1. Proteins are made of chains of amino acids joined by peptide bonds. Chains with fewer than 50 units are called peptides, while larger chains have structural or catalytic functions. 2. There are 20 common amino acids that differ in their variable side chains. Amino acids can be neutral, acidic, basic, or polar depending on their side chain properties. 3. The primary structure of a protein is its amino acid sequence. Secondary structures include alpha helices and beta sheets. Tertiary structure describes the overall 3D shape, while quaternary involves interactions of multiple protein molecules.

1. 1
Biomolecules:
Amino Acids, Peptides, and Proteins

2. 2
Proteins – Amides from Amino Acids
• Amino acids contain a basic amino group and an
acidic carboxyl group
• Joined as amides between the NH2 of one amino
acid and the CO2H the next
• Chains with fewer than 50 units are called peptides
• Proteins: large chains that have structural or catalytic
functions in biology

3. 3
Structures of Amino Acids
• In neutral solution, the COOH is ionized and the
NH2 is protonated
• The resulting structures have “+” and “-” charges
(a dipolar ion, or zwitterion)
• They are like ionic salts in solution

4. 4
The Common Amino Acids
• 20 amino acids form amides in proteins
• All are α-amino acids - the amino and carboxyl
are connected to the same C
• They differ by the other substituent attached to
the α carbon, called the side chain, with H as the
fourth substituent except for proline
• Proline, is a five-membered secondary amine,
with N and the α C part of a five-membered ring

5. 5
The Structure of Amino Acids
H2N
C
CO2H
R
H
R – can be a series of functional groups

6. 6
Abbreviations and Codes
Alanine A, Ala
Arginine R, Arg
Asparagine N, Asn
Aspartic acid D, Asp
Cysteine C, Cys
Glutamine Q, Gln
Glutamic Acid E, Glu
Glycine G, Gly
Histidine H, His
Isoleucine I, Ile
Leucine L, Leu
Lysine K, Lys
Methionine M, Met
Phenylalanine F, Phe
Proline P, Pro
Serine S, Ser
Threonine T, Thr
Tryptophan W, Trp
Tyrosine Y, Tyr
Valine V, Val

7. 7
Learning the Names and Codes
• The names are not systematic so you learn them by
using them (They become your friends)
• One letter codes – learn them too
– If only one amino acid begins with that letter, use it (Cys,
His, Ile, Met, Ser, Val)
– If more than one begins with that letter, the more common
one uses the letter (Ala, Gly, Leu, Pro, Thr)
– For the others, some are phonetic: Fenylalanine, aRginine,
tYrosine
– Tryp has a double ring, hence W
– Amides have letters from the middle of the alphabet (Q –
Think of “Qtamine” for glutamine; asparagine -contains N
– “Acid” ends in D and E follows (smallest is first: aspartic
aciD, Glutamic acid E)

8. 8
Neutral Hydrocarbon Side Chains
H2N CHC
CH3
OH
O
H2N CHC
CH
OH
O
CH3
CH2
CH3
H2N CHC
CH2
OH
O
HN
C OH
O
H2N CHC
H
OH
O
H2N CHC
CH
OH
O
CH3
CH3
Glyciene
Phenylalanine
Isoleuciene
Valine
Proline
Alanine
H2N CHC
CH2
OH
O
CHCH3
CH3
Leuciene

9. 9
-OH, SH (Nucleophiles) and -S-CH3
Cysteine C, Cys
Methionine M, Met
Serine S, Ser
Threonine T, Thr
Tyrosine Y, Tyr
H2N CHC
CH2
OH
O
OH
H2N CHC
CH
OH
O
OH
CH3
H2N CHC
CH2
OH
O
OH
H2N CHC
CH2
OH
O
SH
Serine
Threonine
Tyrosine
Cysteine
Methionine
H2N CHC
CH2
OH
O
CH2
S
CH3

10. 10
Acids and Amides
Aspartic acid D, Asp
Glutamic Acid E, Glu
Asparagine N, Asn
Glutamine Q, Gln
H2N CHC
CH2
OH
O
CH2
C
OH
O
H2N CHC
CH2
OH
O
CH2
C
NH2
O
H2N CHC
CH2
OH
O
C
OH
O
H2N CHC
CH2
OH
O
C
NH2
O
Glutamic Acid
Glutamine
Aspartic Acid Asparagine

