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Key Concepts

• Properties of the 20 amino acids that occur in peptides and proteins

are crucial to the structure and function of proteins.
o stereochemistry
o relative hydrophobicity or polarity
o hydrogen bonding properties
o ionization properties
o other chemical properties

• Condensation of 2 amino acids forms the peptide bond, the amide

linkage holding amino acid residues in peptide and protein

• Properties of the peptide bond have major consequences in terms of

the 3-dimensional structures of proteins

There's an excellent website on amino acids being developed here in the

Department of Biochemistry and Molecular Biophysics; parts of it are still
under construction, but there are links to various very useful parts of it here in
these notes, and indeed parts of it may be used in class.


• Proteins are polymers of α -amino acids:

• There are 20 different amino acids found in proteins and they differ by
the nature of the R group.

• Both the α -amino group (amino group substituent on the α C) and the
α -carboxyl group (carboxyl substituent on the α C) are ionizable.
o α -COOH group: a weak acid, can DONATE its proton, with a
pKa of about 2-3. What's the conjugate base form of the
carboxyl group? Which form is charged, and is it a positive or
a negative charge?
o α -NH2 group: a weak base (there's an unshared pair of electrons
on the N; the neutral amino group can ACCEPT a proton).
What's the conjugate acid form of the amino group? Which
form is charged, and is it a positive or a negative charge?
o pKas of α -amino and α -carboxyl groups are different for
different amino acids, and also are altered if they're the terminal
groups on a chain of amino acids, i.e., a peptide or protein.

• The nonionic form shown above does NOT occur in water. WHY

Predominant form in H2O is the zwitterion: .

Stereochemistry of the amino acids

• α -carbon is asymmetric (has four different substituents) except for one

amino acid, for which the R group is a hydrogen atom.
• amino acids occur as enantiomers (nonsuperimposable complete
mirror images)
• L-amino acids are the naturally occurring enantiomers found in all
• There are naturally occurring D-amino acids, but not in proteins (found
in some bacterial cell wall peptide structures, in some peptide antibiotics,
etc.) (D_L)

• Perspective formulas show stereochemistry; projection formulas CAN be

written "correctly", with convention that horizontal bonds project out of
paper and vertical bonds behind plane of paper, but often biochemists
use projection formulas casually (inaccurately), knowing that if it's in a
protein, it's always an L-amino acid. (See also Fig. 5-3 in Lehninger

• Fig. 5-4 (Nelson & Cox: Lehninger Principles of Biochemistry):

Absolute configurations of D-glyceraldehyde as the reference
compound for α -amino acids. D- and L- apply only to the absolute
configuration around the chiral α carbon; 2 of the 20 amino acids
(threonine and isoleucine) have a second chiral center, requiring the RS
system to describe their structures accurately, but we aren't going to
worry about using the RS system here.

Which of the amino acids does NOT have a chiral center, so has no D/L

Amino Acid Abbreviations

amino acid (or 3-letter 1-letter Mnemonic for 1-
residue in protein) abbreviation abbreviation letter abbreviation
Glycine Gly G Glycine
Alanine Ala A Alanine
Valine Val V Valine
Leucine Leu L Leucine
Isoleucine Ile I Isoleucine
Proline Pro P Proline
Methionine Met M Methionine
Phenylalanine Phe F Fenylalanine
tWyptophan (or tWo
Tryptophan Trp W
Tyrosine Tyr Y tYrosine
Serine Ser S Serine
Threonine Thr T Threonine
Cysteine Cys C Cysteine
Aspartic Acid Asp** D asparDic acid
Glutamic Acid Glu* E gluEtamic acid
Asparagine Asn** N asparagiNe
Glutamine Gln* Q Q-tamine
Histidine His H Histidine
Lysine Lys K (before L)
Arginine Arg R aRginine

* Glx = either acid or amide (when it isn't known which it is)

**Asx = either acid or amide (when it isn't known which it is)

Properties of Amino Acid Side Chains

Side chains ("R groups") provide proteins with unique structural and
functional properties.
Additional C atoms in R groups (after the α C) designated by successive
Greek letters: β , γ , δ , ε , as shown in the structure of the amino acid
LYSINE (Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed., p.
Side chain classes

• The side chains of the amino acids play an essential role in determining
the properties of proteins.

