Light Scattering Coupled to Refractive Index and UV Absorption Measurements in Studies of Membrane ProteinsAbsorption Measurements in Studies of Membrane Proteins

Homo- and Hetero Associations

Ewa Folta-Stogniew

Biophysics Resource; Keck Laboratory Yale School of Medicine

• Light Scattering Technologies

– Static and dynamic light scattering

– Parameters derived from SLS and DLS measurements

• Flow Mode Light Scattering Applications in Studies of Membrane Proteins

– Determination of an oligomeric state of membrane proteins from SEC-LS/UV/RI measurement

– Determination of complex formation and stoichiometry from SEC-LS measurements

• Capabilities and limitation of SEC-LS/RI/UV measurements

Li ht S tt i E i tLight Scattering Experiments

sample cell

Monochromatic

Laser Light Io Icellg o I1

I

2

detector at angle

ComputerI

detector at angle 2

Light Scattering Experiments

• Static (classical)time-averaged intensity of scattered light

• Dynamic (quasielastic)fluctuation of intensity of scattered light with time

Parameters derived: Parameters derived:

light

Measurements:• batch mode a a e e s de ed

• Molar Mass (weight-average) accuracy ~5%

• (1/2) root mean square radii

for (1/2)> (20) ~ 15 nm

a a e e s de ed• DT translation diffusion coefficient

• Rh hydrodynamic radius (Stokes radius) Uncertainty of ~10% for monodisperse sample

• “in-line” mode combined with a fractionation step,

i.e. chromatography, mainly Size Exclusion Chromatography, Flow Field Fractionation

( g ) ( )

• A2 second virial coefficient

Rayleigh-Debye-Zimm formalism Stokes-Einstein

cAPMR

cKw 2

2)(

1)(

*

hT R

kTD6

Rayleigh-Debye-Zimm formalism Stokes-Einstein

D t l ti l diff i ffi i tR() Rayleigh ratio (excess scattered light)c sample concentration (g/ml)Mw weight-average molecular weight (molar mass)A2 second virial coefficient (ml-mol/g2)P() form factor (angular dependence)K optical constant [4π2n2 (dn/dc)2 /(λo4NA)]

DT translational diffusion coefficientk Boltzmann constantT temperatureRh radiusη solvent viscosity

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

Three Detector monitoring

Define Peaks

LS UV QELS dRI

e

1.0

rela

tive

scal

e

0.5

volume (mL)0.0 5.0 10.0 15.0 20.0 25.0

0.0

RI UV at 280 nm LS at 90°

( )

cAPMR

cK22)(

1)(

*

Rayleigh-Debye-Zimm formalism

PMR w 2)()(

R() Rayleigh ratio (excess scattered light)c sample concentration (g/ml)Mw weight-average molecular weight (molar mass)A2 second virial coefficient (ml mol/g2)

1/(Mw)control graph

A2 second virial coefficient (ml-mol/g2)P() form factor (angular dependence)K optical constant [4π2n2 (dn/dc)2 /(λo4NA)]

res ults graph

-51.495x 10

-51.500x 10

)(*RcK

LS dRI

e

1.0

K*c

/R(t

heta

)

-51.485x 10

-51.490x 10

rela

tive

scal

e

0.5

s in ²( th e ta/2)0.0 0.2 0.4 0.6 0.8

volum e (m L)12.0 14.0 16.0 18.0

0.01 1

SEC/LS results: Molar Mass Distribution Plot

molar mass vs. volume

BSA S200 110708a P N

BSA

Monomer: 66 kDa

BSA_S200_110708a_P_N

ol)

51.5x10 Weight-average molar mass

Measured every 2 μl

mol

ar m

ass

(g/m

o

45.0x10

51.0x10

volume (mL)0.0 5.0 10.0 15.0 20.0 25.0

m

0.0

UV trace; A280nm(RI trace)

volume (mL)

Rayleigh-Debye-Zimm formalismProtein 47 kDa (monomer) well characterized porin detergent

cAPMR

cKw 2

2)(

1)(

*

R() Rayleigh ratio (excess scattered light)c sample concentration (g/ml)

detergent

dodecyl maltoside (C12M) MW = 511 g/mol

0.5g/L i.e. 0.05%

CMC = 0.008% micelle size 50-70 kDa

p (g/ )Mw weight-average molecular weight (molar mass)A2 second virial coefficient (ml-mol/g2)P() form factor (angular dependence)K optical constant [4π2n2 (dn/dc)2 /(λo4NA)]

Protein+lipid+detergent

0.8Peak ID - PRNC__DC

90°AUX1AUX2

Protein+lipid+detergentProtein+lipid+detergent

0.4

0.6

90°

Det

ecto

r

AUX2

empty micelleempty micelle

-0.2

0.0

0.2

AU

X, 9

LS @ 90 degree

RI

UV @ 280 nm

Protein+det.+lipids

Protein+det.+lipids

Protein

10.0 12.0 14.0 16.0 18.0 20.0Volume (mL)

