)

Method to release the immobilized biological molecules from biosensor tip and visualized them by EM (A way of validating inhibitors ndash Secondary screens)

Removing Biosensor tip Dip into lt2microl buffer+decoupling reagent

Prepare EM grid

EM Grids may be negative stained or transferred to blot apparatus for CryoEM

Untransitioned Prepore Visualized

Early Experiments Show Hydrophobic Attraction Nanodisk Formation Blocks Attraction

Unprotected NanoDiskProtected

Lipid Nanodisks

Scaffold protein organizes phospholipidsforming a variable diameter disk

Lipids Membrane Scaffold Protein

Transitioned Pore ProteinNanodisk Protected

PA Transitioned Pore

Nanodisk

Removal from solid surface (BLItz tip) indicating that

insertion of PA in to lipid nanodisc confirmed by TEM

Negative Stain EM Field

Additional Applications

Tetanus Toxin

Nanodisk

TeNTAvg n=64

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry

Naik S1 Kumru OS Cullom M Telikepalli SN Lindboe E Roop TLJoshi SB Amin D Gao P Middaugh CR Volkin DB Fisher MT Protein Sci 2014 Oct23(10)1461-78

Attached Biotinylated GroEL to strepavidin coated BLI tip

Collected binding data upon mAb denaturation ndash exposing hydrophobic sites on mAb

Imaged GroEL and GroEL Conjugates when released from tip

Established a proof of concept for a tracking assay to detect structurally alteredandor aggregated species of pharmaceutically relevant proteins

Method to release the immobilized biological molecules from biosensor tip and visualized them by EM (A way of validating inhibitors ndash Secondary screens)

Removing Biosensor tip Dip into lt2microl buffer+decoupling reagent

Prepare EM grid

EM Grids may be negative stained or transferred to blot apparatus for CryoEM

Untransitioned Prepore Visualized

Early Experiments Show Hydrophobic Attraction Nanodisk Formation Blocks Attraction

Unprotected NanoDiskProtected

Lipid Nanodisks

Scaffold protein organizes phospholipidsforming a variable diameter disk

Lipids Membrane Scaffold Protein

Transitioned Pore ProteinNanodisk Protected

PA Transitioned Pore

Nanodisk

Removal from solid surface (BLItz tip) indicating that

insertion of PA in to lipid nanodisc confirmed by TEM

Negative Stain EM Field

Additional Applications

Tetanus Toxin

Nanodisk

TeNTAvg n=64

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry

Naik S1 Kumru OS Cullom M Telikepalli SN Lindboe E Roop TLJoshi SB Amin D Gao P Middaugh CR Volkin DB Fisher MT Protein Sci 2014 Oct23(10)1461-78

Attached Biotinylated GroEL to strepavidin coated BLI tip

Collected binding data upon mAb denaturation ndash exposing hydrophobic sites on mAb

Imaged GroEL and GroEL Conjugates when released from tip

Established a proof of concept for a tracking assay to detect structurally alteredandor aggregated species of pharmaceutically relevant proteins

Untransitioned Prepore Visualized

Early Experiments Show Hydrophobic Attraction Nanodisk Formation Blocks Attraction

Unprotected NanoDiskProtected

Lipid Nanodisks

Scaffold protein organizes phospholipidsforming a variable diameter disk

Lipids Membrane Scaffold Protein

Transitioned Pore ProteinNanodisk Protected

PA Transitioned Pore

Nanodisk

Removal from solid surface (BLItz tip) indicating that

insertion of PA in to lipid nanodisc confirmed by TEM

Negative Stain EM Field

Additional Applications

Tetanus Toxin

Nanodisk

TeNTAvg n=64

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry

Naik S1 Kumru OS Cullom M Telikepalli SN Lindboe E Roop TLJoshi SB Amin D Gao P Middaugh CR Volkin DB Fisher MT Protein Sci 2014 Oct23(10)1461-78

Attached Biotinylated GroEL to strepavidin coated BLI tip

Collected binding data upon mAb denaturation ndash exposing hydrophobic sites on mAb

Imaged GroEL and GroEL Conjugates when released from tip

Established a proof of concept for a tracking assay to detect structurally alteredandor aggregated species of pharmaceutically relevant proteins

Early Experiments Show Hydrophobic Attraction Nanodisk Formation Blocks Attraction

Unprotected NanoDiskProtected

Lipid Nanodisks

Scaffold protein organizes phospholipidsforming a variable diameter disk

Lipids Membrane Scaffold Protein

Transitioned Pore ProteinNanodisk Protected

PA Transitioned Pore

Nanodisk

Removal from solid surface (BLItz tip) indicating that

insertion of PA in to lipid nanodisc confirmed by TEM

Negative Stain EM Field

Additional Applications

Tetanus Toxin

Nanodisk

TeNTAvg n=64

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry

Naik S1 Kumru OS Cullom M Telikepalli SN Lindboe E Roop TLJoshi SB Amin D Gao P Middaugh CR Volkin DB Fisher MT Protein Sci 2014 Oct23(10)1461-78

