3 | Amino Acids,

Peptides, and Proteins

© 2017 W. H. Freeman and Company

 CHAPTER 3
Amino Acids, Peptides,
and Proteins

Learning goals:
• Structure and naming of amino acids
• Structure and properties of peptides
• Ionization behavior of amino acids and peptides
• Methods to characterize peptides and proteins
 Proteins:
Main Agents of Biological Function
• Catalysis
– Enzymes

• 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)
 Amino Acids:
Building Blocks of Protein
• Proteins are linear 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
 Amino Acids Share Common Structural Features

• The α carbon is bonded to four different groups: a carboxyl group, an amino

group, an R group, and a hydrogen atom (In glycine, the R group is another
hydrogen atom).
• Amino acids share many features, differing only at the R substituent.
• The α-carbon atom is thus a chiral center (The α carbon always has four
substituents and is tetrahedral.
• Amino acids have two possible stereoisomers (called enantiomers).
– Optically active (they rotate the plane of planepolarized light)
 Stereoisomerism in α-amino acids
• The absolute configurations of amino acids are specified by the D, L system.
• Another system of specifying configuration around a chiral center is the RS
system (systematic nomenclature of organic chemistry; > 1 chiral C).
• The amino acid residues in proteins are L stereoisomers.
Amino Acids can be classified by R group
• The R groups in this class of amino acids are nonpolar and hydrophobic.
• Glycine has the simplest structure (no real contribution to interactions driven by
the hydrophobic effect).
• Proline has an aliphatic side chain with a distinctive cyclic structure.
• The secondary amino (imino) group of proline residues is held in a rigid
conformation that reduces the structural flexibility of polypeptide regions
containing proline.
• Aromatic R groups are relatively nonpolar (hydrophobic). All can contribute to
the hydrophobic effect.
• The hydroxyl group of tyrosine can form hydrogen bonds, and it is an important
functional group in some enzymes.
• Tyrosine and tryptophan are significantly more polar than phenylalanine
• These amino acid side chains absorb UV light at 270–280 nm (most proteins)
• The absorbance, A, is directly proportional to the concentration.
 hydroxyl group
sulfhydryl group

amide group

• The R groups of these amino acids are more soluble in water, or more
hydrophilic (hydrogen bonds with water).
• Cysteine is readily oxidized to form a covalently linked dimeric amino acid
called cystine (formation of disulfide bonds).
• The disulfide-linked residues are strongly hydrophobic.
• Disulfide bonds play a special role in the structures of many proteins.
 (Basic) (Acidic)

• Each amino acid has a second

carboxyl group

• The most hydrophilic R groups (either positively or negatively charged)

• Amino group, guanidinium group, & aromatic imidazole group
• Histidine may be positively charged (protonated form) or uncharged at pH 7.0.
• facilitate many enzyme-catalyzed reactions by serving as proton
donors/acceptors.
 Uncommon Amino Acids Also Have
Important Functions
• In addition to the 20 common amino acids, proteins may contain residues
created by postsynthetic modification of common residues.
• 4-Hydroxyproline (plant cell wall proteins and collagen), 5-hydroxylysine
(collagen), 6-N-methyllysine (myosin), γ-Carboxyglutamate (blood-clotting
protein prothrombin & Ca+-binding protein), desmosine (a derivative of four Lys
residues, in the fibrous protein elastin)
• Selenocysteine and pyrrolysine
– special cases (not created through a postsynthetic modification).
– during protein synthesis through an unusual adaptation of the genetic code
– Selenocysteine contains selenium rather than the sulfur of cysteine
– Pyrrolysine is found in a few proteins in several methanogenic (methane-producing)
archaea and in one known bacterium, and plays a role in methane biosynthesis.
• Some amino acid residues in a protein may be covalently modified to alter the
protein’s function (modification as a protein regulatory strategy)
– phosphorylation, methylation, acetylation, adenylylation, ADP-ribosylation
• Ornithine and citrulline (key intermediates in the biosynthesis of arginine)
 Amino Acids Can Act as Acids and Bases

• The amino and carboxyl groups of amino acids function as weak acids and bases.
– Dual (acid-base) nature → amphoteric
• When dissolved in water at neutral pH, it exists in solution as the dipolar ion, or
zwitterion.

