Crooked Cell

8/2/2019 Crooked Cell

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Mystery of the Crooked Cell:Investigate Sickle Cell Anemia

Using Gel Electrophoresis

Module developed at BostonUniversity School of Medicine

Presented by Dr. Dan Murray


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Outline

Sickle Cell Anemia

Central Dogma of Biology

Genetic Code

Hemoglobin

Electrophoresis


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Sickle Cell Anemia


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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


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Central Dogmaof Biology


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Central Dogma of Biology

Transcription:Conversion ofinformation fromDNA to mRNA

Translation:Conversion ofinformation fromRNA to protein


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The Genetic Code


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The Genetic Code

start


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Met

5 3mRNA sequence

N-terminus C-terminu

Protein chains always beginwith Met

Protein sequence

The Genetic Code

AUG AAC AAU GCG CCG GAG GAA GCG GAG

53 orientationcorresponds toN-term C-term orientation

Met---AsnMet---Asn---AsnMet---Asn---Asn---AlaMet---Asn---Asn---Ala---Pro---Glu---Glu---Ala---Glu


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Hemoglobin


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Hemoglobin

Multi-subunit protein (tetramer)

2 and 2 subunits

Heme

One per subunit

Has an iron atom

Carries O2

In red blood cells


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Sickle Cell Hemoglobin

GUG CAC CUG ACU CCU GAG GAG AAGval his leu thr pro glu glu lys1 2 3 4 5 6 7 8

GUG CAC CUG ACU CCU GUG GAG AAGval his leu thr pro val glu lys1 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.


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Sickle Cell Hemoglobin

Significant change

in structure caused

by the single mutation


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A Possible Cure for

Sickle Cell Anemia?During fetal development, a differentgene (gamma) produces hemoglobin

Expression of gamma gene stopsnaturally during development

Research efforts focused on stopping

silencing of gamma geneWould provide sickle cell patients withgood hemoglobin


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Electrophoresis


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Gel Electrophoresis

Method for separating molecules (DNA,

proteins, etc.) on the basis of physical orchemical properties such as:

(1) size

(2) shape

(3) electrical charge


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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


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Electrophoresis of Proteins

More complex than DNA electrophoresis

Different proteins have different charges

Proteins vary widely in shape

Polyacrylamide is usually the gel

medium


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Protein ElectrophoresisNon-Denaturing conditions

Non-denaturing (native): no pretreatment ofproteins 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


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Non-Denaturing Electrophoresis of

Normal and Mutant Hemoglobin

Charge, Size, Shape

Q. Which of the above properties will be different fornormal Hemoglobin (HgA) and mutantHemoglobin (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.


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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 andGlu about same size

HgA more compact

than HgS


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Protein Structure


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Protein Structure

1 = Primary Structure

2 = Secondary Structure3 = Tertiary Structure

4 = Quaternary Structure


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Primary Structure

Definition - Sequence of amino acids in aprotein

Example Primary structure of the enzymelysozyme:

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.


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Secondary Structure

Definition Regular patterns of relativelysmall segments of a protein held togethermainly by H-bonds

-helix -structure

http://www.ultranet.com/~jkimball/BiologyPages/S/SecondaryStructure.html

Examples:


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Tertiary Structure

Definition Overall 3-D shape of a protein.Two basic types are globularand fibrous.

Globular (Pepsin) Fibrous(Collagen)

http://www.ultranet.com/~jkimball/BiologyPhttp://dwb.unl.edu/Teacher/NSF/C10/C10Links/

main.chem.ohiou.edu/~wathen/chem302/ rotein.htmla es/S/Secondar Structure.html

Examples:


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Quaternary Structure

Definition

Overall 3-D shape of a multi-subunitprotein

Rabbit muscle

glycogenphosphorylase

Example:


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All Levels of Structure

htt ://sosnick.uchica o.edu/ rec uastru.html


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Protein Electrophoresis

Denaturing conditions

Proteins treated with SDS (anionic detergent) beforeelectrophoresis (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

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