Transcript Proteins
Proteins
Protein Function
Catalysis
Structure
Movement
Defense
Regulation
Transport
Antibodies
Monomers—
Amino Acids
R-groups
Hydrophilic
Hydrophobic
Uncharged
Charged
Large
Small
Confer unique
chemical properties
on each aa
PROTEIN LEVELS OF STRUCTURE
PRIMARY STRUCTURE
Is a unique characteristic of every protein
Is encoded by the nucleotide sequence of
DNA
Is thus a form of genetic information
Is read from the amino terminus to the
carboxyl terminus
Nature of Protein Sequences
Sequences and composition reflect the
function of the protein:
Membrane proteins have more hydrophobic
residues.
Homologous proteins from different
organisms have similar sequences.
e.g., cytochrome c is highly conserved
Cytochrome c
SECONDARY STRUCTURE I: THE -HELIX
Helix
If N-terminus is at bottom,
then all peptide N-H bonds
point “down” and all
peptide C=O bonds point
“up”.
N-H of residue n is Hbonded to C=O of residue
n+4.
Secondary Structure II: The -Strand
approx.
3.4 A
Several -strands assemble into a
-sheet (a tertiary structural element)
TERTIARY STRUCTURE
3-D structure.
Form follows function!!
Native vs denatured
Determinants of tertiary structure
Amino acid sequence
Environment in which the protein resides
Stabilizing Interactions
Hydrogen Bonds
Electrostatic interactions (“salt-bridges” or
ion pairs)
van der Waals interactions (dipole-dipole
and dispersion)
Hydrophobic interactions
Disulfide bridges
Protein Denaturation
•Denaturants--Anything
stabilizing interactions
•
•
•
•
Heat
Salts
pH
Organic solvents
that can disrupt
Quaternary Structure
ANTIBODIES
Extremely specific
Definitions:
Antigen
Epitope (antigenic determinant)
Hapten
FLUORESCEIN – a hapten
Antibody Structure
Antigen
binding
site
V
V
V
Light
Chain
Antigen
binding
site
V
SS
SS
Heavy Chains
Light
Chain
Antibody Structure
Antibody Structure
Recognition and Binding
The N-terminal
region of antibody
light chains and
heavy chains form
the antigen binding
site
The variability in
amino acid sequence
provides the
structural basis for
the diversity of
antigen-binding sites
Antigen Binding
Antigen 1
Antigen 3
Polyclonal vs Monoclonal Abs
107-109 genetically distinct lymphocytes,
each producing a single type of Ab.
Polyclonal—normal immune response.
Several Abs, recognition of various
epitopes with varying affinities.
Monoclonal
Monoclonal Ab Production
Given:
Normal cells—Mortal
Transformed cells—Immortal
Two Pathways of DNA Synthesis
Major
Salvage—Requires HGPRT
8-azaguanine—HGPRT poison.
Aminopterin---Interferes w/ major pathway
PEG---promotes cell fusion
HAT Selection
1)
2)
3)
Select HGPRT- mutant
myeloma by treatment with 8azaguanine
Fuse HGPRT- mutant myeloma
with normal cells using PEG
Select with aminopterin
1)
2)
3)
4)
Normal?
Myeloma?
Hybridoma?
Screen for desired
monoclonal.
MAbs in the Lab
Macs extremely useful in molecular biology
and medicine
Applications
Affinity columns
Western blots
ELISA (Enzyme Linked ImmunoSorbent
Assay)
Back
The Future?
Single Chain Antibodies
Catalytic antibodies
Bifunctional antibodies
Etc.
Features of a single-chain antibody (sFv).
N
VL
VH
Linker
C
The linker typically consists
of a flexible/soluble peptide
(for example, [GGGGS]6)
CL
CH1 CH2
CH3
CH1 CH2
CH3
CL
Consists of the variable light
(VL) chain of an antibody
joined via a linker to the
variable heavy (VH) domain.
The sFv maintains the
antigen binding specificity
(but not always the affinity)
of the parent antibody.
Back