STRUCTURE PECULIARITIES OF α-CRYSTALLIN STUDIED BY SMALL ANGLE NEUTRON AND X-RAY SCATTERING

TN Murugova1 OI Ivankov15 AI Kuklin13 KO Muranov2 NB Poliansky2 VM Garamus4 AV Krivandin2

1FLNP JINR Dubna Russia2Institute of Biochemical Physics of RAS Moscow Russia (the Tasking)3Moscow Institute of Physics and Technology Dolgoprudny Russia4Helmholtz-Zentrum Geesthacht Zentrum fuumlr Material- und Koumlstenforschung GmbH Geesthacht Germany5Taras Shevchenko National University of Kyiv Kyiv Ukraine

Function

bullThe main component of the vertebrate eye lens (300 mgml)

bull Provides a proper refractive index of the lens

bullChaperon-like activity (forms soluble complexes with destabilized proteins and prevents their unspecific aggregation and uncontrolled denaturation)

bullSlows down the age-dependent loss of the lens transparency (cataract)

Problem

bullHow does this protein protect the lensbullWhat is the mechanism of its binding of target proteins bullWhat is the role of oligomeric structure and subunit

exchange in this mechanism

Knowledge of the quaternary structure of α-crystallin is a missing link in defining a structure-function relationship and the possible role of the a-crystallin subunits in the lens and other organs in normal and abnormal conditions

The quaternary structure of α-crystallin is not known to date

Structure oligomeric protein

Monomers αA- and αB-crystallin20 kDa

Oligomer20-40 monomers400-1000 kDa

Models of α-crystallin structure

Aquilina J A and Morris A M Evidence for specific subunit distribution and interactions in the quaternary structure of α-crystallin2010httprouoweduauscipapers175

Open micellar configurationB GROTH-VASSELLI ET AL Exp Eye Res (1995) 61 249-253

Single particle reconstructions of native a-crystallin from Electron microscopyDA Haley et al J Mol Biol (2000) 298 261plusmn272

Micellar three-layer modelWalsh et al THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol 266 pp20079-200841991

3-layer modelTardieu et al I Mol Biol (1986) 192711 -724

A model of αB-crystallin with bound α-lactalbumin Electron microscopyScale bar represents 100 Aring J Horwitz Exp Eye Res (2003) 76 145-153

Why Small Angle Scattering

bullPolydisperse system variable size and number of monomers

bullDynamic system monomer exchange between oligomers

bullEfforts to crystallize α-crystallin have failed

bullThe aggregate is too large for high resolution 2D NMR

Small angle scattering allows to study structure of macromolecules (10-1000 Aring) in solution

ShapeSizeMolecular massVolume

Distance distribution function p(r)(indirect Fourier transform)

001 01

1E-3

001

01

1

10

SANS SAXSIn

tens

ity a

u

q A-1

Curves of SANS and SAXS for the same sample of α-crystallin (concentration 9 mgml)

0 50 100 150 20000

02

04

06

08

10

p(r)

r A

SANS SAXS

Dmax

Distance distribution functions for α-crystallin molecule calculated from SANS and SAXS curves by indirect transform

max max

2 2

0 0

( ) ( ) D D

gR p r r dr p r dr max

0

(0) 4 ( )D

I p r dr

Program GNOM httpwwwembl-hamburgdebiosaxssoftwarehtml

Function

bullThe main component of the vertebrate eye lens (300 mgml)

bull Provides a proper refractive index of the lens

bullChaperon-like activity (forms soluble complexes with destabilized proteins and prevents their unspecific aggregation and uncontrolled denaturation)

bullSlows down the age-dependent loss of the lens transparency (cataract)

Problem

bullHow does this protein protect the lensbullWhat is the mechanism of its binding of target proteins bullWhat is the role of oligomeric structure and subunit

exchange in this mechanism

Knowledge of the quaternary structure of α-crystallin is a missing link in defining a structure-function relationship and the possible role of the a-crystallin subunits in the lens and other organs in normal and abnormal conditions

The quaternary structure of α-crystallin is not known to date

Structure oligomeric protein

Monomers αA- and αB-crystallin20 kDa

Oligomer20-40 monomers400-1000 kDa

Models of α-crystallin structure

Aquilina J A and Morris A M Evidence for specific subunit distribution and interactions in the quaternary structure of α-crystallin2010httprouoweduauscipapers175

Open micellar configurationB GROTH-VASSELLI ET AL Exp Eye Res (1995) 61 249-253

Single particle reconstructions of native a-crystallin from Electron microscopyDA Haley et al J Mol Biol (2000) 298 261plusmn272

