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Amino acids, peptides and proteins: Structure and naming of amino acids
1. Amino Acids, Peptides,
Proteins
• Structure and naming of amino acids
• Structure and properties of peptides
• Ionization behavior of amino acids and peptides
• Purification and assay methods
• Peptide sequencing and chemical synthesis
• Protein sequence analysis
2. Proteins: Main Agents of
Biological Function
• Catalysis:
–enolase (in the glycolytic pathway)
–DNA polymerase (in DNA replication)
• Transport:
–hemoglobin (transports O2 in the blood)
–lactose permease (transports lactose across the cell membrane)
• Structure:
–collagen (connective tissue)
–keratin (hair, nails, feathers, horns)
• Motion:
–myosin (muscle tissue)
–actin (muscle tissue, cell motility)
3. Amino Acids: Building Blocks of
Protein
• Proteins are heteropolymers of -amino acids
• Amino acids have properties that are well
suited to carry out a variety of biological
functions:
– Capacity to polymerize
– Useful acid-base properties
– Varied physical properties
– Varied chemical functionality
4. Amino Acids: Atom Naming
• Organic nomenclature: start from one end
• Biochemical designation: start from
-carbon and go down the R-group
5. Most -Amino Acids are Chiral
• The -carbon has always
four substituents and is
tetrahedral
• All (except proline) have an
acidic carboxyl group, a
basic amino group, and an
alpha hydrogen connected
to the -carbon
• Each amino acid has an
unique fourth substituent R
• In glycine, the fourth
substituent is also hydrogen
6. Amino Acids: Classification
Common amino acids can be placed in five
basic groups depending on their R substituents:
• Nonpolar, aliphatic (7)
• Aromatic (3)
• Polar, uncharged (5)
• Positively charged (3)
• Negatively charged (2)
13. Not incorporated by ribosomes
Arise by post-translational modifications of
proteins
Reversible modifications, esp.
phosphorylation is important in regulation
and signaling
Uncommon Amino
Acids in Proteins
14. Ionization
At acidic pH, the carboxyl
group is protonated
and the amino acid is
in the cationic form
At neutral pH, the
carboxyl group is
deprotonated but the
amino group is
protonated. The net
charge is zero; such
ions are called
Zwitterions
At alkaline pH, the amino
group is neutral –NH2
and the amino acid is
in the anionic form.
15. Substituent effects on pKa Values
-carboxy group is much more acidic than in carboxylic acids
-amino group is slightly less basic than in amines
16. Amino Acids Can
Act as Buffers
Amino acids with
uncharged side-chains,
such as glycine, have two
pKa values:
The pKa of the -carboxyl
group is 2.34
The pKa of the -amino
group is 9.6
It can act as a buffer in
two pH regimes.
17. Amino Acids Carry a Net Charge
of Zero at a Specific pH
•Zwitterions predominate at pH values between the pKa
values of amino and carboxyl group
•For amino acid without ionizable side chains, the Isoelectric
Point (equivalence point, pI) is
• At this point, the net charge is zero
– AA is least soluble in water
– AA does not migrate in electric field
2
2
1 pK
pK
pI
18. Ionizable Side Chains Can Show
Up in Titration Curves
• Ionizable side chains
can be also titrated
• Titration curves are
now more complex
• pKa values are
discernable if two pKa
values are more than
two pH units apart
Why is the side-chain
pKa so much higher?
19. How to Calculate the pI When the
Side-chain is Ionizable?
