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Foundations of Biochemistry: Amino Acids
Protein Building Blocks
Cindy McKinney, Ph.D.
Reading Assignment
• Chapter 6, Lieberman and Marks
Lecture Objectives
• Identify the basic structural properties of an amino acid, and identify the
side chains of the 20 amino acids commonly found in proteins.
• Predict the ionization of an amino acid at pH 7.0, including the side chain
if it is an ionizable group (side chain pKa values will be provided where
appropriate).
• Recognize the nine essential amino acids and define why they are
“essential.”
• Define disulfide formation between two free cysteine side chains, and
predict how disulfide formation affects protein primary structure.
• Compare and contrast the chemical properties of each of the four weak
forces: hydrogen bonds, dipole-dipole interactions, hydrophobic
interaction, ionic interactions.
• Define “biomolecular recognition” and relate how the four week forces
govern the interaction of biomolecules.
Proteins are made from Amino Acids
Primary Protein Structure
Linked aa chains (S-S bridge) of
two polypeptides
Why should doctors care about amino acids? Clinical relevance?
Disease Examples (aa defects):
• HOMOCYSTINURIA: error in methionine metabolismMAPLE SYRUP URINE DISEASE:
inability to metabolize branched chain amino acids
• SICKLE CELL ANEMIA: a single non-conservative amino acid (aa) mutation in hemoglobin
(Hb) affects hemoglobin structure and function
• CYSTIC FIBROSIS: most common aa mutation is Δ508F making a defective channel protein
Therapeutic Example (mechanism of drug action):
• PHARMACOLOGY: many drugs work by reacting with key amino acid side chains in
enzymes and proteins
Amino acids are the basic structural unit of all proteins
• There are 20 common amino acids; 8 are essential
A 'free' amino acid (a single amino acid) always has:
-an amino group -NH2,
-a carboxyl group -COOH
-a hydrogen -H
-a chemical group or side chain -"R”
These are all joined to a central α-carbon atom (see
following diagram)
When used as the building blocks for polypeptide chains
(protein) they will be linked to each other through the NH2
and COOH groups (shown later).
The Basics: Structure of an Amino Acid
α carbon
Note:
• There are 20 common amino acids used in proteins
• Protonated amino group (physiological pH)
• C-terminal carboxyl group
• R-side chain from alpha carbon---yields distinct
molecules because R can be: Non-polar, aromatic
uncharged polar, sulfur containing, charged (basic
or acidic)
Peptide bond
Classification of AA side chains-1
• Non-polar (aliphatic) “R” groups—hydrophobic (do not like contact with
water/aqueous solutions; because of their aversion to water are frequently found in
the interior (core) of a protein where they are “protected” from water
 Glycine (Gly; G) is the simplest and smallest of all amino acids, and the only
one which is not optically active since it has a single hydrogen (H) atom as it's
R side chain.
 Alanine (Ala; A) has a methyl group as it's R side chain.
 Valine (Val; V) has a slightly longer R side chain --there is a branch.
Note: As the aliphatic side chains get longer they are also more hydrophobic
 Leucine (Leu; L) is very similar to valine except it has another methyl group
attached to the R side chain
 Isoleucine (Ile; I) similar to leucine & valine except that the orientation of the
atoms in the R side chain is slightly different. Isoleucine also has two centers
of asymmetry.
 Proline (Pro; P) different from the other amino acids--- the R side chain is a 5
member ring derived from bonding to the α-carbon and the amino group.
This
structure effects the architecture of proteins. Considered aliphatic, it does
not “mind” being in contact with water as much as the others.
Classification of AA side chains-2
Structures of aliphatic amino acids:
R
R
R
α
α
R
α
R
α
α
R=ring
α
proline
Classification of AA side chains-3
Aromatic Amino Acids: the R group is an aromatic ring;
aromatic rings are hydrophobic
 Phenylalanine (Phe; F) It contains a phenyl ring attached to a
methylene group; the phenyl ring makes F a hydrophobic aa.
