NCMH, Nottingham, UKhttp://www.nottingham.ac.uk/ncmh
Svedberg 125th anniversary
Advances in conformational analysisin heterogeneous systems
Steve Harding
Haug triangle and “scaling relations” or power law coeffs
[] ~ M0; s ~M0.67; Rg ~ M0.33
[] ~ M1.8; s ~M0.15; Rg ~ M1.0
[] ~ M0.5-0.8; s ~M0.4-0.5; Rg ~ M0.5-0.6
ks/[] ~ 1.6
ks/[] ~ 1.6ks/[] ~ 0.3
ks: Gralén coefficient
Power law plot - example
Galactomannansa=0.74+0.01
Picout, Ross-Murphy, Jumel & Harding (2002)Biomacromolecules 3, 761-767
[] ~ Ma
Rollings J (1992) in Laser Light Scattering inBiochemistry (Harding, Sattelle & Bloomfield eds)
Change in Conformation
Conformation Zoning Diagram
Pavlov, Rowe & Harding (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
Conformation Zoning Diagram
HYDFIT plot – flexibility determination, Lp
“Bushin-Bohdanecky” relation
2/1
2/1
3/10
3/10
3/12 2w
L
p
Lw M
M
LBMA
M
“Yamakawa-Fujii” relation
....22
843.13
12/1
32
2/1
0
00
pL
w
pL
w
A
L
LM
MAA
LM
M
N
vMs
Garcia de la Torre & Ortega (2007), Biomacromolecules 8, 2462-2475
Konjac glucomannan, Lp ~ 13nm(Kok et al, 2009)
Structure and heterogeneity of
gliadin: a hydrodynamic evaluationS. Ang et al, Eur. Biophys. J. (2009)
ELLIPS1
www.nottingham.ac.uk/ncmh
Conformation analysis in a polydisperse protein system –gliadin
Conformation analysis in a dimerising protein system – neurophysin
or
ELLIPS3
www.nottingham.ac.uk/ncmh
a/b
b/c
R
Shape parameters R and are fromsedimentation, viscosity andfluorescence measurements
Conformation analysis in a dimerising protein system – neurophysin
= {3M}/{NAkTth}R = ks/[]
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
R
R
Conformation analysis in an aggregated monoclonalantibody system – effect of bioprocessing
Freeze-thaw bioprocessed IgG4
Lu, Harding, Rowe, Davis, Fish, Varley, & Mulot,
(2008) J.Pharm Sci, 97, 948-957
Monomer – is there a link betweenconformation change and aggregation? –need s and other data to answer this
Conformation analysis in an aggregated monoclonalantibody system – effect of bioprocessing
Freeze-thaw bioprocessed IgG4
Lu, Harding, Rowe, Davis, Fish, Varley, & Mulot, S.
(2008) J.Pharm Sci, 97, 948-957
Bead model – “cusp”shape for IgE, 1990
Modelled on s=7.26S,Rg= 6.8nm
.. and iterated from crystalstructure of a hinge deletedIgG mutant
A model of chimeric IgG3 wild type
A model of chimerichinge deleted IgG3HM5.
More recent strategies use even more data: s, Rg,Dmax, [] and crystal structure of the domains
Modellingalgorithm:SOLPRO
Monomer – is there a link betweenconformation change and aggregation? –need s and other data to answer this
Conformation analysis in an aggregated monoclonalantibody system – effect of bioprocessing
Freeze-thaw bioprocessed IgG4
10 15 20 25
0.0
0.2
0.4
0.6
0.8
1.0N
orm
alis
edD
etec
tor
Res
po
nse
Elution Time (min)
LS 90o
DRIDPV
Chicken FibrinogenMw = 335000 g/mol (0.1 %)[] = 27.7 ml/g (0.3 %)
….on-line intrinsic viscosity measurement
20 25 30 35 40 45
0.0
0.2
0.4
0.6
0.8
1.0
LS 90o
DRIDPV
Norm
alis
edD
etec
tor
Res
ponse
Elution Time (min)
Mw = 396000 g/mol (0.1 %)[] = 107.6 ml/g (1%)
Mw = 120000 g/mol (0.1 %)[] = 46.2 ml/g (0.3 %)
Mw = 13150 g/mol (0.6 %)[] = 12.1 ml/g (0.7 %)
Mw = 620 g/mol (7 %)[] = 2.8 ml/g (0.4 %)
Monomer – is there a link betweenconformation change and aggregation? –need s and other data to answer this
Conformation analysis in an aggregated monoclonalantibody system – effect of bioprocessing
Freeze-thaw bioprocessed IgG4