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ate Identification and Determination of Binding Kin E + S ES real world or massive complexity of living organisms both the E or t proteins, DNA, RNA, carbohydrates, lipids, any number of chemical ors and metabolites OR entire cells binding to one another through eractions at cell surface receptors that are comprised of all of th Molecule A + Molecule B AB Complex

Substrate Identification and Determination of Binding Kinetics E + SES In the real world or massive complexity of living organisms both the E or the S

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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)

Chromogenic and Flourogenic Synthetic Substrates for Culture Media

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

The general structure for the library was H-XX-azole-XX-N2

Building a Library

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.

Identifying Inhibitors Using Phage Display

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.

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