AMINO ACIDS
The building blocks for proteins
Garrett and Grisham, Biochemistry, Third Edition
What You Need to Know
• Four basic classes of amino acids
– What are general characteristics and importance for each class?
– acid-base properties?
– How do they form peptide bonds?
– What other way can they be classified?
• Biological characteristics of some rare amino acids?
– i.e. Hydroxylysine, hydroxyproline, gamma-aminobutyric acid
(GABA), histamine
• What are the acid base properties of amino acids?
• What are the spectroscopic properties of amino acids?
• Understand the fundamental structural pattern of amino acids in
proteins.
• Understand protein structure nomenclature.
Garrett and Grisham, Biochemistry, Third Edition
AMINO ACIDS USED IN LIVIING ORGANISMS
GENERAL CONSIDERATIONS
• There are a host of amino acids yet only
20 unique amino acids are used to make
proteins
• Amino acids in proteins contain a central
tetrahedral carbon atom
• Amino acids polymerize in fabricating
proteins via peptide bonds
Garrett and Grisham, Biochemistry, Third Edition
Amino Acids
Building Blocks of Proteins
Anatomy of an amino acid. Except for proline and its derivatives, all of the
amino acids commonly found in proteins possess this type of structure.
Amino Acids Can Join Via Peptide Bonds
The -COOH and NH3+ groups of two
amino acids can
react with the
resulting loss of a
water molecule to
form a covalent
amide bond.
What is the Fundamental Structural Pattern in Proteins?
Peptide formation is the creation of an amide bond between
the carboxyl group of one amino acid and the amino group of
another amino acid.
Garrett and Grisham, Biochemistry, Third Edition
The Peptide Bond
The trans conformation of the peptide bond.
Garrett and Grisham, Biochemistry, Third Edition
What is the Fundamental Structural Pattern in Proteins?
(a) The peptide bond has partial double bond character. One of
the postulated resonance forms is shown here.
Garrett and Grisham, Biochemistry, Third Edition
What is the Fundamental Structural Pattern in Proteins?
(b) The peptide bond has partial double bond character. One of
the postulated resonance forms is shown here.
Garrett and Grisham, Biochemistry, Third Edition
What is the Fundamental Structural Pattern in Proteins?
(c) The peptide bond is best described as a resonance hybrid
of the forms shown on the two previous slides.
Garrett and Grisham, Biochemistry, Third Edition
The Peptide Bond
• Is usually found in the trans conformation
• Has partial (40%) double bond character
• Is about 0.133 nm long - shorter than a typical
single bond but longer than a double bond
• Due to the double bond character, the six
atoms of the peptide bond group are always
planar
• N partially positive; O partially negative
Garrett and Grisham, Biochemistry, Third Edition
What is the Fundamental Structural Pattern in Proteins?
• Proteins are unbranched polymers of amino acids
• Amino acids join head-to-tail through formation of
covalent peptide bonds
• Peptide bond formation results in release of water
• The peptide backbone of a protein consists of the
repeated sequence –N-Cα-Co• “N” is the amide nitrogen of the amino acid
• “Cα” is the alpha-C of the amino acid
• “Co” is the carbonyl carbon of the amino acid
Garrett and Grisham, Biochemistry, Third Edition
What is the Fundamental Structural Pattern in Proteins?
The coplanar relationship of the atoms in the amide group is
highlighted here by an imaginary shaded plane lying between
adjacent α-carbons.
Garrett and Grisham, Biochemistry, Third Edition
20 Amino Acids IN PROTEINS
On the basis of their R groups the
amino acids can be categorized as:
Non-polar amino acids
Polar, uncharged amino acids
Polar, charged amino acids
Garrett and Grisham, Biochemistry, Third Edition
Note + charge on histidine which
has a side chain pKa = 6.4
Thus, 10% of His side chains are
positively charged at pH 7.4.