The rings can stack on each other in a protein.
 Tyrosine (Tyr; Y) contains a hydroxyl group at the end of the
phenyl ring--- it can form H-bonds. Tyrosine is less
hydrophobic than phenylalanine.
 Tryptophan (Trp; W) has an indole ring attached to the
methylene group. The indole ring is highly hydrophobic.
Phenylalanine
H-bonds
Tyrosine
Classification of AA side chains-4
Sulfur containing Amino Acids
 Cysteine (Cys; C) contains a sulphydryl group (-SH). This is extremely reactive,
and can form hydrogen bonds. Cysteine --- very important to protein
structure because it can form disulphide bridges (S-S) the -SH group of
cysteine can form hydrogen bonds however, the aliphatic part of the side
chain makes it quite hydrophobic.
 Methionine (Met; M) is a special amino acid it is the "start" amino acid in
the process of translation (protein synthesis), and therefore, begins every
single protein made. It also has a sulphur atom, this time it is in a thio-ether
linkage, that can not form S-S bonds. Methionine has a highly hydrophobic R
side-chain.
Cysteine
Methionine
Classification of AA side chains-5
Hyrophillic Amino Acids—these aa “like” water but can be neutral,
acidic or basic at physiological pH
• Charged Negative Amino Acids (Acidic)—highly polar and
negatively charged
 Aspartate (Asp; D) is really aspartic acid. It is called aspartate
because it is usually negatively charged at physiological pH ---it is
named for the carboxylate anion. (Compare acetic acid and
acetate.)
 Glutamate (Glu; E) is also called glutamic acid. The R side chain of
glutamate also has a carboxylate group which has a negative
charge at physiological pH
Classification of AA side chains-6
• Basic Amino Acids—R side chain (+) at physiological pH
 Lysine (Lys; K) Has long side chin  although the side chain
appears to be a hydrophobic hydrocarbon chain--- it is very
polar because of the terminal amino group…therefore
classified as a hydrophillic amino acid.
 Arginine (Arg; R) has the largest R side chains. The guanidino
group attached to the side chain it has a high pKa value thus
positively charged at physiological pH
 Histidine (His; H) contains an imidazole ring…this often sits
inside the active site of an enzyme and helps bonds to be made
or broken as the enzyme works (because it can exist in two
states -uncharged or positively charged)
Arginine
Histidine
Lysine
Classification of AA side chains-7
• Neutral Amino Acids---not charged at physiological pH BUT all




contain polar R groups that can form H-bonds (classified as hydrophillic)
Serine (Ser; S) contains an aliphatic chain with a hydroxyl group---a
hydroxylated the hydroxylated version of alanine. The -OH group makes
the aa highly reactive and hydrophillic (readily forms hydrogen bonds)
Threonine (Thr; T) another neutral amino acid that contains a highly
reactive (and highly hydrophillic) –OH group. This is an aa that contains
two centers of asymmetry (two asymetric carbon atoms) (also found in
Ile).
Asparagine (Asn; N) is the amide derivative of Aspartic acid. When the
carboxylate side chain is amidated the resulting amide is uncharged.
Note
terminal amide group on N as opposed to the carboxyl group on
aspartat
Glutamine (Gln; Q) similar to Asparagine containing a terminal amide
instead of a carboxyl group as in glutamate. These two are called the
amide derivatives of their parent amino acids.
amide
Amino Acids---Characteristics
• Each aa has a non-polar side chain that does not gain or lose protons or
participate in H-bond formation
• “R” side chain is best characterized as “oily” or lipid-like that promotes
hydrophobic (excludes water) interactions.
• Non-polar aa in proteins: In aqueous solutions or a polar environment, the
hydrophobic side chains cluster together in the interior of the protein (see
right hand figure)
• Proline: Differs from other aa in that the R side chain and α-amino N form a
rigid , five-membered ring structure. The unique geometry of proline
contributes to the fibrous structure of collagen.