11. 11
Amines
Arginine R, Arg
Histidine H, His
Lysine K, Lys
Tryptophan W, Trp
H2N CHC
CH2
OH
O
CH2
CH2
CH2
NH2
H2N CHC
CH2
OH
O
N
NH
H2N CHC
CH2
OH
O
CH2
CH2
NH
C
NH2
NH
H2N CHC
CH2
OH
O
HN
Tryptophan
Arginine
Lysine Histidine

12. 12
Chirality of Amino Acids
• Glycine, 2-amino-acetic acid, is achiral
• In all the others, the α carbons of the amino
acids are centers of chirality
• The stereochemical reference for amino acids is
the Fischer projection of L-serine
• Proteins are derived exclusively from L-amino
acids

13. 13
Types of side chains
• Neutral: Fifteen of the twenty have neutral side
chains
• Acidic Amino Acids: Asp and Glu have a second
COOH and are acidic
• Basic Amino Acids: Lys, Arg, His have additional
basic amino groups side chains (the N in
tryptophan is a very weak base)
• Polar Amino Acids: Cys, Ser, Tyr (OH and SH)
are weak acids that are good nucleophiles

14. 14
Notes on Histidine
• Contains an imidazole ring that is partially protonated
in neutral solution
• Only the pyridine-like, doubly bonded nitrogen in
histidine is basic. The pyrrole-like singly bonded
nitrogen is nonbasic because its lone pair of electrons
is part of the 6 π electron aromatic imidazole ring (see
Section 24.4).

15. 15
Essential Amino Acids
• All 20 of the amino acids are necessary for protein synthesis
• Humans can synthesize only 10 of the 20
• The other 10 must be obtained from food

16. 16
Isoelectric Points
• In acidic solution, the carboxylate and amine are
in their conjugate acid forms, an overall cation
• In basic solution, the groups are in their base
forms, an overall anion
• In neutral solution cation and anion forms are
present
• This pH where the overall charge is 0 is the
isoelectric point, pI

17. 17
pI Depends on Side Chain
• The 15 amino acids thiol, hydroxyl groups or
pure hydrocarbon side chains have pI = 5.0 to
6.5 (average of the pKa’s)
• D and E have acidic side chains and a lower pI
• H, R, K have basic side chains and higher pI

18. 18
Electrophoresis
• Proteins have an overall pI that depends on the
net acidity/basicity of the side chains
• The differences in pI can be used for separating
proteins on a solid phase permeated with liquid
• Different amino acids migrate at different rates,
depending on their isoelectric points and on the
pH of the aqueous buffer

19. 19
Titration Curves of Amino Acids
• If pKa values for an amino acid are known the
fractions of each protonation state can be
calculated (Henderson-Hasselbach Equation)
• pH = pKa – log [A-
]/[HA]
• This permits a titration curve to be calculated or
pKa to be determined from a titration curve

20. 20
Titration Curves

21. 21
Synthesis of Amino Acids
• Bromination of a carboxylic acid by treatment
with Br2 and PBr3 (Section 22.4) then use NH3 or
phthalimide (24.6) to displace Br

22. 22
The Amidomalonate Synthesis
• Based on malonic ester synthesis (see 22.8).
• Convert diethyl acetamidomalonate into enolate
ion with base, followed by alkylation with a
primary alkyl halide
• Hydrolysis of the amide protecting group and the
esters and decarboxylation yields an α-amino

23. 23
Reductive Amination of α-Keto
Acids
• Reaction of an α-keto acid with NH3 and a
reducing agent produces an α-amino acid

24. 24
Enantioselective Synthesis of
Amino Acids
• Amino acids (except glycine) are chiral and pure
enantiomers are required for any protein or peptide
synthesis
• Resolution of racemic mixtures is inherently
ineffecient since at least half the material is
discarded
• An efficient alternative is enantioselective synthesis

25. 25
Chemical Resolution of R,S Amino
Acids
• Convert the amino group into an amide and
react with a chiral amine to form diastereomeric
salts
• Salts are separated and converted back to the
amino acid by hydrolysis of the amide

