Transcript Biomolecules: Peptides and Proteins Lecture 5, Medical Biochemistry Lecture 5 Outline • Overview of amino acids, peptides and the peptide bond • Discuss the levels.

Biomolecules:
Peptides and Proteins
Lecture 5, Medical Biochemistry
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
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 Ca atom
Levels of Protein Structure
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
Protein Structure Levels (cont)
• TERTIARY: the overall threedimensional 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
Disulfide bonds
Form between two intraor interchain cysteine
residues, product called
cystine
- Stabilizes/creates protein
conformation
- Prevalent in extracellular/
secreted proteins
Ex: INSULIN
Stabilizing Forces
1. Electrostatic/ionic
2. Hydrogen bonds
3. Hydrophobic interactions
4. Disulfide bonds
2o Structure:
a-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
End-on view of a-helix
b-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.
Anti-Parallel
Parallel
Super-secondary structure
examples
b-bend
Super-secondary structures
commonly found in some
DNA-binding proteins
Domains, examples:
b-Barrel
Bundle
Saddle
Ex: Tertiary Structure
Ex: Quaternary Structure
Myoglobin
b-subunit Hemoglobin
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
Sequence Comparison Examples
(Surface helix)
Myoglobin
Hba (horse)
Hbb (horse)
Hba (human)
Hbb (human)
Hbg (human)
Hbd (human)
(Internal helix)
Myoglobin
Hba (horse)
Hbb (horse)
Hba (human)
Hbb (human)
Hbg (human)
Hbd (human)
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.
Heme Structure
Heme-Fe2+
Protein-Heme Complex
with bound oxygen
Hemoglobin Properties
• At the tertiary level, the surface residues of the a
and b subunits form complementary sites that
promote tetramer formation (a2b2), 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
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)
Protein Separation by SDSPolyacrylamide 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)
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.
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.
Ion Exchange Chromatography
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
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:
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 crossover analysis of sequence data banks, Mass
Spec methodology, and living cells