• Amino acids contain at least two ionizable

protons, each with its own pKa.
• The carboxylic acid has an acidic pKa and will
be protonated at an acidic (low) pH:
−COOH ↔ COO− + H+
• The amino group has a basic pKa and will be
protonated until basic pH (high) is achieved:
−NH4+ ↔ NH3 + H+
Amino Acids Have Characteristic Titration Curves

• At low pH, the amino acid exists in a positively charged form (cation).
• At high pH, the amion acid exists in a negatively charged form (anion).
Cation → Zwitterion → Anion

Amino Acids Can Act as Buffers.

 Chemical Environment Affects pKa Values
• -carboxy group is much more acidic than in carboxylic acids.
• -amino group is slightly less basic than in amines.
 Amino Acids Carry a Net Charge of Zero
at a Specific pH (the pI)
• Zwitterions predominate at pH values between the pKa values of the amino
and carboxyl groups.
• For amino acids without ionizable side chains, the Isoelectric Point
(equivalence point, pI) is:
pK1 + pK 2
pI =
2
• At this point, the net charge is zero.
– AA is least soluble in water.
– AA does not migrate in electric field.
• Titration Curves Predict the Electric Charge of Amino Acids.
• Ionizable side chains can be also titrated and influence the pI of the
amino acid.
For glycine,
 Amino Acids Polymerize to Form Peptides
• Peptides are small condensation products of amino acids.
• They are “small” compared with proteins (Mw < 10 kDa).
• Dipeptide, tri- , tetra- , penta- , etc.
• Numbering (and naming) starts from the amino terminus (N-terminal).

serylglycyltyrosylalanylleucine
peptide bond Ser-Gly-Tyr-Ala-Leu
SGYAL
 Peptides: A Variety of Functions
• Hormones and pheromones
– insulin (think sugar metabolism)
– 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)
 Conjugated Proteins Are Covalently Bound to
a Nonprotein Entity
• Some proteins contain permanently associated chemical
components in addition to amino acids (conjugated proteins).

TABLE 3-4 Conjugated Proteins

Class Prosthetic group Example

Lipoproteins Lipids β1-Lipoprotein of blood

Glycoproteins Carbohydrates Immunoglobulin G
Phosphoproteins Phosphate groups Casein of milk
Hemoproteins Heme (iron porphyrin) Hemoglobin
Flavoproteins Flavin nucleotides Succinate dehydrogenase
Metalloproteins Iron Ferritin
Zinc Alcohol dehydrogenase
Calcium Calmodulin
Molybdenum Dinitrogenase
Copper Plastocyanin
Studying Proteins and Peptides Sometimes Requires
Purification from a Mixture
• To study a protein in detail, the researcher must be able to separate it from
other proteins in pure form. A pure preparation is essential before a protein’s
properties and activities can be determined.
• The source of a protein is generally tissue or microbial cells. The first step in any
protein purification procedure is to break open these cells, releasing their
proteins into a solution called a crude extract.
• Separation relies on differences in physical and chemical properties:
– Charge, size, affinity for a ligand, solubility, hydrophobicity, thermal stability
• The extract is subjected to treatments that separate the proteins into different
fractions based on a property such as size or charge, a process referred to as
fractionation.
• Early fractionation steps in a purification utilize differences in protein solubility,
which is a complex function of pH, temperature, salt concentration, and etc.
• The solubility of proteins is lowered in the presence of some salts, an effect
called “salting out.” The addition of certain salts ((NH4)2SO4) in the right amount
can selectively precipitate some proteins, while others remain in solution.
– After salting-out, salt must be removed by dialysis.
 Column Chromatography

• The most powerful methods for

fractionating proteins make use of
column chromatography, which takes
advantage of differences in protein
charge, size, binding affinity, and etc.
• Column chromatography allows
separation of a mixture of proteins
over a solid phase (porous matrix)
using a liquid phase to mobilize the
proteins.
• Proteins with a lower affinity for the
solid phase will wash off first; proteins
with higher affinity will retain on the
column longer and wash off later.

