1. Characterization of Protein- & Biomolecule-based Biointerfaces
Prof. Prabhas Moghe
November 13, 2006
125:583

2. Outline Methods for assessing concentration of biomolecules
ELISA
Radiolabeling
Fluorometry
Methods for assessing thickness, conformation, organization of biomolecules at interfaces
Circular Dichroism
FTIR Spectroscopy (Reviewed)
Atomic force microscopy (Reviewed)
Ellipsometry
Total Internal Reflection Fluorescence (TIRF) (Reviewed)
Methods for estimating protein or ligand affinity to interface
Scatchard Analysis (Radiolabeling)
Surface Plasmon Resonance (SPR) (Reviewed)

3. Direct Determination Using Labeled Species
Need to label
Usually need to remove solution from material
Advantages:
Sensitivity
Can provide information on one species in a “soup”
Disadvantages:
Need to label particular species of interest
Need to process samples
Some people don’t like to work with radioactivity
Fluorescence labeling may alter properties of solute + +

4. FTIR vs. Radiolabeleing

5. Data analysis: the Langmuir isotherm
Basic equation:
Assumptions:
Distinct adsorption sites
Single type (binding energy) of site
Independence of sites
Solute doesn’t change form after adsorption

6. Determining Amounts of Biomolecules on Surfaces: Preliminaries
Useful properties of biomolecules (absorbance; fluorescence; birefringence)
Labeling (use of fluorophore or radiolabeling)
Typical amounts

7. What is an ELISA? Enzyme-linked immunosorbent assay
Name suggests three components
Antibody (immuno)
Allows for specific detection of analyte of interest
Solid phase (sorbent)
Allows one to wash away all the material that is not specifically captured
Enzymatic amplification
Allows you to turn a little capture into a visible color change that can be quantified using an absorbance plate reader

8. Capture and Detection Antibodies

9. Sandwich ELISA

10. Competitive ELISA
Less is more. More antigen in your sample will mean more antibody competed away, which will lead to less signal

11. Enzymes with Chromogenic Substrates
High molar extinction coefficient (i.e., strong color change)
Strong binding between enzyme and substrate (low KM)
Linear relationship between color intensity and [enzyme]

12. If you’re lucky…

13. X-ray photoelectron spectroscopy (aka ESCA)

14. Paper # 1

15. RGD peptide immobilization

16. Ellipsometry
Polarization:
Equipment Schematic:
Measure:

17. Ellipsometry
Ellipsometry consists of the measurement of the change in polarization state of a beam of light upon reflection from the sample of interest. The exact nature of the polarization change is determined by the sample's properties (thickness and refractive index). The experimental data are usually expressed as two parameters Y and D. The polarization state of the light incident upon the sample may be decomposed into an s and a p component (the s-component is oscillating parallel to the sample surface, and the p-component is oscillating parallel to the plane of incidence). The intensity of the s and p component, after reflection, are denoted by Rs and Rp. The fundamental equation of ellipsometry is then written:

18. Ellipsometry through Substrate Layers (Advanced review)

20. Back to RGD peptide example

21. Determining Characteristics of Adsorbed Biomolecular Structure

22. Single molecule protein detection approaches
Piehler, COSB, 15:4-14, 2005

23. Circular dichroism (CD)
Dichroism is the phenomenon in which light absorption differs for different directions of polarization. Linear dichroism involves linearly polarized light where the electric vector is confined to a plane. Circularly polarized light contrasts from linearly polarized light. In linearly polarized light the direction of the electric vector is constant and its magnitude is modulated; in circularly polarized light the magnitude
is constant and the direction is modulated, as shown below.

24. Circular Dichroism Principle
The differential absorption of radiation polarized in two directions as function of frequency
is called dichroism. When applied to plane polarized light, this is called linear dichroism; for circularly polarized light, circular dichroism. We can think of linear polarized light as the result of two equal amplitudes of opposite circular polarization. After passing through an optically active sample, circularly polarized light will be changed in two aspects. The two components are still circularly-polarized, but the magnitudes of the counter-rotating E-components will no longer be equal as the molar extinction coefficients for right- and left-polarized light are unequal. The direction of the E-vector no longer traces a circle - instead it traces an ellipse. There will also be a rotation of the major axis of the ellipse due to differences in refractive indices.
The optics for making circularly polarized light uses a linear polarizer P and a quarter-wave retarder R. Circularly polarized light can be decomposed in the sum of two mutually perpendicular linearly polarized waves that are one quarter of a wavelength out of phase. With Ey retarded on quarter of a wave relative to Ez, we have right circularly polarized light as diagrammed here. If Ez were retarded one quarter of a wave relative to Ey, then the circularly polarized light would be left-handed.

25. CD (continued) 2 related properties
Optical rotation: differential transmission of circularly polarized light in left- and right-hand directions
Circular dichroism: differential absorption of left- and right-circularly polarized light

26. Circular dichroism spectroscopy is particularly good for:
* determining whether a protein is folded, and if so characterizing its secondary structure, tertiary structure, and the structural family to which it belongs
* studying the conformational stability of a protein under stress -- thermal stability, pH stability, and stability to denaturants -- and how this stability is altered by buffer composition or addition of stabilizers and excipients
* determining whether protein-protein interactions alter the conformation of protein. Small conformational changes have been seen, for example, upon formation of several different receptor/ligand complexes.
Secondary structure can be determined by CD spectroscopy in the far-uv spectral region ( nm). At these wavelengths the chromophore is the peptide bond, and the signal arises when it is located in a regular, folded environment. Alpha-helix, beta-sheet, and random coil structures each give rise to a characteristic shape and magnitude of CD spectrum. The approximate fraction of each secondary structure type that is present in any protein can thus be determined by analyzing its far-uv CD spectrum as a sum of fractional multiples of such reference spectra for each structural type.
Like all spectroscopic techniques, the CD signal reflects an average of the entire molecular population. Thus, while CD can determine that a protein contains about 50% alpha-helix, it cannot determine which specific residues are involved in the alpha-helical portion.

28. CD of proteins and nucleic acids

29. CD of adsorbed T4 lysozyme

30. Characterizing Interactions of Biomolecules on Surfaces Estimation of ligand affinity to substrates Ligand-Receptor Binding Ligand-Cell Binding Ligand-Substrate Binding (Paper)

31. Binding of labeled ligands to receptors
Concentration of monovalent ligand, L (moles/volume or M)
Concentration of monovalent receptor, R (#/cell)
Concentration of Complex, C (#/cell)
Concentration of cells=n (#/unit volume)

32. Binding of labeled ligands to receptors, continued
Let u be the scaled/dimensionless number of ligand-receptor complexes, =C/RT
ueq u

33. Scatchard Plot to estimate KD

34. Paper # 2: Affinity of biomolecular binding to substrates

36. Experimental System

38. Adsorption Model

39. Kinetic Model for Binding

40. Fitting the best model to experimental data