There is a wide diversity in the chemical properties of amino acid side

chains, but they can be grouped into classes, sometimes with overlapping
"membership" (e.g., tyrosine is both aromatic and hydroxyl-containing).
Other classifications are also possible (for example, the 5 classes in
textbook, Fig. 5-5, discussed below). You are expected to know all 20
amino acid structures and their R group properties, including ionization
properties (see table below with "generic" pKa values for groups in
peptides and proteins and links to titration curves, and the PDF of proton
dissociation reactions).

Side Chain Class Amino Acids

glycine, alanine, valine,
leucine, isoleucine
Cyclic proline
phenylalanine, tyrosine,
Hydroxyl-Containing serine, threonine, tyrosine
Sulfur-Containing cysteine, methionine
Basic histidine, lysine, arginine
aspartic acid, glutamic acid,
Acidic and Their Amides
asparagine, glutamine
• Structures and classification below are from Nelson & Cox:
Lehninger Principles of Biochemistry, 3rd ed., Fig. 5-5. States of
ionization are the PREDOMINANT forms found at pH 7.
• Nonpolar, aliphatic R groups
o Gly: quite water-soluble (as is Pro)
o Ala, Val , Leu and Ile: increasing hydrophobicity with
increasing number of C atoms in hydrocarbon chain
o Pro: cyclic (--> unusual properties)
 shares many properties with the aliphatic group
 rigidity of ring plays critical role in protein structure (more
about that later)
o Met: methyl thioether (S-containing)
 quite hydrophobic
 Met's terminal methyl group important in metabolism

• Aromatic R groups
o Phe: phenyl group (linked to β -CH2, so Phe = alanine with a
phenyl substituent on the methylene C)
 VERY hydrophobic.
o Trp: indole functional group on β C
 electronegative atom in ring system
 not as hydrophobic as Phe
 hydrogen bonding capability (donor? acceptor? how
many hydrogen bonds?)
o Tyr: phenylalanine with aromatic OH group (phenolic OH) =
 ionizable (pKa around 10; loss of proton gives phenolate
 hydrogen bonding capability (donor? acceptor? how
many hydrogen bonds?)
 Tyr R group is the least hydrophobic of the 3 aromatic
amino acid side chains.

• Polar, uncharged R groups

o Ser and Thr: aliphatic OH groups, not ionizable in pH range
 pKa values so high that under any biologically reasonable
pH conditions they're polar but not ionizable.
 hydrogen bonding capability (donor? acceptor? how
many hydrogen bonds?)
o Asn and Gln: amide functional groups
 VERY polar, but NOT ionizable
 hydrogen bonding capability (donor? acceptor? how
many hydrogen bonds?)
o Cys: thiol (also called a sulfhydryl group) -- not very polar, and
IS ionizable
 sulfur atom makes protonated -SH group more
hydrophobic than an aliphatic OH group
 thiol DOES lose its proton in physiologically relevant pH
range (pKa about 8.5)
 generates -S (thiolate anion is quite hydrophilic due to the

• Positively charged R groups (sometimes called "basic" R groups)

o Arg: guanidino group
 VERY high pKa (~12+), so a very weak acid (stronger
 carries + charge all across physiological pH range
 resonance forms of guanidino group stabilize protonated
form (charge is delocalized)
 hydrogen bonding capability (donor? acceptor? how
many hydrogen bonds?)
o Lys: ε -amino group (a primary amine)
 pKa about 10
 protonated form (predominates at physiological pH)
carries + charge
 hydrogen bonding capability (donor? acceptor? how
many hydrogen bonds?)
o His: imidazole functional group (has 2 N atoms in 5-membered
unsaturated ring)
 pKa about 6-6.5
 protonated form carries + charge, but at pH 7
predominant form is neutral (despite textbook's
categorization as "positively charged")
 very important player in catalytic activity of many enzymes
 hydrogen bonding capability, and also proton

• Negatively charged R groups (sometimes called "acidic" R groups)

o Asp and Glu: side chain carboxyl groups
 pKa values around 4
 predominant form at physiological pH = carboxylate
 hydrogen bonding capability (donor? acceptor? how
many hydrogen bonds?)
Relative hydrophobicity/hydrophilicity of amino acid R groups

• Table 12.2 (Berg, Timoczko and Stryer, Biochemistry, 5th ed.):

