Protein Sequencing Protein Evolution: Exam Review

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Lecture 7

Protein Sequencing Protein Evolution Exam Review

Overview of Protein Sequencing


(1) Purify Protein (2) Determine number of PP End group analysis (Dansyl chloride rxn)

(3) Fragment PP into smaller peptides Enzymes (Trypsin, Chymotrypsin, etc.) Chemical (CNBr)

(4) Determine the sequence Edman Degradation with PITC (5) Assemble a sequence

(6) Elucidate S-S bonds Amino acid composition

Q12. You must cleave the following peptide into smaller fragments. Which of the proteases listed in the table would be likely to yield the most fragments? The fewest? NMTQGRCKPVNTFVHEPLVDVQNVCFKE

Q15. Separate cleavage reactions of a polypeptide by CNBr and chymotrypsin yield fragments with the following amino acid sequences. What is the the sequence of the intact polypeptide? CNBr treatment Chymotrypsin 1. Arg-Ala-Tyr-Gly-Asn 1. Met-Arg-Ala-Tyr 2. Leu-Phe-Met 2. Asp-Met-Leu-Phe 3. Asp-Met 3. Gly-Asn

Q17. Treatment of a polypeptide with 2-mercaptoethanol yields two PP: 1. Ala-Val-Cys-Arg-Thr-Gly-Cys-Lys-Asn-Phe-Leu 2. Tyr-Lys-Cys-Phe-Arg-His-Thr-Lys-Cys-Ser Treatment of the intact PP with trypsin yields fragments with the following aa compositions: 3. (Ala, Arg, Cys2, Ser, Val) 4. (Arg, Cys2, Gly, Lys, Thr, Phe) 5. (Asn, Leu, Phe) 6. (His, Lys, Thr) 7. (Lys, Tyr) Indicate the positions of disulfide bonds in the intact PP.

Sequencing by Mass Spectrometry


Electrospray Ionization Mass Spectrometry

ESI-MS spectrum of horse heart apomyoglobin


Q. Two successive peaks in the mass spectrum have measure m/z ratios of 1414.0 and 1542.3. What is the original apomyoglobin molecule? p1= (M+z)/z p2= (M+z-1)/z-1 M= 16,975D (16,951 D in table 5-1)

Tandem Mass Spectrometry in amino acid sequencing

http://us.expasy.org/
Tools and software packages Proteomics Similarity searches (BLAST)

Also try:

http://www.ncbi.nlm.nih.gov/BLAST/

Protein/ protein-protein BLAST (blastp)

Protein Evolution Species variation in homologous proteins


The primary structures of a given protein from related species closely resemble one another. If one assumes, according to evolutionary theory, that related species have evolved from a common ancestor, it follows that each of their proteins must have likewise evolved from the corresponding ancestor.

A protein that is well adapted to its function, that is, one that is not subject to significant physiological improvement, nevertheless continues to evolve.

Neutral drift: changes not effecting function

Homologous proteins (evolutionarily related proteins) Compare protein sequences: Conserved residues, i.e invariant residues reflect chemical necessities. Conserved substitutions, substitutions with similar chemical properties (Asp for Glu), (Lys for Arg), (Ile for Val) Variable regions, no requirement for chemical reactions etc.

Amino acid difference matrix for 26 species of cytochrome c


Man,chimp Rhesus monkey Horse Donkey cow,sheep dog gray whale rabbit kangaroo Chicken penguin Duck Rattlesnake turtle Bullfrog Tuna fish worm fly silk moth Wheat Bread mold Yeast Candida k. 0 1 12 11 10 11 10 9 10 13 13 11 14 15 18 21 27 31 43 48 45 51 0 11 10 9 10 9 8 11 12 12 10 15 14 17 21 26 30 43 47 45 51 Average differences 0 1 3 6 5 6 7 11 12 10 22 11 14 19 22 29 46 46 46 51 0 2 5 4 5 8 10 11 9 21 10 13 18 22 28 45 46 45 50 10.0 0 3 2 4 6 9 10 8 20 9 11 17 22 27 45 46 45 50 0 3 5 7 10 10 8 21 9 12 18 21 25 44 46 45 49

0 2 6 9 9 7 19 8 11 17 22 27 44 46 45 50

5.1 0 6 8 8 6 18 9 11 17 21 26 44 46 45 50 0 12 10 10 21 11 13 18 24 28 47 49 46 51

0 2 3 19 8 11 17 23 28 46 47 46 51

0 3 20 8 12 18 24 27 46 48 45 50

0 17 7 11 17 22 27 46 46 46 51

9.9 14.3 0 12.6 22 0 24 10 0 26 18 15 0 29 24 22 24 31 28 29 32 46 46 48 49 47 49 49 48 47 49 47 47 51 53 51 48


Rattlesnake

18.5 0 14 45 41 45 47 0 25.9 45 0 47 54 0 47.0 47 47 41 0 47 50 42 27 0


Bread mold

gray whale

Man,chimp

cow,sheep

Tuna fish

worm fly

kangaroo

Chicken,

Bullfrog

silkworm

penguin

monkey

Donkey

rabbit

turtle

Horse

dog

Duck

Wheat

Candida

Yeast

Phylogenetic tree
Indicates the ancestral relationships among the organisms that produced the protein. Each branch point indicates a common ancestor. Relative evolutionary distances between neighboring branch points are expressed as the number of amino acid differences per 100 residues of the protein. PAM units or Percentage of Accepted Mutations

PAM values differ for different proteins.

Although DNA mutates at a assumed constant rate. Some proteins cannot accept mutations because the mutations kill the function of the protein and thus are not viable.

Mutation rates appear constant in time

Although insects have shorter generation times that mammals and many more numbers of replication, number of mutations appear to be independent of the number of generations but dependent upon time

Cytochrome c amino acid differences between mammals, insects and plants note the similar distances

Evolution through gene duplication


Many proteins within an organism have sequence similarities with other proteins. These are called gene or protein families. The relatedness among members of a family can vary greatly. These families arise by gene duplication. Once duplicated, individual genes can mutate into separate genes. Duplicated genes may vary in their chemical properties due to mutations. These duplicate genes evolve with different properties. Example the globin family.

Genealogy of the globin family


Hemoglobin: is an oxygen transport protein it must bind and release oxygen as the cells require oxygen

Myoglobin:

22 22 22 22

is an oxygen storage protein it binds oxygen tightly and releases it when oxygen concentrations are very low

The globin family history


1. Primordial globin gene acted as an Oxygen-storage protein. 2. Duplication occurred 1.1 billion years ago. lower oxygen-binding affinity, monomeric protein. 3. Developed a tetrameric structure two and two chains increased oxygen transport capabilities. (22). 4. Mammals have fetal hemoglobin with a variant chain i.e. (22). 5. Human embryos contain another hemoglobin (22). 6. Primates also have a chain with no known unique function. (22).

Modular Construction of some proteins


1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 11 12

Modules (sequence motifs): ~ 40 -100 residues

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