A li ti f Li ht S tt iApplication of Light Scattering · 2019. 10. 6. · Dimerization of FIR FIR: human c-myc FarUpStream Element (FUSE) Binding Prot ein (FBP) Interacting Repressor

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



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)


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


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)


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


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



High salt buffer: 300 mM KClLow salt buffer: 100 mM KCl


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)


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


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


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)



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.




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



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




WT 102 kDWT monomer = 102 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.




binding to quartz surface of DAD flow cell

LS 6 LS 7 LS 8 LS 9 LS 10 LS 11


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




no binding to glass surface of DAWN flow cell

LS 6 LS 7 LS 8 LS 9 LS 10 LS 11


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


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


Extracellular ligand binding domain (LBD) of the metabotropic glutamate g g ( ) p g




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






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





lt h


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





lt h


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

[email protected]

Documents

A li ti f Li ht S tt iApplication of Light Scattering · 2019. 10. 6. · Dimerization of FIR FIR: human c-myc FarUpStream Element (FUSE) Binding Prot ein (FBP) Interacting Repressor