Chem 153A Discussion
Week 4
TA: Alexa Novales
Email: novalesnoelle@gmail.com
Discussion Sections:
Thu 9-9:50, Thu 2-2:50, Fri 12-12:50
Some Helpful Things to Keep in Mind
• What is binding?
• Small molecules can bind to proteins by noncovalent interactions, usually
transient and reversible
• Same noncovalent interactions that dictate protein structure!
• A molecule that binds to a protein reversibly is called a ligand
• A region in the protein where the ligand binds is called the binding
site
• In enzymes, the ligand is called the substrate and the binding site is
called the active site or catalytic site
• Ligand binding has some specificity– proteins can’t bind everything!
Quantitative Description of Binding
• The kinetics of such a process is typically described by the
dissociation rate Kd
dissociation
P [L]
Kd =
=
association
[PL]
↓ Kd = ↓ dissociation = ↑ association = ↑ affinity
Fractional Analysis
• Θ (theta) = fraction of binding sites on
the protein that are occupied
• Θ = occupied binding sites/total
binding sites
• Refer to lecture slides for the
complete derivation of the fractional
saturation equation
came from the
previous slide!
Based on what we know now, which protein
has higher affinity for the ligand?
Right shift = decreased affinity
Left shift = increased affinity
Cooperativity
• Proteins that display cooperativity have multiple binding sites
• Binding at one site affects binding at other sites
• Positive Cooperativity: first binding event increases the affinity at other
binding sites
• Negative Cooperativity: first binding event reduces affinity at other binding
sites
• Cooperativity is easy to spot on fractional saturation plots
• Sigmoidal (S-shaped) = positive cooperativity
• Hyperbolic, reaches 100% saturation = noncooperative
• Hyperbolic, does not reach 100% saturation = negative cooperativity
Hemoglobin and Myoglobin Saturation
Green
Myoglobin
Saturation (Θ)
Hemoglobin
If this was not Hb/Mb, then x would be
[L] (mM)
pO2 (Torr)
Orange
• Sigmoidal
• Hyperbolic
• Cooperativity
• No cooperativity
• Multiple binding • Can have
sites
multiple binding
sites (binding of
one site does not
affect affinity!)
You’ll learn in lecture what
affects affinity for oxygen;
practice determining which way
the curves shift in those
different conditions!
Structural Changes Upon O2 Binding
• In the absence of oxygen, the heme group is
puckered because of the larger radius of the Iron
atom
• Oxygen binding to the heme group reduces the
radius of the Iron, forming a planar heme
• Because of this bond between the iron and the
Proximal His, formation of a planar heme pulls
His F8 toward the heme group
• This results in movement of the F helix– crucial
for Hb function!
Hb undergoes a structural change upon
binding oxygen
Oxygen binding triggers a
T→R conformational
change:
T= Tense State
Lower Affinity
“Deoxyhemoglobin”
R= Relaxed State
Higher Affinity
“Oxyhemoglobin”
• The T→R transition alters ~50
weak noncovalent interactions
(the same ones we covered when
learning about protein structure!)
that lie at the interface of the Hb
subunits
• Keep in mind that BOTH
conformations bind oxygen, just
at different affinities!
Small changes in the position of the F helix alter
the contacts at the interface between subunits
Helices reposition in the subunits when Hb
goes from the T state to the R state
• To look at some of the examples of repositioning, refer to Dr. Gober’s
lecture slides!
T and R State Conformations
• The essential feature of Hb’s T→R transition is that its subunits are so
tightly coupled that the tertiary structural changes within one subunit
bring about quaternary changes in the entire tetrameric protein
The concerted model
suggests that there is
a shift in equilibrium
between the T and R
states– both can exist
when fully bound or
fully unbound, but
there will always be
favorability towards
one.
T= Tense State
More stable
Lower Affinity
“Deoxyhemoglobin”
R= Relaxed State
More flexible
Higher Affinity
“Oxyhemoglobin”
The sequential model
suggests that the shift from
T→R occurs in a stepwise
fashion upon subunit
binding– fully unbound Hb is
always in the T state and will
sequentially shift into the R
state upon occupying the
binding sites.
Remember that these are
both models-- we aren’t
100% certain how
cooperativity works, but
these will do for now!
Week 4 Takeaways
• Proteins can bind small molecules called ligands
• Protein-ligand binding is maintained through noncovalent interactions
• Same noncovalent interactions we’ve learned previously
• Noncovalent interactions allow ligand binding to be transient and reversible
• Kd is a measure of binding affinity
• The lower, the better!
• Cooperativity is when a single binding event affects subsequent binding
events
• Know the difference between positive and negative cooperativity
• Hb exudes positive cooperativity
• Know the T vs R states of Hb