Attached Biotinylated GroEL to strepavidin coated BLI tip

Collected binding data upon mAb denaturation ndash exposing hydrophobic sites on mAb

Imaged GroEL and GroEL Conjugates when released from tip

Established a proof of concept for a tracking assay to detect structurally alteredandor aggregated species of pharmaceutically relevant proteins

Lipid Nanodisks

Scaffold protein organizes phospholipidsforming a variable diameter disk

Lipids Membrane Scaffold Protein

Transitioned Pore ProteinNanodisk Protected

PA Transitioned Pore

Nanodisk

Removal from solid surface (BLItz tip) indicating that

insertion of PA in to lipid nanodisc confirmed by TEM

Negative Stain EM Field

Additional Applications

Tetanus Toxin

Nanodisk

TeNTAvg n=64

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry

Naik S1 Kumru OS Cullom M Telikepalli SN Lindboe E Roop TLJoshi SB Amin D Gao P Middaugh CR Volkin DB Fisher MT Protein Sci 2014 Oct23(10)1461-78

Attached Biotinylated GroEL to strepavidin coated BLI tip

Collected binding data upon mAb denaturation ndash exposing hydrophobic sites on mAb

Imaged GroEL and GroEL Conjugates when released from tip

Established a proof of concept for a tracking assay to detect structurally alteredandor aggregated species of pharmaceutically relevant proteins

Transitioned Pore ProteinNanodisk Protected

PA Transitioned Pore

Nanodisk

Removal from solid surface (BLItz tip) indicating that

insertion of PA in to lipid nanodisc confirmed by TEM

Negative Stain EM Field

Additional Applications

Tetanus Toxin

Nanodisk

TeNTAvg n=64

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry

Naik S1 Kumru OS Cullom M Telikepalli SN Lindboe E Roop TLJoshi SB Amin D Gao P Middaugh CR Volkin DB Fisher MT Protein Sci 2014 Oct23(10)1461-78

Attached Biotinylated GroEL to strepavidin coated BLI tip

Collected binding data upon mAb denaturation ndash exposing hydrophobic sites on mAb

Imaged GroEL and GroEL Conjugates when released from tip

Established a proof of concept for a tracking assay to detect structurally alteredandor aggregated species of pharmaceutically relevant proteins

Removal from solid surface (BLItz tip) indicating that

insertion of PA in to lipid nanodisc confirmed by TEM

Negative Stain EM Field

Additional Applications

Tetanus Toxin

Nanodisk

TeNTAvg n=64

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry

Naik S1 Kumru OS Cullom M Telikepalli SN Lindboe E Roop TLJoshi SB Amin D Gao P Middaugh CR Volkin DB Fisher MT Protein Sci 2014 Oct23(10)1461-78

Attached Biotinylated GroEL to strepavidin coated BLI tip

Collected binding data upon mAb denaturation ndash exposing hydrophobic sites on mAb

Imaged GroEL and GroEL Conjugates when released from tip

Established a proof of concept for a tracking assay to detect structurally alteredandor aggregated species of pharmaceutically relevant proteins

Additional Applications

Tetanus Toxin

Nanodisk

TeNTAvg n=64

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry

Naik S1 Kumru OS Cullom M Telikepalli SN Lindboe E Roop TLJoshi SB Amin D Gao P Middaugh CR Volkin DB Fisher MT Protein Sci 2014 Oct23(10)1461-78

Attached Biotinylated GroEL to strepavidin coated BLI tip

Collected binding data upon mAb denaturation ndash exposing hydrophobic sites on mAb

Imaged GroEL and GroEL Conjugates when released from tip

Established a proof of concept for a tracking assay to detect structurally alteredandor aggregated species of pharmaceutically relevant proteins

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry

Naik S1 Kumru OS Cullom M Telikepalli SN Lindboe E Roop TLJoshi SB Amin D Gao P Middaugh CR Volkin DB Fisher MT Protein Sci 2014 Oct23(10)1461-78

Attached Biotinylated GroEL to strepavidin coated BLI tip

Collected binding data upon mAb denaturation ndash exposing hydrophobic sites on mAb

Imaged GroEL and GroEL Conjugates when released from tip

Established a proof of concept for a tracking assay to detect structurally alteredandor aggregated species of pharmaceutically relevant proteins

Acknowledgements

Anthrax pore transloconR John Collier NIH SR37AI022021Bythe JanowaikBrian Pentulete

Edward Gogol

Principle Investigator ndashNIH R01AI090085Mark T Fisher

Graduate Students Hiro KatayamaSubhashchandra NaikSusan Brock

Postdoctoral fellowsNarahari AkkaladeviSrayanta Mukerjee

Research TechniciansLynn Chollet-Hinton

Application ScientistWesley McGinn-Straub

Na ZhangPhilip Gao

Thank You

Questions

Thank You

Questions