Module No.

BIOCHEMISTRY

Amino Acids and Peptides 1

Dr. Bato August 17, 2016
 + Levine sign
 initially enzymes measured CK-MB
Outline and troponin I
I. Clinical Cases
A. Clinical Case 1 – SCID
B. Clinical Case 2 – Progeria II. PROTEOMICS & PROTEOME
C. Clinical Case 3 – Myasthenia Gravis Proteomics
D. Clinical Case 4 – Muscular Dystrophy → study of all the proteins in the body which are mostly
E. Clinical Case 5 – Prion Disease unknown function, but we do know that it exists
F. Clinical Case 6 – Acute Myocardial Infarction
II. Proteomics & Proteome
→ Study of proteome
III. Protein  Protein + genome
A. Properties and Functions  Coined by Marc Wilkins (1994)
B. Protein Translation → Analogy to genomics
C. Posttranslational Modifications → Coined in 1997
D. Protein Folding → Interdisciplinary domain benefitting from the human
E. How does body destroy unwanted or damaged proteins?
e.1 Ubiquitin-Proteasome Proteolytic Pathway
genome project
e.2 Lysosomal Degradative Pathway → Experimental analysis: protein purification & mass
e. Amino Acids spectrophotometry
A. Properties → Genomics, transcriptomics, metabolomics
B. Side Chains III. PROTEINS
C. Hydropathy
D. pKa A. Properties & Funtions
E. pl CHARACTERISTICS
F. Peptide Bond → Polypeptides
f. Protein Structure → One of the most important biomolecule
g. Chromatography → Physically and functionally complex
A. Techniques by chromatographic bed shape
→ Made from amino acid
a.1 Column
FUNCTIONS
a.2 Planar
→ Enzymes
B. Techniques using separation mechanism
→ Motor proteins
b.1 Size exclusion
b.2 Ion exchange
→ Receptor proteins
b.3 Hydrophobic interaction → Structural proteins
b.4 Affinity → Storage proteins
C. Techniques based on mobile phase type → Gene regulatory proteins
c.1 High-pressure liquid → Transport proteins
→ Signal proteins
→ Special purpose proteins
LEGEND
Lecture Powerpoint, Audio, Textbook AMINO ACIDS

I. CLINICAL CASES
Severe Combined  immunoglobulin defect/ adenine
Immunodeficiency deaminase enzyme defect
(SCID)  6 month old male infant
 recurrent fever, otitis media, sore
throat, upper respiratory infection
 bubble boy (grew up in an incubator)
 died due to Epstein-Barr virus
Progeria  defective Lamin A
 irreversible aging
 fatal
 athralgia (severe muscle pain) &
myalgia (muscle pain) associated with a
facie of an old man
Myasthenia  autoantibodies against Acetylcholine B. PROTEIN TRANSLATION
Gravis receptor (acetylcholine cannot bind to the  Process whereby biological cells generate new
AchR) proteins; it is balanced by the loss of cellular proteins
 no weakness in morning via degradation or export’
 profound muscle weakness in the  TRANSLATION  assembly of amino acids by
afternoon associated with ptosis ribosomes
Muscular  defect in dystrophin (protein located a) TRANSCRIPTION
Dystrophy between the sarcolemma & outermost  mRNA chain is generated, with one strand of the
layer of myofilaments DNA double helix in the genome as a template
 difficulty in standing associated with
Gower sign b) TRANSLATION
 hypertrophic cal muscles  synthesis of proteins from RNA
Prion Disease  Bovine Spongiform encephalopathy In eukaryotes, translation occurs in the cytoplasm,
 Creutzfeld-Jacob Disease where the ribosomes are located
 Kuru  In activation, the correct amino acid (AA) is joined to
 Scrapie the correct transfer RNA (tRNA). While this is not, in
 neurodegenerative syndrome the technical sense, a step in translation, it is required
 difficulty in standing without support for translation to proceed.
Acute Myocardial  coronary artery blockage causing
Infarction luminal obstruction with noted heart
muscle death
 65 y.o with severe chest pain
associated with diaphoresis (10/10)

[Brodit, Bueno, Canlas, Choy, Francisco, Huevas] Checked by: [Gayados] Page 1 of 8
[Biochemistry] [Amino Acid & Peptides] Module #1, Lecture #4

C. POSTTRANSLATIONAL MODIFICATION

 covalent and generally enzymatic modification of proteins

during or after protein biosynthes
 an occur on the amino acid side chains or at the protein's C-
or N- termini.

