amyloid
DESCRIPTION
Sample preparation (etc) for MAS SSNMR of biomembranes. David Middleton School of Biological Sciences. amyloid. biomembranes. crystalline proteins. Sample preparation (etc) for MAS SSNMR. What the world sees. Reality. Solid-state NMR techniques for biomembranes. - PowerPoint PPT PresentationTRANSCRIPT
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