chp 5 ligand binding biochem

ligand

small molecule/ion that reversibly binds to a larger molecule (protein/DNA)

myoglobin

stores O₂

monomer (1x subunit)

1x heme => 1 O₂ binding site

common motif as hemoglobin

hemoglobin

tetramer (4x subunits)

4 hemes, 4 O₂ binding sites

α₂β

common structural motif as myoglobin

heme prosthetic group (oxygen and CO can bind)

Fractional Saturation (Y)

Y=# of occupied binding sites/total # of binding sites

=[P:L]/[[P:L]+[P]

P=protein

L=ligand

P:L=protein bound ligand

quantitative description of ligand binding (association)

P+L←→P:L

association P to L - keq [P:L]/[P][L]

association constant - ka=[P:L]/[P][L]

assumptions

• [L]> > [P:L]

• [L]~constant

• no ICE tables

larger Ka →

more P:L

aka more saturation

kd=

[P][L]/[PL]=dissociation constant=1/ka

relationship between Y and kd

Y=L/L+Kd

kd=[L] at ½ Y

small Kd

=> low association => high association => large Ka

cooperative binding

bind one ligand which affects affinity for binding in 2nd ligand

low affinity

structure of hemoglobin

tetramer α₂β₂

cooperative binding

α-β interferences

• hydrophobic interactions

• H bonding

• salt bridges (ionic interaction)

2 states of hemoglobin

T state

R state

T state

low affinity (deoxygenated) conformation

no O₂ bounds

stabilized by salt bridges/ionic interactions

R state

O₂ bound (oxygenated)

high affinity for O₂

conformation change from T state after O₂ binds \

change in α₁β₂ and α₂β₁

stabilization of T state by

ion pair interactions

only present in T state

• α₁β₂ and α₂β₁ salt bridge

+ O₂ disrupts bridge

• conformation change

T→R transition linked to

oxygen binding

in R state binding affinity for O2 higher

induced fit model : conformation change causes better ligand binding

allosteric proteins

multi-subunit proteins that undergo ligand dependent changes in binding affinity

binding a ligand at one site, affects binding to another site

if allosteric ligand =active ligand → homotopic

if allosteric ligand is not an active ligand → heterotopic

cooperativity qualitatively

proteins = dynamic

ligand binds => stabilizes conformation that favors additional ligand binding

• increases affinity for ligand at additional site

hill coefficient

(\log\left(\frac{Y}{1-Y}\right)=n\log-\log kd

n= degree of cooperativity

• n=1, noncooperative

• n>1 positive cooperativity

• n<1 neg cooperativity

n_max=min # of binding sites in protein

Monod Wyman Changeaux model for cooperativity

all or nothing

limitation → treats all subunits equally

Koshland’s model for cooperativity

sequential model

change induces change

more generizable

look at subunits with independent functions

Hb also transports

H+ - pH dependent transporter

Hb binds in T state (stabilizing salt bridge found at low pH)

very important for O2 binding and release

Hb also transports, mech ??

CO2 away from respiring tissues

CO2 in T state transported out of cell tissues

biophosphoglycerate (BPG or DPG)

stabilizes T state

bind 1 BPG tetramer

high [BPG] , T-State → low affinity O2, faster release

fetal hemoglobin

ɑ₂ɤ₂ tetramer

different than adult Hb

binds O2 with higher affinity than adult Hb

how do negative regulatory factors work together

CO2, BPG, H+ reduce O2 affinity in Hb

BPG is better at reducing O2 affinity than CO

Hb diseases

sickle cell anemia (hemoglobin aggregates and red cells distort and block capillaries

alpha thalassemia (beta chains only, forming hemoglobin that is not cooperative

beta thalassemia (alpha chains only, form insoluable aggregates resulting in few red cells—anemia)