Application of Light Scattering in a Core Facility Setting
Ewa Folta-Stogniew
Yale University
Biophysics Resource of Keck Laboratory: Yale School of Medicine
Mission:
quantitative characterization of interactions between
biomolecules using in solution biophysical methods
Common questions:
• how tight is the binding ? ( binding affinity: Kd, Ka)
• how many of each molecule are in the complex ? (stoichiometry)
• how fast does the complex form? (kinetics)
• is the binding event enthalpy or entropy-driven? (thermodynamics)
List of technologies:
• Size Exclusion Chromatography coupled with Light Scattering (SEC/LS)
• Dynamic Light Scattering (DLS)
• Isothermal MicroCalorimeter (ITC)
• Spectrofluorometer
• Stopped-Flow Spectrofluorometer
• Surface Plasmon Resonance (SPR) Sensor [BiaCore Biosensor; T100]
• Composition Gradient Static Light Scattering (CGSLS)
• Asymmetric flow Field-Flow Fractionation (AFFF)
http://info.med.yale.edu/wmkeck/biophysics/
Biophysics Resource of Keck Laboratory: Yale School of Medicine
Mission:
quantitative characterization of interactions between
biomolecules using in solution biophysical methods
Common questions:
• how tight is the binding ? ( binding affinity: Kd, Ka)
• how many of each molecule are in the complex ? (stoichiometry)
• how fast does the complex form? (kinetics)
• is the binding event enthalpy or entropy-driven? (thermodynamics)
List of technologies:
• Size Exclusion Chromatography coupled with Light Scattering (SEC/LS)
• Dynamic Light Scattering (DLS)
• Isothermal MicroCalorimeter (ITC)
• Spectrofluorometer
• Stopped-Flow Spectrofluorometer
• Surface Plasmon Resonance (SPR) Sensor [BiaCore Biosensor; T100]
• Composition Gradient Static Light Scattering (CGSLS)
• Asymmetric flow Field-Flow Fractionation (AFFF)
http://info.med.yale.edu/wmkeck/biophysics/
Typical SEC(AFFF); MALLS system
SEC column
waste or
or AFFF
collection
sample
HPLC
UV/Vis
UV
LS
RI
system
detector
detector
detector
DLS+SLS
0.1 µ m pre - filtered buffer
0.1 µ m “in - line” filter
Computer
FL
detector
BSA
SEC/LS results: Molar Mass Distribution Plot
Monomer: 66 kDa
Weight-average molar mass
Measured every 2 μl
UV trace; A280nm
(RI trace)
Is that ALL?
BSA
SEC/LS results: Molar Mass Distribution Plot
Monomer: 66 kDa
Weight-average molar mass
Measured every 2 μl
UV trace; A280nm
(RI trace)
The streptococcal C1 bacteriophage lysin, PlyC,
Holoenzyme is a multimeric protein:
50.3 kDa, “catalytic” subunit
Ext. coeff. A0.1%280
= 2.2
8.0 kDa, “binding” subunit
Ext. coeff. A0.1%280
= 0.3
Molar Mass vs. Volume
Molar Mass (g/mol)
1.6x105
PlyC062705a_01_P_N_178
PlyC062705b_01_P_N_178
PlyC062705c_01_P_N_178
PlyC062705d_01_P_N_178
PlyC062705e_01_P_N_178
PlyC062705f_01_P_N_178
1.2x105
SEC/LS
8.0x104
MW= 114.0 0.4 kDa
4.0x104
PlyC 1 big+8 small
0.0
10.0
12.0
14.0
16.0
predicted MW = 114.3 kDa
18.0
Volume (mL)
SEC/LS accuracy ~3 % , i.e. ~ 3kDa for PlyC
PlyC
1 big+8 small
MW = 114.3 kDa
Ext. coeff. A0.1%280
= 1.2
PlyC_bis
2 big+2 small
MW = 116.6 kDa
Ext. coeff. A0.1%280
= 2.0
Nelson D, Schuch R., Chahales P., Zhu S., and Fischetti V. A. (2006) PlyC: A multimeric bacteriophage lysin. Proceedings of the National Academy of Sciences 103: 10765-10770
“on-line” determination of extinction coefficient
a
from UV/RI ratio ~ A0.1%280
Evaluated models:
1 big+8 small
MW=
PlyC
model (1+8)
2 big+2 small
MW=
PlyC_bis
model (2+2)
Ext. coeff. A0.1%280
= 1.2
0.1%
= 2.0
Ext. PlyCB.
coeff. AThe
Octameric
eight
subunits arranged in a
280 PlyCB
ring as observed in the crystal structure of PlyC.
y = 1.2389x
R2 = 0.9861
3
observed UV/RI ratio
2.5
Protein
Ext.
coeff.
