chapter 5: introduction to studying protein
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CHAPTER 5: INTRODUCTION TO STUDYING PROTEINIntroduction to Biotechnology, BIOL1414Austin Community College, Biotechnology Dept
LEARNING OUTCOMES Describe the structure of proteins, including the significance of amino
acid R-groups and their impact on the three-dimensional structure of proteins.
Explain the steps of transcription and translation in protein synthesis. Discuss the role of naturally occurring proteins and recombinant
proteins in biotechnology. Differentiate proteins that function as part of structure, as antibodies,
and as enzymes. Describe the structure of antibodies and explain the relationship
between antibodies and antigens. Discriminate among the classes of enzymes and discuss the effect of
reaction conditions on enzyme activity. Summarize polyacrylamide gel electrophoresis and identify its
usefulness for studying proteins.
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CENTRAL DOGMA DNA codes for RNA which codes for proteins that
confer traits.
Click to see VideoAnimation
THE IMPORTANCE OF PROTEINS IN
BIOTECHNOLOGYThe ability to synthesize and modify peptides
or proteins is crucial to the production of virtually every biotechnology product Food processing – the creamy in ice creamTextile and leather goods – bio-bleachesDetergents – enzymes to dissolve stainsPaper manufacturing and recycling – reduce
negative environmental impactsAdhesives – barnacles and musselsBioremediation – proteins used to clean up
harmful waste
PROTEINS AS BIOTECH PRODUCTS Therapeutic Protein
• used to treat a disease that is caused by a gene that fails to produce a necessary protein or that produces a dysfunctional protein
Protein Synthesis in a Eukaryotic Cell. In a eukaryotic cell, DNA is located within chromosomes in the nucleus. The mRNA transcripts carry the DNA code out to the ribosomes, which translate the code into a strand of amino acids.
PROTEIN SYNTHESIS:
TRANSCRIPTION Protein synthesis is a
two-step process:
First Step - Transcription Genetic code must be
rewritten onto a messenger molecule
RNA STRUCTURE
Uracil instead of thymine
Ribose sugar instead of deoxyribose sugar
Single strandedVarious shapesCan leave the
nucleus
RNA STRUCTURETRANSCRIPTION & TRANSLATION
REQUIRES 3 TYPES OF RNA mRNA – RNA copy of DNA that carries genetic
information from the nucleus to the ribosomes rRNA – makes up the ribosomes tRNA – carries amino acids to ribosomes for
protein synthesis
TRANSCRIPTION RNA polymerase binds to a promoter region on
double stranded DNA and unzips the double helix.
TRANSCRIPTION Free RNA nucleotides pair with the
complementary DNA of the template strand
TRANSCRIPTION
Introns are spliced out
7 methyl guanosine cap
Poly-A tail
Travels to the ribosomes in the cytoplasm
mRNA is Processed mRNA leaves nucleus
ribosome
nucleus
LET’S PUT IT ALL TOGETHER Transcription Animation
YOUR TURN!
PROTEIN SYNTHESIS:
TRANSLATION
Protein synthesis is a two-step process:
Second Step - Translation
mRNA nucleotide code is rendered into a sequence of amino acids
TRANSLATIONA protein is a string of amino
acids held together by peptide bonds and do most of the work in a cell
TRANSLATION
TRANSLATION
BACTERIAL PROTEIN SYNTHESIS
Animation on protein synthesis
TRANSLATION
Important Definitions A codon is
composed of 3 RNA nucleotides
Each codon codes for one amino acid
Protein does the work in a cell
TRANSLATION
TRANSLATION
TRANSLATION Use the Genetic Code to translate the
following mRNA strand: CGAGAAGUC
TRANSLATION Always begins at a start codon and ends at
a stop codon. The region between the start and stop
codons is called the open reading frame (ORF)
PRACTICE
Click on the animation to transcribe and translate a gene.
This is great practice! HINT! This will be on your exam!
Click to see animation
TRANSLATION INITIATION mRNA attaches to
the small subunit of a ribosome
tRNA anticodon pairs with mRNA start codon
Large ribosomal subunit binds and translation is initiated
tRNA anticodon
amino acid
TRANSLATION ELONGATION Anticodon of tRNA carrying next amino acid binds
to codon on mRNA A peptide bond joins the amino acids and the first
tRNA is released.
TRANSLATION TERMINATION Amino acid chain continues until a stop
codon is read. The amino acid chain is released and all of the translation machinery is recycled to translate another protein.
LET’S PUT IT ALL TOGETHER
Click on the animation below
Translation Video
Translation Animation
PUTTING IT ALL TOGETHER….
5’-GATCTGAATCGCTATGGC-3’ 3’-CTAGACTTAGCGATACCG-5’mRNA 5’-GAUCUGAAUCGCUAUGGC-
3’ CUAGACUUAGCGAUACCG Asp, Leu, Asn, Arg, Tyr, Gly
Coding:Template:mRNA:tRNA:amino acid:
PROTEIN STRUCTURE Once the amino acid
chain is released from the ribosome, a number of modifications are made in order for the protein to perform it’s intended function.