Amino Acids---Characteristics-2
•
•
•
•
•
•
The amino acids with uncharged polar side chains have zero net charge at neutral pH
Serine, threonine, and tyrosine each contain a polar hydroxyl (-OH) group that may be
involved in H-bond formation
Side chains of the polar OH group of serine, threonine, and rarely tyrosine can serve as
an attachment site for phosphate (PO4) group
The side chains of asparagine and glutamine each contain a carbonyl group and an
amide group, both of which can participate in hydrogen bond formation.
Disulfide bonds: The –SH side chain of cysteine is a component of many enzyme active
sites
In proteins, the –SH groups of two cysteines can become oxidized to create a dimer
(cystine) that contains a covalent cross-link or disulfide bond (-S-S-).
Amino Acids---Characteristics-3
• Aspartic and glutamic acid are proton donors
• At physiologic pH (7.4), the side chains of these amino acids are fully
ionized, containing a negatively charged carboxyl (–COO-) group. They
are therefore called aspartate or glutamate to emphasize these acids
as being negatively charged at physiological pH.
Amino Acids---Characteristics-4
• The R groups of Histidine, Lysine and Arginine are proton (H+) acceptors
• At physiological pH the side chains of lysine and arginine are fully ionized
and positively charged.
• Histidine is weakly basic
– When incorporated into a protein, its R group can be either positively
charged or neutral depending on the ionic environment provided by the
polypeptide chain
– This property of histidine contributes to its role in the function of
proteins such as hemoglobin
Titration of an amino acid with a non-ionizable side
chain
Glycine Titration Curve
Isoelectric point (pI)=no net charge
Two pKas noticeable on graph: pKa1= -COOH group and pKa2= -NH3
Titration of an amino acid with a ionizable side chain
Titration Curve of Histidine
There are three pKas available: the α-COOH, the α-NH3 and the R side group (indole ring)
This pKa depends on the R group structure
Amino Acids Interactions
Peptide A
Peptide B
• the hydrophobic effect, non-polar side chains will gather together in the
protein interior whenever possible.
• Aromatic side chains rarely exposed to polar / aqueous environment
Amino Acids Interactions-2
Hydrogen Bonds/Electrostatic Interactions
Peptide A
Peptide B
• Electronegative atoms (e.g., O, N) will attract electropositive H+ atoms
This not a covalent bond, but an electrostatic interaction between
atoms
Amino Acids Interactions-3
Electrostatic Interactions
Peptide A
atoms with a formal positive or negative
charge will attract one another
Again, not a covalent bond, but an
electrostatic interaction between oppositely
charged atoms
Peptide B
Amino Acids Interactions-4
Disulfide Bridges: cysteine + cysteine
• Cysteine may exist as a free sulfydryl (-SH)
group or it may form a covalent disulfide (S-S)
bond
• Disulfide bonds play a key role in protein
structure and function – they hold different
parts of a protein together (ex: insulin)
H+ removed
• Disulfide bonds are sometimes considered part
of the primary structure of proteins, but they
contribute to secondary/tertiary structure (by
holding polypeptide chain in proximity)
• Disulfide bond formation: enzymatic process
carried out when proteins are made.
It is carefully regulated.