• Chromatographic methods are typically enhanced by the use of HPLC, or

high-performance liquid chromatography.
Separation by Charge: Ion Exchange
• In cation-exchange chromatography,
the solid matrix has negatively
charged groups.
• Proteins with a net positive charge
migrate through the matrix more
slowly than those with a net
negative charge.

At pH<pI, an amino acid has a net positive charge.

At pH>pI, it has a net negative charge.
Separation by Size: Size Exclusion
= Gel filtration chromatography
Separation by Binding: Affinity
Proteins Can Be Separated and Characterized by
Electrophoresis

• Separation in analytical scale is commonly done by electrophoresis.

– The electric field pulls proteins according to their charge.
– The gel matrix hinders mobility of proteins according to their size and
shape.
– The gel is commonly polyacrylamide, so separation of proteins via
electrophoresis is often called polyacrylamide gel electrophoresis, or
PAGE.
• Proteins can be visualized as well as separated, permitting a researcher to
estimate quickly the number of different proteins in a mixture or the
degree of purity of a particular protein preparation.
– Proteins are visualized by adding a dye such as Coomassie blue. Also,
silver staining, fluorescent dyes can be used.
• Electrophoresis can be used to determine crucial properties of a protein
such as its isoelectric point and approximate molecular weight.
• The migration of a protein in a gel during electrophoresis is a function of its size
and charge.
• Non-denaturing (=Native) PAGE: separated by size & charge
• Denaturing (SDS) PAGE: separated by only size
– SDS (sodium dodecyl sulfate) – a detergent
– SDS binds to proteins and facilitate unfolding (→denature).
– SDS gives all proteins a uniformly negative charge.
– The rate of movement will only depend on size: small proteins will move faster.
SDS-PAGE Can Be Used to Calculate the Molecular
Weight of a Protein

이동거리와 단백질의 분자량 (size)은 반비례합니다!

Isoelectric Focusing Can Be Used to Determine
the pI of a Protein
Isoelectric Focusing and SDS-PAGE Are Combined in
2D Electrophoresis

• Thousands of cellular proteins can be

resolved using this technique.
• Individual protein spots can be cut out
of the gel and identified by mass
spectrometry (➔ Proteomics)
 Specific Activity Describes the Purity of the
Protein of Interest
• Proteins in a complex mixture often require more than one purification to
completely isolate the protein of interest.
• During purification, determination of the location of the protein of interest can
be performed by tracking its function.
• The function of the protein is called the “activity.”
• The ratio of activity to total protein concentration is called the “specific activity.”
• In a successful purification step, the loss of nonspecific (unwanted) protein is
much greater than the loss of activity; therefore, specific activity increases even
as total activity falls.

Same activity
Different specific activity
(→increasing)

Protein is purified
 The Structure of Proteins: Primary Structure

• Primary structure of a protein is the sequence of amino acid residues.

• Secondary structure refers to particularly stable arrangements of amino acid
residues giving rise to recurring structural patterns.
• Tertiary structure describes all aspects of the three-dimensional folding of a
polypeptide.
• When a protein has two or more polypeptide subunits, their arrangement in
space is referred to as quaternary structure.
 The Function of a Protein Depends on
Its Amino Acid Sequence
• Proteins with different functions always have different amino acid sequences.
• It is essential to further biochemical analysis that we know the sequence of the
protein we are studying.
• The actual sequence is generally determined from the DNA sequence.
• Edman degradation (classical method):
– successive rounds of N-terminal modification, cleavage, and identification
– can be used to identify protein with known sequence
• Mass spectrometry (modern method):
– MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus
the amino acid sequence
– can be used to determine posttranslational modifications
Direct protein sequencing

Fred Sanger (1950s) determined the

sequence of the protein insulin.
Edman’s Degradation for Protein Sequencing
• N-terminal amino acids can be sequenced by Edman’s Degradation.
Enzymes called proteases catalyze the hydrolytic
cleavage of peptide bonds
• Some proteases cleave only the peptide bond adjacent to particular amino
acid residues.

