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

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Characterization of Protein- & Biomolecule-based

Biointerfaces

Prof. Prabhas MogheNovember 13, 2006

125:583

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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)

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Direct Determination Using Labeled Species

• Need to label• Usually need to remove solution from

material

+ +

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FTIR vs. Radiolabeleing

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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

f

b=

Cs

Cs,max

=Cbulk

K +Cbulk

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Determining Amounts of Biomolecules on Surfaces:

Preliminaries• Useful properties of biomolecules

(absorbance; fluorescence; birefringence)

• Labeling (use of fluorophore or radiolabeling)

• Typical amounts

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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

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Capture and Detection Antibodies

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Sandwich ELISA

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Competitive ELISA

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

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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]

v =kcat E⎡⎣ ⎤⎦ S⎡⎣ ⎤⎦KM + S⎡⎣ ⎤⎦

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If you’re lucky…

Sample Standard Curve

y = -0.0583Ln(x) + 0.3858

R2 = 0.99190

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.1 1 10 100

Concentration (ug/mL)

Aborbance (490 nm)absorbance

Log. (absorbance)

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X-ray photoelectron spectroscopy (aka ESCA)

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Paper # 1

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RGD peptide immobilization

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Ellipsometry

Polarization: Equipment Schematic:

Measure:

ρ =

Rp

Rs

=Rp

Rs

⋅exp i δ p −δs( )⎡⎣

⎤⎦

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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 and . 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:

ρ =RP

Rs

= tanψ eiΔ , where

tanψ is amplitude change upon reflection;Δ is phase shift

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Ellipsometry through Substrate Layers

(Advanced review)

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Back to RGD peptide example

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Determining Characteristics of Adsorbed Biomolecular Structure

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Single molecule protein detection approaches

Piehler, COSB, 15:4-14, 2005

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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.

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Circular Dichroism Principle

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.

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.

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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

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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 (190-250 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.

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CD of proteins and nucleic acids

Nucleic acids: Proteins:

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CD of adsorbed T4 lysozyme

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Characterizing Interactions of Biomolecules on Surfaces

Estimation of ligand affinity to substrates

Ligand-Receptor BindingLigand-Cell Binding

Ligand-Substrate Binding (Paper)

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Binding of labeled ligands to receptors

R + LÉ

kr

kf

C dC(t)

dt=kf R(t).L(t).−krC(t)

RT =R(t) +C(t)

Lo =L(t) + (n

NAv

).C(t) dC(t)

dt=kf RT −C(t)[ ] Lo −

nNAV

⎛

⎝⎜⎞

⎠⎟.C(t)

⎡

⎣⎢

⎤

⎦⎥−krC(t)

If ligand depletion is ignored,dC(t)

dt=kf RT −C(t)[ ]Lo −krC(t) C(t) =Co exp −kf Lo + kr{ } +

kf LoRT

kf Lo + kr

⎛

⎝⎜

⎞

⎠⎟ 1−exp − kf Lo + kr( )t{ }⎡⎣

⎤⎦

At equilibrium, k f RT −Ceq⎡⎣ ⎤⎦gLo =krCeq

Ceq =RT Lo

KD + Lo

; KD =kr

kf

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)

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Binding of labeled ligands to receptors, continued

• Let u be the scaled/dimensionless number of ligand-receptor complexes, =C/RT

ueq

u

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Scatchard Plot to estimate KD

Ceq

L=−

1KD

Ceq +RT

KD

⎛

⎝⎜⎞

⎠⎟

Bound ligand

Free ligand= −1 / KD( ).(Bound ligand) + (RT / KD )

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Paper # 2:Affinity of biomolecular binding to

substrates

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Experimental System

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Adsorption Model

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Kinetic Model for Binding

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Fitting the best model to experimental data

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