Est.
2
UV/RI ratio
1.5
P_C_1big_8small
1
P_C_2big_2small
0.5
0
0
0.5
1
1.5
est. ext. coeff.
2
2.5
UV/RI ratio
observed
computed
residual
^2
Apo
1.026
1.279
1.271
0.000
BAM
1.788
2.147
2.215
0.005
BSA
0.700
0.821
0.867
0.002
CA
1.737
2.273
2.152
0.015
OVA
0.730
0.919
0.904
0.000
Ti
0.928
1.070
1.150
0.006
PlyC
(1+8)
1.204
1.600
1.491
0.012
PlyC_bis
(2+2)
2.000
1.600
2.478
0.770
a Philo
J S, Aoki K. H., Arakawa T., Narhi L. O., and Wen J. (1996) Dimerization of the Extracellular Domain of the Erythropoietin (EPO) Receptor by EPO: One High-Affinity and One Low-Affinity
Interaction. Biochemistry 35: 1681-1691
Nelson D, Schuch R., Chahales P., Zhu S., and Fischetti V. A. (2006) PlyC: A multimeric bacteriophage lysin. Proceedings of the National Academy of Sciences 103; 10765-10770
Dimerization of FIR
FIR: human c-myc FarUpStream Element (FUSE) Binding Protein (FBP) Interacting Repressor (FIR)
FIR protein fragment: first two RRM domains
FIR: 23.4 kDa monomer; seen as a dimer in the X-ray structure
27.0
MW disstributions
3.526.0
26000
3
24000
4.9 mg/ml
Abs 295nm
MW [kDa]
25.0
2.5
224.0
1 mg/ml
20000
1.5
0.14 mg/ml
18000
MW [Da]
protein alone
23.0
MW [Da]
16000
AUC results
MW [Da]
14000
MW [Da]
1
0.522.0
0
21.0
13.5
2.2 mg/ml
22000
0
14.5
15.5
100
16.5
200
volume [ml]
12000
17.5
300
400
500
FIR [uM]
Crichlow G V, Zhou H., Hsiao H. H., Frederick K. B., Debrosse M., Yang Y., Folta-Stogniew E. J., Chung H. J., Fan C., De la Cruz E. M., Levens D., Lolis E., and Braddock D. (2008) Dimerization of
FIR upon FUSE DNA binding suggests a mechanism of c-myc inhibition. EMBO J 27: 277-289
Dimerization of FIR depends on DNA binding event
FIR protein: 23 kDa monomer
ssDNA fragment upstream of the P1 promoter, known as FUSE;
8 kDa
FIR+DNA complex; task: determine stoichiometry of the FIR+DNA complex in solution
FIR_DNA; 58 kDa ;
2:1 binding
66 kDa
43 kDa
Molar Mass vs. Volume
Molar Mass (g/mol)
8.0x10 4
55
FIR+DNA 0.65 mg/ml
FIR+DNA 0.32 mg/ml
FIR+DNA 1.59 mg/ml
6.0x10 4
FIR alone
35
4.0x10 4
FIR+DNA
2.0x10 4
0.0
11.0
15
12.0
13.0
14.0
15.0
16.0
0
Volume (mL)
FIR-DNA complexes
MW (kDa)
FIR+DNA (2:1) complex
54.7
FIR+DNA (2:2) complex
62.8
Observed MW
57.7
30
60
90
120
150
[FIR] (µM)
Concentration dependent measurements reveal that in
solution the dimerization is driven by DNA binding
Crichlow, G. V., Zhou, H., Hsiao, H-h., Frederick, K. B.,, Debrosse, M., Yuande Yang, Y., Folta-Stogniew, E. J., Chung, H-J., Chengpeng Fan, C., De La Cruz, E., Levens, D., Lolis, E.,
and Braddock, D. (2008) “Dimerization of FIR upon FUSE binding suggests a mechanism of c-myc inhibition”, EMBO J 27: 277-289
Dimerization of FIR depends on DNA binding event
FIR protein: 23 kDa monomer; seen as a dimer in X-ray structure
ssDNA fragment upstream of the P1 promoter, known as FUSE;
8 kDa
FIR+DNA complex; task: determine stoichiometry of the FIR+DNA complex in solution
FIR_DNA; 58 kDa ;
2:1 binding
66 kDa
43 kDa
complex stoichiometry from UV/RI measurements
Molar Mass vs. Volume
8.0x10 4
FIR+DNA 0.65 mg/ml
FIR+DNA 0.32 mg/ml
FIR+DNA 1.59 mg/ml
6.0x10 4
DNA
(UV/RI=2.34)
2
UV/RI
Molar Mass (g/mol)
2.5
4.0x10 4
1.5
FIR:DNA (2:2; 1:1)