Amino Acids Interactions-5
Ionizable R side chains play a significant role in protein structure and function
Note: when solution pH=pKa, the side chain is 50% ionized
Weak Forces: Understanding
Protein Structure and Function
Four weak forces at play:
1) van der waals
2) Hydrogen bonds
3) Ionic interactions/bonds
4) Hydrophobic interactions (water excluding)
van der Waals Interactions
• induced electrical interactions between
closely approaching atoms/ molecules as
their negatively-charged electron clouds
fluctuate instantaneously in time
•depends on the distance between
interacting atoms
• each interaction provides about 0.4 to 4 kJ
of energy/ molecule
• many vdw interactions in a system
provides a large energy potential
Hydrogen Bonds
Examples between water molecules and serine residues in a protein
Ionic Bonds
Charged based attraction
Hydrophobic Interactions
H2O becomes organized
• Tendency of water or polar molecules to
exclude nonpolar groups or molecules
• Interacting hydrophobic molecules have
vdw interactions, but not a primary
energetic / thermodynamic consideration
• Entropy-driven process: water is more
organized when it surrounds non-polar
molecules
• Drives the creation and maintenance of
macromolecular structures: formation of
lipid bilayers
outside
inside -
Protein: Primary Structure
• Primary structure of a protein = linear sequence
of amino acids linked by peptide bonds
Note: water loss
during peptide bond
formation
What would a tri-peptide look like ?
Variations in Primary Structure
Amino acid linear sequence (N-terminal to C-terminal linked through peptide bonds)
affect the secondary and tertiary structure.
-protein structure even with the same sequence can vary among species
-protein structure can also vary between
>tissues (isoforms)
> stage of development (fetal Hb vs. adult Hb)
> individuals
These changes are tolerated if:
> confined to non-critical regions (variant) of the protein (not active enzyme sites)
> conservative substitutions of one amino acid for another of similar structure
> confer an advantage to protein function
Polymorphisms in Protein Structure
Variations can arise by mutations in DNA (point mutations, indels)
may be an obvious dysfunction or disease
A single point mutation in an invariant protein region has dire
consequences
when these changes occur frequently in a population
=polymorphism
Currently about 33% of loci in human genome appear to be
Polymorphic
sickle cell point mutation is a stable polymorphism in population
> heterozygote provides some resistance to malaria
Protein Families and SuperFamilies
Divergent evolution: gene duplications are
often associated with an ancestral gene; one
protein may retain the original function while
the duplicated protein may derive a new or
related function
Example: globin family (different
proteins/function)
Paralogs: myoglobin,α-globin, β-globin, γ-globin,
δ-globin
A model for the evolution of β-globin genes in mammals based on Phylogeny. The gene tree
is drawn within the constraints of a species tree (21). The ancient gene duplication event
(indicated by an arrow) gave rise to two ancestral genes, A and B. A was the progenitor of
marsupial ω-globin and the β-like globin genes of birds. B gave rise to the β-like globin genes
of mammals. Genes or pseudogenes that may be expected to occur are indicated by ?.
To simplify the diagram, not all of the avian β-like globin genes (as exemplified by the chicken)
are shown.
PNAS 98: 1101-1106
Posttranslational Modification in Proteins
A few amino acids may be modified after translation from the mRNA
is completed---result of enzymatic activity on specific amino acids
≥ 100 different post translation modifications recognized
These modifications may serve:
1) regulatory role
2) target/anchor a protein in a membrane
3) target a protein for degradation
4) enhance a protein’s interaction with another protein
Posttranslational Modification in Proteins
Types of posttranslational modifications (textbook figure 6.13)
1) Glycosylation: modification of amino acids at O-links or N-links
N-linked small chain carbohydrates found in cell surface proteins
protect cell from proteolysis or immune attack.
2) Fatty Acylation or Prenylation: many membrane proteins contain
linked lipid groups that interact with lipids in membranes.
3) Regulatory Modifications: ADP-ribosylation, Phosphorylation, or
Acetylation in many cases used to regulation activity of enzymatic
proteins
4) Modifications of R group chains: blood clotting COOH added at γC
of glutamate
5) Selenocysteine: required in a few enzymes for activity
HSe-CH2-CH-COONH3+
Question
• A glutamate is substituted for a valine in Hb
causing sickle cell anemia. This is a nonconservative replacement. However, the
substitution of an aspartate for a glutamate is
a conservative replacement. How would you
define non-conservative and conservative?
What effect do you think this nonconservative replacement might have on
protein structure?