Solution Properties of antibodies:

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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


s
o
20,w

1S=10
-
13
sec


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


s
o
20,w

1S=10
-
13
sec


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


1
st

demonstration
that IgE is cusp
shaped


Davies, Harding,
Glennie & Burton, 1990



Bead model, s=7.26 Svedbergs, R
g
=
6.8nm

…by comparing hydrodynamic properties
with those of hingeless mutant IgGMcg



Consistent with function….


Bead model, s=7.26 Svedbergs, R
g
= 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)=1.039

P(bead) = 1.039

P(ellipsoid)=1.045

P(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/f
o

(see d2dbt8 notes)


f/f
o


=conformation parameter x hydration term


f/f
o

=


P

x
(1 +

d
/
r
o
vbar)
1/3




Can be measured from the diffusion coefficient or from
the sedimentation coefficient


f/f
o


= constant x {1/vbar
1/3
} x {1/ M
1/3
} x {1/D
o
20,w
}



f/f
o


= constant x {1/vbar
1/3
} x (1
-
vbar.
r
o
) x M
2/3

x {1/s
o
20,w
}


Experimental measurement of
f/f
o

for IgGFab

Experimental measurement of
f/f
o

for IgGFab

Estimation of time
-
averaged hydration,
d
app

for the domains+whole antibody



d
app

=

{[(
f/f
o
)/P]
3

-

1}
r
o
vbar


Fab' domain

P(bead model) = 1.023

f/f
o

(calculated from s
o
20,w

and M) = 1.22
+
0.01

d
app

= 0.51 g/g

Fc domain

P(bead model) = 1.039

f/f
o

(calculated from s
o
20,w

and M) = 1.29
+
0.02

d
app

= 0.70 g/g

Intact antibody = 2 Fab's + 1 Fc.

Consensus hydration
d
app

~ 0.59 g/g



we can now estimate P(experimental)
for the intact antibody





P(experimental)

=
f/f
o

x
(1 +

d
app
/r
o
vbar)
-
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, Walters

Burton & 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

C

These 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 [
h



viscosity increment
n


剡摩畳 潦⁧祲慴楯渠o
g



Mittelbach factor G


And work is ongoing in the NCMH in conjunction with
other laboratories