Polarity scale for amino acid residues based on free energy changes
for moving a residue from a hydrophobic environment (dielectric
constant = 2) into H2O.
• Similar trends for relative hydrophobicities in text Table 5-1 (diff.
numerical scale, and not arranged in order of relative polarity)
• Depending on how transfer experiments are done, different absolute
numbers can be obtained, but the general trends of relative polarity are
o Phe, Met, Ile, Leu, Val are very hydrophobic
o Arg, Asp, Lys, Glu, Asn, Gln, and His are quite hydrophilic
o The rest are in between -- neither very polar nor very hydrophobic
• Fig. 5-7: Reversible oxidation of 2 cysteine side chain thiols to form
cystine, or re-reduction to 2 thiols
o disulfide bonds between 2 Cys residues in a (usually
extracellular) protein
o often a critical structural feature in extracellular proteins (stabilize
folded structures, in interior of protein structure)
o When found in intracellular proteins, usually have a functional
Ionization Properties of Amino Acid Functional Groups (in PEPTIDES

• weak conjugate acid/base groups in peptides and proteins crucial to

o only one α -amino and one α -carboxyl group on a peptide or
proteins (at the termini of the chain) because the rest of the α -
amino and α -carboxyl groups are tied up in amide bonds holding
monomers together in polymer (more later)
o side chain ionizable groups (only 7 of the 20 amino acids)
• PDF of the acid dissociation reactions for functional groups of amino
acid residues in peptides and proteins
• ionization states of side chain weak acid groups control charges on
• Note: local environment in peptide or protein determines actual pKa of
that specific group, so the ranges shown below (and the rather arbitrary
"generic" values, rounded off for simplicity) are only the usual expected
ranges for pKa values for the functional groups in peptides and proteins;
the pKa of a specific group in a specific protein can lie significantly
outside the expected range if the local environment is unusual.
• links in table below are to titration curves for that amino acid or
functional group

Group usual pKa range, in peptides & proteins

(approx."generic"pKa )
α -Carboxyl (terminal group of
~3.0 - 4.0 (generic 3.0)
peptide or protein)
Asp, Glu (side chain carboxyl) ~4.0 - 4.5 (generic 4.0)
His (imidazole) ~6.0 - 7.4 (generic 6.5)
Cys (thiol, SH) ~8.5 - 9.0 (generic 8.5)
Tyr (phenolic OH) ~9.5 - 10.5 (generic 10.0)
α -Amino (terminal group of
~8.0 - 9.0 (generic 8.0)
peptide or protein)
Lys (ε -amino) ~9.8 - 10.4 (generic 10.0)
Arg (guanidino) ~12.0 - 12.5 (generic 12.0)

Isoelectric point (pI)

• pI = "isoelectric pH" = "isoelectric point" = pH at which the NET

charge on a molecule is ZERO.
o If pH < pI, net charge is positive (more + than - charges)
o If pH > pI, net charge is negative (more - than + charges)
• pI = the pH exactly halfway between the two pKa values surrounding
the zero net charge equivalence point on the titration curve
(examples to be analyzed in class: Gly and His)

• Fig. 5-10. Titration curve of glycine (Nelson & Cox: Lehninger

Principles of Biochemistry, 3rd ed.)
• Fig. 5-12b. Titration curve of histidine (Nelson & Cox: Lehninger
Principles of Biochemistry, 3rd ed.)
• Molecular separations based on charge properties (paper
electrophoresis of amino acids as an example)
• paper strip soaked in buffer, in contact with 2 reservoirs with electrodes
connected to a power supply

Buffer Buffer
reservoir #1 reservoir #2
(anode; O (cathode;
anions move cations move
toward it) toward it)

origin (sample of an amino acid applied)

• When a voltage is applied, in which direction will the amino acid

move? What do you need to know to answer that question?
o ________________________

o ________________________
• e.g., Histidine: (sample dissolved in buffer, applied at origin above,
and voltage applied)

buffer pH net charge direction of migration


Ultraviolet absorbance of amino acid side chains

• Aromatic amino acids (Trp, Tyr, Phe) absorb light in the near
ultraviolet region of the spectrum (250-300 nm).
• Trp has highest molar absorptivity, followed by Tyr, with Phe making
only a small contribution.
• Disulfide bonds (between Cys residues in proteins) also absorb in the uv
range, but much less than the aromatics.
• Fig. 5-6 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed.):
Absorbance of ultraviolet light by aromatic amino acids
Posttranslational modifications of amino acid side chains