26. 26
Enzymic Resolution
• Enzymes selectively catalyze the hydrolysis of
amides formed from an L amino acid (S chirality
center)

27. 27
Enantioselective Synthesis of
Amino Acids
• Chiral reaction catalyst creates diastereomeric
transition states that lead to an excess of one
enantiomeric product
• Hydrogenation of a Z enamido acid with a chiral
hydrogenation catalyst produces S enantiomer
selectively

28. 28
Peptides and Proteins
• Proteins and peptides are amino acid polymers in
which the individual amino acid units, called residues,
are linked together by amide bonds, or peptide bonds
• An amino group from one residue forms an amide
bond with the carboxyl of a second residue

29. 29
Peptide Linkages
• Two dipeptides can result from reaction between A
and S, depending on which COOH reacts with which
NH2 we get AS or SA
• The long, repetitive sequence of NCHCO
atoms that make up a continuous chain is called the
protein’s backbone
• Peptides are always written with the N-terminal amino
acid (the one with the free NH2 group) on the left and
the C-terminal amino acid (the one with the free
CO2H group) on the right
• Alanylserine is abbreviated Ala-Ser (or A-S), and
serylalanine is abbreviated Ser-Ala (or S-A)

30. 30
Covalent Bonding in Peptides
• The amide bond that links different amino acids
together in peptides is no different from any
other amide bond (see Section 24.4). Amide
nitrogens are nonbasic because their unshared
electron pair is delocalized by interaction with
the carbonyl group. This overlap of the nitrogen
p orbital with the π orbitals of the carbonyl group
imparts a certain amount of double-bond
character to the C–N bond and restricts rotation
around it. The amide bond is therefore planar,
and the N–H is oriented 180° to the C=O.

31. 31
Covalent Bonding in Peptides

32. 32
Disulfides
• Thiols in adjacent chains can form a disulfide RS–SR
through spontaneous oxidation
• A disulfide bond between cysteine residues in different
peptide chains links the otherwise separate chains
together, while a disulfide bond between cysteine
residues in the same chain forms a loop

33. 33
Structure Determination of Peptides:
Amino Acid Analysis
• The sequence of amino acids in a pure protein is
specified genetically
• If a protein is isolated it can be analyzed for its
sequence
• The composition of amino acids can be obtained
by automated chromatography and quantitative
measurement of eluted materials using a
reaction with ninhydrin that produces an intense
purple color

34. 34
Amino Acid Analysis
Chromatogram

35. 35
Peptide Sequencing: The
Edman Degradation
• The Edman degradation cleaves amino acids
one at a time from the N-terminus and forms a
detectable, separable derivative for each amino
acid

36. 36
Peptide Sequencing: C-Terminal
Residue Determination
• Carboxypeptidase enzymes cleave the C-
terminal amide bond
• Analysis determines the appearance of the first
free amino acid, which must be at the carboxy
terminus of the peptide

37. 37
Peptide Synthesis
• Peptide synthesis requires that different amide bonds
must be formed in a desired sequence
• The growing chain is protected at the carboxyl
terminal and added amino acids are N-protected
• After peptide bond formation, N-protection is removed

38. 38
Carboxyl Protecting Groups
• Usually converted into methyl or benzyl esters
• Removed by mild hydrolysis with aqueous
NaOH
• Benzyl esters are cleaved by catalytic
hydrogenolysis of the weak benzylic C–O bond

39. 39
Amino Group Protection
• An amide that is less stable than the protein
amide is formed and then removed
• The tert-butoxycarbonyl amide (BOC) protecting
group is introduced with di-tert-butyl dicarbonate
• Removed by brief treatment with trifluoroacetic
acid

40. 40
Peptide Coupling
• Amides are formed by
treating a mixture of an acid
and amine with
dicyclohexylcarbodiimide
(DCC)

41. 41
Overall Steps in Peptide Synthesis

42. 42
Automated Peptide Synthesis: The
Merrifield Solid-Phase Technique
• Peptides are connected to beads of
polystyrene, reacted, cycled and cleaved at the
end