UV/RI=0.72
1
2.0x10 4
0.5
0.0
11.0
12.0
13.0
14.0
15.0
16.0
Volume (mL)
FIR:DNA (2:1)
UV/RI=0.47
0
0
20
40
60
80
FIR
(UV/RI=0.14)
MW complex (kDa)
FIR-DNA complexes
MW (kDa)
FIR+DNA (2:1) complex
54.7
FIR+DNA (2:2) complex
62.8
Observed MW
57.7
FIR:DNA (2:1)
55 kDa
FIR:DNA (2:2)
63 kDa
Crichlow, G. V., Zhou, H., Hsiao, H-h., Frederick, K. B.,, Debrosse, M., Yuande Yang, Y., Folta-Stogniew, E. J., Chung, H-J., Chengpeng Fan, C., De La Cruz, E., Levens, D., Lolis, E.,
and Braddock, D. (2008) “Dimerization of FIR upon FUSE binding suggests a mechanism of c-myc inhibition”, EMBO J 27: 277-289
Multiple oligomeric states for reconstituted KtrAB K+ Transporter
KtrAB ion transporter:
complex of KtrB membrane protein and KtrA RCK domain (regulating and conductance of K+)
KtrB: integral membrane protein isolated in the presence of detergent (DDM) as a
polypeptide:detergent(lipid) complex
220 156
1.0
66 kDa
300
A280
200
0.6
absorbace at 280 nm
polypeptide+detergent+lipids
polypeptide
0.4
150
100
0.2
50
0.0
0
12
14
16
18
MW [kDa]
250
0.8
20
Volume [mL]
Protein
KtrB
(monomer 49kDa)
Polypeptide
[kDa]
Oligomeric
state
Full
complex
[kDa]
Grams of
detergent/lipids per
gram of polypeptide
98
dimer
238
1.4
Albright R A, Ibar J. L. V., Kim C. U., Gruner S. M., and Morais-Cabral J. H. (2006) The RCK Domain of the KtrAB K+ Transporter: Multiple Conformations of an Octameric Ring. Cell 126: 1147-1159
Multiple oligomeric states for reconstituted KtrAB K+ Transporter
KtrAB ion transporter:
complex of KtrB membrane protein and KtrA RCK domain (regulating and conductance of K+)
KtrA RCK domain : basic assembly dimer, higher order oligomers: tetramer or octamer
66 kDa
220 kDa
2
120
A280
100
1
80
60
40
Molar Mass [kDa]
140
20
0
0
12
13
14
elution volume [ml]
Albright R A, Ibar J. L. V., Kim C. U., Gruner S. M., and Morais-Cabral J. H. (2006) The RCK Domain of the KtrAB K+ Transporter: Multiple Conformations of an Octameric Ring. Cell 126: 1147-1159
Multiple oligomeric states for reconstituted KtrAB K+ Transporter
KtrAB ion transporter
complex : KtrAB
octameric KtrA + dimeric KtrB
(8:2) model polypeptide = 228 kDa
octameric KtrA + 2x dimeric KtrB
(8:4) model polypeptide = 325 kDa
complex
polypeptide
KtrA Abs 280 nm
KtrB Abs 280nm
KtrAB Abs 280nm
500
1.0
300
0.5
200
Mw (kDa)
A280
400
octameric KtrA + 2x dimeric KtrB (8:4) model
polypeptide = 325 kDa
octameric KtrA + dimeric KtrB (8:2) model
polypeptide = 228 kDa
100
0.0
0
12
14
16
18
Elution volume (ml)
Buffer: 25 mM Tris, 150 mM NaCl, 1 mM DTT, 1 mM NADH, 1 mM DDM
Multiple oligomeric states for reconstituted KtrAB K+ Transporter
KtrAB ion transporter
complex : KtrAB
A280
1.0
octameric KtrA + dimeric KtrB
(8:2) model polypeptide = 228 kDa
octameric KtrA + 2x dimeric KtrB
(8:4) model polypeptide = 325 kDa
Dimer:octamer=0.4
Dimer:octamer=0.9
Dimer:octamer=2.2
Dimer:octamer=3.4
dimer:octamer
KtrB:KtrA
complex
Elution
volume
(ml)
Total
mass of
complex
(kDa)
Polypeptide
(kDa)
lipids
(kDa)
0.4
8:2
14.23
486
228
256
0.9
8:2
14.05
521
240
281
2.2
8:4
13.99
552
302
261
3.7
8:4
13.91
560
299
251
0.5
0.0
12
14
16
18
Elution volume (ml)
dimer:octamer
KtrB:KtrA
Excess
KtrB
dimer?
