Post on 18-Jul-2015
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Biolayer Interferometry Methodology
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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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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