a combined approach to size, heterogeneity, conformation ... combined approach to size,...
TRANSCRIPT
Steve HardingNational Centre for Macromolecular Hydrodynamics
A combined approach to size,heterogeneity, conformation & flexibility
of bio-macromolecules
Size (mol wt.) & heterogeneity:
• SEC-MALLs – mol wt distributions
• AUC sedimentation equilibrium analysis – M* & distributions
• AUC sedimentation velocity analysis – g*(s) & distributions
• Size (mol wt.) and heterogeneity: SEC-MALLs
• Size (mol wt.) and heterogeneity: SEC-MALLs
mucin
• Size (mol wt.) and heterogeneity: SEC-MALLs
• Size (mol wt.) and heterogeneity: Analytical ultracentrifuge
Sedimentation Velocity Sedimentation Equilibrium
Air Solvent
Solution
conc, c
distance, r
Top view, sector ofcentrifuge cell
Rate of movement ofboundary sed. coeff
Centrifugal force
conc, c STEADY STATEPATTERN
FUNCTION ONLYOF MOL. WEIGHTPARAMETERS
distance, r
Centrifugal forceDiffusion
so20,w
1S=10-13sec
• Size (mol wt.) and heterogeneity: Analytical ultracentrifuge
M* analysis of sedimentation equilibrium
Creeth JM & Harding SE (1982) J. Biochem. Biophys. 7, 25-34
Mw,app
cell bottom
Chitosan G213
Sedimentation equilibrium M* plot
Ball, Harding & Mitchell (1988)
SEC - sedimentationequilibrium mol. wtdistribution: alginate
Sedimentation velocity g*(s) plot
Numerical solutions tothe Lamm equation byClaverie et al (1975) &implemented by Todd &Haschemayer (1981)
Lamm (1923) equation:
DLS analysis: Contin plot
R. Tester, T Patel, S. Harding, Carbohydrate Research (2006)
Sedimentation velocity g*(s) plot: starch
……consider antibodies used in therapies, and theirstate of aggregation after bioprocessing
SAN02 Freeze-thaw (1.16mg/mL)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0 10 20 30 40 50
s(S)
g*(
s)
Control
10cycles
20cycles
25cycles
Size distribution by sedimentation velocity of a bioprocessed IgG4 antibody
SAN03 pure
0
5
10
15
20
25
0.1 1 10 100 1000 10000
Vo
lum
e(%
)
Size (d.nm)
Size Distribution by Volume
Record 108: san 003 pure Record 109: san 003 pure Record 110: san 003 pure
Record 111: san 003 pure Record 112: san 003 pure Record 113: san 003 pure
size, d (nm)
Am
ou
nt
(%)
Size distribution by DLS of a bioprocessed IgG4 antibody
…provides an excellent assessment of the quality/ the extent of heterogeneity
…this preparation is even worse!
Also, changes in sedimentation coefficient of monomer may reflectchange in conformation
Molecular conformation & flexibility:
• general conformation – conformation zoning
• flexibility – persistence length using global methods
• whole body or ellipsoid representations
• bead model representations
Rg ~ M0.5-0.6Rg ~ M1.0Rg ~ M0.33
so20,w~ M0.4-0.5so
20,w~ M0.15so20,w~ M0.67
[] ~ M0.5-0.8[] ~ M1.8[] ~ M0
CoilRodSphere
• General molecular conformation: Haug triangle and power law coeffs
Mark-Houwink-Kuhn-Sakurada Power law plot
Galactomannansa=0.74+0.01
Picout et al (2003) Biomacromolecules 2,1301-1309
Rollings J (1992) in Laser Light Scattering inBiochemistry (Harding, Sattelle & Bloomfield eds)
Change in Conformation
Rg ~ M0.5-0.6Rg ~ M1.0Rg ~ M0.33
so20,w~ M0.4-0.5so
20,w~ M0.15so20,w~ M0.67
[] ~ M0.5-0.8[] ~ M1.8[] ~ M0
CoilRodSphere
ks/[] ~1.6ks/[] <1ks/[] ~1.6
Conformation Zoning:
Pavlov et al. (1997). Trends inAnalytical Chemistry, 16, 401-405.