Elution
volume
(ml)
8:2 model (228 kDa)
computed
MW for
complex
(kDa)
difference
from
model
(kDa)
correct
model ?
8:4 model (325 kDa)
computed
MW for
complex
(kDa)
difference
from
model
(kDa)
0.4
14.23
228
0
Yes
250
-75
0.9
14.05
240
12
Yes
264
-61
correct
model ?
2.2
Yes
13.99
274
46
302
-24
Yes
3.7
Yes
13.91
271
43
299
-27
Yes
Effects of detergent on oligomeric state of KtrA RCK domain
220 kDa
66 kDa
1.2
(octamer)
0.8
100
A280
KtrA RCK domain no detergent
0.4
50
0.0
Molar Mass [kDa]
150
0
10
12
14
16
elution volume [ml]
220 kDa
66 kDa
0.5
150
0.4
A280
(tetramer and monomer) + micelle
0.3
A280
MMpp
0.2
MMcomplex
100
50
0.1
0.0
0
14
15
16
elution volume [ml]
17
18
Molar Mass [kDa]
KtrA RCK domain plus detergent
Determination of dimerization constant from SEC-LS
measurements
Input:
Results:
•
•
SEC/LS analyses at several eluting
concentrations
Determination of dimerization
constant
Determination of dimerization constant from SEC-LS measurements
Nucleobindin 1 (NUCB1) is a widely expressed multidomain calcium-binding protein whose precise
physiological and biochemical functions are not well understood;
soluble form of NUCB1 (sNUCB1) ;
Monomer
51 kDa
Determination of dimerization constant from SEC-LS measurements
Nucleobindin 1 (NUCB1) is a widely expressed multidomain calcium-binding protein whose precise
physiological and biochemical functions are not well understood;
soluble form of NUCB1 (sNUCB1) ;
Monomer
120
100
12. 5 uM
Mw (kDa)
80
25 uM
60
25 uM
40
40 uM
20
60 uM
0
0.01
1
100
[total protein concentration] uM
0.001 mg/ml
1 μg/ml
1 mg/ml
51 kDa
Determination of dimerization constant from SEC-LS measurements
Nucleobindin 1 (NUCB1) is a widely expressed multidomain calcium-binding protein whose precise
physiological and biochemical functions are not well understood;
soluble form of NUCB1 (sNUCB1) ;
Monomer
120
100
Mw (kDa)
80
1 ml/min
60
40
0.5 ml/min
20
0
0.01
1
100
[total protein concentration] uM
0.001 mg/ml
1 μg/ml
1 mg/ml
51 kDa
protein conc (M) Mw (kDa)
1.27E-08
55.3
2.63E-08
65.26
3.06E-08
53.93
4.67E-08
63.67
6.89E-08
69.29
7.06E-08
63.65
1.52E-07
73
1.61E-07
74.32
4.10E-06
97.84
7.88E-06
99.16
1.26E-05
99.97
2.00E-05
100.6
1.58E-06
95.04
3.15E-07
75.95
2.43E-06
95.68
7.75E-07
90.23
weight-average Mw (kDa)
110
100
90
80
70
60
50
40
-9
2M = D
-8
-7
-6
-5
-4
[sNUCB1] (M)
Mw = f m M m + f d M d = M m (2 − f m )
Ka =
(1 − f m )
[ D]
=
[ M ]2 2( f m ) 2 ct
fm =
− 1 + 1 + 8 K a ct
4 K a ct
Kapoor N, Gupta R., Menon S. T., Folta-Stogniew E., Raleigh D. P., and Sakmar T. P. (2010) Nucleobindin 1 is a calcium-regulated guanine nucleotide
