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

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

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

Central Dogma of Biology

Transcription: Conversion of information from DNA to mRNA Translation: Conversion of information from RNA to protein

The Genetic Code

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

Hemoglobin

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

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

Sickle Cell Hemoglobin Significant change in structure caused by the single mutation

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

Electrophoresis

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

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

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

Protein Electrophoresis Non-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 NameChargeMassShape Protein Q Protein R +230kD 44 42kD

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.

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

Protein Structure

1  = Primary Structure 2  = Secondary Structure 3  = Tertiary Structure 4  = Quaternary Structure

Primary Structure Definition - Sequence of amino acids in a protein Example – Primary structure of the enzyme lysozyme: Lys-Val-Phe-Gly-Arg...Gly-Cys-Arg-Leu Note: By convention, amino acid sequences are written starting with the amino terminus.

Secondary Structure Definition – Regular patterns of relatively small segments of a protein held together mainly by H-bonds  -helix  -structure Examples:

Tertiary Structure Definition – Overall 3-D shape of a protein. Two basic types are globular and fibrous. Globular (Pepsin) Fibrous (Collagen) main.chem.ohiou.edu/~wathen/chem302/protein.htmlages/S/SecondaryStructure.html Examples:

Quaternary Structure Definition – Overall 3-D shape of a multi-subunit protein Rabbit muscle glycogen phosphorylase Example:

All Levels of Structure

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 SDS treatment Charge Mass +3 30kD  4 42kD Charge Mass  kD  kD