Determination of the oligomeric state of modified proteins from SEC-LS/UV/RI analysis

1. Glycosylated proteins

2. Proteins conjugated with polyethylene glycol

3. Membrane protein present as a complex with lipids and detergents

Input: Results:

• Polypeptide sequence

• Chemical nature of the modifier

• Oligomeric state of the polypeptide

• Extend of modification (grams of modifier /gram of polypeptide)

“three detector method”

Yutaro Hayashi, Hideo Matsui and Toshio Takagi (1989) Methods Enzymol,172:514-28Jie Wen, Tsutomu Arakawa and John S. Philo (1996) Anal Biochem, 240:155-66Ewa Folta-Stogniew (2006) Methods in Molecular Biology: New and Emerging Proteomics Techniques, pp. 97–112

Determination of the oligomeric state of modified proteins from SEC-LS/UV/RI analysis

))((* UVLSkMW MWp Molecular Weight (polypeptide)2)(RIpMW

ε extinction coefficient

LS light scattering intensity

UV absorbance (ε)

RI refractive index change

k calibration constant

“three detector method”

Yutaro Hayashi, Hideo Matsui and Toshio Takagi (1989) Methods Enzymol,172:514-28Jie Wen, Tsutomu Arakawa and John S. Philo (1996) Anal Biochem, 240:155-66Ewa Folta-Stogniew (2006) Methods in Molecular Biology: New and Emerging Proteomics Techniques, pp. 97–112

Determination of the oligomeric state of detergent solubilized proteins:

polypeptide+lipids+detergent complexes of unknown detergent+lipids content

Proteins: 47 kDa porin LamB trimer = 141 kDa33 kDa hemolysin -HL heptamer = 231 kDa

detergent

dodecyl maltoside (C12M) MW = 511 g/mol

0.5g/L i.e. 0.05%

CMC = 0.008% micelle size 50-70 kDa

Protein 47 kDa (monomer) well characterized porin detergent

Rayleigh-Debye-Zimm formalism

detergent

dodecyl maltoside (C12M) MW = 511 g/mol

0.5g/L i.e. 0.05%

CMC = 0.008% micelle size 50-70 kDa

cAPMR

cKw 2

2)(

1)(

*

R() Rayleigh ratio (excess scattered light)c sample concentration (g/ml)p (g/ )Mw weight-average molecular weight (molar mass)A2 second virial coefficient (ml-mol/g2)P() form factor (angular dependence)K optical constant [4π2n2 (dn/dc)2 /(λo4NA)]

0.8Peak ID - PRNC__DC

90°AUX1AUX2

Protein+lipid+detergent

0.4

0.6

90°

Det

ecto

r

AUX2

empty micelle

-0.2

0.0

0.2

AU

X, 9

LS @ 90 degree

RI

UV @ 280 nm

Protein+det.+lipids

Protein+det.+lipids

Protein

10.0 12.0 14.0 16.0 18.0 20.0Volume (mL)

475 kDa 220 kDa 66 kDa 43 kDa

1.2

]

300A280 LamBMMpp LamB

A 280 0.8

Mpp

[kD

a

200

A

0.4 MM

100

12 14 16 18 20 220.0 0

elution volume [mL]

porin monomer = 47 kDa

MW = 149 3 kDa trimer 51 6 10

Molar Mass vs. Volume PRNB__DCPRNC__DC51.6x10

__PRNA__DC

51.2x10

(g/m

ol)

48.0x10

Mol

ar M

ass

(

44.0x10

0.010.0 12.0 14.0 16.0 18.0 20.0

Volume (mL)

Determination of the oligomeric state of detergent solubilized proteins:

polypeptide+lipids+detergent complexes of unknown detergent+lipids content

Proteins: 47 kDa porin LamB trimer = 141 kDa

33 kDa hemolysin -HL heptamer = 231 kDa

detergent

dodecyl maltoside (C12M) MW = 511 g/mol

0.5g/L i.e. 0.05%

CMC = 0.008% micelle size 50-70 kDa

monomer = 33 kDa

MW = 225 13 kDa heptamer

3001.2 MMpp -HLA280 HL

Mpp

[kD

a]

200

A28

0 0.8280

MM

100

A

0.4

l ti l [ L]

12 14 16 18 20 2200.0

elution volume [mL]

Proteins: 47 kDa porin LamB trimer = 141±3 kDa (141 kDa)

33 kDa hemolysin HL heptamer = 215±20 kDa (231 kDa)

475 kDa 220 kDa 66 kDa 43 kDa

33 kDa hemolysin -HL heptamer = 215±20 kDa (231 kDa)

475 kDa 220 kDa 66 kDa 43 kDa

1.2

Da]

300A280 LamBMMpp LamBMMpp -HL

A28

0

0.4

0.8

MM

pp [k

D

100

200A280 HL

12 14 16 18 20 220.0 0

elution volume [mL]