Micellar three-layer modelWalsh et al THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol 266 pp20079-200841991

3-layer modelTardieu et al I Mol Biol (1986) 192711 -724

A model of αB-crystallin with bound α-lactalbumin Electron microscopyScale bar represents 100 Aring J Horwitz Exp Eye Res (2003) 76 145-153

Why Small Angle Scattering

bullPolydisperse system variable size and number of monomers

bullDynamic system monomer exchange between oligomers

bullEfforts to crystallize α-crystallin have failed

bullThe aggregate is too large for high resolution 2D NMR

Small angle scattering allows to study structure of macromolecules (10-1000 Aring) in solution

ShapeSizeMolecular massVolume

Distance distribution function p(r)(indirect Fourier transform)

001 01

1E-3

001

01

1

10

SANS SAXSIn

tens

ity a

u

q A-1

Curves of SANS and SAXS for the same sample of α-crystallin (concentration 9 mgml)

0 50 100 150 20000

02

04

06

08

10

p(r)

r A

SANS SAXS

Dmax

Distance distribution functions for α-crystallin molecule calculated from SANS and SAXS curves by indirect transform

max max

2 2

0 0

( ) ( ) D D

gR p r r dr p r dr max

0

(0) 4 ( )D

I p r dr

Program GNOM httpwwwembl-hamburgdebiosaxssoftwarehtml

Problem

bullHow does this protein protect the lensbullWhat is the mechanism of its binding of target proteins bullWhat is the role of oligomeric structure and subunit

exchange in this mechanism

Knowledge of the quaternary structure of α-crystallin is a missing link in defining a structure-function relationship and the possible role of the a-crystallin subunits in the lens and other organs in normal and abnormal conditions

The quaternary structure of α-crystallin is not known to date

Structure oligomeric protein

Monomers αA- and αB-crystallin20 kDa

Oligomer20-40 monomers400-1000 kDa

Models of α-crystallin structure

Aquilina J A and Morris A M Evidence for specific subunit distribution and interactions in the quaternary structure of α-crystallin2010httprouoweduauscipapers175

Open micellar configurationB GROTH-VASSELLI ET AL Exp Eye Res (1995) 61 249-253

Single particle reconstructions of native a-crystallin from Electron microscopyDA Haley et al J Mol Biol (2000) 298 261plusmn272

Micellar three-layer modelWalsh et al THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol 266 pp20079-200841991

3-layer modelTardieu et al I Mol Biol (1986) 192711 -724

A model of αB-crystallin with bound α-lactalbumin Electron microscopyScale bar represents 100 Aring J Horwitz Exp Eye Res (2003) 76 145-153

Why Small Angle Scattering

bullPolydisperse system variable size and number of monomers

bullDynamic system monomer exchange between oligomers

bullEfforts to crystallize α-crystallin have failed

bullThe aggregate is too large for high resolution 2D NMR

Small angle scattering allows to study structure of macromolecules (10-1000 Aring) in solution

ShapeSizeMolecular massVolume

Distance distribution function p(r)(indirect Fourier transform)

001 01

1E-3

001

01

1

10

SANS SAXSIn

tens

ity a

u

q A-1

Curves of SANS and SAXS for the same sample of α-crystallin (concentration 9 mgml)

0 50 100 150 20000

02

04

06

08

10

p(r)

r A

SANS SAXS

Dmax

Distance distribution functions for α-crystallin molecule calculated from SANS and SAXS curves by indirect transform

max max

2 2

0 0

( ) ( ) D D

gR p r r dr p r dr max

0

(0) 4 ( )D

I p r dr

Program GNOM httpwwwembl-hamburgdebiosaxssoftwarehtml

Structure oligomeric protein

Monomers αA- and αB-crystallin20 kDa

Oligomer20-40 monomers400-1000 kDa

Models of α-crystallin structure

Aquilina J A and Morris A M Evidence for specific subunit distribution and interactions in the quaternary structure of α-crystallin2010httprouoweduauscipapers175

Open micellar configurationB GROTH-VASSELLI ET AL Exp Eye Res (1995) 61 249-253

Single particle reconstructions of native a-crystallin from Electron microscopyDA Haley et al J Mol Biol (2000) 298 261plusmn272

Micellar three-layer modelWalsh et al THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol 266 pp20079-200841991