D. PROTEIN FOLDING

 process by which a protein structure assumes its functional

shape or conformation
 physical process by which a protein chain acquires its native
3-dimensional structure, a conformation that is usually
biologically functional, in an expeditious and reproducible
manner

[Brodit, Bueno, Canlas, Choy, Francisco, Huevas] Checked by: [Gayados] Page 2 of 8
[Biochemistry] [Amino Acid & Peptides] Module #1, Lecture #4

e.2 Lysosomal Degradative Pathway

– ATP-independent
– Lysosomes
– Exogenous peptides

E. HOW DOES BODY DESTROY UNWANTED OR DAMAGED IV. Amino Acids

PROTEINS?
e.1 Ubiquitin-Proteasome Proteolytic Pathway A. Properties
 ATP-dependent -­‐ Organic acid compound that contains both an amino (-
 Cytosol NH) group and a carboxylic acid (-COOH) functional
 Uses “ubiquitin” (evolutionarily conserved 76-amino acid protein group.
that is central to multiple cellular function)
-­‐ Exhibits amphoteric property, allowing it to function as
 Endogenous peptides either base or acid. Excellent for buffering mechanism.
 Ub molecule contains seven lysine residues, and -­‐ Basic structural and functional monomer subunit of
functional selectivity is provided by diverse patterns of proteins and polypeptides
protein linkage to these amino acids
-­‐ There are more than 300 amino acids known in nature,
 Linkage to some lysines leads to passage of the tagged
but there are more or less 20 amino acid found only in
protein to the proteasome for degradation
human.
 fate of Ub-conjugated proteins among the several -­‐ Amino acids that are generally found in proteins are
pathways is determined by the number of Ub moieties
classified as α-amino acid
conjugated and the site of the conjugation linkages on st
-­‐ Selenocysteine is considered the 21 amino acid
the Ub molecule
 A Ub-activating enzyme, E1, binds to Ub and then transfers it
to one of dozens of Ub-conjugating enzymes (E2). -­‐ Identical as Enantiomerism – Optically active
 These act together with one of about 800 different Ubligating stereoisomers. These are organic compounds with
enzymes (E3) to add Ub to a lysine on the doomed protein identical molecular formula having the same number
 Additional Ub moieties are added to the original Ub, forming a and types of atomic elements, but they different in
polyubiquitin chain (atleast four Ubs) terms of their molecular configuration or atomic spatial
 Proteasomes are highly conserved organelles in the arrangement.
cytoplasm and possibly the nucleus: -­‐ Enantiomers: 2 stereoisomers that are
– barrel-shaped complexes whose main (but not only)
function is to digest polyubiquitinated proteins
nonsuperimposable mirror images.
– two types of proteasomes: 20S and 26S -­‐ Isoleucine and threonine have 2 chiral carbons
– The degradative unit of both is a 20S destruction L and D-aa can be converted to each other using
chamber, to which, in the 26S proteasome, two 19S racemase
“caps” are attached
– The capsat the entrance to the proteolytic core
regulate entry
– The 20S proteasomeslack these caps
– The 20S proteasomes are important in degradation of
oxidized proteins
– In 26S proteasomes, polyubiquitinated proteins are
degraded.
 Mutations that interfere with normal proteasomal function
are lethal

[Brodit, Bueno, Canlas, Choy, Francisco, Huevas] Checked by: [Gayados] Page 3 of 8
[Biochemistry] [Amino Acid & Peptides] Module #1, Lecture #4

