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Substrate Identification and Determination of Binding Kinetics
E + S ES
In the real world or massive complexity of living organisms both the E or the Scan be proteins, DNA, RNA, carbohydrates, lipids, any number of chemicalcofactors and metabolites OR entire cells binding to one another through a seriesof interactions at cell surface receptors that are comprised of all of the above.
Molecule A + Molecule B AB Complex
Once we know what system or function we are interested in we can thinkabout the second problem of detecting and isolating important molecularcomplexes.
What are the important tools of the 21st century.1) Massive libraries of genomic sequence information
- Genomic, proteomic, and bioinformatics tools2) 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.
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 processor metabolic function that is of particular interest (i.e. one that has animportant role in human health or disease). Please note that the humanhealth 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)
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 analysis3- 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 proteincomplexes.
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 arealso coupled to MS techniques.
4- Good “old fashion” biochemistry. Application of inhibitors and ormutagenesis to “catch” molecular complexes at intermediate steps.
5- Searching for inhibitors (or lead compounds) using phage display.
In Living Organisms, Metabolites TraditionallyIdentified by Labeling and Chemical Analysis
Metabolite identification Protein Identification
Cell growth in unlabeled media
Induction or stress of system and introductionof 35S for variable defined lengths of time.
Removal of inducer or stress and growth onunlabeled media.
Cell lysis and fractionation followed by 2D gelelectrophoresis and MS.
Cell growth in unlabeled media
Addition of labeled metabolite (e.x. 13C glucose)for variable defined lengths of time (The Pulse).
Addition of excess unlabeled metabolite (The Chase)
Cell lysis and fractionation followed by chemicaland spectroscopic analysis (e.x. NMR, TLC, LC-MS)
Multi-test Identificationswith commercialsignificance.
Rapid detection/identification of pathogenicorganisms in the food industry is of greatimportance. This applies to ANY additive thatwill be ingested. The same technology and chemical/biochemical approaches canbe applied elsewhere (i.e. detection of celltypes and surface receptors).
See Journal of Microbiological Methods 79 (2009) 139–155
Identifying Substrates Using Combinatorial Chemistry
Resin orPEG Bead
Fluorophore
Peptide or polysaccharide
Quencher
The strength of the technology is in the range of poly peptide and polysacchatidelibraries that are available for screening.
Using Combinatorial Chemistry To IdentifyInhibitors of Matrix Metalloproteases (MMPs)
MMPs belong to a family of structurally related zinc containingendoproteinases. Their primary function is degradation of a varietyof extracellular matrix components. They are known to participatein various pathological conditions such as arthritis, cancer andosteoporosis, hence inhibition of MMPs may be very important inclinical treatment
The design of many MMP inhibitors has focused on finding a zincbinding motif, which can chelate the active-site zinc(II)ion effectively,a backbone which can provide hydrogen bond interactions with theenzyme, 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
Resin orPEG Bead
Fluorophore
Substrate
Quencher
Inhibitor
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 andsequences that “stick” can be identified or amplified.
Lysozyme Epitopes Identified by Phage Display
PDB Entrys;1VFB – C chain3HFL – Y chain3HFM – Y chain
All have residues 1-129
Kinetic Characterization of Substrate Binding
• How tight is the binding?
• How many binding sites?
• Is there any cooperation between binding sites?
The basics;
You need to know the basics, do the kinetic constantsmake physiological sense. Alternatively, for an inhibitor ordrug, 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]
n
n
or
In general, binding can be represented by Michaelis Menton kinetics where V is replaced with [ES]
Where n is referred to a cooperativity coefficient
Isothermal Titration Calorimetry (ITC)
Most biochemical reactions, including binding, involve asmall change in heat. This is what ITC measures.
- Moreover, think about the physiological environment, is typical substrate binding driven by entropic contributions?