The protein must fold into it’s appropriate 3-dimensional shape.
Proper folding of the protein is essential for it’s activity because it must bind it’s substrate to perform it’s job.
PROTEIN STRUCTURE
Primary – Peptide bonds in a chain of amino acids
Secondary – Hydrogen bonding between amino acids forms alpha-helices and beta-sheets
Tertiary – three dimensional folding of protein due to disulfide linkages and hydrophobic interactions between alpha-helices and beta-sheets
Quaternary – aggregation of multiple polypeptide chains
PROTEIN STRUCTURE
PROTEIN STRUCTURE Primary – Peptide bonds in a chain of amino
acids
Secondary – Hydrogen bonding between amino acids forms alpha-helices and beta-sheets
PROTEIN STRUCTURE
Tertiary – three dimensional folding of protein due to disulfide linkages and hydrophobic interactions between alpha-helices and beta-sheets
PROTEIN STRUCTURE
Quaternary – aggregation of multiple polypeptide chains
PROTEIN STRUCTURE
PROTEIN STRUCTURE Glycosylation
• A glycoprotein is a protein on which sugar groups have been added
• Increases solubility, orients protein in membrane, extends life of protein
• Occurs in the golgi
PROTEIN STRUCTURE Another group of
proteins, the antibodies is structurally interesting and functionally very importantThe function of an
antibody is to recognize and bind foreign proteins or other molecules called antigens, for removal
Each type of antibody has the same basic shape
ANTIBODIES - APPLICATION Most antibodies are very specific and
bind to distinct regions of molecules called epitopes
In the lab, antibodies can be used to bind certain molecules under study, or for diagnosisA common test used to determine the
presence of a protein in solution is the ELISA – Enzyme Linked Immunosorbent Assay
ENZYMES: PROTEIN CATALYSTS Enzymes are proteins that act as
catalysts Enzymes are involved in virtually every
reaction in a cell Many companies have focused on producing
enzymes for sale The molecules upon which enzymes act are
called substrates
FACTORS THAT AFFECT ENZYME ACTIVITY Amount of substrate in a solution Temperature of a reaction Acidity or alkalinity
WORKING WITH ENZYMES Most proteins are kept at cold temperatures
to prevent degradation They are stored in fridges, cold rooms or
freezers
A technician loads protein samples on a vertical gel. Vertical gel boxes operate in a fashion similar to horizontal gel boxes.
STUDYING PROTEINSSDS-PAGE Animation
Watch this video!How to run SDS-PAGE
Vertical Gel Electrophoresis. Although vertical gel boxes vary from one manufacturer to another, all are basically of the same design. The gel cassettes are snapped or screwed in place (right). Running buffer is added behind the gel, covering the wells. Buffer is poured in the front of the gel cassette to cover the front opening. When the top is placed on the box (left) and the power is turned on, electricity flows from the top (negative charge) to bottom (positive charge). Negatively charged samples move down the gel toward the positive electrode.
STUDYING PROTEINS
STUDYING PROTEINS
SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) Separates proteins in an electrical field based on
molecular size
STUDYING PROTEINS Sodium Dodecyl Sulfate (SDS)
A detergent that denatures the secondary and tertiary structure of the protein
Coats the protein with negative charges
Add SDS
STUDYING PROTEINS Polyacrylamide Gel Electrophoresis (PAGE)
Much tighter gel matrix than agarose, which makes polyacrylamide ideal for separating proteins
STUDYING PROTEINS SDS-PAGE – Proteins separate by size
If run molecular weight marker at the same time, can determine molecular weight of protein
The smaller the peptide chain the faster it moves through the gel
STUDYING PROTEINS SDS-PAGE: Gels of different concentration
STAINING SDS-PAGE The most popular stain for visualizing
protein is Coomassie® Blue Silver stain, however, is much more
sensitiveWhen samples have low concentrations of
protein or DNA, silver-staining is the method of choice.Coomassie: Silver Stain:
QUESTIONS AND COMMENTS?
REVIEW QUESTIONSYour Turn!
Put your name at the top of a sheet of paper, answer these questions and hand in:
1. How many different kinds of amino acids are found in proteins? What distinguishes one amino acid from another?
2. How many polypeptide chains are found in an antibody, and how are they held together in the protein?
3. Distinguish between transcription and translation.4. If a structural gene’s code is “TAC GGC ATG CCC TTA CGC
ATC,” what will the mRNA transcript be? If the mRNA transcript is translated into a peptide, what would the amino-acid sequence of the peptide be?
5. What happens if an enzyme is at a temperature significantly above its optimum temperature? What happens if an enzyme is at a pH significantly above or below its optimum level?
6. What does “PAGE” stand for, and what samples are studied using PAGE? What separates molecules on a PAGE gel?
REFERENCES1. Biotechnology: Science for the New
Millennium. 2012. Ellyn Daugherty.
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