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Solution Properties of antibodies:
Purity
Conformation
Text book representation of antibody structure:
Main tool: Analytical Ultracentrifuge
Sedimentation Velocity Sedimentation Equilibrium
2 types of AUC Experiment:
Air Solvent
Solution
conc, c
distance, r
Rate of movement of boundary sed. coeff
Centrifugal force
conc, c
distance, r
Centrifugal force Diffusion
so20,w
1S=10-13sec
STEADY STATE PATTERN
FUNCTION ONLY OF MOL. WEIGHT PARAMETERS
Sedimentation Velocity Sedimentation Equilibrium
2 types of AUC Experiment:
Air Solvent
Solution
conc, c
distance, r
Rate of movement of boundary sed. coeff
Centrifugal force
conc, c
distance, r
Centrifugal force Diffusion
so20,w
1S=10-13sec
STEADY STATE PATTERN
FUNCTION ONLY OF MOL. WEIGHT PARAMETERS
Solution Properties of antibodies:
Purity
Ultracentrifuge Analysis: IgG4 preparation
Ultracentrifuge Analysis: IgG4 preparation
Solution Properties of antibodies:
Conformation – “Crystallohydrodynamics”
Single Ellipsoids won’t do…
So use the bead model approximation …
Developed by J. Garcia de la Torre and co-workers in Murcia Spain
2 computer programmes: HYDRO & SOLPRO
(please refer to D2DBT7 notes – see the example for lactoglobulin octamers)
Conventional Bead model
Bead-shell model
1st demonstration that IgE is cusp shaped
Davies, Harding, Glennie & Burton, 1990
Bead model, s=7.26 Svedbergs, Rg= 6.8nm
…by comparing hydrodynamic properties with those of hingeless mutant IgGMcg
Consistent with function….
Bead model, s=7.26 Svedbergs, Rg= 6.8nm
High Affinity Receptor
Consistent with function….
High Affinity Receptor
Conventional Bead model
Bead-shell model
Better approach is is to use shell models!
Bead-shell model: Human IgG1
Crystal structure of domains
+ solution data for domains
+ solution data for intact antibody
= solution structure for intact antibody
We call this approach “Crystallohydrodynamics”
Take Fab' domain crystal structure, and fit a surface ellipsoid….
PDB File: 1bbj 3.1Å
Fitting algorithm: ELLIPSE (J.Thornton, S. Jones & coworkers)
Ellipsoid semi-axes (a,b,c) = 56.7, 35.6, 23.1.
Ellipsoid axial ratios (a/b, b/c) = (1.60, 1.42)
Hydrodynamic P function = 1.045: see d2dbt8 notes
Now take Fc domain crystal structure, and fit a surface ellipsoid….
Do the same for Fc
PDB File: 1fc1 2.9Å
Fab’ Fc
Now fit bead model to the ellipsoidal surface
P(ellipsoid)=1.039P(bead) = 1.039
P(ellipsoid)=1.045P(bead) = 1.023
Use SOLPRO computer programme: Garcia de la Torre, Carrasco & Harding, Eur. Biophys. J. 1997
Check the P values are OK
The TRANSLATIONAL FRICTIONAL RATIO f/fo (see d2dbt8 notes)
f/fo =conformation parameter x hydration term
f/fo = P x (1 + ovbar)1/3
Can be measured from the diffusion coefficient or from the sedimentation coefficient
f/fo = constant x {1/vbar1/3} x {1/ M1/3} x {1/Do20,w}
f/fo = constant x {1/vbar1/3} x (1-vbar.o) x M2/3 x {1/so20,w}
Experimental measurement of f/fo for IgGFab
Experimental measurement of f/fo for IgGFab
Estimation of time-averaged hydration, app for the domains+whole antibody
app ={[(f/fo)/P]3 - 1}ovbar
Fab' domain
P(bead model) = 1.023
f/fo (calculated from so20,w and M) = 1.22+0.01
app = 0.51 g/g
Fc domain
P(bead model) = 1.039
f/fo (calculated from so20,w and M) = 1.29+0.02
app = 0.70 g/g
Intact antibody = 2 Fab's + 1 Fc.
Consensus hydration app ~ 0.59 g/g
we can now estimate P(experimental) for the intact
antibody
P(experimental) = f/fo x (1 + appovbar)-1/3
P=1.107 P=1.112 P=1.118
P=1.121 P=1.122 P=1.143
IgG’s: all these compact models give P’s lower than experimental
…so we rule them out!
P = 1.230 P = 1.217
Models for IgG2 & IgG4. Experimental P=1.22+0.03 (IgG2)
=1.23+0.02 (IgG4)
Carrasco, Garcia de la Torre, Davis, Jones, Athwal, WaltersBurton & Harding, Biophys. Chem. 2001
P=1.208(Fab)2
(Fab)2 : P(experimental) = 1.23+0.02
P = 1.263 P = 1.264
“Open” models for IgG1 (with hinge) P(experimental) = 1.26+0.03
P=1.215 P=1.194
P=1.172
A B
CThese are coplanar models for a mutant hingeless antibody, IgGMcg.
P(experimental) = 1.23+0.03
UNIQUENESS PROBLEM:
Although a particular model may give conformation parameter P in good agreement with the ultracentrifuge data, there may be other models which also give good agreement.
This is the uniqueness or “degeneracy” problem.To deal with this we need other hydrodynamic data:
Intrinsic viscosity [] – viscosity increment
Radius of gyration Rg – Mittelbach factor G
And work is ongoing in the NCMH in conjunction with other laboratories
