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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