a li ti f li ht s tt iapplication of light scatteringkeck.med.yale.edu/biophysics/talks/20th...
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A li ti f Li ht S tt iApplication of Light Scattering
Ewa Folta-StogniewYale UniversityYale University
Biophysics Resource of Keck Laboratory: Yale School of Medicine
Mission: quantitative characterization of interactions betweenMission: 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)
g
• Spectrofluorometer • Stopped-Flow Spectrofluorometer • Surface Plasmon Resonance (SPR) Sensor [BiaCore Biosensor; T100]• Composition Gradient Static Light Scattering (CGSLS)• 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 betweenMission: 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)
g
• Spectrofluorometer• Stopped-Flow Spectrofluorometer• Surface Plasmon Resonance (SPR) Sensor [BiaCore Biosensor; T100]• Composition Gradient Static Light Scattering (CGSLS)• Composition Gradient Static Light Scattering (CGSLS) • Asymmetric flow Field-Flow Fractionation (AFFF)
http://info.med.yale.edu/wmkeck/biophysics/
Typical SEC(AFFF); MALLS system
columnSEC
sample
waste or
collectionsamplesample
waste or
collection
waste or
collectionor AFFF
HPLC UV LS RI HPLC UV/Vis LS RI FL
system detector detector
DLS+SLS
detectorsystem detector detector
DLS+SLS
detector detector
Computer 0.1 m pre -filtered buffer
0.1 m “in -line” filter Computer 0.1 m pre -filtered buffer
0.1 m “in -line” filter
SEC/LS results: Molar Mass Distribution PlotBSA
Monomer: 66 kDa
molar mass vs. volume
BSA_S200_110708a_P_N
51.5x10 Weight-average molar mass
Measured every 2 μl
olar
mas
s (g
/mol
)
45 0 10
51.0x10
0.0 5.0 10.0 15.0 20.0 25.0
mo
0.0
45.0x10
UV trace; A280nm
(RI trace)volume (mL)
Is that ALL? SEC/LS results: Molar Mass Distribution Plot
BSA
Monomer: 66 kDa
molar mass vs. volume
BSA_S200_110708a_P_N
51.5x10 Weight-average molar mass
Measured every 2 μl
olar
mas
s (g
/mol
)
45 0 10
51.0x10
0.0 5.0 10.0 15.0 20.0 25.0
mo
0.0
45.0x10
UV trace; A280nm
(RI trace)volume (mL)
The streptococcal C1 bacteriophage lysin, PlyC,
Holoenzyme is a multimeric protein:
50.3 kDa, “catalytic” subunit
8.0 kDa, “binding” subunit
Ext. coeff. A0.1%280 = 2.2
Ext. coeff. A0.1%280 = 0.3
51.2x10
51.6x10
)
Molar Mass vs. Volume PlyC062705a_01_P_N_178PlyC062705b_01_P_N_178PlyC062705c_01_P_N_178PlyC062705d_01_P_N_178PlyC062705e_01_P_N_178PlyC062705f_01_P_N_178
44.0x10
48.0x10
Mol
ar M
ass
(g/m
ol
SEC/LS MW= 114.0±0.4 kDa
0.010.0 12.0 14.0 16.0 18.0
Volume (mL)
PlyC 1 big+8 small predicted MW = 114.3 kDa
SEC/LS accuracy ~3 % , i.e. ~ 3kDa for PlyC
PlyC 1 big+8 small MW = 114.3 kDa
PlyC bis 2 big+2 small MW = 116 6 kDa
Ext. coeff. A0.1%280 = 1.2
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
PlyC_bis 2 big+2 small MW 116.6 kDa Ext. coeff. A 280 = 2.0
“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) Ext. coeff. A0.1%280 = 1.2
y = 1.2389xR2 0 9861
3
2 big+2 small MW= PlyC_bis model (2+2) Ext. coeff. A0.1%280 = 2.0 Octameric PlyCB. The eight PlyCB subunits arranged in a
ring as observed in the crystal structure of PlyC.
R2 = 0.9861
2
2.5
V/R
I rat
io
UV/RI ti
Protein
Ext. coeff. Est. UV/RI ratio
residual^2observed computed
Apo 1 026 1 279 1 271 0 000
0.5
1
1.5
obse
rved
UV UV/RI ratio
P_C_1big_8small
P_C_2big_2small
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
0
0.5
0 0.5 1 1.5 2 2.5
est. ext. coeff.