• Identify species that carries
a net zero charge
• Identify pKa value that
defines the acid strength of
this zwitterion: (pK2)
• Identify pKa value that
defines the base strength of
this zwitterion: (pKR)
• Take the average of these
two pKa values
20. Peptides and Peptide bonds
Peptide bond in
a di-peptide
“Peptides” are
small
condensation
products of
amino acids
They are “small”
compared to
proteins (di, tri,
tetra… oligo-)
21. Peptide Ends are Not the Same
Numbering starts from the amino terminus
AA1 AA2 AA3 AA4 AA5
22. The Three Letter Code
• Naming starts from
the N-terminus
• Sequence is written
as:
Ala-Glu-Gly-Lys
• Sometimes the one-
letter code is used:
AEGK
23. Peptides: A Variety of Functions
• Hormones and pheromones:
– insulin (think sugar)
– oxytocin (think childbirth)
– sex-peptide (think fruit fly mating)
• Neuropeptides
– substance P (pain mediator)
• Antibiotics:
– polymyxin B (for Gram - bacteria)
– bacitracin (for Gram + bacteria)
• Protection, e.g. toxins
– amanitin (mushrooms)
– conotoxin (cone snails)
– chlorotoxin (scorpions)
24. Proteins are:
• Cofactor is a general term for functional non-amino acid component
– Metal ions or organic molecules
• Coenzyme is used to designate an organic cofactors
– NAD+ in lactate dehydrogenase
• Prosthetic groups are covalently attached cofactors
– Heme in myoglobin
• Polypeptides (covalently linked -amino acids) + possibly –
• cofactors,
• coenzymes,
• prosthetic groups,
• other modifications
27. Peptides and Proteins-
Burning Questions
Sequence and composition?
Three-dimensional structure?
Folding Mechanism?
Biochemical role?
Functional regulation?
Molecular interactions with small and macro-molecules?
Structural and sequence relatives?
Cellular and sub-cellular localization?
Physical and chemical properties?
28. Purification – Fractionation of
Protein Mixtures
• Separation relies on differences in physico-
chemical properties
– Solubility – Selective Precipitation (Centrifugation)
– Thermal stability --
– Charge --Electrophoresis, Isoelectric Focusing, IEC
– Size – Dialysis, Sedimentation (Centrifugation), GFC
– Affinity for a ligand – “Pull down” assays (Centrifugation),
AC
– Hydrophobicity (HIC)
• Chromatography is commonly used for
preparative separation
33. Electrophoresis for Protein
Analysis
Separation in
analytical scale is
commonly done by
electrophoresis
– Electric field pulls
proteins according to
their charge
– Gel matrix hinders
mobility of proteins
according to their
size and shape
34. SDS PAGE: Molecular Weight
• SDS – sodium dodecyl
sulfate – a detergent
• SDS micelles binds to,
and unfold all the
proteins
– SDS gives all proteins an
uniformly negative
charge
– The native shape of
proteins does not matter
– Rate of movement will
only depend on size:
small proteins will move
faster
-
36. Spectroscopic Detection of Aromatic
Amino Acids
• The aromatic amino acids
absorb light in the UV
region
• Proteins typically have
UV absorbance maxima
around 275-280 nm
• Tryptophan and tyrosine
are the strongest
chromophores
• Concentration can be
determined by UV-visible
spectrophotometry using
Beers law: A = ·c·l
37. Chapter 8: Summary
In this chapter, we learned about:
• The many biological functions of peptides and
proteins
• The structures and names of amino acids found in
proteins
• The ionization properties of amino acids and
peptides
• The methods for separation and analysis of
proteins
44. 44
Primary Structure of Proteins
The particular sequence of amino acids that is
the backbone of a peptide chain or protein
H3N CH
CH3
C
O
N
H
CH C
O
N
H
CH C
O
N
H
CH C O-
O
CH
CH CH3
CH3
CH2
SH
CH2
CH2
S
CH3
+
Ala-Leu-Cys-Met