-­‐ The α-carbon is ASYMMETRIC – meaning to say that D. pKa

the central α-carbon atom is bounded by 4 different -­‐ Amino acids are known weak acids
groups of molecules or atoms that are molecularly -­‐ Acid strength
oriented in a tetrahedral configuration. -­‐ The larger the Ka, the stronger the acid, because most
of the HA has dissociated into H+ and A–. Conversely,
the smaller the Ka, the less acid has dissociated and,
-­‐ 19 out of the 20 amino acids exhibits chirality except therefore, the weaker the acid.
GLYCINE because there are 2 hydrogen atoms are -­‐ Net charge: alebraic sum of all the (+) and (-) charged
covalently bonded to the α-carbon atom. groups present - depends on the pKa values
-­‐ Altering the charge on the aa by varying the pH
facilitates the physical separation
B. Side Chains
-­‐ Net charge of zero found in zwitterions
-­‐ pKa is affected by the environment
-­‐ A polar environment favors charged form
-­‐ Nonpolar one favors uncharged

E. pl
-­‐ is the pH midway between pKa values of an isoelectric
species
-­‐ zwitterions (dipolar ion)
-­‐ Isoelectric means the molecule has an equal number
of (+) and (-) charges, thus it conforms a Neutral state
-­‐ 0

F. Peptide Bond

-­‐ Amino acids are covalently bonded together by

means of peptide bond (amide linkage) that forms
molecular polypeptide chains.
-­‐ A process called dehydration synthesis will
chemically combine one amino acid to another amino
acid via reaction between the carboxylic acid group of
the preceding (first) amino acid and the amino group of
the succeeding (second) amino acid. This
condensation reaction will lead to the formation of
peptide bond and eventually release water molecule.

C. Hydropathy
-­‐ Important determinant in protein folding that indicates
the relative hydrophilicity or hydrophobicity of each
amino acid
-­‐ High positive values means highly hydrophobic (non-
polar property); high negative ones are hydrophilic Dipeptide = 2 amino acids, 1 peptide bond
(polar property) Tripeptide = 3 amino acids, 2 peptide bonds
Tetrapeptide = 4 amino acids, 3 peptide bonds
Polypeptide = many amino acids, many peptide bonds

[Brodit, Bueno, Canlas, Choy, Francisco, Huevas] Checked by: [Gayados] Page 4 of 8
[Biochemistry] [Amino Acid & Peptides] Module #1, Lecture #4

V. Protein Structure

4 organizational levels of Protein structure

o β- Bends
1. Primary structure
 Generally composed of four amino
-­‐ Defined linear sequence of amino acids in a one dimensional
plane. acids, one of which may be proline,
-­‐ Amino Acid Sequence (Gly-X-Y) the amino acid that causes a kink in
o X-Proline the polypeptide chain. Glycine, the
o Y-HydroxyProline amino acid with the smallest R
group.
 Stabilized by the formation of
hydrogen and ionic bonds.
3. Tertiary structure
-­‐ Assembly of secondary structures into larger functional
polypeptide units that conforms a well-defined three-
dimensional molecular structure. Stabilized by:

 D – Disulfide bridges
 I – Ionic Bonds
 S – Salt Bridges
 H – Hydrogen bonds

-­‐ 3-D spatial behavior: helices, sheets, bends, turns, and

loops
-­‐ Domains: protein structure sufficient to perform a
particular chemical or physical task such as substrate
2. Secondary structure or ligand binding
-­‐ Primary polypeptide structure folds 3 to 30- amino acid -­‐ Triple Helical Bond
residues forming geometrically ordered units via -­‐ Final geometric shape that a protein assumes
intramolecular hydrogen bonding.
-­‐ The way in which the primary structure of a 4. Quaternary structure
polypeptide chain folds (Left Handed Manner) -­‐ Consist of two or more interacting polypeptide chains
each of which is commonly referred to as a oligomeric
• 3 types: protein subunit
o α Helix
-­‐ Triple helix wounded in a Right Handed Manner
 Right-handed α Helix are more
stable due to L-amino acids • Monomer: single polypeptide chain
• Dimer: 2 polypeptide chains
 Spiral structure, consisting of a tightly • Homodimer: 2 copies of the same polypeptide
packed, coiled polypeptide backbone
core, with the side chains of the • Heterodimer: 2 copies of different polypeptide
component amino acids extending
outward from the central axis to avoid
interfering sterically with each other.
 Stabilized by extensive hydrogen
bonding between peptide-bond carbonyl
oxygens and amide hydrogens that are
part of the polypeptide backbone.
 Each turn contains 3.6 amino acids