3-layer modelTardieu et al I Mol Biol (1986) 192711 -724

A model of αB-crystallin with bound α-lactalbumin Electron microscopyScale bar represents 100 Aring J Horwitz Exp Eye Res (2003) 76 145-153

Why Small Angle Scattering

bullPolydisperse system variable size and number of monomers

bullDynamic system monomer exchange between oligomers

bullEfforts to crystallize α-crystallin have failed

bullThe aggregate is too large for high resolution 2D NMR

Small angle scattering allows to study structure of macromolecules (10-1000 Aring) in solution

ShapeSizeMolecular massVolume

Distance distribution function p(r)(indirect Fourier transform)

001 01

1E-3

001

01

1

10

SANS SAXSIn

tens

ity a

u

q A-1

Curves of SANS and SAXS for the same sample of α-crystallin (concentration 9 mgml)

0 50 100 150 20000

02

04

06

08

10

p(r)

r A

SANS SAXS

Dmax

Distance distribution functions for α-crystallin molecule calculated from SANS and SAXS curves by indirect transform

max max

2 2

0 0

( ) ( ) D D

gR p r r dr p r dr max

0

(0) 4 ( )D

I p r dr

Program GNOM httpwwwembl-hamburgdebiosaxssoftwarehtml

Models of α-crystallin structure

Aquilina J A and Morris A M Evidence for specific subunit distribution and interactions in the quaternary structure of α-crystallin2010httprouoweduauscipapers175

Open micellar configurationB GROTH-VASSELLI ET AL Exp Eye Res (1995) 61 249-253

Single particle reconstructions of native a-crystallin from Electron microscopyDA Haley et al J Mol Biol (2000) 298 261plusmn272

Micellar three-layer modelWalsh et al THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol 266 pp20079-200841991

3-layer modelTardieu et al I Mol Biol (1986) 192711 -724

A model of αB-crystallin with bound α-lactalbumin Electron microscopyScale bar represents 100 Aring J Horwitz Exp Eye Res (2003) 76 145-153

Why Small Angle Scattering

bullPolydisperse system variable size and number of monomers

bullDynamic system monomer exchange between oligomers

bullEfforts to crystallize α-crystallin have failed

bullThe aggregate is too large for high resolution 2D NMR

Small angle scattering allows to study structure of macromolecules (10-1000 Aring) in solution

ShapeSizeMolecular massVolume

Distance distribution function p(r)(indirect Fourier transform)

001 01

1E-3

001

01

1

10

SANS SAXSIn

tens

ity a

u

q A-1

Curves of SANS and SAXS for the same sample of α-crystallin (concentration 9 mgml)

0 50 100 150 20000

02

04

06

08

10

p(r)

r A

SANS SAXS

Dmax

Distance distribution functions for α-crystallin molecule calculated from SANS and SAXS curves by indirect transform

max max

2 2

0 0

( ) ( ) D D

gR p r r dr p r dr max

0

(0) 4 ( )D

I p r dr

Program GNOM httpwwwembl-hamburgdebiosaxssoftwarehtml

Why Small Angle Scattering

bullPolydisperse system variable size and number of monomers

bullDynamic system monomer exchange between oligomers

bullEfforts to crystallize α-crystallin have failed

bullThe aggregate is too large for high resolution 2D NMR

Small angle scattering allows to study structure of macromolecules (10-1000 Aring) in solution

ShapeSizeMolecular massVolume

Distance distribution function p(r)(indirect Fourier transform)

001 01

1E-3

001

01

1

10

SANS SAXSIn

tens

ity a

u

q A-1

Curves of SANS and SAXS for the same sample of α-crystallin (concentration 9 mgml)

0 50 100 150 20000

02

04

06

08

10

p(r)

r A

SANS SAXS

Dmax

Distance distribution functions for α-crystallin molecule calculated from SANS and SAXS curves by indirect transform

max max

2 2

0 0

( ) ( ) D D

gR p r r dr p r dr max

0

(0) 4 ( )D

I p r dr

Program GNOM httpwwwembl-hamburgdebiosaxssoftwarehtml

Distance distribution function p(r)(indirect Fourier transform)

001 01

1E-3

001

01

1

10

SANS SAXSIn

tens

ity a

u

q A-1

Curves of SANS and SAXS for the same sample of α-crystallin (concentration 9 mgml)

