Ahmed Group Lecture 6 Cell and Tissue Survival Assays Lecture 6.
Lecture 6
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Transcript of Lecture 6
Lecture 6
Protein-protein interactions
Affinities (cases of simple and cooperative binding)
Examples of Ligand-protein interactions
Antibodies and their generation
0 2 4 6 8 100
0.2
0.4
0.6
0.8
U1 r( )
U2 r( )
U4 r( )
U6 r( )
r
1/r2
1/r6
1/r
Long-range and short-range interactions
Even without NET CHARGES on the molecules, attractive interactions always exist. In the presence of random thermal forces all charge-dipole or dipole-dipole interactions decay steeply (as 1/r4 or 1/r6)
1/r4
Interatomic interaction: Lennard-Jones potential describes both repulsion and attraction
Uo 1
U x( ) Uo x12
2x6
0.6 0.8 1 1.2 1.4 1.6 1.8
1
0
1
2
U x( )
x
600
1200 )/(2)/()( rrErrErEp
r = r0 (attraction=minimum)
r = 0.89r0
r = r0
steric repulsion
Bond stretching is often considered in the harmonic approximation:
202
1 )()( xxxU
desolvdispstericeltot EBAqE
Van der Waals
Here is a typical form in which energy of interactions between two proteins or protein and small molecule can be written Ionic pairs +
H-bondingremoval of waterfrom the contact
What determines affinity and specificity?
Tight stereochemical fitand Van der Waals forces Electrostatic interactionsHydrogen bondingHydrophobic effect
All forces add up giving the total energy of binding:
Gbound– Gfree= RT
lnKd
Simple binding
LRRL
]][[][ RLKLR b
off
onb k
k
RL
LRK
]][[
][
][][][
][][
LRRLR
totalRLR
B
Receptor occupancy:
][1
][
LK
LKB
b
b
bd K
K1
][
][
LK
LB
d
Mass action:
(Langmuir isotherm)kinetic parametersequilibrium parameter
1/koff = residence time
in the bound state
][
][
LK
LB
d
Receptor occupancy is a hyperbolic function of [L ](Langmuir adsorption isotherm)
B1 x(
B2 x(
B3 x(
L0 10 20 30 40 50
0
0.2
0.4
0.6
0.8
1
)
)
)
Kd = 1Kd = 3
Kd = 10
Bmax
Kd has the dimension of concentration and should be measured in the same units as L (M).
Note that for a shallow curve it is hard to say where it saturates
97% of O2 is carried in the form of Oxyhemoglobin (HbO2)
3% - dissolved in plasma
P1/2 = 28 mm Hg
When PO2 changes from 100 to 40 mm Hg, the saturation decreases from 98 to 75%
physiological range
Oxygen and Hemoglobin
From G. Hummer
CO binds to the porphyrin ring of heme exactly where O2 binds
nn
n
KRL
RL 1
][][
][
What if the binding to multiple sites on the same receptor is strictly interdependent (i.e. cooperative)?
nntotn
n
KRLRL
RL 1
][][
][
nn
n
tot
nn LK
L
R
RLB
][
][
][
][
Hill equation, n is Hill coefficient 0 1 2 3 4 50
0.2
0.4
0.6
0.8
1
B1 x( )
B2 x( )
B3 x( )
L
n=2n=4
n=1
rearrange
Probability of binding to one
site ~[L]
Probability of binding
simultaneously to n sites ~[L]n
Myoglobin, n = 1
Hemoglobin, n = 2.8
pO2 (kPa)
0 2 4 6 8 100
0.2
0.4
0.6
0.8
1
B1 x( )
B3 x( )
nn
n
n LK
LB
][
][
pO2 in tissues
Hemoglobin vs Myoglobin
Cooperativity is due to tight intersubunit interactions
xkxB
d
n
d
n
xkxB
n – Hill coefficient
independent binding
cooperative binding
Protein Kinase A spatially organizes ATP and peptide chain to facilitate the phosphorylation reaction
(old book)
Intracellular signaling adapter domains SH2 and SH3
Proline-rich sequenceSegment containing phosphotyrosine
Fig 16-11 Fig 16-23
PDZ domains spatially organize ion channel/receptor complexes in synapses
“Postsynaptic density” complex
(old book)
Fatty acid binding protein (FABP)
Fig. 10-24
Common theme: hormones promote dimerization of receptors
Fig. 16-7
The Growth Hormone sequentially binds to two receptors
first binding event second receptor is then recruited
Fig 15-3
Binding of the Epidermal Growth Factor (EGF) leads to receptor dimerization not by cross-linking but by exposing ‘sticky’ loops
Fig. 16-17
Antibody (IgG)
CDR = complementarity determining region
The lymph system and lymph nodesSee Chapter 24
Clonal selection of B lymphocytes: prolifereation and differentiation of these cells is induced by an encounter with an antigen recognized by the surface receptor
The immunoglobulin fold and the hypervariable regions
Fig. 24-12
Variability of sequence in hypervariable loops
The antigen recognition site
Fig. 24-13
Light chain coding regions:
VLCL
100 5 variants
Heavy chain coding regions:
VHD CH
100 30 4 variants
therefore, total number of combinations is ~ 6,000,000
Combinatorial diversity of antibodies
see Lodish (4th edition)
V – variableC – constant
The recognition site exposes flexible loops typically with many polar residues