Transcript A Chemical Approach To Illustrate the Principal of Signal

A Chemical Approach To Illustrate the
Principal of Signal Transduction
Cascades Using the Avidin-Biotin System
Margherita Morpurgo, Heike Hofstetter,
Edward A. Bayer, and Meir Wilchek
J. Am. Chem. Soc. 1998, 120, 12734-12739
Introduction
In nature, cellular functions are propagated by
cascades of molecules, which interact with one another
for signal transduction. Generally, the sequential process
is initiated by the binding of an extracellular signal to a
receptor culminating in one or more specific cellular
responses
In this way, a signal, for example, can be
transferred from the outside of the cell en route to the
nucleus—a process mediated by ligands, e.g., hormones,
cytokines, and growth factors.
Goal
To determine whether a cascade can be
formed artificially, whereby the binding of
one molecule would depend on the
“signaling” of another.
Two molecules that display differing affinities for the same
binding site of a protein:
[Simple diagram]
Consequently, one step will be dependent on a previous
one, thus enabling us to trigger a cascade of binding,
e.g., to construct an organized system of protein
multilayers.
Background
Method based on the Avidin-Biotin system:
Avidin-biotin is based on the high affinity of the
cofactor Biotin for the protein Avidin and the ease of
spectrophotometric measurement
Used the egg-white protein avidin, deglycosylated
(DG) avidin, and streptavidin which bind to two
different ligands, biotin and 4-hydroxyazobenzene-2carboxylic acid (HABA), with different affinities, 10-15
and 10-6 M, respectively.
Background
Based on the binding of the dye HABA to Avidin and the ability
of Biotin to displace the dye in stoichiometric proportions
Binding of HABA to avidin is accompanied by an instantaneous
shift in the absorption spectrum from Ïmax 348 nm of the free
HABA to Ïmax 500 nm of the complexed tautomer.
Furthermore, avidin can be forced to catalyze the hydrolysis of
HABA derivatives, in which the hydroxyl function is blocked with
a protecting group (e.g., an acetyl moiety), to accommodate the
azo dye in its tautomeric form at the binding site.
The Plan, Stan:
HABAylated avidin
+ biotin
Biotin (blue)
binds avidin
and expells
HABA
Anti-HABA
antibodies
+ avidin
B
Biotinylated
antibody
Multilayer Assemblies
OHHydrolysis
A HABA-containing affinity
label was designed, such that
the dye would remain covalently
attached to the binding site of avidin.
Cyclic
HABA
Cyclic HABA
added to avidin.
Reaction
monitored by
UV-Vis spec.
Gradual shift in absorption
from 328 nm to 504 nm
w/biotin
w/o biotin
Biotin was added to the red HABAylated avidin:
immediate shift to 356 nm observed
Yet, the orange color (of HABA) continued to be associated
with the protein, indicating HABA was still covalently bound
to colorless avidin.
More evidence of HABA covalently
binding avidin
m/z = 14 289
~single subunit
of avidin
Avidin + HABA
m/z = 14 727
Difference: 14 727
-14 289
438
~ mass of cyclic
HABA
Matrix-Assisted Light Desorption Ionization (MALDI) Mass
Spectra of avidin before and after reaction with cyclic HABA.
To determine
which avidin
residue was
modified and
covalently bound
to the HABA
moiety, the protein
was subjected to
trypsin digestion.
HPLC analysis of the tryptic
digest of HABAylated avidin.
356 nm, adsorption
maxima of HABA
220 nm,
adsorption
maxima of the
peptides.
Lys-111
Amino Acid Sequence of HABAylated Avidin (orange)
Cyclic HABA can be hydrolyzed by avidin, which enables
the attachment of the HABA moiety to an appropriate
residue in or near the binding site of avidin.
HABA with side chain
Adjacent
monomer
on avidin
Binding site
of avidin
(one monomer)
w/ biotin
w/o biotin
Polyclonal antibodies against
HABA were examined for their
interaction with the HABAylated
avidin in the presence and absence
of biotin.
Mission Complete
HABAylated
avidin
+ biotin B
Biotin (blue)
binds avidin
and expells
HABA
Biotin-saturated
HABAylated
Avidin
+ Biotinylated
antibody
B
In Summary…
• Used the avidin-biotin system to demonstrate the interplay
of molecular recognition and oriented protein assembly
which serves as a prerequisite for the principle of signal
transduction
• Biotin served as the effector or trigger of the cascade
• Avidin has four binding sites: limited amounts of biotin can
be added to displace only one or two HABA molecules to
trigger the cascade vectorially in different dimensions
Conclusion
• System can be considered a chemical mimic of signal
transduction, which, in nature, can also be modulated in
different ways to enhance the response, following the
initial triggering by a hormone or another effector
• This concept of chemical mimicry and the assembly
of protein multilayers is appropriate for application in
numerous fields, such as medicine, diagnostics,
biosensors, nanotechnology, and artificial intelligence,
thus expanding the scope of the avidinbiotin system.
Questions?