Protein Fingerprinting and Evolution

Ms. Haut

Analyze protein profiles from a variety of fish

Use acrylamide electrophoresis to separate proteins by size

Compare biochemical and phylogenetic relationships

Protein Fingerprinting

Making Proteins

DNA TAC GGATCG AGATGA

mRNA AUGCCUAGCUCUACU

tRNA UACGGAUCGAGAUGA

Amino Acid Tyr Gly Ser Arg STOP

Levels of Protein Organization

4o3o

2o1o

Protein Size Comparison Break protein complexes into individual

proteins

Denature proteins using detergent and heat

Separate proteins based on size

Protein Size

Size measured in kilodaltons (kDa)

Dalton = mass of hydrogen molecule

= 1.66 x 10-24 gram

Average amino acid = 110 daltons

Muscle Contains Proteins of Many Sizes

Protein kDa Function

Titin 3000 Center myosin in sarcomere

Dystrophin 400 Anchoring to plasma membrane

Filamin 270 Cross-link filaments

Myosin heavy chain

210 Slide filaments

Spectrin 265 Attach filaments to plasma membrane

Nebulin 107 Regulate actin assembly

-actinin 100 Bundle filaments

Gelosin 90 Fragment filaments

Fimbrin 68 Bundle filaments

Actin 42 Form filaments

Tropomysin 35 Strengthen filaments

Myosin light chain

27 Slide filaments

Troponin (T.I.C) 30, 19, 17 Mediate contraction

Thymosin 5 Sequester actin monomers

Actin and Myosin Actin

5% of total protein 20% of vertebrate muscle

mass 375 amino acids = 42 kDa Forms filaments

Myosin Tetramer two heavy subunits

(220 kDa) two light subunits (20 kDa) Breaks down ATP for muscle

contraction

Campbell, 2007.

Protein Fingerprinting First carried out in 1956 to show that

sickle-cell anemia was caused by a change in a single amino acid of the hemoglobin protein

The method we will use SDS-PAGE was developed in 1970 by U.K. Laemmli

Protein Electrophoresis

Proteins are charged molecules whose sequences are determined by DNA

Polyacrylamide gels are used to separate small molecules such as proteins Agarose gels separate large molecules

The percentage of acrylamide gel to be used depends on the size range of the proteins of interest. (the higher the % = the denser the matrix and better the separation of small molecules)

http://departments.oxy.edu/biology/Franck/Bio222/Lectures/Feb1lecture.htm

Protein Electrophoresis

Proteins are separated by size—smaller proteins travel farther in the matrix

Useful Information Gathered from Protein Sequence Relationship to other proteins (protein families)

Example: Viral protein that produces cancer is nearly identical to normal cellular growth factor.

Evolution of organisms (phylogenetic trees).

Information for creating antibodies: specific regions can be identified.

Information for making DNA probes.

Traditional Systematics and Taxonomy Classification

Kingdom Phylum Class Order Family Genus Species

Traditional classification based upon traits: Morphological Behavioral

Can biomolecular evidence be used to determine evolutionary relationships?

Biochemical Similarities Traits are the result

of: Structure Function

Proteins determine structure and function

DNA codes for proteins that confer traits

Biochemical Differences

Changes in DNA leads to proteins with: Different functions Novel traits Positive, negative or no

effects

Genetic diversity provides pool for natural selection = evolution

Evolution and Classification of Fishes Evolutionary

trees Show

evolutionary lineages of different species over time

http://bio.winona.edu/berg/ILLUST/evoltree.gif

Protein gel electrophoresis

Protein gel from protein fingerprinting experiment.  Each blue band represents a distinct protein.  The pattern of bands gives information about the composition of a sample

http://www.bridgewater.edu/~sbaron/Bio%20325%20Pics.htm

Species Sequence

Beef, Horse, Pig .......val.gln.lys.cys.ala.gln.cys.his.thr.val.glu.lys....

Salmon .......val.gln.lys.cys.ala.gln.cys.his.thr.val.glu.....

Chicken .......val.gln.lys.cys.ser.gln.cys.his.thr.val.glu.....

Silkworm .......val.gln.arg.cys.ala.gln.cys.his.thr.val.glu.....

Yeast phe.lys.thr.arg.cys.glu.leu.cys.his.thr.val.glu.....

Rhodospirillum rubrum

lys.cys.leu.ala.cys.his.thr.phe.asp.glu.gly.ala.asp.lys....

Common Sequence --------------.....cys.X.Y.cys.his.thr.......

Variations in amino acid sequence in one part of the cytochrome c molecule

Variations in amino acid sequence in one part of the cytochrome c molecule

http://www.rtis.com/nat/user/elsberry/evobio/evc/argresp/sequence.html

Fish Classification Chondrichthyes (cartilaginous fishes)

Cartilaginous skeleton, thick skin w/o scales, no swim bladders or lungs

Sharks, skates, rays

Osteichthyes (bony fishes) Bony skeleton, true scales, paired fins w/ moveable

rays

Agnatha (jawless fishes) Eel-like, jawless fishes with parasitic and scavenging

lifestyles No scales or paired fins

Phylogenetic Tree

http://www.fao.org/documents/show_cdr.asp?url_file=/DOCREP/V7180E/v7180e04.htm

Phylogenetic tree for fishes

Which fishes should you select?

Choose fish specimens that will provide striking and distinct results

Select some closely related fishes (salmon/trout) and some more distantly related fishes (get exotic—shark, tuna)

Other aquatic organisms—mollusks—scallops, octopus; arthropods—clams, oysters, crab, shrimp

Protein Fingerprinting Procedures

Day 2

Day 3Day 1

What’s in the Sample Buffer?• Tris buffer to provide

appropriate pH

• SDS (Sodium Dodecyl Sulfate) detergent to dissolve proteins and give them a negative charge

• Glycerol to make samples sink into wells

• Bromophenol Blue dye to visualize samples

http://departments.oxy.edu/biology/Franck/Bio222/Lectures/Feb1lecture.htm

Why Heat the Samples?

• Heating the samples denatures protein complexes, allowing the separation of individual proteins by size

s-s SDS, heat

Proteins with SDS

+

–

How Does an SDS-PAGE Gel Work?

Negatively charged proteins move to positive electrode

Smaller proteins move faster

Proteins separate by size

+

s-s SDS, heat

Proteins with SDS

–

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) SDS Detergent (Sodium

Dodecyl Sulfate) Solubilizes and denatures

proteins Adds negative charge to

proteins

Heat denatures proteins

O S

O

O

O

-

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH3

SDS

Why Use Acrylamide Gels to Separate Proteins? Acrylamide gel give a tight matrix Ideal for protein separation Smaller pore size than agarose Proteins much smaller than DNA

Average amino acid = 110 DaAverage nucleotide pair = 649 Da1 kilobase of DNA = 650 kDa1 kilobase of DNA encodes 333 amino acids = 36 kDa

Gel Analysis

Lane1. Kaleidoscope Markers2. Shark3. Salmon4. Trout5. Catfish6. Sturgeon7. Actin and Myosin

Standard

Molecular Weight Estimation

0

50

100

150

200

250

0 20 40 60

Distance (mm from well)

Siz

e in

kD

a

kDa mm203 8.5135 12.086 18.5

41 28.0

33 34.0

19 41.5

8 44.5

Molecular Weight Analysis

Ready Gel Assembly

Step 1Step 2

Step 3 Step 4

Step 1