The Specificity of Some Common Methods for Fragmenting Polypeptide

TABLE 3-6
Chains
Reagent (biological source)a Cleavage pointsb
Trypsin (bovine pancreas) Lys, Arg (C)
Submaxillary protease (mouse submaxillary gland) Arg (C)
Chymotrypsin (bovine pancreas) Phe, Trp, Tyr (C)
Staphylococcus aureus V8 protease (bacterium S. aureus) Asp, Glu (C)
Asp-N-protease (bacterium Pseudomonas fragi) Asp, Glu (N)
Pepsin (porcine stomach) Leu, Phe, Trp, Tyr (N)
Endoproteinase Lys C (bacterium Lysobacter enzymogenes) Lys (C)
Cyanogen bromide Met (C)
aAll reagents except cyanogen bromide are proteases. All are available from commercial sources.
bResidues furnishing the primary recognition point for the protease or reagent; peptide bond cleavage occurs on either the
carbonyl (C) or the amino (N) side of the indicated amino acid residues.
Mass Spectrometry Offers an Alternative Method to
Determine Amino Acid Sequences
Electrospray ionization mass Obtaining protein sequence information
spectrometry of a protein with tandem MS (MS/MS)
 Protein Sequences as Clues to Evolutionary
Relationships
• Sequences of proteins with identical functions from a wide range of species
can be aligned and analyzed for differences.
• Differences indicate evolutionary divergences.
• Analysis of multiple protein families can indicate evolutionary relationships
between organisms, ultimately the history of life on Earth.
• When a series of related nucleic acid or protein sequences are compared, a
consensus sequence is the one that reflects the most common base or amino
acid at each position.

• Members of a family of proteins that

share a common ancestor are called
homologous proteins, or homologs.
• Paralogs: two proteins in a family
present in the same species
• Orthologs: homologs from
different species
Evolutionary tree derived from amino A consensus tree of life
acid sequence comparisons

• The tree is based on analyses of many

• The tree is based on the GroEL family different protein sequences and
of proteins. additional genomic features
Problems

a. maximum protonation occurs at the lowest pH (the highest [H]).

b. at the first pKa, or pK1 (2.34), it is half deprotonated. The average net charge is (+1)+(0)/2 = ½
c. -amino group is half-deprotonated at its pKa, or pK2 (9.60).
d. pH = pKa + log ([A-]/[HA]). If pH = pKa, log ([A-]/[HA]) = 0. [A-]/[HA] = 1.  [A-] = [HA]; half-deprotonated
e. -amino group is half-deprotonated at its pKa.
f. II and IV; in the pKa regions
g. III; at the isoelectric point (pI)
h. III; the pH at which 1.0 equivalent of OH- has been added, pH 5.97
i. V; pH 11.3
j. III; at pI (5.97)
k. V; both groups are fully deprotonated, with a neutral amino group and a negatively charged carboxyl group
l. II; the carboxyl group is half ionized at pH = pK1.
m. III
n. V
o. I, III, and V;
Problems

Assuming that the average Mr per residue is 110 (corrected for loss of water in formation of the peptide
bond), a protein containing 682 residues has an Mr of approximately (682 x 110) 75,000.

a. For carboxyl-terminal pK1 = 2.34. For amino-terminal, pK2=9.67. For pKa values of R groups, Glu (4.25), His
(6.00), and Arg (12.48) residues

b. Find the two ionizable groups with pKa values that “straddle” the point at which net peptide charge = 0
(here, two groups that ionize near pH 8): the -amino group of Glu and the His imidazole group.

The protein has four subunits, with molecular masses of 160, 90, 90, and 60 kDa. The two 90 kDa subunits
(possibly identical) are linked by one or more disulfide bonds.
Problems

a. specific activity = fold

b. Step 4, ion-exchange chromatography; this gives the greatest increase in specific activity
c. Step 3, pH precipitation; two-thirds of the total activity from the previous step was lost here.
d. Yes. The specific activity did not increase further after step 5. SDS-PAGE is an excellent, standard way of
checking homogeneity and purity.
Problems

Protein C has a net negative charge because there are more Glu and Asp residues than Lys, Arg, and His residues.
Protein A has a net positive charge. Protein B has no net charge at neutral pH.
A cation-exchange column has a negatively charged polymer, so protein C interacts most weakly with the column
and is eluted first, followed by B, then A.