dissociation inhibitor of G{alpha}i1. J.Biol.Chem. 285; 31647-31660
protein conc (M) Mw (kDa)
1.27E-08
55.3
2.63E-08
65.26
3.06E-08
53.93
4.67E-08
63.67
6.89E-08
69.29
7.06E-08
63.65
1.52E-07
73
1.61E-07
74.32
4.10E-06
97.84
7.88E-06
99.16
1.26E-05
99.97
2.00E-05
100.6
1.58E-06
95.04
3.15E-07
75.95
2.43E-06
95.68
7.75E-07
90.23
Kd=0.26µM ± 0.03 µM (12%)
weight-average Mw (kDa)
110
100
90
80
70
60
50
40
-9
2M = D
-8
-7
-6
-5
-4
[sNUCB1] (M)
Mw = f m M m + f d M d = M m (2 − f m )
Ka =
(1 − f m )
[ D]
=
[ M ]2 2( f m ) 2 ct
fm =
− 1 + 1 + 8 K a ct
4 K a ct
Kapoor N, Gupta R., Menon S. T., Folta-Stogniew E., Raleigh D. P., and Sakmar T. P. (2010) Nucleobindin 1 is a calcium-regulated guanine nucleotide
dissociation inhibitor of G{alpha}i1. J.Biol.Chem. 285; 31647-31660
Samples
Protein
+
sNUCB1-Ca2
+
sNUCB1(W333Ter)-Ca2
Monomer
(kDa)
51
37
Oligomeric State in Solution
Molecular Weight (kDa)
Dimerization Constant
AUC
SEC/MALLS
AUC
SEC/MALLS
98.9 ± 0.41
35.2 ± 0.05
99 ± 1*
37± 2
0.26 ± 0.03 µM
*Average from four SEC/MALLS analyses ± StDev
sNUCB1(W333Ter)-Ca2+ truncation mutant, which lacks the lucine zipper domain
Kapoor N, Gupta R., Menon S. T., Folta-Stogniew E., Raleigh D. P., and Sakmar T. P. (2010) Nucleobindin 1 is a calcium-regulated guanine nucleotide dissociation inhibitor of G{alpha}i1.
J.Biol.Chem. 285; 31647-31660
Samples
Protein
+
sNUCB1-Ca2
+
sNUCB1(W333Ter)-Ca2
Monomer
(kDa)
51
37
Oligomeric State in Solution
Molecular Weight (kDa)
Dimerization Constant
AUC
SEC/MALLS
AUC
SEC/MALLS
98.9 ± 0.41
35.2 ± 0.05
99 ± 1*
37± 2
0.26 ± 0.03 µM
Shape Analysis
Rh (DLS)
6.2 ± 0.1
3.0 ± 0.1
f/fo
2.03
1.59
*Average from four SEC/MALLS analyses ± StDev
sNUCB1(W333Ter)-Ca2+ truncation mutant, which lacks the lucine zipper domain
Kapoor N, Gupta R., Menon S. T., Folta-Stogniew E., Raleigh D. P., and Sakmar T. P. (2010) Nucleobindin 1 is a calcium-regulated guanine nucleotide dissociation inhibitor of G{alpha}i1.
J.Biol.Chem. 285; 31647-31660
Determination of dimerization constant from SEC-LS measurements
SecA protein
(nanomotor promotes protein translocation in eubacteria)
conflicting reports about whether SecA functions as a monomer or dimer
WT
monomer = 102 kDa
DS8 deletion mutant monomer = 101 kDa
D11 deletion mutant
1
2
3
4
5
monomer = 100 kDa
6
7
8
9
10
11
Met Leu Ile Lys Leu Leu Thr Lys Val Phe Gly
The two subunits in the crystal structure of B. subtilis SecA
The first nine residues of each subunit are shown in yellow and bluea.
