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• Proteins often consist of multiple domains
– Usually different functions (eg. catalysis, regulation, targeting)
– Often can be physically separated
• Non-covalent interactions: 4 structure
• One polypeptide with multiple ‘independent’ subdomains
• Protein structures fall into a limited number of categories
– Classified according to 2 structure composition
• a, b, a/b, a + b
– Conserved motifs seen, with limited variation, in a number of
proteins
• Note: conservation of structure is a great way to determine an
evolutionary relationship…better than function or sequence
• Protein folding is complex
– How does a protein “know” how to fold?
• Completely due to amino acids (some proteins may need assistance
from molecular “chaperones”)
– Studying protein folding
• Often through denaturation/renaturation curves: how stable is a
protein? How quickly does it (un)fold?
– Several imperfect models
Reversible binding involving proteins
1. Interactions between proteins
2. Protein/DNA
3. Protein/small molecule ligand
Reversible binding involving proteins
(Ch. 5)
1. Interactions between proteins
– Different from 4° structure
•
•
•
Lower affinity (in general)
Reversible
Potential for numerous partners
4° Structure
Hemoglobin
Four ‘separate’ polypeptide chains
One ‘protein’
Function as a whole
Protein-protein
interaction
Antibody (green)/Antigen (red)
Two different proteins
Found apart
Reversible binding
1. asdf
2. Protein vs. “small” molecule
– Protein acts as a carrier for the molecule
•
•
Hemoglobin/O2
Metallochaperones
– Enzymes
•
Catalyze a reaction involving the substrate
3. Protein-DNA interactions
Principles of reversible interactions
• Affinity of protein for ligand is very specific
– eg. high affinity for Mg2+, low affinity for Zn2+
– eg. fumarase: distinguishes stereoisomers of tartaric
acid
• Ligand binding site is usually complementary to
the ligand BUT ligand binding can cause drastic
conformational changes
– Induced fit
– Conformational changes result in tighter binding but
strain both protein and ligand
C
C
C
C
INACTIVE PKA
cAMP binding results in conformational
change: regulatory subunits no longer
bind catalytic: ACTIVE PKA
Principles of reversible interactions
• Enzymes
– Ligands = substrate and product
– Induced fit stress can drive catalysis
Quantification of protein-ligand
interactions (non-catalytic)
P + L ↔ PL
Ka =
Reversible: represent as equilibrium
[PL]
[P][L]
Association constant
(don’t confuse with Ka/pKa)
High Ka: [complex] is relatively high
ie. protein has a high affinity for the ligand
[PL]
Ka * ([L]) = [P]
Amount of complex depends on
concentration of free ligand as well
as the affinity (Ka)
Quantification of protein-ligand
interactions
• Work with dissociation constants
PL ↔ P + L
Kd = [P][L]
[PL]
Equilibrium equation describing
dissociation
Note that Kd = 1/Ka
Quantification of protein-ligand
interactions
• Assume [L] >> [P]
– Few proteins (binding sites), lots of the ligand
– ie. conc. of free ligand doesn’t change (much) even if
all ligand-binding sites are filled
• Fraction of ligand binding sites filled (q)
q=
[L]
[L] + Kd
q=
[L]
[L] + Kd
When [L] = Kd, q = 0.5
**When [L] = Kd (note: no matter what [P] is (remember
assumption, though)), half of the binding sites will be
filled
Lower Kd: need less ligand to fill binding sites
Lower Kd corresponds to higher
affinity/stronger binding
% of sites filled vs. [L]
Units of Kd: concentration (M, mM, mM, etc)
Fraction of binding sites occupied
q
Protein x with three
different ligands
Max binding (q = 1.0, all binding
sites filled/saturated)
1
50% of saturation
0.5
0
Kd1
Kd2
Kd3
Case study: oxygen binding in
myoglobin and hemoglobin
• Oxygen is poorly soluble
in water (blood)
• Iron (Fe2+)/O2 complex is
soluble
– But free iron is toxic
• Use proteins containing
an iron cofactor
– Myoglobin
– Hemoglobin
Iron is part of a heme prosthetic group:
permanent association with protein
Iron has six coordination sites
Four bind heme nitrogens
One binds protein histidine
“proximal” histidine
One can bind O2
Structure of myoglobin
• Extremely compact
• ~75% a helix (no b
structure)
– Eight helical segments
– Four terminate in
proline
• Interior: hydrophobic
except for two
histidines