• chemical modifications AFTER biosynthesis of proteins

• occur for a few amino acid residues in some proteins
• Some examples (see also Fig. 5-8, Nelson & Cox: Lehninger Principles
of Biochemistry, 3rd ed.)):
O-Phosphoserine 4-Hydroxyproline 5-Hydroxylysine γ -carboxyglutamate
• reversible phosphorylation and dephosphorylation of Ser, Thr, and Tyr
residues very important in covalent regulation of activity of some
enzymes and many biosignalling proteins, including some hormone
receptors and transcription factors
• 4-hydroxyproline & 5-hydroxylysine important in structure of collagen
(fibrous protein in connective tissue)
• γ -carboxyglutamate important in a number of proteins whose function
involves Ca2+ binding, including several proteins involved in blood

Chemical Reactions of Amino Acids

• All amino acids have at least two reactive groups: the α− amino and α -
carboxyl groups and these groups can react with a variety of reagents.
Here are two examples:

• A particularly interesting example is the green fluorescent protein (GFP)

from the Pacific Northwest jellyfish Aequorea victoria, which has
generated intense interest as a marker for gene expression and
localization of gene products. The chromophore, which results from the
spontaneous cyclization and oxidation of the sequence -Ser65-Tyr66-
Gly67- , is unusual because it does not involve a non-protein
chromophore, as is usually the case for colored proteins. The
chromophore is buried in the interior of GFP.

The Peptide Bond

• Peptides and proteins:polymers of amino acids joined bypeptide bonds

• amide linkages from condensation of α -carboxyl group of one amino
acid with α -amino group of another amino acid

• process repeated many times --> linear chain of amino acids, a

polypeptide chain
• convention: sequence written from left to right starting with residue with
free α -amino group (the N-terminal or amino terminal amino acid
residue) and ending with the residue containing the free α -carboxyl
group (the C-terminal or carboxyl terminal residue),
e.g., NH2-Glu-Gly-Ala-Lys-COOH = EGAK
• average residue mass ~110 (average Mr of the 20 amino acids minus Mr
of H2O)
• a polypeptide chain with 100 amino acid residues would have a Mr of
about 11,000)
• small peptides (a "few" amino acid residues) = oligopeptides

Peptide bond formation endergonic (∆ Go' ~21 kJ/mol)

• (How would a cell make the reaction go in the direction of

condensation in an aqueous environment? no details needed here for
biochemical mechanism -- that's covered in BIOC 411)
• peptide bonds metastable in aqueous environment -- equilibrium lies far
in direction of hydrolysis, but RATE of hydrolysis very slow in absence
of catalyst
• Enzymes that catalyze peptide bond hydrolysis = peptidases or
proteases, e.g., (specific examples of proteases) your digestive proteases
like trypsin and pepsin

Ionization properties of peptides

• analyzed the same way as for free amino acids

• one α -amino group (pKa approx. 8) and one α -carboxyl group (pKa
approx. 3), plus any ionizable side chains on residues in the peptide
• To figure out approximate net charge of a peptide at a given pH:
o make yourself notes on the sequence to keep track of what you're
o add up charges on all the ionizable groups

Example: Fig. 5-14 (Nelson & Cox: Lehninger Principles of Biochemistry, 3rd
ed.): pentapeptide SGYAL = Ser-Gly-Tyr-Ala-Leu
= Serylglycyltyrosylalanylleucine
Amino Acid Analysis

• Sequence of amino acids in a protein is dictated by the sequence of

nucleotides in the gene encoding that protein:

(from Berg, Tymoczko & Stryer, Biochemistry, 5th ed., p. 28)

• Each protein (unique sequence) has unique amino acid composition.

• Can chemically hydrolyze (hot 6N HCl) a pure protein to generate the
free amino acids and determine its amino acid composition
• Because side chains of the amino acids have different properties, can
separate and quantitate all 20 amino acids using a variety of
chromatographic techniques, as illustrated below.
Peptide bond has resonance structures --> partial double bond character

• Due to the partial double bond character of the peptide bond, the O, C, N
and H atoms are nearly planar and there is no rotation about the peptide
bond (peptide). As we shall see later, the planarity of the these elements
has important consequences for the three dimensional structure of

• Generally, the two Cα groups are in a trans configuration, which

minimizes steric interaction (cis/trans).