Mystery of the Crooked Cell:

1. Mystery of the Crooked Cell: Investigate Sickle Cell Anemia Using Gel Electrophoresis Module developed at Boston University School of Medicine Presented by Dr. Dan Murray

2. Outline • Sickle Cell Anemia • Central Dogma of Biology • Genetic Code • Hemoglobin • Electrophoresis

3. Sickle Cell Anemia

4. Sickle Cell Anemia • Genetic Disease • Heterozygous individuals – carriers • Homozygous individuals – diseased • Hemoglobin • Found in red blood cells • Carries oxygen to tissues • SCA Results from Defective Hemoglobin • Hemoglobins stick together • Red blood cells damaged • Complications from low oxygen supply to tissues • Pain, organ damage, strokes, increased infections, etc. • Incidence highest among Africans and Indians • Heterozygotes protected from Malaria

5. Central Dogma of Biology

6. Central Dogma of Biology Transcription: Conversion of information from DNA to mRNA Translation: Conversion of information from RNA to protein

7. The Genetic Code

8. The Genetic Code start

9. The Genetic Code • Protein chains always begin with Met • 53 orientation corresponds to • N-term  C-term orientation mRNA sequence 5’ 3’ AUG AAC AAU GCG CCG GAG GAA GCG GAG Met---Asn---Asn---Ala Met---Asn---Asn---Ala---Pro---Glu---Glu---Ala---Glu Met---Asn Met---Asn---Asn Met N-terminus C-terminus Protein sequence

10. Hemoglobin

11. Hemoglobin • Multi-subunit protein (tetramer) • 2  and 2  subunits • Heme • One per subunit • Has an iron atom • Carries O2 • In red blood cells

12. Sickle Cell Hemoglobin Normal mRNA GUG CAC CUG ACU CCU GAG GAG AAG val his leu thr pro glu glu lys 1 2 3 4 5 6 7 8 Normal protein Mutation (in DNA) Mutant mRNA GUG CAC CUG ACU CCU GUG GAG AAG val his leu thr pro val glu lys 1 2 3 4 5 6 7 8 Mutant protein Glutamate (glu), a negatively charged amino acid, is replaced by valine (val), which has no charge.

13. Sickle Cell Hemoglobin Significant change in structure caused by the single mutation http://webpub.alleg.edu/dept/bio/bio221/Discussion/hbBoth.gif

14. A Possible Cure for Sickle Cell Anemia? • During fetal development, a different gene (gamma) produces hemoglobin • Expression of gamma gene stops naturally during development • Research efforts focused on stopping silencing of gamma gene • Would provide sickle cell patients with good hemoglobin

15. Electrophoresis

16. Gel Electrophoresis Method for separating molecules (DNA, proteins, etc.) on the basis of physical or chemical properties such as: (1) size (2) shape (3) electrical charge

17. Electrophoresis of DNA • Gels are made of agarose or polyacrylamide • DNA samples loaded, voltage applied • Negatively charged DNA migrates toward “+” electrode • Smaller DNA fragments migrate faster

18. Electrophoresis of Proteins • More complex than DNA electrophoresis • Different proteins have different charges • Proteins vary widely in shape • Polyacrylamide is usually the gel medium

19. Protein ElectrophoresisNon-Denaturing conditions • Non-denaturing (native): no pretreatment of proteins before electrophoresis • Proteins retain normal shape • Proteins retain normal charge • Proteins separated on basis of charge, size, and shape +2 30kD 4 42kD

20. Non-Denaturing Electrophoresis of Normal and Mutant Hemoglobin Charge, Size, Shape Q. Which of the above properties will be different for normal Hemoglobin (HgA) and mutant Hemoglobin (HgS)? A. Charge: Yes, HgA has one “–” more than HgS. Size: No, HgA and HgS are the same size. Shape: Yes, the shapes are different.

21. Migration Rates of Normal and Mutant Hemoglobin Which Hg migrates faster during electrophoresis? HgA has one more “” than HgS  Amino acids Val and Glu about same size NA NA HgA more compact than HgS 

22. Protein Structure

23. Protein Structure • 1 = Primary Structure • 2 = Secondary Structure • 3 = Tertiary Structure • 4 = Quaternary Structure

24. Primary Structure Definition - Sequence of amino acids in a protein Example – Primary structure of the enzyme lysozyme: 1 2 3 4 5 126 127 128 129 Lys-Val-Phe-Gly-Arg...Gly-Cys-Arg-Leu Note: By convention, amino acid sequences are written starting with the amino terminus.

25. Secondary Structure Definition – Regular patterns of relatively small segments of a protein held together mainly by H-bonds Examples: -helix -structure http://www.ultranet.com/~jkimball/BiologyPages/S/SecondaryStructure.html

26. Tertiary Structure Definition – Overall 3-D shape of a protein. Two basic types are globular and fibrous. Examples: Fibrous (Collagen) Globular (Pepsin) http://www.ultranet.com/~jkimball/BiologyPhttp://dwb.unl.edu/Teacher/NSF/C10/C10Links/main.chem.ohiou.edu/~wathen/chem302/protein.htmlages/S/SecondaryStructure.html

27. Quaternary Structure Definition – Overall 3-D shape of a multi-subunit protein Example: Rabbit muscle glycogen phosphorylase http://bmbiris.bmb.uga.edu/wampler/tutorial/prot4.html

28. All Levels of Structure http://sosnick.uchicago.edu/precpquastru.html

29. Protein Electrophoresis Denaturing conditions • Proteins treated with SDS (anionic detergent) before electrophoresis (SDS-PAGE) • SDS molecules bind to the Protein • Proteins lose normal shape • Proteins all have same charge/mass ratio • Proteins are separated on basis of size only ChargeMass +3 30kD 4 42kD ChargeMass 300 30kD 420 42kD SDS treatment