Protein Structure and Analysis

1. Protein Structure and Analysis

2. Types of proteins • Enzymes • Eg. Phosphoglucomutase, telemerase, DNA polymerase • Functional proteins • eg. Collagen (bones and tendons), keratin (hair). • Receptors/messenger proteins • eg. cAMP, insulin, p30 etc.

3. Overview of transcription and translation • DNA  RNA  protein.

4. DNA  RNA  protein. • Choice of 4 nucleotides on RNA  20 different amino acids possible in any protein. • Amino acid abbreviations (One letter code system is now preferred). • Triplet of nucleotides (on mRNA) is called a codon. Sometimes more than one combination (or triplet) can specify a particular amino acid.

5. Genetic code is “degenerate”

6. 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 • 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.

7. Orders of Protein Structure Sequence of amino acids ie. Primary Structure • Spontaneously fold into sheets or wrap into coils ie. Secondary structure. • Tertiary structure depends on the secondary structure eg. Form fibers or globular proteins. • Quaternary structure is the name given to the whole protein if it is composed of several subunits.

8. Secondary, tertiary and quaternary structure • A “ribbon” diagram showing two molecules of transthyretin docked together. The diagram illustrates both a-helix and b-sheet levels of protein structure.

9. Correlation of structure, function and disease. • 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 • “Mistakes” in coding or incorporation of amino acids are the basis of many diseases • sickle cell anemia • abnormal growth and division eg. BRAC1, BRAC2, p53

10. Leucine Zipper motif

11. Antibodies 101! Antibodies are yet another type of protein! • Specific antibody will combine with its specific antigen to give an antibody-antigen complex. • Antigen : “………any foreign substance that elicits an immune response • The site on small site on the antigen to which the antibody binds is called the epitope.

12. Antibodies : “………an immunoglobulin capable of specific combination with the antigen that caused its production in a susceptible animal……….” • “Y” shaped molecules that consist of four polypeptide chains ie. Two identical copies of “heavy” chain and two copies of the “light” chain.

13. Structure of an antibody

14. Peptide Antigens • 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.

15. Protein Methods Studying proteins involves methods for detecting, identifying and quantifying proteins. • Gel electrophoresis; 1and 2 dimensional forms • Western and Southern blotting • ELISAs; immunofluoresence, immunolocalization etc. • Protein sequencing • Protein engineering and production

16. Protein Gel Electrophoresis • 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.

17. Two-dimensional electrophoresis for immuno-detection of actin.

18. Protein (Western) blotting • -   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.

19. Western blotting • 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.

20. Immunohistochemistry in situ • Immunohistochemistry of breast tumor tissue showing expression of BRAC1 antigen using purified mAb expressed to purified protein.

21. Immunocytochemistry • Expression of polycyclic aromatic hydrocarbon adducts in HeLa cells cultured in vitro

22. 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)  p- nitrophenol (colorless) (yellow) SUBSTRATE COLOR

23. ELISA procedure

24. In situ modification of ELISA procedure

25.   Protein Engineering • Improve protein’s characteristics to enhance function; • 1. Increased stability : resistance to degradation, pH change, temperature, oxidation and contamination. • 2. Enhanced enzyme activity : change in substrate specificity etc.

26. 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.

27. Protein Sequencing • - linear order of amino acids in a protein can be elucidated by a process called Edman Degradation. • Amino acids that make up the amino terminal end of the polypeptide strand are determined one at a time by chemical degradation. • Method has been automated and is conducted by an amino acid sequenator.

28. What do you do with the data? • NCBI database • Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. • The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. • BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families. • More later!!

29. What else do you do with the data? Proteins and rDNA technology • Advances in agricultural, medical and environmental biotechnology rely on recombinant DNA technologies. • -   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).