VI. Chromatography
-­‐ Discovered by Mikhail Tsvet in 1900
-­‐ Based on thin-layer chromatography of separating
plant extracts
-­‐ Depends on the relative affinity of different proteins for
o β Pleated sheets a given stationary phase and for the mobile phase
 Often visualized as broad arrows -­‐ Association between each protein and the matrix is
 Can be formed from two or more weak and transient
separate polypeptide chains that are
arranged either antiparallel to each other
-­‐ Proteins interacting more strongly with the stationary
or parallel to each other phase are retained longer
 Peptide backbone highly extended -­‐ Length of time that a protein is associated with the
 aa residues form a zigzag or pleated stationary phase is a function of the composition of
pattern both stationary and mobile phases
-­‐ Optimal separation achieved by manipulation of the
composition of the 2 phases

[Brodit, Bueno, Canlas, Choy, Francisco, Huevas] Checked by: [Gayados] Page 5 of 8
[Biochemistry] [Amino Acid & Peptides]
A. Techniques by Chromatographic bed shape
a.1 Column
-­‐ Stationary phase: column containing small spherical
beads of modified cellulose, acrylamide, or silica
whose surface has been coated with chemical
functional groups
-­‐ The matrix interacts with proteins based on
1) charge
2) hydrophobicity
3) ligand-binding properties
-­‐ A protein mixture is applied to the column
-­‐ Liquid mobile phase is percolated through it
-­‐ Small portions of the mobile phase or eluant are
collected
a.2 Planar
-­‐ Separation of mixtures in a filter paper based on
partition of a solute between 2 solvents one of which is
immobilized by the substance paper.
B. Techniques using separation mechanism
b.1 Size Exclusion
-­‐ Also known as gel filtration or gel permeation
-­‐ Separates proteins based on their Stokes radius
(sphere diameter) Stoke radius is a function of
molecular mass and shape A tumbling elongated
protein occupies a larger volume than a spherical
protein of the same mass
-­‐ Employs porous beads
-­‐ Proteins with large Stokes radii remain in the eluent
and emerge before proteins that have smaller Stokes
radii and are able to enter the porous beads
-­‐ Proteins emerged via descending order of their Stokes
radii
b.2 Ion Exchange
-­‐ Proteins interact with the stationary phase via charge-
charge interactions
-­‐ Proteins with net (+) charge at a given pH adhere to
beads with negatively charged functional groups such
as carboxylates or sulfates (cation exchangers)
-­‐ Negatively charged proteins adhere to tertiary or
quaternary amines (anion exchangers)
-­‐ Proteins compete with monvalent ions for binding
hence “ion exchange”
-­‐ Example: negatively charged proteins bind to
diethylaminoethyl (DEAE) cellulose via replacing the
Cl- or CH3COO
-­‐ Bound proteins are selectively displaced by gradually
raising the concentration of the monovalent ions in the
mobile phase
-­‐ Proteins elute in inverse order of strength of their
interactions with stationary phase S
-­‐ equential elution achieved via pH manipulation
b.3 Hydrophobic Interaction Chromatography
-­‐ Separates via tendency to associate with a stationary
phase matrix coated with hydrophobic groups (phenyl
Sepharose, octyl Sepharose)