Polar, acidic (negatively charged) amino acids
Polar, basic (positively charged) amino acids
AMINO ACID R GROUPS
SUMMARY OF FUNCTIONS
• Non polar
a) establish folding pattern (globular)
b) promote hydrophobic interactions
• Polar,
uncharged and charged
a) establish folding pattern (fibrous)
b) promote specific interactions
His, Ser, Lys, Asn, Cys
Several Amino Acids Occur in
Proteins as a result of
posttranslational modifications
•
•
•
•
Hydroxylysine, hydroxyproline - collagen
Carboxyglutamate - blood-clotting proteins
Pyroglutamate – in bacteriorhodopsin
GABA, Histamine, serotonin
Garrett and Grisham, Biochemistry, Third Edition
Marfan’s Syndrome
• Genetic disorder (dominant trait)
• Fibrillin protein is mutated
• Secondary to mutated fibrillin is
excessive transforming
growth factor beta (TGF-β)
• Weakens tissue causing
features of Marfans (long limbs,
aortic root dialation, depressed
sternum, hyper-flexible joints
•Narrow jaws and high arched palate
creating dental and orthodontal
problems
• partially dislocated lens (80% of
patients), early onset glaucoma
Garrett and Grisham, Biochemistry, Third Edition
Garrett and Grisham, Biochemistry, Third Edition
Garrett and Grisham, Biochemistry, Third Edition
Garrett and Grisham, Biochemistry, Third Edition
Titration of glutamic acid.
Acid-Base Properties of Amino Acids
• Amino Acids are Weak Polyprotic Acids
• Each amino acid has at least two
chemical groups capable of acting as an
acid or base.
• Details of these transformations are
shown on the next slides.
Garrett and Grisham, Biochemistry, Third Edition
pKa Values of the Amino Acids
You should know these numbers and know
what they mean!
• Alpha carboxyl group - pKa = 2
• Alpha amino group - pKa = 9
• These numbers are approximate, but
entirely suitable for our purposes.
Garrett and Grisham, Biochemistry, Third Edition
The ionic forms of an amino acid, shown without consideration of any ionizations on
the side chain.
pKa Values of the Amino Acid
R Groups
• Arginine, Arg, R: pKa(guanidino
group) = 12.5
• Aspartic Acid, Asp, D: pKa = 3.9
• Cysteine, Cys, C: pKa = 8.3
• Glutamic Acid, Glu, E: pKa = 4.3
• Histidine, His, H: pKa = 6.0
Garrett and Grisham, Biochemistry, Third Edition
pKa Values of the Amino Acid
R Groups, cont.
•
•
•
•
Lysine, Lys, K: pKa = 10.5
Serine, Ser, S: pKa = 13
Threonine, Thr, T: pKa = 13
Tyrosine, Tyr, Y: pKa = 10.1
Garrett and Grisham, Biochemistry, Third Edition
Titration of glycine, a simple
amino acid. The isoelectric
point, pI, the pH where the
molecule has a net charge of
0, is defined as (pK1+ pK2)/2.
Titration of lysine.
Reactions of Amino Acids
• Carboxyl groups form amides & esters
• Amino groups form Schiff bases and
amides
• Side chains show unique reactivities
– Cys residues can form disulfides and
can be easily alkylated
– Few reactions are specific to a single
kind of side chain
Garrett and Grisham, Biochemistry, Third Edition
Spectroscopic Properties
• Phe, Tyr, and Trp absorb at UV
wavelengths
• Absorbance at 280 nm is a good
diagnostic device for amino acids
• Absorbance at 190 nm is characteristic of
peptide bonds and is therefore a good
method for detecting proteins which
contain few aromatic amino acids.
Garrett and Grisham, Biochemistry, Third Edition
The ultraviolet
absorption spectra of
the aromatic amino
acids at pH 6. (From
Wetlaufer, D.B.,
1962. Ultraviolet
spectra of proteins
and amino acids.
Advances in Protein
Chemistry 17:303–
390.)
Amino Acid Purification Methods
• Chromatographic Methods
– Ion exchange
– Reversed Phase
Garrett and Grisham, Biochemistry, Third Edition
Cation (a) and anion (b)
exchange resins commonly
used for biochemical
separations.