45. 45
Secondary Structure – Alpha
Helix
• Three-dimensional arrangement of amino acids
with the polypeptide chain in a corkscrew shape
• Held by H bonds between the H of –N-H group and
the –O of C=O of the fourth amino acid along the
chain
• Looks like a coiled “telephone cord”
46. 46
Secondary Structure – Beta
Pleated Sheet
• Polypeptide chains are arranged
side by side
• Hydrogen bonds form between
chains
• R groups of extend above and
below the sheet
• Typical of fibrous proteins such
as silk
47. 47
Secondary Structure – Triple
Helix
• Three polypeptide chains woven
together
• Glycine, proline, hydroxy proline
and hydroxylysine
• H bonding between –OH groups
gives a strong structure
• Typical of collagen, connective
tissue, skin, tendons, and cartilage
48. 48
Learning Check P1
Indicate the type of structure as
(1) primary (2) alpha helix
(3) beta pleated sheet (4) triple helix
A. Polypeptide chain held side by side by H bonds
B. Sequence of amino acids in a polypeptide chain
C. Corkscrew shape with H bonds between amino acids
D. Three peptide chains woven like a rope
49. 49
Solution P1
Indicate the type of structure as
(1) primary (2) alpha helix
(3) beta pleated sheet (4) triple helix
A. 3 Polypeptide chain held side by side by H bonds
B. 1 Sequence of amino acids in a polypeptide chain
C. 2 Corkscrew shape with H bonds between amino acids
D. 4 Three peptide chains woven like a rope
50. 50
Tertiary Structure
• Specific overall shape of a protein
• Cross links between R groups of amino acids in
chain
disulfide –S–S– +
ionic –COO– H3N–
H bonds C=O HO–
hydrophobic –CH3 H3C–
51. 51
Learning Check P2
Select the type of tertiary interaction as
(1) disulfide (2) ionic
(3) H bonds (4) hydrophobic
A. Leucine and valine
B. Two cysteines
C. Aspartic acid and lysine
D. Serine and threonine
52. 52
Solution P2
Select the type of tertiary interaction as
(1) disulfide (2) ionic
(3) H bonds (4) hydrophobic
A. 4 Leucine and valine
B. 1 Two cysteines
C. 2 Aspartic acid and lysine
D. 3 Serine and threonine
54. 54
Quaternary Structure
• Proteins with two or more chains
• Example is hemoglobin
Carries oxygen in blood
Four polypeptide chains
Each chain has a heme group to
bind oxygen
55. 55
Learning Check P3
Identify the level of protein structure
1. Primary 2. Secondary
3. Tertiary 4. Quaternary
A. Beta pleated sheet
B. Order of amino acids in a protein
C. A protein with two or more peptide chains
D. The shape of a globular protein
E. Disulfide bonds between R groups
56. 56
Solution P3
Identify the level of protein structure
1. Primary 2. Secondary
3. Tertiary 4. Quaternary
A. 2 Beta pleated sheet
B. 1 Order of amino acids in a protein
C. 4 A protein with two or more peptide
chains
D. 3 The shape of a globular protein
E. 3 Disulfide bonds between R groups
57. 57
Protein Hydrolysis
• Break down of peptide bonds
• Requires acid or base, water and heat
• Gives smaller peptides and amino
acids
• Similar to digestion of proteins using
enzymes
• Occurs in cells to provide amino
acids to synthesize other proteins and
tissues
58. 58
Hydrolysis of a Dipeptide
H3N CH
CH3
C
O
N
H
CH C
O
CH2
OH
OH
+
H3N CH
CH3
COH
O
+ CH C
O
CH2
OH
OH
H3N
H2O, H+
+
+
heat
59. 59
Denaturation
Disruption of secondary, tertiary and quaternary protein
structure by
heat/organics
Break apart H bonds and disrupt hydrophobic
attractions
acids/ bases
Break H bonds between polar R groups and
ionic bonds
heavy metal ions
React with S-S bonds to form solids
agitation
Stretches chains until bonds break
60. 60
Applications of Denaturation
• Hard boiling an egg
• Wiping the skin with alcohol swab for injection
• Cooking food to destroy E. coli.