-­‐ Proteins with exposed hydrophobic surfaces adhere to
the matrix via hydrophobic interactions enhanced by a
mobile phase of high ionic strength
-­‐ Nonadherent proteins are washed first
-­‐ Polarity of the mobile phase is then decreased by
gradually lowering salt concentration
-­‐ If interaction between protein and stationary phase is
particularly strong, ethanol or glycerol is added to
[Brodit, Bueno, Canlas, Choy, Francisco, Huevas]
Module #1, Lecture #4
decrease polarity and weaken hydrophobic
interactions
b.4 Affinity Chromatography
-­‐ Exploits the high selectivity of most proteins for their
ligands
-­‐ Enzymes may be purified using immobilized
substrates, products, coenzymes, or inhibitors
-­‐ In theory, only proteins interacting with immobilized
ligands adhere
-­‐ Bound proteins eluted either by competition with
soluble ligand or less selectively by disrupting protein-
ligand interactions using urea, guanidine HCl, mildly
acidic pH, high salt concentration
-­‐ Stationary phase matrices available commercially
contain ligands such as NAD+ or ATP analogs
-­‐ Most powerful and widely applicable affinity matrices
are used for recombinant protein purifications
-­‐ Uses a Ni2+ matrix that binds proteins with an
attached polyhistidine tag and a glutathione matrix that
binds a recombinant protein linked to glutathione S-
transferase.
C. Techniques based on mobile phase type
c.1 High Pressure Liquid Chromatography
-­‐ Employs incompressible silica or alumina microbeads
as stationary phase and pressures of up to a few
thousand psi
-­‐ Incompressible matrices permit both high flow rates
and enhanced resolution
-­‐ Resolve complex mixtures of lipids or peptides whose
properties differ only slightly
-­‐ Reversed phase HPLC exploits a hydrophobic
stationary phase of aliphatic polymers 3 -18 carbon
atoms in length
-­‐ Peptide mixtures are eluted using a gradient of a
water-miscible organic solvent such as acetonitrile or
methanol
c.2 Polyacrylamide Gel Electrophoresis (PAGE)
-­‐ Most widely used method for determining protein purity
-­‐ Uses sodium dodecyl sulfate
-­‐ Electrophoresis separates charged biomolecules
based on the rates at which they migrate in an applied
electrical field
-­‐ Acrylamide is polymerized and cross-linked to form a
porous matrix
-­‐ SDS denatures and binds to proteins at a ratio of one
molecule of SDS per 2 peptide bonds
-­‐ 2-mercaptoethanol or dithiothreitol used to reduce or
break disulfide bonds
-­‐ Large number of anionic SDS molecules overwhelms
the charge contributions of the aa functional groups
-­‐ Charge to mass ratio of each SDS-polypeptide
complex is equal
-­‐ The physical resistance encountered by the
polypeptide determines the rate of migration
c. 3 Isoelectric Focusing
-­‐ Ionic buffers called ampholytes and an applied electric
field are used to generate a pH gradient within a
polyacrylamide marix
-­‐ Applied proteins migrate until they reach the region of
the matrix where the pH at which a molecule’s net
charge is 0
-­‐ Used in conjunction with SDS-PAGE
-­‐ Separates polypeptides based on pI in one dimension
and based on Mr in the second
Checked by: [Gayados] Page 6 of 8
[Biochemistry] [Amino Acid & Peptides] Module #1, Lecture #4

