Lesson 7: Ligand Binding and Antibodies

enzymes bind

substrates

receptors bind

signaling molecules

lectins bind

carbohydrates

transporters bind

small molecules --> deliver them elsewhere

binding site

part of protein that binds ligands

ligand

molecule that binds to a protein specifically and reversibly

ligand binding is (covalent or non-convalent)

noncovalent

fractional saturation (definition and symbol)

θ; fraction of total protein bound to ligand

fractional saturation (equation)

θ = [PL]/([PL] + [P])

saturation curve

plots θ as a function of ligand concentration

saturation curve shape (and why)

hyperbolic because saturation is reached at a certain ligand concentration --> all binding sites are filled

Kd

equilibrium constant for ligand binding; concentration of ligand where θ = 0.5

relationship between Kd and affinity

inversely related; low Kd --> high affinity

prosthetic group

non-protein organic group permanently associated with protein & essential to activity

Kd in proteins that bind gas

Kd = p50

fractional saturation curve of proteins that bind gas

uses pressure instead of concentration

antibodies

immunoglobulins that bind viruses, bacteria, and foreign molecules to target them for destruction

IgG

immunoglobulin G; major antibody in blood

IgG structure

4 polypeptide chains linked by disulfide bridges; 2 heavy chains and 2 light chains in a Y shape

base of IgG Y-structure (chain type + terminal)

C-terminals of heavy chains

arms of IgG Y-structure (chain type + terminal)

N-terminals of all 4 chains

FAB region (definition and location)

antigen binding fragments; ends of arms where IgG binds to antigen

FC region (location)

base of IgG

IgG’s protease cleavage sites

where the base of IgG bends into 2 arms

constant region (definition)

amino acid sequence conserved across IgG

variable region (definition)

variable amino acid sequence conferring specificity to IgG

immunoglobulin fold

structural motif of immunoglobulins, found multiple times in light and heavy chains; β-sheet regions joined by disulfide bonds and small helices

antigen binding sites made of…

large, flexible loops of the light and heavy variable regions

antigen binding sites (variability: where are they the same and different?)

vary from IgG to IgG, but the identical on 2 arms of the same IgG

epitope

aka antigenic determinant; binding site on antigen for IgG

hypervariable loops

amino acid variation in short segments of variable region; 10^8 possibilities

what causes hypervariable loops

random genetic changes during β-lymphocyte development

Western Blot

uses antibodies to identify presence of proteins in an electrophoretically separated sample

stages of Western Blot

SDS-PAGE -> electroblotting -> immunoblotting -> detection

electroblotting

aka Western Transfer; transfer the protein from PAGE to a thin membrane, sandwich in casette, then an electric field moves proteins through the gel and traps them on the membrane

immunoblotting

apply antibody to probe the membrane for specific proteins

detection

uses densitometry to quantify protein density

blocking

applying blocking buffer to the electroblotted membrane to prevent antibodies from binding to the membrane itself

monoclonal antibody

recognizes one specific epitope; can recognize a linear epitope on a denatured protein

polyclonal antibody

targets multiple epitopes on the same antigen; often only recognize conformational epitopes

linear epitope

epitope consists of primary linear sequence; independent of conformation

conformational epitope

3D epitope found only on the native conformation

Fc region of Western antibody shows…

what animal the antibody originated from

Fc region is used for… (2)

1) epitope for secondary antibody 2) often linked to a reporter enzyme that produces color/light changes

densitometry

measures the density of a protein band using color change and analysis software

ELISA

enzyme-linked immunoabsorbent assay; detects presence/absence of a protein via washing and binding

ELISA steps

substrate added --> primary antibody added --> secondary antibody added --> stop solution --> quantification

primary antibody

antibody with high affinity for target protein epitope binds to target protein

secondary antibody

antibody conjugated to an enzyme binds to primary antibody

stop solution

blocks enzymatic reaction and causes color change

how is protein concentration quantified in ELISA

microplate reader quantifies [protein] using absorbance