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

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

Central Dogma of Biology

Transcription:

Conversion of information from DNA to mRNA

Translation:

Conversion of information from RNA to protein

The Genetic Code

The Genetic Code

start

The Genetic Code

Protein chains always begin with Met 53 orientation corresponds to N-term C-term orientation

mRNA sequence

AUG AAC AAU GCG CCG GAG GAA GCG GAG

N-terminus

Met---Asn---Asn---Ala---Pro---Glu---Glu---Ala---Glu Met---Asn---Asn---Ala Met---Asn Met---Asn---Asn Met

Protein sequence

C-terminus

Hemoglobin

Hemoglobin
Multi-subunit protein (tetramer)

2 and 2 subunits

Heme
One per subunit Has an iron atom Carries O2

In red blood cells

Sickle Cell Hemoglobin

Normal mRNA

Normal protein

GUG CAC CUG ACU CCU GAG GAG AAG val his leu thr pro glu glu lys 1 2 3 4 5 6 7 8
Mutation (in DNA)

Mutant mRNA

Mutant protein

GUG CAC CUG ACU CCU GUG GAG AAG val his leu thr pro val glu lys 1 2 3 4 5 6 7 8

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

Name Protein Q Protein R

Charge
+2 4

Mass
30kD 42kD

Shape

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?
Normal Mutant (HgA) (HgS) Charge Size Shape Reason
HgA has one more than HgS NA

NA

Amino acids Val and Glu about same size

HgA more compact than HgS

Protein Structure

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 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

Tertiary Structure
Definition Overall 3-D shape of a protein. Two basic types are globular and fibrous. Examples: 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

Quaternary Structure
Definition Overall 3-D shape of a multi-subunit protein

Example:

Rabbit muscle glycogen phosphorylase

All Levels of Structure

http://sosnick.uchicago.edu/precpquastru.html

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 Charge Mass Charge Mass SDS treatment 300 30kD 420 42kD

+3
4

30kD

42kD