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(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
MW disstributions27.0
2.5
3
3.5
m
22000
24000
26000
4.9 mg/ml2.2 mg/ml1 mg/ml
25.0
26.0
Da]
1
1.5
2
Abs
295
nm16000
18000
200001 mg/ml0.14 mg/mlMW [Da]MW [Da]MW [Da]
23.0
24.0
MW
[kD
protein aloneAUC results
0
0.5
13.5 14.5 15.5 16.5 17.5
volume [ml]
12000
14000[ ]
MW [Da]
21.0
22.0
0 100 200 300 400 500
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
FIR [uM]
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
48.0x10Molar Mass vs. Volume
66 kDa 43 kDaFIR_DNA; 58 kDa ; 2:1 binding
55
p y p
44.0x10
46.0x10
rMas
s (g
/mol
)
FIR+DNA 0.65 mg/mlFIR+DNA 0.32 mg/mlFIR+DNA 1.59 mg/ml
35
FIR alone FIR+DNA
0.0
42.0x10
11.0 12.0 13.0 14.0 15.0 16.0
Mol
ar
Volume (mL) 0 30 60 90 120 150
15
Volume (mL)
[FIR] (M)0 30 60 90 120 150
Concentration dependent measurements reveal that in solution the dimerization is driven by DNA binding
FIR-DNA complexes MW (kDa)FIR+DNA (2:1) complex 54.7FIR+DNA (2:2) complex 62 8
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
solution the dimerization is driven by DNA bindingFIR+DNA (2:2) complex 62.8Observed MW 57.7
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
48.0x10Molar Mass vs. Volume
66 kDa 43 kDaFIR_DNA; 58 kDa ; 2:1 binding
p y p
complex stoichiometry from UV/RI measurements
2 5
44.0x10
46.0x10
rMas
s (g
/mol
)
FIR+DNA 0.65 mg/mlFIR+DNA 0.32 mg/mlFIR+DNA 1.59 mg/ml
1
1.5
2
2.5
UV
/RI
DNA (UV/RI=2.34)
FIR:DNA (2:2; 1:1) UV/RI 0 72
0.0
42.0x10
11.0 12.0 13.0 14.0 15.0 16.0
Mol
ar
Volume (mL)
0
0.5
1
0 20 40 60 80
UFIR:DNA (2:1) UV/RI=0.47
UV/RI=0.72
Volume (mL)
FIR-DNA complexes MW (kDa)FIR+DNA (2:1) complex 54.7FIR+DNA (2:2) complex 62 8
MW complex (kDa) FIR (UV/RI=0.14)
FIR:DNA (2:1) FIR:DNA (2:2)
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
FIR+DNA (2:2) complex 62.8Observed MW 57.7
55 kDa 63 kDa
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 l tid d t t(li id) lpolypeptide:detergent(lipid) complex
0.8
1.0
250
300220 156 66 kDa
0.8
1.0
250
300220 156 66 kDa
A 280
0.4
0.6
MW
[kD
a]
100
150
200
absorbace at 280 nmpolypeptide+detergent+lipids polypeptideA 2
800.4
0.6
MW
[kD
a]
100
150
200
absorbace at 280 nmpolypeptide+detergent+lipids polypeptide
V l [ L]12 14 16 18 20
0.0
0.2
0
50
V l [ L]12 14 16 18 20
0.0
0.2
0
50
Volume [mL]
Protein Polypeptide[kDa]
Oligomeric state
Full complex
[kDa]
Grams of detergent/lipids per gram of polypeptide
Volume [mL]
Protein Polypeptide[kDa]
Oligomeric state
Full complex
[kDa]
Grams of detergent/lipids per gram of polypeptide[kDa] gram of polypeptide
KtrB(monomer 49kDa) 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
[kDa] gram of polypeptide
KtrB(monomer 49kDa) 98 dimer 238 1.4
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
220 kDa 66 kDa2
[kD
a]
100
120
140
A 280 1
Mol
ar M
ass
20
40
60
80
elution volume [ml]12 13 14
00
20
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
Effects of detergent on oligomeric state of KtrA RCK domain
220 kDa 66 kDa
0.8
1.2
[kD
a]
150
220 kDa 66 kDa
KtrA RCK domain no detergent
A28
0
0.4
Mol
ar M
ass
50
100g
(octamer)
220 kDa 66 kDa
elution volume [ml]10 12 14 16
0.0 0
00.3
0.4
0.5
s [k
Da]
100
150
A280
KtrA RCK domain plus detergent
(tetramer and monomer) + micelleA
280
0.1
0.2
Mol
ar M
ass
50
100MMpp
MMcomplex
elution volume [ml]14 15 16 17 18
0.0 0
Determination of dimerization constant from SEC-LS ete at o o d e at o co sta t o S C Smeasurements
Results:Input: Results:• Determination of dimerization
constant
Input:• SEC/LS analyses at several eluting
concentrations
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 102 kDWT monomer = 102 kDa
DS8 deletion mutant monomer = 101 kDa
D11 deletion mutant monomer = 100 kDa
1 2 3 4 5 6 7 8 9 10 11
Met Leu Ile Lys Leu Leu Thr Lys Val Phe Gly
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
The two subunits in the crystal structure of B. subtilis SecAThe first nine residues of each subunit are shown in yellow and bluea.
