Chapter 5: Introduction to Studying Protein

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.