aPicture
taken from : Or, E., A. Navon, and T. Rapoport. 2002. Dissociation of the dimeric SecA ATPase during protein translocation across the bacterial membrane. EMBO J. 21:4470-4479
SecA protein
WT
monomer = 102 kDa
DS8 deletion mutant monomer = 101 kDa
D11 deletion mutant
monomer = 100 kDa
Low salt buffer:
High salt buffer:
10 mM Tris pH 7.5, 5 mM Mg2+, 100
mM KCl
Molar Mass vs. Volume
10 mM Tris pH 7.5, 5 mM Mg2+, 300
Molar Mass vs. Volume
DS8_120407e_01_P_N
D11_062507a_01_P_N_temp
WT_062507a_01_P_N
2.5x105
Molar Mass (g/mol)
Molar Mass (g/mol)
2.0x105
1.5x105
1.0x105
5.0x104
0.0
12.0
14.0
16.0
Volume (mL)
18.0
mM KCl
2.5x10
5
2.0x10
5
1.5x10
5
1.0x10
5
5.0x10
4
0.0
12.0
14.0
D11_120607e_01_P_N
W T _120607e_01_P_N
DS8_120607e_01_P_N
16.0
Volume (mL)
18.0
D11 deletion mutant
mono= 101 kDa
High salt buffer:
10 mM Tris pH 7.5, 5 mM Mg2+, 300
Molar Mass vs. Volume
2.5x10
5
mM KCl,
0.71 mg/mlD11_120607c_01_P_N
7.0 µM
Mw=106 kDa
D11_120607b_01_P_N
0.085 mg/ml
0.83
µM
Mw=103 kDa
D11_120607a_01_P_N
0.045 mg/ml 0.44 µM
Mw=103 kDa
0.019 mg/ml 0.18 µM
Mw=102 kDa
1.5x105
1.0x105
5.0x104
MW changes w ith concentration
0.0
12.0
14.0
16.0
Volume (mL)
18.0
220
MW weight average (kDa)
Molar Mass (g/mol)
2.0x105
D11 high salt
160
D11 high salt
D11 high salt
D11 high salt
100
40
0.01
0.1
1
[protein] (uM)
10
100
Low salt buffer:
D11 deletion mutant
mono= 101 kDa
10 mM Tris pH 7.5, 5 mM Mg2+, 100
Molar Mass vs. Volume 0.69 mg/ml
2.5x10
5
0.17 mg/ml
0.083 mg/ml
0.037 mg/ml
0.014 mg/ml
D11_062507b_01_P_N
6.8 µM
Mw=163
D11_062507e_01_P_N
1.7 µM
Mw=137
D11_062507d_01_P_N
kDa
kDa
Mw=129 kDa
Mw=115 kDa
Mw=105 kDa
0.81 µM
0.36 µM
0.14 µM
1.5x105
1.0x105
5.0x104
MW changes w ith concentration
0.0
12.0
14.0
16.0
Volume (mL)
18.0
220
MW weight average (kDa)
Molar Mass (g/mol)
2.0x105
mM KCl,
D11 low salt
160
D11 low salt
D11 low salt
D11 low salt
D11 low salt
100
40
0.01
0.1
1
[protein] (uM)
10
100
D11 deletion mutant
mono= 101 kDa
Low salt buffer:
10 mM Tris pH 7.5, 5 mM Mg2+, 100
Molar Mass vs. Volume 0.69 mg/ml
2.5x10
5
0.17 mg/ml
0.083 mg/ml
0.037 mg/ml
0.014 mg/ml
Molar Mass (g/mol)
2.0x105
mM KCl,
D11_062507b_01_P_N
6.8 µM
Mw=163
D11_062507e_01_P_N
1.7 µM
Mw=137
D11_062507d_01_P_N
kDa
kDa
Mw=129 kDa
Mw=115 kDa
Mw=105 kDa
0.81 µM
0.36 µM
0.14 µM
1.5x105
1.0x105
5.0x104
MW changes w ith concentration
14.0
16.0
Volume (mL)
Mw = f m M m + f d M d = M m (2 − f m )
2M = D
(1 − f m )
[ D]
Ka =
=
[ M ]2 2( f m ) 2 ct
fm =
− 1 + 1 + 8 K a ct
4 K a ct
18.0
220
MW weight average (kDa)
0.0
12.0
D11 low salt
160
D11 low salt
D11 low salt
D11 low salt
D11 low salt
100
40
0.01
0.1
1
[protein] (uM)
10
100
WT
monomer = 102 kDa
DS8 deletion mutant monomer = 101 kDa
D11 deletion mutant
Low salt buffer:
monomer = 100 kDa
100 mM KCl
Molar Mass vs. Volume
High salt buffer:
Molar Mass vs. Volume
DS8_120407e_01_P_N
D11_062507a_01_P_N_temp
WT_062507a_01_P_N
2.5x105
Molar Mass (g/mol)
1.0x105
5.0x104
0.0
12.0
14.0
16.0
18.0
2.5x10
5
2.0x10
5
1.5x10
5
1.0x10
5
5.0x10
4
0.0
12.0
WT
DS8
D11
16.0
18.0
Volume (mL)
Kd= <1e-9
Kd=7±1e-8 M
Kd=3.5±0.2e-6 M
WT
DS8
D11
240
Kd= 2.2±0.2e-6 M
Kd= 2.41±0.05e-5 M
Kd> 2.4e-4 M
weight-average MW (kDa)
240
220
200
180
160
140
120
100
80
1e-9
D11_120607e_01_P _N
W T _120607e_01_P _N
DS8_120607e_01_P _N
14.0
Volume (mL)
weight-average MW (kDa)
Molar Mass (g/mol)
2.0x105
1.5x105
