Protein Structure/Function
C483 Spring 2013
1. Proteins segments which fold first can promote the folding of other
sections of the protein into the native conformation by a process known as
A) renaturation.
B) stabilization.
C) hydrophobic interaction.
D) disulfide bridge formation.
E) cooperativity.
2. Hydrophobic amino acid sequences in myoglobin are responsible for
A) covalent bonding to the heme prosthetic group.
B) the folding of the polypeptide chain.
C) the irreversible binding of oxygen.
D) A and B above.
3. A hyperbolic binding curve differs from a sigmoidal binding curve in that
the hyperbolic curve
A) has a single equilibrium constant for binding.
B) always has a higher dissociate constant than a sigmoidal curve.
C) shows cooperativity.
D) suggests multiple ligand binding.
E) All of the above.
4. Conditions in the tissues which enhance the delivery of oxygen by
hemoglobin are the presence of
A) more carbon dioxide present.
B) 2,3 BPG present.
C) lower pH.
D) All of the above.
E) A and B above.
5. True/False The hydrophobic crevice of globin prevents complete electron
transfer to the oxygen so that the electron returns to the iron atom when oxygen
dissociates.
6. True/False Myoglobin has a greater affinity for oxygen than hemoglobin.
Announcements
• MLK Jr—No Discussion Next week
• Discussion worksheets will be posted
• Exam preparation
– Review summaries
– Review Notes
– Homework Problems
• Study Guide for more practice
• Applications from Discussion
Experiments in Protein Folding
• Heat
Denaturation
• Renaturation
– Often reversible
– Primary
sequence
contains folding
information
RNase A
• What happens to
RNase A when
– 2-mercaptoethanol
is added?
– When urea is
added
– When both are
added?
Chaotropic agent
Scientists Draw Conclusions
Protein Folding
• Importance of
Hydrophobicity
• Cooperativity
• Domains fold
separately
– Less than 200
aa is relatively
fast
Chaperones
• Global energy
minimum versus local
energy minimum
• Prevent misfolding or
correct misfolding
Case Study: Hemoglobin
•
•
•
•
•
Myoglobin
Hemoglobin
Heme
Oxygen delivery
Oxygen binding
Structure of Myoglobin
• Noncovalent binding
• Hydrophobic pocket
• Proximal and distal His
Reversible Oxygen Binding
Structure of Hemoglobin
• Oligomer of four units resembling Mb
• a2b2 tetramer
• Treated as dimer of ab units
Binding Constants and Curves
• Simple mechanism is best!
• From point of view of association:
Protein + Molecule of Interest
Complex
Ka = [Complex]/[Free Protein][M]
• Take home message:
– Higher Ka means greater percentage bound
• If Ka greater than 108, “tight binding”
• If Ka less than 104, “little binding”
– Applies to protein/protein interactions (4.9)
Dissociation Constants
• Assume excess of
molecule of
interest
• KD = [P][M of I]
[Bound P]
Plot of % bound
protein as a function
of amount of
molecule of interest
Hyperbolic curve for linear scale
Becomes sigmoidal for semi-log scale
Apply to Myoglobin
• Y = fraction bound (level
of saturation)
• Linear scale
• Myoglobin is half
saturated at 2.8 torr O2
• In tissue, myoglobin is
nearly saturated
Binding Curve of Hemoglobin
• Hemoglobin is halfsaturated at 26 torr O2
• Hemoglobin has less
affinity for oxygen
• Hb saturated in lungs
• When it reaches tissues,
oxygen is released
• Steep in important
region
• But why sigmoidal?
Conformational Equilibrium
• R
T
• Relaxed state binds
oxygen well (low halfsaturation)
• Tense state binds
oxygen poorly
• Equilibrium lies toward
Tense state
Hb Conformations
• Binding of Oxygen
changes shape of unit
• Shape of subunit affects
shapes of other
subunits
– Oxygen-bound unit
causes other subunits to
become relaxed
– The rich become richer
– Cooperative binding
Observed Curve
• At low [O2], curve looks
like T
• At high [O2], curve looks
like R
• The more oxygen that
gets bound, the better
more oxygen gets bound
• Oxygen binding and offloading in the range of
tissue oxygen
Why is Tight Favored?
• Allosteric regulation
• 2,3-BPG
• Holds all subunits toward
the “Tight” conformation
• No 2,3-BPG leads to halfsaturation at 12 torr
• Physiological problem?
Bohr Effect
• pH also effects oxygen
binding
– Anaerobic exercise
– High CO2 concentration
• Lower pH leads to
protonation of protein
• Ion pairs form that
stabilize the deoxy
(unbound) form
Answers
1.
2.
3.
4.
5.
6.
E
B
A
D
T
T