Operation of a cation exchange column,
separating a mixture of Asp, Ser, and Lys.
a) The cation exchange resin in the beginning,
Na+ form.
(b) A mixture of Asp, Ser, and Lys is added to
the column containing the resin.
(c) A gradient of the eluting salt (e.g., NaCl) is
added to the column. Asp, the least positively
charged amino acid, is eluted first.
(d) As the salt concentration increases, Ser is
eluted.
(e) As the salt concentration is increased
further, Lys, the most positively charged of the
three amino acids, is eluted last.
Chromatographic
fractionation of a synthetic
mixture of amino acids on
ion exchange columns
A second column with
different buffer conditions is
used to resolve the basic
amino acids.
Reversed phase chromatography
of amino acids
Garrett and Grisham, Biochemistry, Third Edition
The Sequence of Amino Acids in a Protein
•
•
•
•
Is a unique characteristic of every protein
Is encoded by the nucleotide sequence of DNA
Is thus a form of genetic information
Is read from the amino terminus to the
carboxyl terminus
Garrett and Grisham, Biochemistry, Third Edition
Amino acid
composition:
Frequencies of the
various amino acids in
proteins for all the
proteins in the
SWISS-PROT protein
knowledgebase.
These data are
derived from the
amino acid
composition of more
than 100,000 different
proteins (representing
more than 40,000,000
amino acid residues).
The range is from
leucine at 9.55% to
tryptophan at 1.18%
of all residues.
Garrett and Grisham, Biochemistry, Third Edition
Proteins - Large and Small
Garrett and Grisham, Biochemistry, Third Edition
Protein Structure (nomenclature)
 Peptide=short polymers of amino acids
NOTE: each unit=amino acid residue (dipeptide, tripeptide, tetrapeptide)
If >12 residues=oligopeptide
If several dozen residues=polypeptide
 Proteins are composed of 1 or more polypeptide chains
1=monomeric proteins
2 or more = multimeric proteins (homomultimeric or heteromultimeric)
 Multimeric proteins are usually designated by greek letters and
subscripts
i.e.
2=homodimer
2b2=heteromultimer (tetramer) (4 polypeptides of 2 kinds)
Garrett and Grisham, Biochemistry, Third Edition
Protein Structure
 Protein Shape: 2 general classes
 globular
 fibrous
 Primary Structure: amino acid sequence
(NH2 to COO end)
 Secondary Structure: characteristic patterns
of the polypeptide chain extending along 1
dimension - caused by hydrogen bonding
interactions between adjacent amino acid
residues, i.e. helical or pleated sheets
Garrett and Grisham, Biochemistry, Third Edition
Protein Structure
 Tertiary Structure: when the polypeptide
chains bend and fold to assume a more
compact three-dimensional shape
 Quaternary Structure: subunit organization in
proteins
 Conformation Possibilities: rotational
possibilities create many possible orientations
for the protein chain
 proteins have many biological functions, thus
they can be classified by their biological roles
Garrett and Grisham, Biochemistry, Third Edition
Proteins - Large and Small
• Insulin - A chain of 21 residues, B chain of 30
residues -total mol. wt. of 5,733
• Glutamine synthetase - 12 subunits of 468
residues each - total mol. wt. of 600,000
• Connectin proteins - alpha - MW 2.8 million
• beta connectin - MW of 2.1 million, with a
length of 1000 nm -it can stretch to 3000 nm
Garrett and Grisham, Biochemistry, Third Edition
Proteins - Large and Small
Garrett and Grisham, Biochemistry, Third Edition
Proteins - Large and Small
From Table 4.2 Size of protein molecules.
Molecular weights: Insulin, 5,733;
Cytochrome c, 12,500;
Ribonuclease, 12,640;
Lysozyme, 13,930;
Myoglobin, 16,980.
Garrett and Grisham, Biochemistry, Third Edition
Proteins - Large and Small
From: Table 4.2 Size of Protein Molecules
Molecular weights: Hemoglobin, 64,500;
Immunoglobulin, 149,900;
Glutamine synthetase, 600,000.
Garrett and Grisham, Biochemistry, Third Edition