Garland R. Marshall, Washington University, St. Louis, MO
Shedding Light on GPCR Signal
Transduction
Garland R. Marshall, Washington University, St. Louis, MO
The Human
Eye
Rhodopsin is the GPCR protein in the membrane
of the photoreceptor cell of the eye.
eye
Garland R. Marshall
Department of Biochemistry and Molecular Biophysics
Light-Sensitive
Protein
Washington University, St. Louis
Outer Segment
Of Rod
Bio5325 Protein Course, November 16, 2006
The Retina
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Amplification Cascade
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
cytoplasm
(Menon, et al. Physiol Rev. 2001, 81, 1659.)
Bio5325 Protein Course, November 16, 2006
(K. Palczewski et al., Science, 289,
289, 739,
2000)
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
QUESTION ONE: How does photoisomerization of 11-cis-retinal
trigger change in TM helix packing?
DOGMA: Relief of steric strain caused by photoisomerization is
driving force for TM helix reorganization.
HYPOTHESIS: 11-cis retinal acts as stopper stabilizing TM helix
organization seen in dark-adapted state; photoisomerization
removes stabilization and TM6 is able to rotate and change
conformation of intracellular loops to crate binding site for α subunit of transducin.
transducin.
Garland R. Marshall, Washington University, St. Louis, MO
Saam , J., Tajkhorshid
Saam,
Tajkhorshid,, E.,
Hayashi, S., and Schulten
Schulten,,
K. (2002) Biophys
Biophys.. J. 83
83,,
3097-3112
MD up to 10 ns
(1 ns was ~ 4.5 days on
128 processors of the
Cray T3E)
LiTERATURE:
LiTERATURE: Two groups have use molecular simulation (10
nsec)) of rhodopsin in lipid bilayers to model photoisomerization.
nsec
photoisomerization.
Bio5325 Protein Course, November 16, 2006
Simplified calculations
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
Rotations of individual TM helices in R* around Tx helical axis
Helix 1
Tz
4
2
3
1
7
5
6
Relative energy, kcal/mol
Tx
Ty
Helix 3
Helix 4
Helix 5
Helix 6
80
60
40
20
0
-180
YZ
Helix 2
100
XY
-150
-120
-90
-60
-30
0
30
60
90
120
150
Deviation from Tx value in starting m odel, ! Tx, degrees
XZ
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
EPR Spectrum Provides Structural Information
Environmental variables
Side-chain mobility
Side-chain accessibility
Structural information
(proteins)
Secondary structure
Tertiary interactions
Backbone dynamics
Secondary structure
Solvent accessibility
Surface electrical
potential
Side-chain proximity
Inter-residue distance
Movement
of helix 6
of
rhodopsin
estimated (R*) = EPR(R*) x crystal structure (R) / EPR (R)
Val139
to
Lys248
Glu249
Val250
Thr251
Arg252
EPR
R
12-14(Å)
15-20
15-20
12-14
15-20
crystal structure
R
8.5
11.5
10.35
8.33
11.5
EPR
R*
23-25
15-20
12-14
23-25
23-25
estimated
R*
15.6
11.5
8.5
15.6
15.6
model
R*
14.4
11.5
10.1
13.8
14.8
EPR measurement: Farrens et al. Science 274, 768-770 (1996)
Crystal structure of R: Palczewski et al. Science 289, 739-745 (2000)
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
139-248
139-249
139-250
139-251
139-252
R
12-14
15-20
15-20
12-14
15-20
R*
23-25
15-20
12-14
23-25
23-25
SL positions
Experimental data
Distances, Å
R
8.5
11.5
10.3
8.3
11.5
Estimated distances, Å
R*
15.6
11.5
8.5
15.6
15.6
Garland R. Marshall, Washington University, St. Louis, MO
Extracellular view on
TM helical region of R
(in green) overlapped
over TM helical region
of R* before (in
magenta) and after (in
red) relaxing and
introducing the
disulfide bridge C204C276
Arimoto et al. Biophys
Biophys.. J.
