Solution Properties of antibodies: Purity Conformation.

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