CORN LAW amino acid with L configuration

Chapter 5
Proteins function as:
1. Enzymes:biological catalysts
2. Regulators of catalysis-hormones
3. Transport and store i.e. O
2
, metal ions sugars, lipids, etc.
4. Contractile assemblies
Muscle fibers
Separation of chromosomes etc.
5. Sensory
Rhodopsin nerve proteins

6. Cellular defense immuoglobulins
Antibodies
Killer T cell
Receptors
7. Structural
Collagen
Silk, etc.
Function is dictated by protein structure!!

1. Primary structure
1  = Amino acid sequence, the linear order of
AA’s.
Remember from the N-terminus to the C-terminus
Above all else this dictates the structure and function of the protein.

2. Secondary structure
2  = Local spatial alignment of amino acids without regard to side chains.
Usually repeated structures
Examples: a helix, b sheets, random coil, or b turns

3. Tertiary Structure
3  = the 3 dimensional structure of an entire peptide.
Great in detail but vague to generalize. Can reveal the detailed chemical mechanisms of an enzyme.

4. Quaternary Structure
4  two or more peptide chains associated with a protein.
Spatial arrangements of subunits.
Chapter 5.3 is how to determine a protein’s primary structure.
“Protein Chemistry ”

F. Sanger won the Nobel prize for protein sequencing.
It took 10 years, many people, and it took 100 g of protein!
Today it takes one person several days to sequence the same insulin.
1021 AA bglactosidase 1978

Above all else, purify it first!! Chapter 5.3 then 5.1 and 5.2
1. Prepare protein for sequencing a. Determine number of chemically different polypeptides.
b. Cleave the protein’s disulfide bonds.
c. Separate and purify each subunit.
d. Determine amino acid composition for each peptide.

2. Sequencing the peptide chains: a. Fragment subunits into smaller peptides

50
AA’s in length.
b. Separate and purify the fragments c. Determine the sequence of each fragment.
d. Repeat step 2 with different fragmentation system.

3. Organize the completed structure.
a. Span cleavage points between sets of peptides determined by each peptide sequence.
b. Elucidate disulfide bonds and modified amino acids.
At best, the automated instruments can sequence about 50 amino acids in one run!
Proteins must be cleaved into smaller pieces to obtain a complete sequence.

End Group Analysis
How many peptides in protein?
Bovine insulin should give 2 N-terminii and 2 Cterminii
N-terminus
1-Dimethylamino - naphthalene-5-sulfonyl chloride
Dansyl chloride
Reacts with amines: N-terminus + Lys (K) side chains

Disadvantage with the Dansyl-chloride method is that you must use 6M HCl to cleave off the derivatized amino acid, this also cleaves all other amide bonds (residues) as well.
Edman degradation with Phenyl isothiocyanate, PITC

Edman degradation has been automated as a method to sequence proteins. The PTH-amino acid is soluble in solvents that the protein is not. This fact is used to separate the tagged amino acid from the remaining protein, allowing the cycle of labeling, degradation, and separation to continue.
Even with the best chemistry, the reaction is about
98% efficient. After sufficient cycles more than one amino acid is identified, making the sequence determination error-prone at longer reads.

.

Carboxypeptidase cleavage at the C-terminus
NH
R n-2
CH
O
R n-1
C NH CH
O
C
R n
NH CH
O
C O
H
2
O Carboxypeptidase
NH
R n-2
CH
O
R n-1
C NH CH
O
C O
R n
H
3
N CH
O
C O
Carboxypeptidase A Rn

R, K, P
Rn-1

P
If the Tyr-Ser bond is more resistant to cleavage than the Leu-Tyr, the Ser and the Tyr will appear simultaneously and the C-terminus would still be in doubt.

Permits separation of polypeptide chains
Prevents refolding back to native structure
Performic acid oxidation
Changes cystine or cysteine to Cystic acid
Methionine to Methionine sulfone
2-Mercaptoethanol, dithiothreitol, or dithioerythritol
Keeps the equilibrium towards the reduced form
-S-S2SH

The amino acid composition of a peptide chain is determined by its complete hydrolysis followed by the quantitative analysis of the liberated amino acids.
Acid hydrolysis (6 N HCl) at 120 o C for 10 to 100 h destroys Trp and partially destroys Ser, Thr, and Tyr.
Also
Gln and Asn yield Glu and
Asp
Base hydrolysis 2 to 4 N
NaOH at 100 o C for 4 - 8 h.
Is problematic, destroys Cys
Ser, Thr, Arg but does not harm Trp.

In order to quantitate the amino acid residues after hydrolysis, each must be derivatized at about 100% efficiency to a compound that is colored. Pre or post column derivatization can be done.
o -Phthalaldehyde (OPA)
O
CH
+
CH
O
2-mercaptoethanol
HS CH
2
CH
2
OH +
S CH
2
N
CH
2
OH
O
R
CH C O
Amino acid
R
H
3
N CH
O
C O
These can be separated using
HPLC in an automated setup

Leu, Ala,Gly, Ser, Val, Glu, and Ile are the most common amino acids
His, Met, Cys, and Trp are the least common.
Ratios of polar to non-polar amino acids are indicative of globular or membrane proteins.
Certain structural proteins are made of repeating peptide structures i.e. collagen.