• Heat used to cauterize blood vessels
• Autoclave sterilizes instruments
• Milk is heated to make yogurt
63. 63
Learning Check P5
Tannic acid is used to form a scab on a burn.
An egg becomes hard boiled when placed in
hot water. What is similar about these two
events?
64. 64
Solution P5
Acid and heat cause a denaturation of protein.
They both break bonds in the secondary and
tertiary structure of protein.
65. Biology/Chemistry of Protein Structure
Primary
Secondary
Tertiary
Quaternary
Assembly
Folding
Packing
Interaction
S
T
R
U
C
T
U
R
E
P
R
O
C
E
S
S
66. Protein Assembly
• occurs at the ribosome
• involves dehydration
synthesis and
polymerization of amino
acids attached to tRNA:
NH - {A + B A-B + H O} -COO
• thermodynamically
unfavorable, with E =
+10kJ/mol, thus coupled to
reactions that act as sources
of free energy
• yields primary structure
2 n
3
+ -
67. Primary Structure
• linear
• ordered
• 1 dimensional
• sequence of amino acid
polymer
• by convention, written
from amino end to
carboxyl end
• a perfectly linear amino
acid polymer is neither
functional nor
energetically favorable
folding!
primary structure of human insulin
CHAIN 1: GIVEQ CCTSI CSLYQ LENYC N
CHAIN 2: FVNQH LCGSH LVEAL YLVCG ERGFF YTPKT
68. Protein Folding
• tumbles towards conformations
that reduce E (this process is
thermo-dynamically favorable)
• yields secondary structure
• occurs in the cytosol
• involves localized spatial
interaction among primary
structure elements, i.e. the
amino acids
• may or may not involve
chaperone proteins
69. Secondary Structure
• non-linear
• 3 dimensional
• localized to regions of an
amino acid chain
• formed and stabilized by
hydrogen bonding,
electrostatic and van der
Waals interactions
70. Ramachandran Plot
• Pauling built models based on the
following principles, codified by
Ramachandran:
(1)bond lengths and angles – should
be similar to those found in
individual amino acids and small
peptides
(2) peptide bond – should be planer
(3) overlaps – not permitted, pairs of
atoms no closer than sum of their
covalent radii
(4) stabilization – have sterics that
permit hydrogen bonding
• Two degrees of freedom:
71. Protein Packing
• occurs in the cytosol (~60% bulk
water, ~40% water of hydration)
• involves interaction between
secondary structure elements and
solvent
• may be promoted by chaperones,
membrane proteins
• tumbles into molten globule states
• overall entropy loss is small enough
so enthalpy determines sign of E,
which decreases (loss in entropy
from packing counteracted by gain
from desolvation and reorganization
of water, i.e. hydrophobic effect)
• yields tertiary structure
72. Tertiary Structure
• non-linear
• 3 dimensional
• global but restricted to the
amino acid polymer
• formed and stabilized by
hydrogen bonding, covalent (e.g.
disulfide) bonding, hydrophobic
packing toward core and
hydrophilic exposure to solvent
• A globular amino acid polymer
folded and compacted is
somewhat functional (catalytic)
and energetically favorable
interaction!
73. Protein Interaction
• occurs in the cytosol, in close proximity to other folded
and packed proteins
• involves interaction among tertiary structure elements of
separate polymer chains
• may be promoted by chaperones, membrane proteins,
cytosolic and extracellular elements as well as the
proteins’ own propensities
• E decreases further due to further
desolvation and reduction of surface area
• globular proteins, e.g. hemoglobin,
largely involved in catalytic roles
• fibrous proteins, e.g. collagen,
largely involved in structural roles
• yields quaternary structure
74. Quaternary Structure
• non-linear
• 3 dimensional
• global, and across distinct
amino acid polymers
• formed by hydrogen
bonding, covalent bonding,
hydrophobic packing and
hydrophilic exposure
• favorable, functional
structures occur frequently
and have been categorized
