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
Sickle Cell AnemiaGenetic 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
Met
5’ 3’mRNA sequence
N-terminus C-terminus
• Protein chains always begin with Met
Protein sequence
The Genetic Code
AUG AAC AAU GCG CCG GAG GAA GCG GAG
• 53 orientation corresponds to N-term C-term orientation
Met---AsnMet---Asn---AsnMet---Asn---Asn---AlaMet---Asn---Asn---Ala---Pro---Glu---Glu---Ala---Glu
HemoglobinMulti-subunit protein (tetramer) 2 and 2 subunits
HemeOne per subunitHas an iron atomCarries O2
In red blood cells
Sickle Cell Hemoglobin
GUG CAC CUG ACU CCU GAG GAG AAGval his leu thr pro glu glu lys 1 2 3 4 5 6 7 8
GUG CAC CUG ACU CCU GUG GAG AAGval his leu thr pro val glu lys 1 2 3 4 5 6 7 8
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 changein structure caused by the single mutation
http://webpub.alleg.edu/dept/bio/bio221/Discussion/hbBoth.gif
A Possible Cure for Sickle Cell Anemia?
During fetal development, a different gene (gamma) produces hemoglobinExpression of gamma gene stops naturally during developmentResearch efforts focused on stopping silencing of gamma geneWould provide sickle cell patients with good hemoglobin
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 polyacrylamideDNA samples loaded, voltage appliedNegatively charged DNA migrates toward “+” electrodeSmaller DNA fragments migrate faster
Electrophoresis of ProteinsMore complex than DNA electrophoresisDifferent proteins have different chargesProteins vary widely in shape
Polyacrylamide is usually the gel medium
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
Name Charge Mass Shape
Protein Q
Protein R
+2 30kD
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 NA
Normal (HgA)
Mutant (HgS) Reason
Charge
Size
Shape
HgA has one more “” than HgSAmino acids Val and Glu about same sizeHgA 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:
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.
Secondary StructureDefinition – Regular patterns of relatively small segments of
a protein held together mainly by H-bonds
-helix -structure
http://www.ultranet.com/~jkimball/BiologyPages/S/SecondaryStructure.html
Examples:
Tertiary Structure Definition – Overall 3-D shape of a protein. Two basic types are globular
and fibrous.
Globular (Pepsin) Fibrous (Collagen)
http://www.ultranet.com/~jkimball/BiologyPhttp://dwb.unl.edu/Teacher/NSF/C10/C10Links/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
http://bmbiris.bmb.uga.edu/wampler/tutorial/prot4.html
Example:
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
300 30kD
420 42kD