0.5 1.0 1.5 2.0 2.5-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5lo
g(1
0-11 k sM
L)
log (1012
[s]/M L)
A
B
C
D
E
0.5 1.0 1.5 2.0 2.5-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5lo
g(1
0-11 k sM
L)
log (1012
[s]/M L)
A
B
C
D
E
A: very stiff rod
B: with limitedflexibility
C: semi-flexible
D: random coil
E: globular orbranched
0.5 1.0 1.5 2.0 2.5-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
log
(10-1
1 k sML)
log (1012
[s]/ML)
A
B
C
D
E
Bovine glycogen ●
Pectins ■
Pullulans ▲
A: very stiff rod
B: with limitedflexibility
C: semi-flexible
D: random coil
E: globular orbranched
Measure of flexibility: persistence length Lp
Theoretical limits: 0 (random coil) → ∞ (perfect rod)
Practical limits ~ 2nm → 200nm
2/1
2/1
3/10
3/10
3/12 2w
L
p
Lw M
M
LBMA
M
....22
843.13
12/1
32
2/1
0
00
pL
w
pL
w
A
L
LM
MAA
LM
M
N
vMs
“Bushin-Bohdanecky” relation
“Yamakawa-Fujii” relation
• Molecular conformation: flexibility analysis
“Hydfit” or Global analysis: Garcia de la Torre &Ortega, Biomacromolecules (2007)
Konjac glucomannan, Lp ~ 13nm (Kok et al, 2009)
Conformation Zoning:
Pavlov et al. (1997). Trends inAnalytical Chemistry, 16, 401-405.
0.5 1.0 1.5 2.0 2.5-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5lo
g(1
0-11 k sM
L)
log (1012
[s]/M L)
A
B
C
D
E
0.5 1.0 1.5 2.0 2.5-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5lo
g(1
0-11 k sM
L)
log (1012
[s]/M L)
A
B
C
D
E
A: very stiff rod
B: with limitedflexibility
C: semi-flexible
D: random coil
E: globular orbranched
• Molecular conformation: ellipsoid representation
ELLIPS algorithms www.nottingham.ac.uk/ncmh
Universal_Param: Calculates shape parameter from s, D, [], BELLIPS1 Evaluates a/b for prolate or oblate ellipsoid fromshape parameterELLIPS2 Evaluates shape parameter from (a,b,c) or (a/b, b/c)ELLIPS3 Evaluates (a/b, b/c) from combinations of hydrationindependent shape functions.ELLIPS4 Evaluates (a/b, b/c) from electro-optic decaycombined with other hydrodynamic data.ELLIPSDRAW 3D plot of ellipsoid from (a/b, b/c)COVOL Evaluates B from (a/b, b/c)
Structure and heterogeneity of
gliadin: a hydrodynamic evaluationS. Ang et al, Eur. Biophys. J. (2009)
• Molecular conformation: ellipsoid representation
ELLIPS algorithms www.nottingham.ac.uk/ncmh
Universal_Param: Calculates shape parameter from s, D, [], BELLIPS1 Evaluates a/b for prolate or oblate ellipsoid fromshape parameterELLIPS2 Evaluates shape parameter from (a,b,c) or (a/b, b/c)ELLIPS3 Evaluates (a/b, b/c) from combinations of hydrationindependent shape functions.ELLIPS4 Evaluates (a/b, b/c) from electro-optic decaycombined with other hydrodynamic data.ELLIPSDRAW 3D plot of ellipsoid from (a/b, b/c)COVOL Evaluates B from (a/b, b/c)
• Molecular conformation: ellipsoid representation
ELLIPS algorithms www.nottingham.ac.uk/ncmh
Universal_Param: Calculates shape parameter from s, D, [], BELLIPS1 Evaluates a/b for prolate or oblate ellipsoid fromshape parameterELLIPS2 Evaluates shape parameter from (a,b,c) or (a/b, b/c)ELLIPS3 Evaluates (a/b, b/c) from combinations ofhydration independent shape functions.ELLIPS4 Evaluates (a/b, b/c) from electro-optic decaycombined with other hydrodynamic data.ELLIPSDRAW 3D plot of ellipsoid from (a/b, b/c)COVOL Evaluates B from (a/b, b/c)
R
a/b
b/c
a/b
b/c
R
Shape parameters R and are fromsedimentation, viscosity andfluorescence measurements
(a/b) = 4, (b/c) = 1
ELLIPS3 applied to neurophysin monomers
dimersb/c b/c
a/b
a/b
(a/b)= 4, (b/c)= 1 (a/b)=2.5, (b/c)= 3
monomers
Neurophysin dimerises – here’s what happens
……whole-body ellipsoids won’t do for complicatedshapes like antibodies……
……whole-body ellipsoids won’t do for complicatedshapes like antibodies…… so use bead modelling
• Molecular conformation: bead modelling
1st demonstrationthat IgE is cuspshaped, 1990
Bead model: s=7.26S,Rg= 6.8nm
• Molecular conformation: bead modelling
Consistent with function….
Bead model, s=7.26 Svedbergs, Rg= 6.8nm
High AffinityReceptor
A model of chimeric IgG3 wild type
A model ofchimeric IgG3 m15with 15aa in hinge.
A model of chimerichinge deleted IgG3HM5.
Conformation of engineered antibodies from s, Rg,Dmax, [] and crystal structure of the domains
Challenge: conformation determination in mixed systems
… here’s our heterogeneous bioprocessed antibody again
Thanks to:
Professors Arthur Rowe, JoseGarcia de la Torre, Simon Ross-Murphy, Georges Pavlov & Drs.Dave Scott & Gordon Morris