Proteins often consist of multiple domains

• 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