SecA proteinWT monomer = 102 kDa
DS8 deletion mutant monomer = 101 kDa
D11 deletion mutant monomer = 100 kDa
High salt buffer:
10 mM Tris pH 7 5 5 mM Mg2+ 300 mM KCl
Low salt buffer:
10 mM Tris pH 7 5 5 mM Mg2+ 100 mM KCl
52.5x10Molar Mass vs. Volume D11_120607e_01_P_N
WT _120607e_01_P_NDS8_120607e_01_P_N
52.5x10Molar Mass vs. Volume DS8_120407e_01_P_N
D11_062507a_01_P_N_tempWT_062507a_01_P_N
10 mM Tris pH 7.5, 5 mM Mg2+, 300 mM KCl10 mM Tris pH 7.5, 5 mM Mg2+, 100 mM KCl
51.5x10
52.0x10
Mas
s (g
/mol
)
51.5x10
52.0x10
Mas
s (g
/mol
)
45.0x10
51.0x10
Mol
ar M
45.0x10
51.0x10
Mol
ar M
0.012.0 14.0 16.0 18.0
Volume (mL)
0.012.0 14.0 16.0 18.0
Volume (mL)
D11 deletion mutant mono= 101 kDa
High salt buffer: 10 mM Tris pH 7.5, 5 mM Mg2+, 300 mM KCl,
52.5x10Molar Mass vs. Volume D11_120607c_01_P_N
D11_120607b_01_P_ND11_120607a_01_P_N
0.71 mg/ml 7.0 M Mw=106 kDa 0.085 mg/ml 0.83 M Mw=103 kDa 0.045 mg/ml 0.44 M Mw=103 kDa 0.019 mg/ml 0.18 M Mw=102 kDa
51.5x10
52.0x10
Mas
s (g
/mol
)
45.0x10
51.0x10
Mol
ar M
MW changes with concentration
0.012.0 14.0 16.0 18.0
Volume (mL)
160
220
rage
(kD
a)D11 high salt
D11 hi h lt
100
MW
wei
ght a
ver D11 high salt
D11 high salt
D11 high salt
400.01 0.1 1 10 100
[protein] (uM)
D11 deletion mutant mono= 101 kDa
Low salt buffer:10 mM Tris pH 7.5, 5 mM Mg2+, 100 mM KCl,
52.5x10
Molar Mass vs. Volume D11_062507b_01_P_ND11_062507e_01_P_ND11_062507d_01_P_N
0.69 mg/ml 6.8 M Mw=163 kDa 0.17 mg/ml 1.7 M Mw=137 kDa 0.083 mg/ml 0.81 M Mw=129 kDa 0 037 mg/ml 0 36 M Mw=115 kDa
51.5x10
52.0x10
ass
(g/m
ol)
0.037 mg/ml 0.36 M Mw=115 kDa 0.014 mg/ml 0.14 M Mw=105 kDa
45.0x10
51.0x10
Mol
ar M
MW changes with concentration
0.012.0 14.0 16.0 18.0
Volume (mL)
160
220
rage
(kD
a)
D11 low salt
D11 low salt
100
MW
wei
ght a
ver
D11 low salt
D11 low salt
D11 low salt
400.01 0.1 1 10 100
[protein] (uM)
D11 deletion mutant mono= 101 kDa
Low salt buffer:10 mM Tris pH 7.5, 5 mM Mg2+, 100 mM KCl,
52.5x10
Molar Mass vs. Volume D11_062507b_01_P_ND11_062507e_01_P_ND11_062507d_01_P_N