81:3285-3293 (2001)
16
15
CORRELATION BETWEEN
MODEL AND EXPERIMENT
Calculated
14
13
Model = ΔTx values of 0°, 0°
and 120° for TM1, TM3 and
TM6
12
11
10
TM7
TM4
TM6
9
8
8
9
10
11
12
13
14
Experim ental (estim ation)
15
16
R2 = 0.94
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Stereoview of
model of R*
with
reconstructed
intracellular
loops in
magenta
Intracellular view at the loops in R (left, blue shadowed ribbons) and in R* (right, red
shadowed ribbons). TM6 is shown in green in R, and in orange in R*
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Activation Model of Rhodopsin
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Garland R. Marshall, Washington University, St. Louis, MO
R ↔ R* for rhodopsin:
rhodopsin: conclusions
Rotation of TM6 induces movement of IC3
R* is flexible enough to tolerate many
disulfide links
R* for a GPCR can be modeled based on
limited amount of experimental data
Nikiforovich GV, Marshall GR. 3D Model for Meta-II Rhodopsin, An Activated
G-Protein-Coupled Receptor. Biochemistry 42:9110-9120 (2003).
Altenbach,, et al. Biochemistry 2001, 40, 15493-15500.Bio5325 Protein Course, November 16, 2006
Altenbach
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Last Minute Update - Structure of R*
Salom et al. PNAS 2006 Oct 31;103(44):16123-8
Garland R. Marshall, Washington University, St. Louis, MO
Discrepancies between R* structure and biophysical studies?
Crystal structure of R* does not show large movement
of helix 6 seen by spin-label studies of Hubbell et al.
Is it possible that crystal packing forces allow
photoisomerization of retinal and spectral shift to MII
spectra without allowing relaxation of TM segments to
open intracellular binding site for transducin
transducin?
?
Crystal structure done on new crystalline form that
could tolerate photactivation ( prior crystal forms
shattered on light activation).
Spectral changes in crystals
Structure of R*
Differences between R and R*
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
Constitutively active mutations in position
111 of angiotensin type 1 receptor
N111G > N111S > N111A, N111C >
N111I, N111Q, N111H, N111K, N111F, N111Y
Feng Y-H, Miura S, Husain A, Karnik SS. Mechanism of Constitutive Activation of the AT1
receptor: Influence of the Size of the Agonist Switch Binding Residue Asn111.
Biochemistry 1998;37:15791-15798.
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
1.
Garland R. Marshall, Washington University, St. Louis, MO
2.
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
3.
Garland R. Marshall, Washington University, St. Louis, MO
4.
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
5.
Garland R. Marshall, Washington University, St. Louis, MO
6.
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
C-terminal amino acid sequences of G-protein α-subunits
Protein
Gtr
Gtc
Ggust
Go
Gi1
Gi2
Gi3
Gz
Gs
Golf
AA #
340-350
343-353
344-354
344-354
344-354
345-355
344-354
345-355
384-394
371-381
AA Sequence
IKENLKDCGLF
IKENLKDCGLF
IKENLKDCGLF
IANNLRGCGLY
IKNNLKDCGLF
IKNNLKDCGLF
IKNNLKECGLY
IQNNLKY IGLC
QRM H L R Q Y E L L
QRM H L R Q Y E L L
7.
