1. Proteins
Proteins are large biological molecules, or macromolecules, consisting of one or more chains of amino acid residues. Proteins perform a vast array of functions within living organisms, including catalyzing metabolic reactions, replicating DNA, responding to stimuli, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in folding of the protein into a specific three-dimensional structure that determines its act

2. Proteins
A polypeptide is a single linear polymer chain derived from the condensation of amino acids. The individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the genetic code specifies 20 standard amino acids; however, in certain organisms the genetic code can include selenocysteine and—in certain archaea—pyrrolysine. Shortly after or even during synthesis, the residues in a protein are often chemically modified by posttranslational modification, which alters the physical and chemical properties, folding, stability, activity, and ultimately, the function of the proteins. Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors. Proteins can also work together to achieve a particular function, and they often associate to form stable protein complexes.

3. Structure of proteins

4. Color conventions

5. CORN LAW amino acid with L configuration
Protein Geometry
CORN LAW amino acid with L configuration

6. Greek alphabet

7. The Polypeptide Chain

8. Covalent structures of proteins
Chapter 5
Covalent structures of proteins
Proteins function as:
1. Enzymes:biological catalysts
2. Regulators of catalysis-hormones
3. Transport and store i.e. O2, metal ions
sugars, lipids, etc.
4. Contractile assemblies
Muscle fibers
Separation of chromosomes
etc.
5. Sensory
Rhodopsin nerve proteins

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

10. There are four levels of 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.

11. There are four levels of protein structure
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

12. 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.

13. Chapter 5.3 is how to determine a protein’s primary structure.
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”

14. Example of each level of protein structure

15. Amino Acids

16. The amino-acid side chains have different tendencies to participate in interactions
Hydrophobic residues: van der Waals interactions – tendency to avoid contact with water and pack against each other  hydrophobic effect
- Ala & Leu are strong helix-favoring residues, Proline are not because its backbone nitrogen isn’t available for H-bond
- Aromatic side chain of Phe participates in weakly polar interactions
Hydrophilic residues : Hydrogen bonds to one another, to peptide backbone, to polar organic molecules, and to water.
- pKa shift: Asp & Glu (57 in hydrophobic interior or nearby (-) charge), Lys (106 in ?)
- His: most versatile, most often found in enzyme active sites, pKa is 6, neutral, proton donator and acceptor

17. Arg: completely protonated at neural, compared to Lys?
- Cys: common in enzyme active site, most powerful nucleophile. Compared to Ser?
Amphipatic residues : both polar and nonpolar character
- Lys: hydrophic charged region, long hydrophobic region (methylene) involved in van der Waals interactions with hydrophobic side chains
- Tyr: pKa is 9, in some enzyme active site, hydroxyl group can be donor and acceptor of H-bond. Aromatic ring can form weakly polar interactions
- Trp: similar to Tyr, indole amide hydrogen don’t ionize.
- Met: least polar among amphipatic residues, thioether sulfur is excellent ligand for metal ions.

18. Genes and Proteins
The genetic code is degenerate
The genetic code

19. Insulin was the first protein to be sequenced
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 b- glactosidase 1978

20. Steps towards protein sequencing
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.

21. Bovine insulin: note the intra- and inter- chain disulfide linkages

22. 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.

23. 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.

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

26. Edman degradation with Phenyl isothiocyanate, PITC
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

27. Edman degradation has been automated as a method to sequence proteins
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.

28. Demonstration of Edman degradation
Use your CD disk- install it and run chapter 5 Edman degradation.

29. Carboxypeptidase cleavage at the C-terminus
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.

30. Cleavage of disulfide bonds
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-S SH

31. Amino acid composition
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 oC 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 oC for h. Is problematic, destroys Cys Ser, Thr, Arg but does not harm Trp.

32. Amino acid analyzer
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.
These can be separated using HPLC in an automated setup

33. Amino acid compositions are indicative of protein structures
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.

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

35. Specific chemical cleavage reagents
Cyanogen Bromide Rn-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.

36. How to assemble a protein 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.

37. H3N-_-_-_-_-_-_-_-_-_-_-_-_-_-_-COO
H3N-_-_-_-_-_-_-_-_-_-_-_-_-_-_-COO K
F - A - M - K
K - F - A - M
Q - M - K
D - I - K - Q - M
G - M - D - I - K
Y - R - G - M
Y - R
Trypsin cleaves after K or R (positively charged amino acids)
Q - M - K
G - M - D - I - K
F - A - M - K
Y - R
Cyanogen Bromide (CN Br) Cleaves after Met i.e M - X
D - I - K - Q - M K
K - F - A - M
Y - R - G - M

38. 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.

39. 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 
Beta chain position 6 contains altered amino acid

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

42. Deoxyhemoglobin aggregates and deforms cell
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

43. 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

44. (evolutionarily related proteins)
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.

45. Amino acid difference matrix for 26 species of cytochrome c
Man,chimp 0
Rh. monkey Average differences
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
Rattlesnake
Horse
gray whale
Bread mold
Man,chimp
cow,sheep
Chicken,
Tuna fish
silkworm
kangaroo
Bullfrog
worm fly
penguin
Candida
monkey
Donkey
rabbit
turtle
Yeast
Wheat
Duck
dog

46. 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

48. 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.

49. 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

50. 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.

51. Hemoglobin: Myoglobin: is an oxygen transport protein
it must bind and release oxygen as the cells require oxygen
Myoglobin:
is an oxygen storage protein
it binds oxygen tightly and releases it when oxygen concentrations are very low

52. 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 a and two b chains increased oxygen transport capabilities.
4. Mammals have fetal hemoglobin with a variant b chain i.e. g (a2g2).
5. Human embryos contain another hemoglobin 2e2.
6. Primates also have a d chain with no known unique function.

54. 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.