Long peptides have to be broken to shorter ones to be sequenced
Endopeptidases cleave proteins at specific sites within the chain.
R n-1
O
NH CH C
R n
O
NH CH C
Scissile Bond
Trypsin R n -1
= positively charged residues R, K; R n

P
Chymotrypsin R n -1
= bulky hydrophobic residues F, W, T; R n

P
Thermolysin R n
= I, M, F, W, T, V; R n -1

P
Endopeptidase V8 R n -1
= E

Cyanogen Bromide R n -1
= M
Cleave the large protein using i.e trypsin, separate fragments and sequence all of them. (We do not know the order of the fragments!!)
Cleave with a different reagent i.e. Cyanogen Bromide, separate the fragments and sequence all of them. Align the fragments with overlapping sequence to get the overall sequence.

1. Write a blank line for each amino acid in the sequence starting with the N-terminus.
2. Follow logically each clue and fill in the blanks.
3. Identify overlapping fragments and place in sequence blanks accordingly.
4. Make sure logically all your amino acids fit into the logical design of the experiment.
5. Double check your work.

H
3
N-
1 2 3 4 5 6 7 8 9 10 11 12 13 14
_
-
_
-
_
-
_
-
_
-
_
-
_
-
_
-
_
-
_
-
_
-
_
-
_
-
_
-COO
K - F - A - M
Q - M - K
D - I - K - Q - M
K
F - A - M - K
G - M - D - I - K
Y - R - G - M
Y - R
Cyanogen Bromide (CN
Trypsin cleaves after K or R
(positively charged amino
Br) Cleaves after Met i.e M - X
D - I - K - Q - M
K
K - F - A - M acids)
Q - M - K
G - M - D - I - K
F - A - M - K
Y - R
Y - R - G - M

There are a variety of ways to purify peptides
All are based on the physical or chemical properties of the protein.
Size
Charge
Solubility
Chemical specificity
Hydrophobicity/ Hydrophylicity
Reverse Phase High Pressure Liquid Chromatography is used to separate peptide fragments.

Peptide mapping: digest protein with an appropriate agent, then separate using two dimensional paper chromatography
Digested Peptide from normal (HbA) and
Sickle cell anemia (Hbs) hemoglobins
HbA V - H - L - T - P E - E - K
HbS V - H - L - T - P V - E - K b 1 2 3 4 5 6 7 8
Beta chain position 6 contains altered amino acid

Red blood cells :
(a) normal
(b) sickle cell
Electrophoretic separation of hemoglobins

Deoxyhemoglobin aggregates and deforms cell. Primary structure changes dictate quaternary structure.
Why did the problem not die out?
Homozygotic Heterzyatic Homozygotic normal sickle cell trait sickle cell gets malaria resistant gets sickle cell to malaria dies dies

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
Rh. 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 0 Average differences
12 11 0
11 10 1 0 10.0
10 9 3 2 0
11 10 6 5 3 0
10 9 5 4 2 3 0 5.1
9 8 6 5 4 5 2 0
10 11 7 8 6 7 6 6 0
13 12 11 10 9 10 9 8 12 0
13 12 12 11 10 10 9 8 10 2 0 9.9
11 10 10 9 8 8 7 6 10 3 3 0 14.3
14 15 22 21 20 21 19 18 21 19 20 17 0 12.6
15 14 11 10 9 9 8 9 11 8 8 7 22 0
18 17 14 13 11 12 11 11 13 11 12 11 24 10 0
21 21 19 18 17 18 17 17 18 17 18 17 26 18 15 0 18.5
27 26 22 22 22 21 22 21 24 23 24 22 29 24 22 24 0
31 30 29 28 27 25 27 26 28 28 27 27 31 28 29 32 14 0 25.9
43 43 46 45 45 44 44 44 47 46 46 46 46 46 48 49 45 45 0
48 47 46 46 46 46 46 46 49 47 48 46 47 49 49 48 41 47 54 0 47.0
45 45 46 45 45 45 45 45 46 46 45 46 47 49 47 47 45 47 47 41 0
51 51 51 50 50 49 50 50 51 51 50 51 51 53 51 48 47 47 50 42 27 0

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 an 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 than mammals and many more rounds of replication, the 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

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.

• is an oxygen transport protein
•it must bind and release oxygen as the cells require oxygen
• is an oxygen storage protein
•it binds oxygen tightly and releases it when oxygen concentrations are very low

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 a and two b chains increased oxygen transport capabilities.
4. Mammals have fetal hemoglobin with a variant b chain i.e. g (a
2 g
2
).
5. Human embryos contain another hemoglobin

2 e
2.
6. Primates also have a d chain with no known unique function.

Protein Evolution is not organismal evolution
Chimpanzee  human are about 99% the same amino acid sequences in proteins!
However:
•Rapid divergence with few mutational changes suggest altered control of gene expression.
•Controlling the amount, where, and when a protein is made.