Substrate Identification and Determination of Binding Kinetics
E + S
ES
Molecule A + Molecule B
AB Complex
In the real world or massive complexity of living organisms both the E or the S
can be proteins, DNA, RNA, carbohydrates, lipids, any number of chemical
cofactors and metabolites OR entire cells binding to one another through a series
of interactions at cell surface receptors that are comprised of all of the above.
Identification of Substrates and Molecular Complexes
How do I identify a physiologically relevant molecular complex?
To answer this we must first begin with a significant cellular process
or metabolic function that is of particular interest (i.e. one that has an
important role in human health or disease). Please note that the human
health is impacted by many things for example- Plant and animal health in food sources (USDA)
- Energy and other environmental pollution sources (DOE, EPA)
- Technological advances in basic chemistry and physics (NSF)
Once we know what system or function we are interested in we can think
about the second problem of detecting and isolating important molecular
complexes.
What are the important tools of the 21st century.
1) Massive libraries of genomic sequence information
- Genomic, proteomic, and bioinformatics tools
2) Microarray analysis
- Determine how the genome responds to disease, or when
your metabolic pathway is active.
3) Cellular imaging through fluorescent markers that tell you when
key players are expressed and where they are going.
General Tools for Identifying Molecular Interactions
1- All types of chromatography.
1- Paper or thin layer chromatography (TLC)
2- HPLC that may or may not be followed by GC-MS analysis
3- Affinity chromatography, Molecule A is tethered to a bead and
you go “fishing” for molecule B. Similar approaches can
be employed for isolation of DNA or RNA protein
complexes.
2- If you have antibodies for molecule A then immuno precipitation can be
used to “pull down” or precipitate molecule B.
3- Combination of chromatography and electrophoresis methods that are
also coupled to MS techniques.
4- Good “old fashion” biochemistry. Application of inhibitors and or
mutagenesis to “catch” molecular complexes at intermediate steps.
5- Searching for inhibitors (or lead compounds) using phage display.
In Living Organisms, Metabolites Traditionally
Identified by Labeling and Chemical Analysis
Metabolite identification
Protein Identification
Cell growth in unlabeled media
Cell growth in unlabeled media
Addition of labeled metabolite (e.x. 13C glucose)
for variable defined lengths of time (The Pulse).
Induction or stress of system and introduction
of 35S for variable defined lengths of time.
Addition of excess unlabeled metabolite (The Chase)
Removal of inducer or stress and growth on
unlabeled media.
Cell lysis and fractionation followed by chemical
and spectroscopic analysis (e.x. NMR, TLC, LC-MS)
Cell lysis and fractionation followed by 2D gel
electrophoresis and MS.
Chromogenic and Flourogenic Synthetic Substrates for Culture Media
Multi-test Identifications
with commercial
significance.
Rapid detection/identification of pathogenic
organisms in the food industry is of great
importance. This applies to ANY additive that
will be ingested. The same technology and
chemical/biochemical approaches can
be applied elsewhere (i.e. detection of cell
types and surface receptors).
See Journal of Microbiological Methods 79 (2009) 139–155
Identifying Substrates Using Combinatorial Chemistry
Quencher
Fluorophore
Peptide or polysaccharide
Resin or
PEG Bead
The strength of the technology is in the range of poly peptide and polysacchatide
libraries that are available for screening.
Using Combinatorial Chemistry To Identify
Inhibitors of Matrix Metalloproteases (MMPs)
MMPs belong to a family of structurally related zinc containing
endoproteinases. Their primary function is degradation of a variety
of extracellular matrix components. They are known to participate
in various pathological conditions such as arthritis, cancer and
osteoporosis, hence inhibition of MMPs may be very important in
clinical treatment
The design of many MMP inhibitors has focused on finding a zinc
binding motif, which can chelate the active-site zinc(II)ion effectively,
a backbone which can provide hydrogen bond interactions with the
enzyme, and one or more side chains which can have effective van
der Waals interaction with MMP subsites.
One such example PDB ID 1SMP – Baumann et al. JMB 1995 248 653-661
Identifying Inhibitors Using Combinatorial Chemistry
Quencher
Fluorophore
Substrate
Inhibitor
Resin or
PEG Bead
Building a Library
The general structure for the library was H-XX-azole-XX-N2
Building a Library
Made a library of 240,000 and got 184 potential hits or “dark beads”.
Similar to phage display technology, the beads can be washed and
sequences that “stick” can be identified or amplified.
Identifying Inhibitors Using Phage Display
Lysozyme Epitopes Identified by Phage Display
PDB Entrys;
1VFB – C chain
3HFL – Y chain
3HFM – Y chain
All have residues 1-129
Kinetic Characterization of Substrate Binding
The basics;
• How tight is the binding?
• How many binding sites?
• Is there any cooperation between binding
sites?
You need to know the basics, do the kinetic constants
make physiological sense. Alternatively, for an inhibitor or
drug, will an effective dose be achievable?
Old Fashion (Low Budget) Binding Experiments
What are the requirements?
- Ability to get a significant amount of protein and substrate, typically
several mg.
- The ability to detect and accurately measure both the protein and the
substrate in solution.
- A gel filtration column that is compatible with the protein and substrate as
well as fraction collector
What is the protocol?
- Equilibrate column with low concentration of substrate/inhibitor and pass
the protein over the column in that buffer.
- Collect fraction prior to protein elution AND fraction with protein.
- Measure protein and substrate in both fractions and compare to determine
the amount bound by the protein.
Old Fashion (Low Budget) Binding Experiments
We all remember from our first Biochemistry class that a dissociation constant is;
[ES]
[E] + [S]
KD=
[E][S]
[ES]
In general, binding can be represented by Michaelis Menton kinetics where V is replaced with [ES]
or
n
n
Where n is referred to a cooperativity coefficient
Isothermal Titration Calorimetry (ITC)
Most biochemical reactions, including binding, involve a
small change in heat. This is what ITC measures.
Isothermal Titration Calorimetry (ITC)
Isothermal Titration Calorimetry (ITC)
Isothermal Titration Calorimetry (ITC)
Isothermal Titration Calorimetry (ITC)
Isothermal Titration Calorimetry (ITC)
Isothermal Titration Calorimetry (ITC)
What if the Heat Change is Small?
PDB 1A30
- Moreover, think about the physiological environment, is typical substrate binding driven by
entropic contributions?
Surface Plasmon Resonance