300 mM KCl
1e-8
1e-7
1e-6
1e-5
[protein]
1e-4
(M)
1e-3
1e-2
220
200
180
160
140
120
100
80
1e-9
1e-8
1e-7
1e-6
1e-5
[protein]
1e-4
(M)
1e-3
1e-2
Thermodynamic linkage for SecA dimerization from SEC/MALLS
Protein
Low salt (100 mM)
ΔG dimer
(kcal/mol)
Kd [M]
High salt (300 mM)
ΔG dimer
(kcal/mol)
Kd [M]
WT
<1x10-9
-12.3
2.2±0.2x10-6
-7.7
DS8
7±1x10-8
-9.7
2.41±0.05x105
-6.3
D11
3.5±0.2x10-6
-7.4
>2.4x10-4
-4.9
D11 high ΔG = -4.9 kcal/mol
-2.5 kcal/mol
DS8 high ΔG = -6.3 kcal/mol
-2.8 kcal/mol
D11 low ΔG = -7.4 kcal/mol
WT high ΔG = -7.7 kcal/mol
-1.4 kcal/mol
WT high ΔG = -7.7 kcal/mol
-7.4 kcal/mol
-3.4 kcal/mol
-6.0 kcal/mol
DS8 low ΔG = -9.7 kcal/mol
-4.9 kcal/mol
-4.6 kcal/mol
-4.6 kcal/mol
-2.6 kcal/mol
WT low ΔG = -12.3 kcal/mol
WT low ΔG = -12.3 kcal/mol
Das S, Stivison E, Folta-Stogniew E, Oliver D. (2008) “Re-examination of the Role of the Amino-Terminus of SecA in Promoting Its Dimerization and Functional State”, J
Bacteriol. (2008) 190;7302-7307.
Thermodynamic linkage for SecA dimerization from SEC/MALLS
Protein
Low salt (100 mM)
Kd [M]
ΔG dimer
(kcal/mol)
High salt (300 mM)
Kd [M]
ΔG dimer
(kcal/mol)
WT
<1x10-9
-12.3
2.2±0.2x10-6
-7.7
DS8
7±1x10-8
-9.7
2.41±0.05x105
-6.3
D11
3.5±0.2x10-6
-7.4
>2.4x10-4
-4.9
Why no AUC data?
Das S, Stivison E, Folta-Stogniew E, Oliver D. (2008) “Re-examination of the Role of the Amino-Terminus of SecA in Promoting Its Dimerization and Functional State”, J
Bacteriol. (2008) 190;7302-7307.
Determination of dimerization constant from SEC-LS measurements
SecA protein
(nanomotor promotes protein translocation in eubacteria)
conflicting reports about whether SecA functions as a monomer or dimer
WT
monomer = 102 kDa
DS8 deletion mutant monomer = 101 kDa
D11 deletion mutant
1
2
3
4
5
monomer = 100 kDa
6
7
8
9
10
11
Met Leu Ile Lys Leu Leu Thr Lys Val Phe Gly
Determination of dimerization constant from SEC-LS measurements
SecA protein
(nanomotor promotes protein translocation in eubacteria)
conflicting reports about whether SecA functions as a monomer or dimer
WT
monomer = 102 kDa
DS8 deletion mutant monomer = 101 kDa
D11 deletion mutant
1
2
3
4
5
monomer = 100 kDa
6
7
8
9
10
11
Met Leu Ile Lys Leu Leu Thr Lys Val Phe Gly
The two subunits in the crystal structure of B. subtilis SecA
The first nine residues of each subunit are shown in yellow and bluea.
aPicture
taken from : Or, E., A. Navon, and T. Rapoport. 2002. Dissociation of the dimeric SecA ATPase during protein translocation across the bacterial membrane. EMBO J. 21:4470-4479
Determination of dimerization constant from SEC-LS measurements
SecA protein
(nanomotor promotes protein translocation in eubacteria)
conflicting reports about whether SecA functions as a monomer or dimer
WT
monomer = 102 kDa
Determination of dimerization constant from SEC-LS measurements
SecA protein
(nanomotor promotes protein translocation in eubacteria)
conflicting reports about whether SecA functions as a monomer or dimer
WT
monomer = 102 kDa
Determination of dimerization constant from SEC-LS measurements
Extracellular ligand binding domain (LBD) of the metabotropic glutamate
mGluR LBD is a homodimer with a glutamate binding pocket in each subunit
expressed in HEK293S cells; yields ~ 25 ug from a single preparation
extracellular ligand-binding domain (LBD), which acts as a detector of glutamate.
