• 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
• – 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
HemoglobinFour ‘separate’ polypeptide chainsOne ‘protein’Function as a whole
Antibody (green)/Antigen (red)Two different proteinsFound apart
4° StructureProtein-protein interaction
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
INACTIVE PKA
C
C
cAMP binding results in conformationalchange: regulatory subunits no longerbind 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 Reversible: represent as equilibrium
Ka = [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
Ka * ([L]) = [PL] [P]
Amount of complex depends on concentration of free ligand as wellas the affinity (Ka)
Quantification of protein-ligand interactions
• Work with dissociation constants
PL ↔ P + L Equilibrium equation describingdissociation
Kd = [P][L] Note that Kd = 1/Ka
[PL]
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 (
= [L]
[L] + Kd
= [L]
[L] + Kd
When [L] = Kd, = 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, M, etc)
Protein x with three different ligands
Max binding (= 1.0, all binding sites filled/saturated)
50% of saturation
1
0.5
0
Fra
ctio
n of
bin
ding
site
s oc
cupi
ed
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 nitrogensOne binds protein histidine “proximal” histidine
One can bind O2
Structure of myoglobin
• Extremely compact• ~75% helix (no
structure)– Eight helical segments– Four terminate in
proline
• Interior: hydrophobic except for two histidines