0.69 mg/ml 6.8 M Mw=163 kDa 0.17 mg/ml 1.7 M Mw=137 kDa 0.083 mg/ml 0.81 M Mw=129 kDa 0 037 mg/ml 0 36 M Mw=115 kDa
51.5x10
52.0x10
ass
(g/m
ol)
0.037 mg/ml 0.36 M Mw=115 kDa 0.014 mg/ml 0.14 M Mw=105 kDa
45.0x10
51.0x10
Mol
ar M
MW changes with concentration
0.012.0 14.0 16.0 18.0
Volume (mL)
160
220
rage
(kD
a)
D11 low salt
D11 low salt)2( fMMfMfM
100
MW
wei
ght a
ver
D11 low salt
D11 low salt
D11 low salt
)2( mmddmmw fMMfMfM
DM 240
0.01 0.1 1 10 100
[protein] (uM)tm
ma cf
fMDK 22 )(2
)1(][][
ta
tam cK
cKf
4811
WT monomer = 102 kDa
DS8 deletion mutant monomer = 101 kDa
D11 deletion mutant monomer = 100 kDa
High salt buffer: 300 mM KClLow salt buffer: 100 mM KCl
D11 deletion mutant monomer = 100 kDa
51 5x10
52.0x10
52.5x10
g/m
ol)
Molar Mass vs. Volume D11_120607e_01_P_NWT _120607e_01_P_NDS8_120607e_01_P_N
51 5x10
52.0x10
52.5x10
g/m
ol)
Molar Mass vs. Volume DS8_120407e_01_P_ND11_062507a_01_P_N_tempWT_062507a_01_P_N
0.0
45.0x10
51.0x10
1.5x10
12 0 14 0 16 0 18 0
Mol
ar M
ass
(g
0.0
45.0x10
51.0x10
1.5x10
12 0 14 0 16 0 18 0
Mol
ar M
ass
(g
12.0 14.0 16.0 18.0Volume (mL)
WT Kd= 2.2±0.2e-6 MDS8 Kd= 2.41±0.05e-5 MD11 Kd> 2.4e-4 M
240
WT Kd= <1e-9DS8 Kd=7±1e-8 MD11 Kd=3.5±0.2e-6 M
240
12.0 14.0 16.0 18.0Volume (mL)
aver
age
MW
(kD
a)
140
160
180
200
220
240
aver
age
MW
(kD
a)
140
160
180
200
220
[protein] (M)1e-9 1e-8 1e-7 1e-6 1e-5 1e-4 1e-3 1e-2
wei
ght-a
80
100
120
[protein] (M)1e-9 1e-8 1e-7 1e-6 1e-5 1e-4 1e-3 1e-2
wei
ght-
80
100
120
Thermodynamic linkage for SecA dimerization from SEC/MALLS
Protein Low salt (100 mM) High salt (300 mM)
Kd [M] ΔG dimer (kcal/mol)
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
WT high ΔG = -7.7 kcal/mol
D11 high ΔG = -4.9 kcal/mol
D11 low ΔG = -7.4 kcal/mol
-2.8 kcal/mol-2.5 kcal/mol
-7.4 kcal/mol WThigh ΔG = -7.7 kcal/mol
DS8 high ΔG = -6.3 kcal/mol-1.4 kcal/mol
-3.4 kcal/mol-6.0 kcal/mol
WT l ΔG = 12 3 kcal/mol
-4.9 kcal/mol
-4.6 kcal/mol
WT ΔG 12 3 k l/ l
DS8 low ΔG = -9.7 kcal/mol
-2.6 kcal/mol-4.6 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.