WT
monomer = 59kDa
dimeric in solution
mutant
monomer = 59kDa
destabilized dimer?
assess concentration dependent distribution of monomer-dimer
molar mass vs. time






1.2x10
molar mass (g/mol)
1.0x10
8.0x10
6.0x10
4.0x10
2.0x10
protein_X_01.vaf 





protein_X_02.vaf 





protein_X_03.vaf 





protein_X_04.vaf
5
5
4
4
4
4
0.0
20.0
22.0
24.0
26.0
time (min)
28.0
30.0
32.0
Determination of dimerization constant from SEC-LS measurements
Extracellular ligand binding domain (LBD) of the metabotropic glutamate
WT
monomer = 59kDa
dimeric in solution
mutant
monomer = 59kDa
destabilized dimer?
assess concentration dependent distribution of monomer-dimer
Mw protein X (kDa)
160
140
Kd=28 ± 5 nM
120
100
80
60
40
20
0
1e-10
1e-8
1e-6
[protein X] (M)
1e-4
Determination of dimerization constant from SEC-LS measurements
Extracellular ligand binding domain (LBD) of the metabotropic glutamate
WT
monomer = 59kDa
dimeric in solution
mutant
monomer = 59kDa
destabilized dimer?
assess concentration dependent distribution of monomer-dimer
140
110
Kd=23 ± 5 nM
120
100
80
60
40
total conc. vs Col 10
conc. 0.5 ml/min vs MW 0.5 ml/min
20
conc. 0.3 ml/min vs Mw 0.3 ml/min
conc. 0.75 ml/min vs Mw 0.75 ml/min
0
1e-8
1e-6
[protein X] (M)
1e-4
Mw protein X (kDa)
Mw protein X (kDa)
160
Kd=23 ± 5 nM
100
90
80
total conc. vs Col 10
conc. 0.5 ml/min vs MW 0.5 ml/min
70
conc. 0.3 ml/min vs Mw 0.3 ml/min
conc. 0.75 ml/min vs Mw 0.75 ml/min
60
1e-8
[protein X] (M)
Determination of dimerization constant from SEC-LS measurements
Extracellular ligand binding domain (LBD) of the metabotropic glutamate
WT
monomer = 59kDa
dimeric in solution
mutant
monomer = 59kDa
destabilized dimer?
assess concentration dependent distribution of monomer-dimer
Mw protein X (kDa)
160
140
Kd=28 ± 5 nM
120
100
80
60
40
20
0
1e-10
1e-8
1e-6
1e-4
[protein X] (M)
concentration= (6.426 ± 0.094) e-7 g/mL
Determination of dimerization constant from SEC-LS measurements
Extracellular ligand binding domain (LBD) of the metabotropic glutamate
WT
monomer = 59kDa
dimeric in solution
mutant
monomer = 59kDa
destabilized dimer?
assess concentration dependent distribution of monomer-dimer
concentration= (6.426 ± 0.094) e-7 g/mL
Concentration range accessible on an analytical SEC/LS system
~1 μg/ml to ~10 mg/ml
110
27.0
100
26.0
90
25.0
12.5uM injections
25 uM injections
40 uM injections
70
60 uM injections
MW [kDa]
Mw (kDa)
Mw[kDa]
80
flow 1 ml/min
24.0
protein alone
23.0
AUC results
60
22.0
50
40
0.01
21.0
0.1
1
10
100
0
100
200
300
400
500
FIR [uM ]
[protein] uM
Concentration range:
~4 orders of magnitude
0.001 mg/ml
1 μg/ml
1 mg/ml
0.1 mg/ml
9 mg/ml
Size Exclusion Chromatography coupled with Light Scattering
•
Fast and accurate determination of molar masses (weight average) in solution
•
Can be used at wide range of protein concentrations from ~ 1μg/ml to >10mg/ml
(correction for non-ideality)
•
The SEC-UV/RI/LS (static and dynamic) data are very information rich and can be
utilized to learn much more about the sample than “just” determination of Mw
•
Determination of stoichiometry of protein complexes:
•
protein-nucleic acid complexes
membrane protein in complexes with lipids and detergents
•
•
•
Provide information about shape (frictional ratio, f/fo)
Determination of dimerization constant
Ken Williams
Director of W.M. Keck Biotechnology Resource Laboratory at Yale University School
of Medicine
NIH
Users of the Biophysics Resource and SEC/LS Service
Services provided by the Biophysics Resource contributed to > 60 publications
(>30 from Yale)
Light Scattering Services contributed to > 45 publications
Full list at:
http://info.med.yale.edu/wmkeck/biophysics/publications_biophysics_resource.pdf
http://info.med.yale.edu/wmkeck/biophysics
[email protected]
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