amyloid biomembranes crystalline proteins

Sample preparation (etc) for MAS SSNMR Sample preparation (etc) for MAS SSNMR of biomembranesof biomembranes

David Middleton

School of Biological Sciences

20%

80%

sample preparation NMR

90%

10%

sample preparation NMR analysis

Reality What the world sees

Sample preparation (etc) for MAS SSNMRSample preparation (etc) for MAS SSNMR

Associated factors (ligands, peptides, prosthetic groups)

Protein purification and preparation of membranes

angles

ppm

static

aligned bilayers

magic angle spinning

Solid-state NMR techniques for biomembranesSolid-state NMR techniques for biomembranes

60 50 40 30 20 10

I(2)

b/g '

G(1/2)a

I(1)

b/g '

I(2)

a/d

A(2)

a/b

A(1)a/b

F(1/2)a/b I(2)

a/b

13C chemical shift (ppm)

I(1)

a/b

180 170 16060

50

40

30

20

10

A (1/2)

a/CO

I(1/2)

a/COF(1/2)

a/CO

G (1/2)a/CO

13C

chem

ica

lshi

ft(p

pm)

-200 -100 0 100 200

kHz

SSNMR

amorphous dispersion

MAS SSNMR of proteins in their natural membranesMAS SSNMR of proteins in their natural membranesMAS SSNMR can be used to study membrane proteins purified from tissue or bacterial cell without removing them from the native membrane environment

Planar or microsomal membranes isolated by centrifugation

Proteins amenable to analysis are usually naturally abundant or can be overexpressed

-Rhodopsin (95 % of total ROS disk membrane protein)-Nicotinic acetylcholine receptor -P-type ion pumps-porins-Transporters (bacterial)

Advantage: straightforward non-perturbing preparation, stable and functional proteinDisadvantage: Not amenable to labelling (eukaryotic), contaminants

Sample handlingSample handlingNative membranes remain fully hydrated and are sedimented by ultracentrifugation to produce a viscous gel.

Gel is packed into an MAS rotor to as high a protein density as possible (aim typically for 10 nmoles or higher in a 50 l volume – i.e., 8-10 mg/ml for a 40 kDa protein).

200 150 100 50 0

13C chemical shift (ppm)

kidney membrane

200 150 100 50 0

13C chemical shift (ppm)

E. coli membrane

13C CP MAS spectra of natural membranes show background signals from lipids and proteins. Spectra have same features regardless of the source of the membranes.

What information can be gained?What information can be gained?The three dimensional structures of ligands (e.g., hormones and drugs) in their binding pockets can be determined by isotopically labelling the ligand but not the protein. Information about ligand structure can be gained without prior knowledge of the receptor structure (although it helps, of course).

24

6

8

102

4

6

8

100

200

400

600

800

1000

key functional groups

NMR

Coordinates of functional groups in binding site

Low-medium affinity

screenscreen

synthesis

high affinity

flexibleligands

constrainedligands

e.g., drug design

Intensity profile of uridine signal

Cytoplasmic side

Y

M

NR

PL

I

V

I

R

D

S

R

K

K I

I

GL

K

MG

V

G

N

L

KSI

A

LG F

F S V

I I G

L F V S

S F N A

G I S

GI I

S T F

L N M F

L I N

I V L S

A L V

Y V V A

I L M

A I F G

I A V

A A V I

A I L

A N L A

F Y P

L G Y I

M G V

I A W V

Q G I

S I S F

V L S

T L V S

A L V

A S I A

Y G S

LL V

TW

G

L

G

F

Y

Q GN

VV

S

RV

K

G

L

EE

N

L

S

N

K

V

L

S

S

G

V

Q

L

A

E

F

A

K

L

A

GF

D

R

V

A

E E

L

G

Y

IEH

S

L

N

MQI

NE

GQ

S

F

FE M

KL

PY

V

PS

EA

RP

L

I

S

S

A

T

AM QL

M

M

A

VF

E

TI KD

7 8 9 10KA

CO2-

S

T

V L L

V I E

L N S

W F F

I Q L

R Y V

S I F

G I L A

L V K

V I P C

Q L I

V R I H

V A L

L I A V

H L V

A L V F

L L A

D S S V

L

A

I

R RI

1KK

R

M DNH3

+F

D

M

N

Q

G

L

A

FF

NA

L

F

G

L

K

E

LG

V

FG

S

N

E

G

T

F

VE

MF

G

S

F

K

V

F

G

D L

2 3

L G Q

S S L I

I A Y

S E N F

N A V

L E S F

4

V T S

I S M S

A M T

M A T A

A G V

M T M Y

5E P

6F

peptide G

GA

Nucleoside transporter NupC from E. coliRecent example: DQ excitation at rotational resonanceRecent example: DQ excitation at rotational resonance

