Molecular Biotechnology Core

A Circular Dichroisms (CD) Spectropolarimeter (Model J-815 from Jasco) is a type of light absorption spectroscopy that can provide information on the structures of optically active biological macromolecules. CD spectra of proteins between 250 and 185 nm can be analyzed for different secondary structural types such as, alpha helix, parallel and antiparallel beta sheet, turn and other random structures.

The CD spectroscopy is a shared resource and is available to researchers of Cleveland Clinic and neighboring institutions. The details about CD spectroscopy are as follows:

CD Capabilities:

• Model J-815 Spectropolarimeter with computer-controlled data acquisition; wavelength range 163-900 nm
• Single position Peltier system with Fluorescence capability
• Dual range titration system for automated pH, ligand and denaturant titrations
• Scanning Emission Monochromator allows spectral EM scans simultaneous with CD measurement (wavelength range 200-750 nm)
• Temperature-controlled circulating water bath (Julabo)

CD Spectropolarimeter can be used for the following applications:

• Secondary structure analysis of proteins in solution
• Protein folding and conformational studies
• DNA/RNA interaction studies
• Temperature controlled kinetic studies
• Thermal denaturation profiles of aqueous proteins

Microscale thermophoresis (MST) has been recently emerged as a powerful technique for quantifying molecular interactions. It is highly sensitive and can quickly yield detectable signals in response to interaction-induced changes of molecular properties, including size, charge, hydration shell and conformation. This technique is based on thermophoresis, which is defined as temperature gradient-induced directed motion of molecules. In a typical MST experiment using the Monolith NT.115, a temperature gradient is induced by an infrared laser and the resulting change in molecular movement is analyzed by fluorescence. Thus, one of the binding-partners must be a biomolecule with covalently attached fluorophore or a fluorescent fusion protein like GFP. MST experiments can also be performed in a label-free setting using intrinsic fluorescence of protein in another Monolith model NT.LabelFree. The interactions can be measured in complex biological fluids like cell lysate and serum without sample purification, which is much needed for other techniques. It also permits studying of the interaction of small molecules and proteins with ease or membrane proteins stabilized in buffers of choice. Thus, its high adaptability over other techniques renders it unique and unparalleled.

MST Application Includes:

• Affinities between biomolecules including proteins, DNA, RNA, peptides, and small molecules
• Stoichiometry of an interaction
• Dissociation constants of multi-component reactions
• Competition between substrates and inhibitors to an enzyme
• Interactions of biomolecules with nanoparticles, vesicles and viruses
• Surface modifications of nanoparticles
• Recognize different binding sites on a target molecule of interest
• The stability of biomolecules in blood, blood serum and blood plasma
• Adsorption of small molecules to lipid membranes or plasma proteins
• Protein aggregating compounds/conditions

What is ITC?
IIsothermal Titration Calorimetry (ITC) is the gold standard for measuring biomolecular interactions. ITC simultaneously determines all binding parameters (n, K, δH and δS) in a single experiment information that cannot be obtained from any other method. When substances bind, heat is either generated or absorbed. ITC is a thermodynamic technique that directly measures the heat released or absorbed during a biomolecular binding event. Measurement of this heat allows accurate determination of binding constants (KD), reaction stoichiometry (n), enthalpy (δH) and entropy (δS), thereby providing a complete thermodynamic profile of the molecular interaction in a single experiment. Because ITC goes beyond binding affinities and can elucidate the mechanism of the molecular interaction, it has become the method of choice for characterizing biomolecular interactions.

Applications include:

• Characterization of molecular interactions of small molecules, proteins, antibodies, nucleic acids, lipids and other biomolecules
• Lead optimization
• Enzyme kinetics
• Assessment of the effect of molecular structure changes on binding mechanisms
• Assessment of biological activity

Interactions between any two molecules can be studied with ITC, including:

• Protein-small molecule
• Protein-protein
• Target-drug
• Enzyme-inhibitor
• Antibody-antigen
• Protein-DNA
• Protein-lipid
• Small molecule-small molecule

Surface Plasmon Resonance (SPR) has been used to monitor macromolecular interactions in real time. Biacore (Cytiva) uses SPR technology for measuring the interactions of macromolecules with each other, and with small molecule ligands. One of the interacting molecules (ligands) is immobilized, for example, on carboxymethylated dextran over a gold surface, while the second partner (analyte) is captured as it flows over the immobilized ligand surface. Most ligands can be directly immobilized onto the surface of the chip via amino groups, carbohydrate moieties, or sulfhydryl groups. Others are immobilized indirectly through the use of biotinylation of the ligand (such as biotinylated peptides or oligonucleotides), or through immobilized monoclonal antibodies (such as anti-GST). Typical amounts of a protein ligand needed for an immobilization reaction is about 1 µg. The immobilized ligands are remarkably resilient and maintain their biological activity.

The bound analytes can be stripped from the immobilized ligand without affecting its activity to allow many cycles of binding and regeneration on the same immobilized surface. Interaction is detected via SPR in real time at high sensitivity, without labeling. The equilibrium rate constant KD (affinity) is measurable in the range of fM to mM. Because the same affinity may reflect different on-rates and off-rates, SPR excels over most other methods of affinity measurements in that it measures on-rates (ka in the range of 103 to 3 × 109 M-1S-1 for proteins and 103 to 5 x107 M-1S-1 for LMW) and off-rates (kd of 10-5 to 10-2 S-1).

The instrument and services are available on a fee-for-service basis to Cleveland Clinic researchers as well as investigators from Case Western Reserve University and other area institutions.
Biacore S200
Biacore S200 is the most sensitive model available from Cytiva. It can be used for measuring the binding parameters of biomolecular interactions (protein-protein, nucleic acids - protein, protein-lipids, protein-small molecule/fragments etc.). Biacore S200 is a label-free interaction analysis system designed to meet the requirements of high sensitivity and short time to results and analysis for:

• Kinetics and affinity
• Rapid screening of small molecules (96\384-well format)
• Competition assays
• Epitope mapping
• Ranking affinities
• Thermodynamics