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 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

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

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

Hemoglobin

Hemoglobin

Multi-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 change

in 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

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 polyacrylamideDNA samples loaded, voltage appliedNegatively charged DNA migrates toward “+” electrodeSmaller DNA fragments migrate faster

Electrophoresis of Proteins

More 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 HgS

Amino acids Val and Glu about same sizeHgA 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

-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:

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

SDS treatment

Charge Mass

+3 30kD

4 42kD

Charge Mass

300 30kD

420 42kD