200 180 160 140 120 100 80 60 40 20 013C chemical shift (ppm)

uridine

1’6

4

Recent example: DQ excitation at rotational resonanceRecent example: DQ excitation at rotational resonance

kDa

66

45

36

2924

20

14

a b c d

Mapping ligand binding sites

6 Å

GalP

200 150 100 50 0

(ppm)

O

OHOHOH

HOOCOMe

*M G

control

0.00 0.05 0.100.0

0.2

0.4

0.6

0.8

1.0 Kd = 0.8 mM

Kd = 0.5 mM K

d = 0.1 mM

[13C

]MG

pea

k in

tens

ity

concentration (M)

O

OHOHOH

OHOOC

NO2

pNPG

How to confirm the selectivity of a ligand

13C chemical shift (ppm)

Switch expression on/off Displace with competitor ligand

methylglucuronide

200 150 100 50 0

(ppm)

O

OHOHOH

HOOCOMe

MAS SSNMR of reconstituted systemsMAS SSNMR of reconstituted systems

Membrane protein reconstitution involves removing the protein of interest from its native membrane and incorporating into a new, well-defined lipid bilayer.

Advantages: eliminates contaminating proteins; can vary lipid composition systematically; study structure and function in isolation

Disadvantages: requires much more work; may lose protein function altogether

Crude membranes with protein of interest

Purification

Detergent solubilisation

Add lipids/remove detergent

Detergent screen (BOG, DDM)

Detergent concentration/CMC

Solubilised protein function

Affinity (His or FLAG tagged)

MAS SSNMR of reconstituted systemsMAS SSNMR of reconstituted systems

Gel filtration

Selective extraction

Choice of lipids

Dialysis (vesicles)

Biobeads (planar)

Functional characterisation

Purification

Detergent solubilisation

Add lipids/remove detergent

MAS SSNMR of reconstituted systemsMAS SSNMR of reconstituted systems

Functional characterisation

b-adrenergic receptor

Adapted from MacLennan and Kranias, 2003

Case study: regulation of cardiac calcium flux

sarcolipin (atrial and skeletal muscle)

phospholamban (ventricular)

The proteins of interestSERCA (skeletal and cardiac muscle)

Reconstitution of SERCA1 for NMR studies

SERCA1 from rabbit skeletal muscle

SR vesicles solubilization

Reconstitution of SERCA and regulatory protein

SSNMR measurements

5 10 15 20 250.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

5 10 15 20 250.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

OD

280

gradient fractions

OD

280

gradient fractions

0.0 0.2 0.4 0.6

0

20

40

60

80

100

norm

alis

ed

act

ivity

[Ca2+] (M)

SERCA1 SERCA1 + PLB

P

S 66

14.2 6.5

20 24 29 36 45

5 10 15 20 250.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

5 10 15 20 250.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

OD

280

gradient fractions

OD

280

gradient fractions

0.0 0.2 0.4 0.6

0

20

40

60

80

100

norm

alis

ed

act

ivity

[Ca2+] (M)

SERCA1 SERCA1 + PLB

P

S 66

14.2 6.5

20 24 29 36 45

Reconstitution of SERCA1 for NMR studies

free lipid

mixed

SERCA

sucrose density gradient

Dynamics of the conserved C-terminus

NH

O

OH

NH

O

NH2O

NH

O

OH

OH

MGINTRELFLNFTIVLITVILMWLLVRSYQY

Ring dynamics will be impaired if the rings interact with SERCA

13C-labelled Tyr

Is the conserved RSYQY sequence of sarcolipin important for interactions with SERCA?

Structural analysis of microcrystalline proteins

GluR2

S1S2J (dimer)

Ionotropic glutamate receptor 2

X-ray structure

allosteric modulator

Reservoir solution

Glass Cover-slip

Vapour

Diffusion

Protein-precipitant mix

High-vacuum grease

Reservoir solution

Vapour

Diffusion

High-vacuum grease

Glass Cover-slip

Glass Rod

Protein-precipitate mix

A B

Structural analysis of crystalline GluR2 S1S2JHanging drop Sitting drop

BB00 and resolution and resolution

13C CP-MAS spectra

400 MHz 800 MHz

BB00 and resolution and resolution

15N CP-MAS spectra

400 MHz 800 MHz

Structural analysis of crystalline GluR2 S1S2J

Structural analysis of crystalline GluR2 S1S2J

Inverted 20-200μl pipette tip

Protein and precipitant

200-1000μl pipette tip

Laboratory film

Centrifugation

Aspirate liquid and pool precipitate by centrifugation

Cut end and transfer to spacer/rotor by centrifugation

Microfuge tube

20-200μl pipette tip

Rotor/

spacer

Laboratory film

Kel-F drive cap

4mm zirconium rotor

Spacer cap

40l Kel-F spacer

insert

Sample

Structural analysis of crystalline GluR2 S1S2J

Separate Expression

13C media

15N media

Pool

Purification

Concentration Dimer interface

Mixed dimers

Structural analysis of crystalline GluR2 S1S2J