Visualizing macromolecules from Biolayer Interferometry analysis using Electron Microscopy

1. 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

2. Outline
E) Prepore to Pore Transition Kinetic profiles – 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

3. Protective Lethal Edema
Antigen Factor Factor
(produced in response to elevated
C02 levels)
http://www.proteinlounge.com/Animation/Mechanism Of AnthraxToxins
Anthrax lethality Arises from the Production of Protein Toxins.

4. Anthrax structure

5. BLI Instrument
ForteBIO.
http://www.blitzmenow.com/about.html
BLITZ
400-750nm

6. Biolayer Interferometry Methodology
foreBIO.http://www.fortebio.com/interactions/Spring_2012/page5.html

7. Biolayer Interferometry Methodology
foreBIO.http://www.fortebio.com/interactions/Spring_2012/page5.html

8. How Do We Create a Bioreactive
Sensor Tip to Display Kinetics
Anthrax Transition ---- Dnm shift Sensogram

9. Coupling Chemistry

10. 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 dextran
matrix 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
O
EDC/NHS
NO O
SHPDEA
C
NH
O
S
S
S
S
N
E126C LFn
Alternatively a SH containing protein --
the prepore itself can be immobilized
on the tip.
SH

11. E126C LFn Attachment to Matrix
C
OH
O C
O
O C
NH
O
EDC/NHS
NO O
SHPDEA
C
NH
O
S
S
S
S
N
E126C LFn
Succinimide
Carboimide
Pyridinyl dithio ethane amide

12. Prepore attachment to E126C LFn
50 mM L-cysteine
Block
E126C LFn
Prepore
L-cysteine blocks remaining SH Groups
PA Binds with Kd = 1nM

13. LFN binding
sites
Section of
Domain-2
unfold
and refolds
Into
extended
structure
Domain-2
Prepore to Pore transitions (overlay)
Slideshow –click for animation.

14. BLI Transition Protocol

15. pH Induced Transitions

16. Kinetic Data of Anthrax pH-Induced
Transitions
pH SPR BLI
(t1/2)* sec k1 (s
−1
) k2 (s
−1
) k3 (s
−1
)
6.75 1.5 0.190 ± 0.005 0.029 ± 0.001 0.005 ± 0.0009
6.5 1.5 0.813 ± 0.016 0.09 ± 0.005 0.006 ± 0.0004
6 <1 0.872 ± 0.021 0.082 ± 0.004 0.010 ± 0.0005
5.5 <1 1.143 ± 0.024 0.076 ± 0.024 0.008 ± 0.0004
5 <1 1.095 ± 0.018 0.025 ± 0.004 0.010 ± 0.0041

18. Generating Image of Macromolecule

19. Problem - The pore aggregates in solution
Crystal structure of the prepore
Low pH
detergent
1M urea +37˚C
Detergent
Liposomes
Standard approaches can’t prevent
aggregation
X
Hydrophobic Residues

20. Idea – capture pore using Chaperonin as stabilizer
(binds hydrophobic tip)
Chaperonin protein (prevents
Protein aggregation. Large
Hydrophobic binding site.)
Toxin Coupled with GroEL
Blocks Hydrophobic Pore Elements

21. Toxin Coupled with GroEL
Viewed by EM
(Katayama et al., NSMB, July 2008)

22. Method to release the immobilized biological molecules from biosensor tip and
visualized them by EM (A way of validating inhibitors – Secondary screens).
Removing Biosensor tip Dip into <2µl buffer+
decoupling reagent
Prepare EM grid
EM Grids may be negative stained or transferred to blot apparatus for CryoEM

23. Untransitioned Prepore Visualized

24. Early Experiments Show Hydrophobic Attraction
Nanodisk Formation Blocks Attraction
Unprotected NanoDisk
Protected

25. Lipid Nanodisks
Scaffold protein organizes phospholipids
forming a variable diameter disk
Lipids Membrane Scaffold Protein

26. Transitioned Pore Protein
Nanodisk Protected
PA Transitioned
Pore
Nanodisk

27. Removal from solid surface (BLItz tip) indicating that
insertion of PA in to lipid nanodisc, confirmed by TEM
Negative Stain EM Field

28. Additional Applications
Tetanus Toxin
Nanodisk
TeNT
Avg n=64

29. 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 TL,
Joshi SB, Amin D, Gao P, Middaugh CR, Volkin DB, Fisher MT.
Protein Sci. 2014 Oct;23(10):1461-78.
Attached Biotinylated GroEL to strepavidin coated BLI tip
Collected binding data upon mAb denaturation – 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 altered
and/or aggregated species of pharmaceutically relevant proteins.

30. Acknowledgements
Anthrax pore translocon
R . John Collier NIH SR37AI022021
Bythe Janowaik
Brian Pentulete
Edward Gogol
Principle Investigator –NIH R01AI090085
Mark T. Fisher
Graduate Students
Hiro Katayama
Subhashchandra Naik
Susan Brock
Postdoctoral fellows
Narahari Akkaladevi
Srayanta Mukerjee
Research Technicians
Lynn Chollet-Hinton
Application Scientist
Wesley McGinn-Straub
Na Zhang
Philip Gao

31. Thank You
Questions