0 50 100 150 20000

02

04

06

08

10

p(r)

r A

SANS SAXS

Dmax

Distance distribution functions for α-crystallin molecule calculated from SANS and SAXS curves by indirect transform

max max

2 2

0 0

( ) ( ) D D

gR p r r dr p r dr max

0

(0) 4 ( )D

I p r dr

Program GNOM httpwwwembl-hamburgdebiosaxssoftwarehtml

Molecular mass M volume V and hydration value

M = (600plusmn17) kDa rarr Vah = Mν = (740plusmn20)103 Aring3 (anhydrous volume)

000 002 004 006 008 010 012 014 016 018 02000000

00002

00004

00006

00008

00010

Iq2

q A-1

Hydrous volume Vh

(Porod invariant) (Fig 3)

22 0 hV I Q2

0

( )where Q q I q dq

Hydration value for α-crystallin026 g of water per g of protein

Iq2 vs q plot for estimation of Porod volume The Porod volume was calculated by program PRIMUS [20]

2 2

(0)A

S

I NM

c

where I(0)=297 cm-2 (from indirect transformation)NA = 60221023 (Avogadro constant)c=9 mgml (concentration of protein in the sample) (average scattering density of protein) [8]ρs = -05481010cm-2 (scattering density of the solvent) (specific volume) [13]

10 219 10 cm

3(0745 0014) cm mg

Shape of α-crystallin

001 011E-4

1E-3

001

01

1

SANS experiment

model of 3-axial ellipsoid (programm FITTER)

3-axial ellipsoidal shell

(programm SASFIT)

Inte

nsi

ty

cm-1

q A-101

01

1

10

SAXS experiment

3-exial ellipsoid (program FITTER)

3-exial ellipsoidal shell (programm SASFIT)

Inte

nsi

ty

au

q A-1

Approximation of SANS and SAXS data for α-crystallin by model of 3-axial ellipsoid and 3-axial ellipsoidal shell

Model ellipsoidal shell for α-crystallin

3D dumm-models

x y z( )

x y z( )

x y z( )ADUMM-model from SANS

BDUMM-model from SAXS

C3-exis ellipsoid from SANS

Program DAMMIF httpwwwembl-hamburgdebiosaxssoftwarehtml

001 01

1E-3

001

01

1

SANSchi2=142

Inte

nsi

ty c

m-1

q A-1

001 003 005 007 00901

1

10

SAXSchi2=127

Inte

nsi

ty r

elu

n

q A-1

Approximation of experimental data by curves for the 3D-damm models

Shape of α-crystallin

001 011E-4

1E-3

001

01

1

SANS experiment

model of 3-axial ellipsoid (programm FITTER)

3-axial ellipsoidal shell

(programm SASFIT)

Inte

nsi

ty

cm-1

q A-101

01

1

10

SAXS experiment

3-exial ellipsoid (program FITTER)

3-exial ellipsoidal shell (programm SASFIT)

Inte

nsi

ty

au

q A-1

Approximation of SANS and SAXS data for α-crystallin by model of 3-axial ellipsoid and 3-axial ellipsoidal shell

Model ellipsoidal shell for α-crystallin

3D dumm-models

x y z( )

x y z( )

x y z( )ADUMM-model from SANS

BDUMM-model from SAXS

C3-exis ellipsoid from SANS

Program DAMMIF httpwwwembl-hamburgdebiosaxssoftwarehtml

001 01

1E-3

001

01

1

SANSchi2=142

Inte

nsi

ty c

m-1

q A-1

001 003 005 007 00901

1

10

SAXSchi2=127

Inte

nsi

ty r

elu

n

q A-1

Approximation of experimental data by curves for the 3D-damm models

3D dumm-models

x y z( )

x y z( )

x y z( )ADUMM-model from SANS

BDUMM-model from SAXS

C3-exis ellipsoid from SANS

Program DAMMIF httpwwwembl-hamburgdebiosaxssoftwarehtml

001 01

1E-3

001

01

1

SANSchi2=142

Inte

nsi

ty c

m-1

q A-1

001 003 005 007 00901

1

10

SAXSchi2=127

Inte

nsi

ty r

elu

n

q A-1

Approximation of experimental data by curves for the 3D-damm models

The summary table of structural parameters of α-crystallin obtained from small angle scattering

Conclusions

bullThe average values of structural parameters of alpha-cryslallin have been obtained

(Volume size hydration value)

bullEllipsoid-like shape

bullThe presence of internal cavity is available

bullIn case of model ellipsoidal shell the monomers form an unilayer shell

Conclusions

bullThe average values of structural parameters of alpha-cryslallin have been obtained

(Volume size hydration value)

bullEllipsoid-like shape

bullThe presence of internal cavity is available

bullIn case of model ellipsoidal shell the monomers form an unilayer shell