WT low ΔG = -12.3 kcal/mol WT low ΔG = -12.3 kcal/mol
Thermodynamic linkage for SecA dimerization from SEC/MALLS
Protein Low salt (100 mM) High salt (300 mM)
Kd [M] ΔG dimer (kcal/mol)
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 102 kD
LS 6 LS 7 LS 8 LS 9 LS 10 LS 11
strip chart: WT_062507b_01.ADF
e (V
)
0.4
0.6
WT monomer = 102 kDa
DS8 deletion mutant monomer = 101 kDa
D11 deletion mutant monomer = 100 kDa
LS 11 LS 12 LS 14 LS 15 LS 16 LS 17 LS 18
dete
ctor
vol
tage
0 4
-0.2
0.0
0.2
1 2 3 4 5 6 7 8 9 10 11
Met Leu Ile Lys Leu Leu Thr Lys Val Phe GlyLS 6
strip chart: D11 062507a 01.ADF
LS 18 UV dRI QELS
volume (mL)0.0 5.0 10.0 15.0 20.0 25.0
-0.4
LS 6 LS 7 LS 8 LS 9 LS 10 LS 11 LS 12 LS 14
strip chart: D11_062507a_01.ADF
r vo
ltage
(V)
2 0
3.0
4.0
5.0
LS 15 LS 16 LS 17 LS 18 UV dRI QELS
volume (mL)0.0 5.0 10.0 15.0 20.0 25.0
dete
cto
0.0
1.0
2.0
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 102 kDWT monomer = 102 kDa
DS8 deletion mutant monomer = 101 kDa
D11 deletion mutant monomer = 100 kDa
1 2 3 4 5 6 7 8 9 10 11
Met Leu Ile Lys Leu Leu Thr Lys Val Phe Gly
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
The two subunits in the crystal structure of B. subtilis SecAThe first nine residues of each subunit are shown in yellow and bluea.
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
binding to quartz surface of DAD flow cell
LS 6 LS 7 LS 8 LS 9 LS 10 LS 11
strip chart: WT_062507b_01.ADF
e (V
)
0.2
LS 6 LS 7 LS 8 LS 9 LS 10 LS 11
strip chart: WT_062507d_01.ADFe
(V)
0.00
0.02
LS 11 LS 12 LS 14 LS 15 LS 16 LS 17 LS 18
dete
ctor
vol
tag
0.0
0.1LS 11 LS 12 LS 14 LS 15 LS 16 LS 17 LS 18
dete
ctor
vol
tag
-0.06
-0.04
-0.02
LS 18 UV dRI QELS
volume (mL)0.0 5.0 10.0 15.0 20.0 25.0
-0.1LS 18 UV dRI QELS
volume (mL)0.0 5.0 10.0 15.0 20.0 25.0
-0.08
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
no binding to glass surface of DAWN flow cell
LS 6 LS 7 LS 8 LS 9 LS 10 LS 11
strip chart: WT_062507b_01.ADF
e (V
)
0.6
0.7
LS 6 LS 7 LS 8 LS 9 LS 10 LS 11
strip chart: WT_062507d_01.ADFe
(V) 0.36
0.38
LS 11 LS 12 LS 14 LS 15 LS 16 LS 17 LS 18
dete
ctor
vol
tag
0.4
0.5
LS 11 LS 12 LS 14 LS 15 LS 16 LS 17 LS 18
dete
ctor
vol
tag
0 28
0.30
0.32
0.34
LS 18 UV dRI QELS
volume (mL)0.0 5.0 10.0 15.0 20.0 25.0
0.3LS 18 UV dRI QELS
volume (mL)0.0 5.0 10.0 15.0 20.0 25.0
0.26
0.28
Determination of dimerization constant from SEC-LS measurements
Extracellular ligand binding domain (LBD) of the metabotropic glutamate g g ( ) p gmGluR 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. g g ( ) g
WT monomer = 59kDa dimeric in solution
mutant monomer = 59kDa destabilized dimer?
assess concentration dependent distribution of monomer-dimer
molar mass vs. time
protein_X_01.vaf protein_X_02.vaf protein_X_03.vaf protein_X_04.vaf
/mol
)
48.0x10
51.0x10
51.2x10
mol
ar m
ass
(g/
44.0x10
46.0x10
8.0x10
time (min)20.0 22.0 24.0 26.0 28.0 30.0 32.0
0.0
42.0x10
Determination of dimerization constant from SEC-LS measurements
Extracellular ligand binding domain (LBD) of the metabotropic glutamate g g ( ) p g
WT monomer = 59kDa dimeric in solution
mutant monomer = 59kDa destabilized dimer?
