Download Protein Structure and Analysis

Protein Structure and Analysis
Types of proteins
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Enzymes
 Eg. Phosphoglucomutase, telemerase, DNA polymerase
Functional proteins
 eg. Collagen (bones and tendons), keratin (hair).
Receptors/messenger proteins
 eg. cAMP, insulin, p30 etc.
Overview of transcription and translation
DNA 
RNA  protein.
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DNA  RNA  protein.
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Choice of 4 nucleotides on RNA  20 different amino
acids possible in any protein.
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Amino acid abbreviations (One letter code system is now
preferred).
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Triplet of nucleotides (on mRNA) is called a codon.
Sometimes more than one combination (or triplet) can
specify a particular amino acid.
Genetic code is “degenerate”
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Each amino acid contains an amino group (-NH2), a carboxyl group (COOH), a hydrogen atom, and a functional group designated R.
R
|
H2 N – C – COOH
|
H
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Proteins are synthesized from the N terminus (free –NH2) to the C
terminus I.e free amino acids are always added to the C terminus end
of the growing protein chain via peptide bonds.
Orders of Protein Structure
Sequence of amino acids ie. Primary Structure
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Spontaneously fold into sheets or wrap into coils ie.
Secondary structure.
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Tertiary structure depends on the secondary structure eg.
Form fibers or globular proteins.
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Quaternary structure is the name given to the whole protein
if it is composed of several subunits.
Secondary, tertiary and quaternary structure
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A “ribbon” diagram showing
two molecules of transthyretin
docked together. The diagram
illustrates both a-helix and bsheet levels of protein structure.
Correlation of structure, function and
disease.
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A single amino acid can have a profound or subtle effect
on protein shape and hence function eg. proline create
“hair-pin bends”, leucine zipper motif
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“Mistakes” in coding or incorporation of amino acids are
the basis of many diseases
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sickle cell anemia
abnormal growth and division eg. BRAC1, BRAC2, p53
Leucine Zipper motif
Antibodies 101!
Antibodies are yet another type of protein!
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Specific antibody will combine with its specific antigen to
give an antibody-antigen complex.
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Antigen : “………any foreign substance that elicits an
immune response
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The site on small site on the antigen to which the antibody
binds is called the epitope.
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Antibodies : “………an immunoglobulin capable of
specific combination with the antigen that caused its
production in a susceptible animal……….”
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“Y” shaped molecules that consist of four polypeptide
chains ie. Two identical copies of “heavy” chain and two
copies of the “light” chain.
Structure of an antibody
Peptide Antigens
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Recombinant DNA technology can use nucleotide
sequence information for an antigen (if known) to derive a
peptide antigen.
Peptide antigens can be used to immunize rabbits, pigs,
goats etc to generate antibodies for industrial, medical,
research uses.
e.g. commercially available urine test kits, viral plasma screen
kits, protein quantification etc.
Protein Methods
Studying proteins involves methods for detecting,
identifying and quantifying proteins.
1.
2.
3.
4.
5.
Gel electrophoresis; 1and 2 dimensional forms
Western and Southern blotting
ELISAs; immunofluoresence, immunolocalization etc.
Protein sequencing
Protein engineering and production
Protein Gel Electrophoresis
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1. One-dimensional polyacrylamide gel electrophoresis
(PAGE).
- proteins visualized as bands by either dye (coomassie
blue) OR radiolabeling and autoradiography.
2. Two-dimensional electrophoresis
 - first dimension separation by charge called isoelectric
focusing.
 - Second dimension uses SDS-PAGE thus separation by
size.
Two-dimensional electrophoresis for
immuno-detection of actin.
Protein (Western) blotting
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- detect the presence and quantity of antigen
- the molecular weight of the antigen
- the efficiency of antigen extraction (from a purification)
- especially useful for insoluble antigens or antigens that
are easily degraded
The blotting procedure involves transferring the separated
proteins to nitrocellulose paper (blotting) for detection
(probing) with specific antibodies.
Western blotting
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Figure shows purified antibody
to detect Stat 1 (Lane 1) and
unpurified antibody (Lane 2)
used to detect the same protein
using Western blotting
technique.
Immunohistochemistry in situ

Immunohistochemistry of
breast tumor tissue showing
expression of BRAC1 antigen
using purified mAb expressed
to purified protein.
Immunocytochemistry
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Expression of polycyclic
aromatic hydrocarbon adducts
in HeLa cells cultured in vitro
Enzyme-linked Immunoabsorbant
Assays (ELISAs)
- can provide useful measurement of antigen, antibody or protein
concentration
- fast and accurate and can determine the absolute amounts of protein in
unknown samples
- ELISA procedures utilize substrates that produce soluble products
alkaline phosphatase

p-nitrophenylphosphate (pNPP)
(colorless)
SUBSTRATE
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p- nitrophenol
(yellow)
COLOR
ELISA procedure
In situ modification of ELISA procedure
Protein Engineering
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Improve protein’s characteristics to enhance function;
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1. Increased stability : resistance to degradation, pH
change, temperature, oxidation and contamination.
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2. Enhanced enzyme activity : change in substrate
specificity etc.
Technology
1. Oligonucleotide-directed mutagenesis uses a short single stranded
15-20 base long oligonucleotide that is complementary to gene to be
mutated.
BUT,
must know the sequence you want to change AND
the mutagenesis procedure is very inefficient. Thus must enrich for the
mutant DNA.
2. Substitute a cassette : DNA region containing the nucleotides to be
changed is removed with a restriction enzyme and replaced with the
cassette then transferred to E. coli for replication.
3. Other methods : PCR-amplified oligonucleotide-directed
mutagenesis.
Protein Sequencing
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- linear order of amino acids in a protein can be elucidated by a
process called Edman Degradation.
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Amino acids that make up the amino terminal end of the polypeptide
strand are determined one at a time by chemical degradation.
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Method has been automated and is conducted by an amino acid
sequenator.
What do you do with the data?
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NCBI database
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Basic Local Alignment Search Tool (BLAST) finds regions of local
similarity between sequences.
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The program compares nucleotide or protein sequences to sequence
databases and calculates the statistical significance of matches.
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BLAST can be used to infer functional and evolutionary relationships
between sequences as well as help identify members of gene families.
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More later!!
What else do you do with the data?
Proteins and rDNA technology
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Advances in agricultural, medical and environmental biotechnology
rely on recombinant DNA technologies.
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- manufacture of products from cloned genes (eg. therapeutic human
proteins)
- analysis and repair of genetic disorders
- production of transgenic plants, animals and bacteria
- bioremediation
- agriculture (increase yields and insect resistance).
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