-­‐ Enhances the speed and efficiency of primary

structure analysis
-­‐ Circumvents obstacles brought about by
aminoterminal blocking group or the lack of key
overlap peptide
-­‐ Only a few segments of the primary structure should
be determined using Edman’s approach
-­‐ DNA sequencing reveals the order in which amino
acids are added but does not provide posttranslational
modification
o Proteolytic processing
o Methylation
o Glycosylation
o Phosphorylation
o Proline and lysine hydroxylation
c. 4 Sanger Amino Acid Sequencing
o Disulfide bond formation
-­‐ Frederick Sanger won the Nobel Prize in 1958 for
c.7 Mass spectrometry
determining the amino acid sequence of insulin
-­‐ Replaced Edman’s sequencing
-­‐ Insulin: 21 – aa residue A chain and 30 – aa residue B
-­‐ Posttranslational modification of proteins identified via
chain linked by disulfide bonds
mass increments
-­‐ Separated both chains by reducing the disulfide bonds
-­‐ Discriminates molecules based solely on mass
and cleaved with trypsin, chymotrypsin, and pepsin
-­‐ Can detect subtle physical changes in proteins that
-­‐ Resulting peptides isolated and treated with acid to
occur during the life cycle of a cell or organism
hydrolyze peptide bonds and generated 2 – 3 aa
-­‐ A sample in a vacuum is vaporized under conditions
-­‐ Each peptide was reacted with 1-fluoro-2,4 -
where protonation can occur, imparting a positive
dinitrobenzene (Sanger’s reagent)
charge
-­‐ While the ε-amino group of lysine reacts with Sanger’s
-­‐ An electrical field propels cations through a magnetic
reagent, amino-terminal lysines can be Medical
field which deflects them at right angles to their original
Biochemistry 3 2 of 4 distinguished from other
direction of flight and focuses them onto the detector
positions because they react to 2 mol of the Sanger’s
-­‐ The magnetic force required to deflect the path of each
reagent
ionic species onto the detector measured as the
current applied to the electromagnet is recorded
c. 5 Edman Amino Acid Sequencing
-­‐ Pehr Edman introduced phenylisothiocyanate
(Edman’s reagent)
-­‐ Phenylthiohydantoin (PTH) derivative can be removed
under mild conditions to generate a new amino
terminal residue
-­‐ Successive rounds of derivatization with Edman’s
reagent can be used to sequence the first 20 – 30
residues
-­‐ Most polypeptides must be cleaved into smaller
peptides prior to sequencing
-­‐ Cleavage is necessary to circumvent posttranslational
modifications rendering a proteins α-amino group
unreactive to Edman’s reagent c.8 3-D structures: (XRAY CRYSTALLOGRAPHY)
-­‐ Following cleavage, the peptides are purified by -­‐ Crystals formed via precipitation of proteins
reversed-phase HPLC and sequenced -­‐ Protein nature confirmed via mounting of crystals on
quartz capillaries and irradiated with 0.15nm
monochromatic xray.
-­‐ Crystals frozen in liquid nitrogen d. Recorded
diffraction patterns
-­‐ Fourier Synthesis: summates wave function (data
analysis)
-­‐ Laue xray crystallography:
-­‐ polychromatic xray diffraction
-­‐ avoids crystal rotation

c.6 Hybrid Approach

-­‐ Edman sequencing provides a partial amino acid
sequence
-­‐ Oligonucleotide primers modeled on this partial
sequence are used to ID the gene and amplify it using
PCR
-­‐ Once the gene is ID, oligonucleotide sequence is
determined to infer the primary structure of the
encoded polypeptide

[Brodit, Bueno, Canlas, Choy, Francisco, Huevas] Checked by: [Gayados] Page 7 of 8
[Biochemistry] [Amino Acid & Peptides] Module #1, Lecture #4

c.9 (NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY)

-­‐ Measures radio frequency electromagnetic energy
absorbance
-­‐ Frequency / chemical shift at which a nucleus absorbs
energy: Function of both the functional group (where it
resides) and the proximity of other NMR-active nuclei
Medical Biochemistry 3 2 of 4
-­‐ 2-D NMR: 3-D representation (determination of
proximity of the nuclei from one another)
-­‐ Analyzes proteins in aqueous solutions (conformation
+ ligand binding / catalysis = possible)

c.10 MOLECULAR MODELING

-­‐ Computer technology for 3-D protein structure
-­‐ Molecular dynamics programs: simulates
conformational dynamics of proteins and manner in
which factors (temperature, pH, ionic strength, amino
acid substitutions) influence motion
-­‐ Molecular docking programs: interactions between a
ligand-enzyme, etc
-­‐ Homology modeling: 3-D structure of a protein used as
template to build a model of the structure of a related
protein

VII. Reference

Powerpoint of Dr. Bato

Liipincott's Illustrated Reviews, 6th Edition

[Brodit, Bueno, Canlas, Choy, Francisco, Huevas] Checked by: [Gayados] Page 8 of 8