Lecture 5: Medical Biochemistry, Biomolecules: Peptides and Proteins

1. Biomolecules:
Peptides and Proteins
Lecture 5, Medical Biochemistry

2. Lecture 5 Outline
• Overview of amino acids, peptides and the
peptide bond
• Discuss the levels of protein structure
• Describe techniques used for analysis of
proteins

3. Planar nature of the peptide bond. The
partial double bond characteristic prevents
free rotation around the C-N bond; keeping it
in the same plane with the attached O and H
atoms. These planar bonds can pivot around
the shared Cα atom

4. Levels of Protein Structure

5. Protein Structure Levels
• PRIMARY: the linear sequence of
amino acids linked together by peptide
bonds
• SECONDARY: regions within
polypeptide chains with regular,
recurring, localized structure stabilized
by H-bonding between constituent
amino acid residues

6. Protein Structure Levels (cont)
• TERTIARY: the overall three-
dimensional conformation of a protein
• QUATERNARY: the three-dimensional
conformation of a protein composed of
multiple polypeptide subunits
• THE PRIMARY AMINO ACID
SEQUENCE IS THE ULTIMATE
DETERMINANT OF FINAL PROTEIN
STRUCTURE

7. Ex: INSULIN
Disulfide bonds
Form between two intra-
or interchain cysteine
residues, product called
cystine
- Stabilizes/creates protein
conformation
- Prevalent in extracellular/
secreted proteins

8. Stabilizing Forces
1. Electrostatic/ionic 3. Hydrophobic interactions
2. Hydrogen bonds 4. Disulfide bonds

10. 2o
Structure: α-helix
each oxygen of a carbonyl
group of a peptide bond
forms a H-bond with the
hydrogen atom attached to
a nitrogen in a peptide
bond 4 amino acids further
along the chain; very stable
structurally; prolines will
disrupt helix formation

11. End-on view of α-helix

12. Parallel
Anti-Parallel
β-sheet
In this secondary structure, each amino acid residue is rotated
180o
relative to its adjacent residue. Occur most commonly in
anti-parallel directions, but can also be found in parallel. H-bonds
between adjacent chains aid in stabilizing the conformation.

13. β-bend
Super-secondary structure
examples

14. Super-secondary structures
commonly found in some
DNA-binding proteins

15. Domains, examples:
Saddleβ-Barrel Bundle

16. Ex: Tertiary Structure Ex: Quaternary Structure

17. Myoglobin β-subunit Hemoglobin

18. Structure of Myoglobin and
Hemoglobin
• The amino acid sequences of myoglobin
and hemoglobin are similar (or, highly
conserved) but not identical
• Their polypeptide chains fold in a similar
manner
• Myoglobin is found in muscles as a
monomeric protein; hemoglobins are found
in mature erythrocytes as multi-subunit
tetrameric proteins. Both are localized to
the cytosol

19. Sequence Comparison Examples
Myoglobin
Hbα (horse)
Hbβ (horse)
Hbα (human)
Hbβ (human)
Hbγ (human)
Hbδ (human)
Myoglobin
Hbα (horse)
Hbβ (horse)
Hbα (human)
Hbβ (human)
Hbγ (human)
Hbδ (human)
(Internal helix)
(Surface helix)

20. Myoglobin Properties
• At the tertiary level, surface residues prevent one
myoglobin from binding complementarily with
another myoglobin; thus it only exists as a
monomer.
• Each monomer contains a heme prosthetic group: a
protoporphryin IX derivative with a bound Fe2+
atom.
• Can only bind one oxygen (O2) per monomer
• The normal physiological [O2] at the muscle is high
enough to saturate O2 binding of myoglobin.

21. Heme-Fe2+ Protein-Heme Complex
with bound oxygen
Heme Structure

22. Hemoglobin Properties
• At the tertiary level, the surface residues of the α
and β subunits form complementary sites that
promote tetramer formation (α2β2), the normal
physiological form of hemoglobin.
• Contains 4 heme groups, so up to 4 O2 can be bound
• Its physiological role is as a carrier/transporter of
oxygen from the lungs to the rest of the body,
therefore its oxygen binding affinity is much lower
than that of myoglobin.
• If the Fe2+ becomes oxidized to Fe3+ by chemicals
or oxidants, oxygen can no longer bind, called
Methemoglobin

23. Biochemical Methods to Analyze
Proteins
• Electrophoresis
• Chromatography: Gel filtration, ion
exchange, affinity
• Mass Spectrometry, X-ray
Crystallography, NMR
• You will not be tested on the sections in
your textbook describing amino acid
separations (Ch 4), peptide/protein
sequencing and synthesis (Ch 5), and
X-ray crystallography/NMR (Ch 6)

24. Protein Separation by SDS-
Polyacrylamide Gel Electrophoresis
Presence of SDS, a detergent,
denatures and linearizes a protein
(Na and sulfate bind to charged
amino acids, the hydrocarbon chain
interacts with hydrophobic residues).
An applied electric field leads to
separation of proteins based on size
through a defined gel pore matrix.
For electrophoresis in the absence
of SDS, separation is based on size,
charge and shape of the protein
(proteins are not denatured and can
potentially retain function or activity)

25. SDS-Polyacrylamide Gel (cont)
Separation of proteins
based on their size is
linear in relation to the
distance migrated in the
gel. Using protein
standards of known
mass and staining of the
separated proteins with
dye, the mass of the
proteins in the sample
can be determined. This
is useful for purification
and diagnostic purposes.

26. Gel filtration
Separation is based on protein size.
Dextran or polyacrylamide beads of
uniform diameter are manufactured
with different pore sizes. Depending
on the sizes of the proteins to be
separated, they will enter the pore if
small enough, or be excluded if they
are too large.
Hydrophobic Chromatography
Proteins are separated based on their
net content of hydrophobic amino
acids. A hydrocarbon chain of 4-16
carbons is the usual type of resin.

27. Separation of proteins based on
the net charge of their constituent
amino acids. Different salt
concentrations can be used to elute
the bound proteins into tubes in a
fraction collector. As shown below,
resins for binding (+) or (-) charged
proteins can be used
Ion Exchange Chromatography

28. Affinity Chromatography
• Based on the target proteins ability to bind a
specific ligand, only proteins that bind to this
ligand will be retained on the column bead. This is
especially useful for immunoaffinity purification
of proteins using specific antibodies for them.
• Example:

29. Protein Structure Methods
• The sequence of a protein (or peptide) is
determined using sophisticated Mass
Spectrometry procedures. The three
dimensional structures of proteins are
determined using X-ray crystallographic and
NMR (nuclear magnetic resonance)
spectroscopic methods.
• Protein sequence data banks useful for
structural and sequence comparisons
• Please note that the new discipline termed
“Proteomics” is evolving to incorporate cross-
over analysis of sequence data banks, Mass
Spec methodology, and living cells