assess concentration dependent distribution of monomer-dimer
Kd=23 ± 5 nM
(kD
a)
120
140
160
pro
tein
X (
60
80
100
1e-10 1e-8 1e-6 1e-4
Mw
0
20
40
[protein X] (M)1e-10 1e-8 1e-6 1e-4
Determination of dimerization constant from SEC-LS measurements
Extracellular ligand binding domain (LBD) of the metabotropic glutamate g g ( ) p g
WT monomer = 59kDa dimeric in solution
mutant monomer = 59kDa destabilized dimer?
assess concentration dependent distribution of monomer-dimer
Kd=23 ± 5 nM140
160
K =23 ± 5 nM110
d
otei
n X
(kD
a)
60
80
100
120
140 Kd=23 ± 5 nM
otei
n X
(kD
a)80
90
100
1 8 1 6 1 4
Mw
pro
0
20
40
60total conc. vs Col 10 conc. 0.5 ml/min vs MW 0.5 ml/min conc. 0.3 ml/min vs Mw 0.3 ml/min conc. 0.75 ml/min vs Mw 0.75 ml/min
1e 8
Mw
pro
60
70
80total conc. vs Col 10 conc. 0.5 ml/min vs MW 0.5 ml/min conc. 0.3 ml/min vs Mw 0.3 ml/min conc. 0.75 ml/min vs Mw 0.75 ml/min
[protein X] (M)1e-8 1e-6 1e-4
[protein X] (M)1e-8
Determination of dimerization constant from SEC-LS measurements
Extracellular ligand binding domain (LBD) of the metabotropic glutamate g g ( ) p g
WT monomer = 59kDa dimeric in solution
mutant monomer = 59kDa destabilized dimer?
lt h
assess concentration dependent distribution of monomer-dimer
Kd=28 ± 5 nM
kDa)
120
140
160
results graph
eta)
/K*c
47.4x10
47.6x10
47.8x10
w p
rote
in X
(
40
60
80
100
sin²(theta/2)0.0 0.2 0.4 0.6 0.8
R(t
he
47.0x10
47.2x10
control graph
1e-10 1e-8 1e-6 1e-4
Mw
0
20
40( )
LS dRI
ve s
cale
0.5
1.0
[protein X] (M)
volume (mL)12.5 13.0 13.5 14.0
rela
tiv
0.0 1 1concentration= (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 g g ( ) p g
WT monomer = 59kDa dimeric in solution
mutant monomer = 59kDa destabilized dimer?
lt h
assess concentration dependent distribution of monomer-dimer
results graph
eta)
/K*c
47.4x10
47.6x10
47.8x10
LS 7 LS 8 LS 9 LS 10 LS 11 LS 13 LS 14 LS 15 LS 16
strip chart: protein_X_02_A220.vaf
or v
olta
ge (V
)
0 054
0.055
0.056
sin²(theta/2)0.0 0.2 0.4 0.6 0.8
R(t
he
47.0x10
47.2x10
control graphLS 16 LS 17 LS 18 UV FM QELS dRI
volume (mL)5.0 10.0 15.0 20.0 25.0
dete
cto
0.053
0.054 ( )LS dRI
ve s
cale
0.5
1.0
volume (mL)12.5 13.0 13.5 14.0
rela
tiv
0.0 1 1concentration= (6.426 ± 0.094) e-7 g/mL
Concentration range accessible on an analytical SEC/LS system
1 / l t 10 / l~1 μg/ml to ~10 mg/ml
80
90
100
110
Mw[kDa]12 5uM injections
25.0
26.0
27.0
a]
50
60
70
80
Mw
(kD
a) 12.5uM injections25 uM injections40 uM injections60 uM injectionsflow 1 ml/min
22.0
23.0
24.0
MW
[kD
a
protein aloneAUC results
400.01 0.1 1 10 100
[protein] uM
21.00 100 200 300 400 500
FIR [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( g g )
• 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)p ( )• 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
http://info.med.yale.edu/wmkeck/biophysics