Transcript Proteins

Proteins
Protein Function
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Catalysis
Structure
Movement
Defense
Regulation
Transport
Antibodies
Monomers—
Amino Acids
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R-groups
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Hydrophilic
Hydrophobic
Uncharged
Charged
Large
Small
Confer unique
chemical properties
on each aa
PROTEIN LEVELS OF STRUCTURE
PRIMARY STRUCTURE
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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
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Sequences and composition reflect the
function of the protein:
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Membrane proteins have more hydrophobic
residues.
Homologous proteins from different
organisms have similar sequences.
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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
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3-D structure.
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Form follows function!!
Native vs denatured
Determinants of tertiary structure
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Amino acid sequence
Environment in which the protein resides
Stabilizing Interactions
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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
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Heat
Salts
pH
Organic solvents
that can disrupt
Quaternary Structure
ANTIBODIES
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Extremely specific
Definitions:
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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
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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
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Given:
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Normal cells—Mortal
Transformed cells—Immortal
Two Pathways of DNA Synthesis
Major
 Salvage—Requires HGPRT
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8-azaguanine—HGPRT poison.
Aminopterin---Interferes w/ major pathway
PEG---promotes cell fusion
HAT Selection
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2)
3)
Select HGPRT- mutant
myeloma by treatment with 8azaguanine
Fuse HGPRT- mutant myeloma
with normal cells using PEG
Select with aminopterin
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2)
3)
4)
Normal?
Myeloma?
Hybridoma?
Screen for desired
monoclonal.
MAbs in the Lab
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Macs extremely useful in molecular biology
and medicine
Applications
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Affinity columns
Western blots
ELISA (Enzyme Linked ImmunoSorbent
Assay)
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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.
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