43. 43
Automated Synthesis
• The solid-phase technique has been automated,
and computer-controlled peptide synthesizers
are available for automatically repeating the
coupling and deprotection steps with different
amino acids
Applied Biosystems®
Synthesizer

44. 44
Protein Classification
• Simple proteins yield only amino acids on hydrolysis
• Conjugated proteins, which are much more common
than simple proteins, yield other compounds such as
carbohydrates, fats, or nucleic acids in addition to amino
acids on hydrolysis.
• Fibrous proteins consist of polypeptide chains arranged
side by side in long filaments
• Globular proteins are coiled into compact, roughly
spherical shapes
• Most enzymes are globular proteins

45. 45
Some Common Fibrous and
Globular Proteins

46. 46
Protein Structure
• The primary structure of a protein is simply the
amino acid sequence.
• The secondary structure of a protein describes
how segments of the peptide backbone orient
into a regular pattern.
• The tertiary structure describes how the entire
protein molecule coils into an overall three-
dimensional shape.
• The quaternary structure describes how different
protein molecules come together to yield large
aggregate structures

47. 47
α-Keratin
• A fibrous structural protein coiled into a right-
handed helical secondary structure, α-helix
stabilized by H-bondsb between amide N–H
groups and C=O groups four residues away a-
helical segments in their chains

48. 48
Fibroin
• Fibroin has a secondary structure called a
β-pleated sheet in which polypeptide
chains line up in a parallel arrangement
held together by hydrogen bonds between
chains

49. 49
Myoglobin
• Myoglobin is a small globular protein containing
153 amino acid residues in a single chain
• 8 helical segments connected by bends to form
a compact, nearly spherical, tertiary structure

50. 50
Internal and External Forces
• Acidic or basic amino acids with charged side chains
congregate on the exterior of the protein where they can
be solvated by water
• Amino acids with neutral, nonpolar side chains
congregate on the hydrocarbon-like interior of a protein
molecule
• Also important for stabilizing a protein's tertiary structure
are the formation of disulfide bridges between cysteine
residues, the formation of hydrogen bonds between
nearby amino acid residues, and the development of
ionic attractions, called salt bridges, between positively
and negatively charged sites on various amino acid side
chains within the protein

51. 51
Enzymes
• An enzyme is a protein that acts as a catalyst for
a biological reaction.
• Most enzymes are specific for substrates while
enzymes involved in digestion such as papain
attack many substrates

52. 52
Cofactors
• In addition to the protein part, many enzymes also have
a nonprotein part called a cofactor
• The protein part in such an enzyme is called an
apoenzyme, and the combination of apoenzyme plus
cofactor is called a holoenzyme. Only holoenzymes have
biological activity; neither cofactor nor apoenzyme can
catalyze reactions by themselves
• A cofactor can be either an inorganic ion or an organic
molecule, called a coenzyme
• Many coenzymes are derived from vitamins, organic
molecules that are dietary requirements for metabolism
and/or growth

53. 53
Types of Enzymes by Function
• Enzymes are usually grouped according to the
kind of reaction they catalyze, not by their
structures

54. 54
How Do Enzymes Work? Citrate
Synthase
• Citrate synthase catalyzes a mixed Claisen
condensation of acetyl CoA and oxaloacetate to
give citrate
• Normally Claisen condensation require a strong
base in an alcohol solvent but citrate synthetase
operates in neutral solution

55. 55
The Structure of Citrate
Synthase
• Determined by X-ray crystallography
• Enzyme is very large compared to substrates,
creating a complete environment for the reaction

56. 56
Mechanism of Citrate
Synthetase
• A cleft with functional groups binds oxaloacetate
• Another cleft opens for acetyl CoA with H 274
and D 375, whose carboxylate abstracts a
proton from acetyl CoA
• The enolate (stabilized by a cation) adds to the
carbonyl group of oxaloacetate
• The thiol ester in citryl CoA is hydrolyzed

57. 57
Protein Denaturation
• The tertiary structure of a globular protein is the
result of many intramolecular attractions that can
be disrupted by a change of the environment,
causing the protein to become denatured
• Solubility is drastically decreased as in heating
egg white, where the albumins unfold and
coagulate
• Enzymes also lose all catalytic activity when
denatured