12 14 16 18 20 22

Three Detector Method

allows determination of mass of detergent/lipids bound to a polypeptide

)()(

2 UVRIAk

dcdn

app

Protein+lipid+detergent

)(

)('UV

RIKdcdn

dcdn

dcdn

ldppapp

δ

Protein

is mass of detergent and/or lipids per 1 gram of polypeptide

Assumption : detergent does not produce any signal in UV

MWcomplex = 285 kDa

MWpolypeptide = 141 kDa

MWcomplex = 271 kDa

MWpolypeptide = 215 kDa

475 kDa 220 kDa 66 kDa 43 kDa

= 1.02 lipids per 1 gram of polypeptide = 0.26 lipids per 1 gram of polypeptide

1 2 300A280 LamB

280 0.8

1.2

ex [k

Da]

200

300280MMcomplex LamBMMcomplex -HLA280 HL

= 1.02

A2

0.4

MM

com

pl

100 = 0.26

elution volume [mL]

12 14 16 18 20 220.0 0

elution volume [mL]

Yernool D, Boudker O., Folta-Stogniew E., and Gouaux E. (2003) Trimeric subunit stoichiometry of the glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus. Biochemistry 42: 12981-12988

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 (49 kDa) and KtrA RCK domain (regulating and conductance of K+)

KtrA RCK domain (16 kDa) : basic assembly dimer, higher order oligomers: tetramer or octamer

0

0.8

1.2

s [k

Da]

100

150

220 kDa 66 kDa

10 12 14 16

A28

0

0.0

0.4

Mol

ar M

ass

0

50

100

elution volume [ml]10 12 14 16

0.4

0.5

a]

150

220 kDa 66 kDa

A28

0

0.1

0.2

0.3

Mol

ar M

ass

[kD

a

50

100A280MMppMMcomplex

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

elution volume [ml]14 15 16 17 18

0.0 0Buffer + DDM

Multiple oligomeric states for reconstituted KtrAB K+ Transporter

KtrAB ion transporter

complex : KtrAB octameric KtrA + dimeric KtrB (8:2) model polypeptide = 228 kDaoctameric KtrA + 2x dimeric KtrB (8:4) model polypeptide = 325 kDa

complexpolypeptideKtrA Abs 280 nm

400

5001.0

KtrB Abs 280nmKtrAB Abs 280nm

octameric KtrA + 2x dimeric KtrB (8:4) model polypeptide = 325 kDa

Mw

(kD

a)

100

200

300

A 280 0.5

octameric KtrA + dimeric KtrB (8:2) model polypeptide = 228 kDa

polypeptide = 325 kDa

Elution volume (ml)12 14 16 18

00.0

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 octameric KtrA + dimeric KtrB (8:2) model polypeptide = 228 kDaoctameric KtrA + 2x dimeric KtrB (8:4) model polypeptide = 325 kDa

1 0 Dimer:octamer=0 4

dimer:octamerKtrB:KtrA

complex Elution volume

(ml)

Total mass of complex

(kDa)

Poly-peptide(kDa)

lipids(kDa)

A28

0

0.5

1.0 Dimer:octamer=0.4Dimer:octamer=0.9Dimer:octamer=2.2Dimer:octamer=3.4

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

A

0.0

dimer:octamerKtrB:KtrA

ExcessKtrB

Elution volume

8:2 model (228 kDa) correct model ?

8:4 model (325 kDa) correct model ?

Elution volume (ml)12 14 16 18

KtrB:KtrA KtrB dimer?

volume (ml)

model ? model ?

computed MW for complex (kDa)

difference from model(kDa)

computed MW for complex (kDa)

difference from model(kDa)

0.4 14.23 228 0 Yes 250 -750.4 14.23 228 0 Yes 250 75

0.9 14.05 240 12 Yes 264 -61

2.2 Yes 13.99 274 46 302 -24 Yes

3.7 Yes 13.91 271 43 299 -27 Yes

Three detector approach

• fast and accurate determination of oligomeric state of detergent solubilized membrane g gproteins

• only protein sequence needed for determination of association state

• the amount of detergent and lipids associated with the polypeptide can be determined from a single SEC-LS/RI/UV analysis

• suitable for variety of detergentsy g

• can be used at wide range of protein concentrations from ~ 10μg/ml to >10mg/ml (correction for non-ideality)

Ken Williams

Director of W.M. Keck Biotechnology Resource Laboratory at Yale University School of Medicine

NIH

1S10 RR014776 01 “SEC/L Li ht S tt i I t t ti ”1S10 RR014776-01 “SEC/Laser Light Scattering Instrumentation” 1S10 RR023748-01 "Asymmetric flow FFF and Composition Gradient/Light Scattering System"

Users of SEC/LS Service

Light Scattering Services contributed to> 40 publications

Full list at: http://info.med.yale.edu/wmkeck/biophysics/publications_biophysics_resource.pdf

http://info.med.yale.edu/wmkeck/biophysics

Ewa.Folta-Stogniew@yale.edu