Protein Binding Flashcards
Rate of association and dissociation equations? Relationship between them at equilibrium
Rate of association = k1[P][L]; k1is association rate constant
Rate of dissociation = k-1[PL]; k-1 is dissociation rate constant
At equilibrium: k1[P][L] = k-1[PL]
Association and dissociation equilibrium constants (Ka and Kd)
Association equilibrium constant Ka = [PL] / ([P][L]) = k1 / k-1
Dissociation equilibrium constant Kd = [P][L] / [PL] = k-1 / k1
Ka x Kd = ?
1
Can you get from [PL] to Kd
[PL] = Ka[P][L] = [P][L] / Kd
Ligand bound fraction
θ = [PL] / ([P]+[PL]) = Ka[P][L]/([P]+Ka[P][L]) = Ka[L]/(1+Ka[L]) = [L]/(Kd+[L])[Ptotal] = [P]+[PL]
θ= bound fraction
θ= [PL]/[Ptot]
what kind of curve is the ligand binding curve
hyperbolic curve
what does the Kd value correspond to
corresponds to the [L] at which bound fraction is 50%
How can we measure [L], [P] or [PL]
- Free L and bound PL have different absorbance or fluorescence at a certain wavelength
- Free P and bound PL have different absorbance or fluorescence at a certain wavelength
Scatchard linear equation and what can it be used on
Deals with homogenous binding sites
Starts with the
θ = [L]/ (Kd+[L])
then replace θ with θ/n
Scatchard Linear equation:
θ/[L] = n/Kd - θ/Kd
or
θ/[L] = nKa - θKa
Average occupancy per ligand-binding site
θ/n
Scatchard linear plot slope, y-intercept, x-intercept
Slope = -Ka or -1/Kd
y-intercept= nKa or n/Kd
X-intercept= n
Double reciprocal equation? When does it work?
Works when there is only n= 1 binding site; θ is occupancy 0-1
1/[PL] = Kd/([Ptotal][L]) +1/[Ptotal]
Double reciprocal linear plot slope, y-intercept, x-intercept
Slope= Kd/[Ptotal]
y-intercept is 1/[Ptotal]
x-intercept is -1/Kd or -1Ka
Cooperative binding rate of association and dissociation? Equilibrium relationship?
rate of association = k1 [P][L]^n
Rate of dissociation= k-1 [PLn]
These two rates are equal at equilibrium
Association and dissociation equilibrium constant (Ka & Kd)
Association equilibrium constant Ka = [PLn]/([P[L]n) = k1/ k-1 Dissociation equilibrium constant Kd = [P][L]n/[PLn] = k-1 / k1
Fraction of Ligand-bound protein for cooperative binding
Hill plot
Equilibrium dialysis
Establish equilibrium. Protein is retained in the membrane bag. Measure free [L] outside; and bound and free [PL] + [L] inside bag. Free [L] is the same on both side of dialysis membrane. The difference must be [PL]
Kd can therefore be determined with a series of initial [L] inside.
Isothermal Titration Calorimetry (ITC)
ITC measures heat exerted or absorbed by adjusting the electric current to the sample cell to maintain a constant temperature with the reference cell.
Protein is in the sample cell.
Concentrated ligand is injected to the sample cell. Binding gives or takes heat
ITC directly measures DH, Ka, stoichiometry, and indirectly derives DG and DS.
Surface Plasmon Resonance (SPR)
SPR measures both k1 and k-1 so also gets kinetic information
MicroScale Thermophoresis (MST)
Upon binding ligand Fnorm increases because diffusion becomes slower (larger mass of the PL complex).
Therefore, plot Fnorm as a function of ligand concentration
Kd can be derived from the midpoint of the curve!!!