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Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Rhodopsin
(500nm)
hν
MII
(380nm)
MI
(490nm)
Garland R. Marshall, Washington University, St. Louis, MO
transducin
Gtα
Gtα (340-350)
bind&stabilize
0.06
dark_1
light_1
dark_2
light_2
0.05
rhodopsin
0.04
absorbance
Meta II
stabilization
assay
0.03
MII
0.02
MI
0.01
0
-0.01
-0.02
300
350
400
450
500
550
600
650
wavelength (nm)
Bio5325 Protein Course, November 16, 2006
NOE identifies pairs of protons that are in close proximity. (A) Schematic diagram of a peptide
chain, (B) A highly simplified NOESY spectrum
(L. Stryer, Biochemistry , W. H. Freeman, New York, 1995) Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
TRNOE Data on Rhodopsin-Bound
Rhodopsin-Bound α-Peptide
Extent of NMR Proton Assignments -
Garland R. Marshall, Washington University, St. Louis, MO
RhodopsinRhodopsin- α -Peptide Transfer NOE
NMR Experiment
A
B
Phe350Hδ/344LeuHδ
Phe350Hδ/341LysHβδγ
96.4% Light vs. 94.6% Dark
Number of Observable NOE Constraints short-range - 38 Light vs. 31 Dark
long and medium - 98 Light vs. 2 Dark
No Distance Constraint Violations (>0.3Å)
Intrapeptide CHARMM Energy
-186.7±1.2 kcałmol
F1(PPM)
Phe350Hδ/344Leuγ
Cross-peaks in the aromatic-aliphatic region showing interactions
between F350 aromatic protons and side-chain protons of L349, L344,
and K341 from the NOESY spectra of Gtα
Gtα(340-350) in the presence of
the dark-adapted (A) and photoexcited rhodopsin (B)
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Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
TrNOE structure of Gtα(340-350) bound to photoactivated rhodopsin(R*)
Phe350Hδ/349Leuδ
Garland R. Marshall, Washington University, St. Louis, MO
Constrained Peptides
Peptide Analog
EC50
(µM)
relative
activity
IKENLKDCGLF (native sequence)
500
1.0
Icyclo(KENLKDCGLD) (1)
300
1.67
Icyclo(KENLKDCGLE) (2)
120
4.17
Icyclo(DENLKDCGLF(pNH)) (3)
6000
0.083
Icyclo(EENLKDCGLF(pNH)) (4)
75
6.7
Lys341
Asp341
(Glu341)
Asp350
(Glu350)
Phe350
Are the conformation restraints imposed in the C-terminal tail sufficient for triggering
complete activation of Gα
Gα ?
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
CALCULATED
INTERACTION ENERGY
IN WATER
ARE Π-CATION INTERACTIONS WORTH THEIR SALT?
QUESTION TWO: What are dominant intermolecular forces
stabilizing R*-bound conformation of α -peptide?
What is missing?
π-cation interaction
benzene…methylammonium
salt bridge
acetate…methylammonium
DOGMA: Π - cation interactions can energetically dominate salt
bridge interactions even in water (Gallivan
(Gallivan and Dougherty, 2000).
HYPOTHESIS: Π - cation interaction in R*-bound α-peptide is
shielded/neutralized by adjacent salt bridge.
In H2O
LITERATURE: Recent theoretical and experimental studies
stress role of counter-ion in determining strength of Π - cation
interaction (Bartoli and Roelens,
Roelens, 2002).
Gallivan and Dougherty, JACS 122:870, 2000
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
Salt Bridge and π-Cation Interaction in Rhodopsin-Bound
Rhodopsin-Bound
Conformation of α -Peptide
Relative Effects of
Dielectric on π -Cation
and Salt-Bridge
Interactions
interaction stronger
π-cation interaction
QM Calculations
Using
SM5.42R/HF/6-31+G*
Methodology to
Explore Effects of
Different Solvent
Environments
Gallivan and Dougherty, JACS
122:870, 2000
Bio5325 Protein Course, November 16, 2006
Salt bridge
3.1Å
Phe350
2.9Å
Lys341
(dielectric constant) -1
H2O
vacuum
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Garland R. Marshall, Washington University, St. Louis, MO
Bio5325 Protein Course, November 16, 2006
π-cation
Garland R. Marshall, Washington University, St. Louis, MO
Electrostatic potential of various π-systems and
their π-cation binding energies
benzene
p-F
phenol
aniline
p-CN
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
Binding Affinity versus Peptide Property
3.8
-Log (EC50)
3.8
R2 = 0.72
3.6
3.6
3.4
3.4
3.2
3.2
R2 = 0.59
3
3
2.8
2.8
10
15
20
25
30
35
0.8
0.4
0.2
0
-0.2
-0.4
-0.6
-0.8
(negative correlation!)
3.8
-Log (EC50)
0.6
Hammett sigma constant
Binding energy (kcal/mol)
(negative correlation!)
Counter-ion
effect on πcation
binding
interaction
energy
Ac: acetate
DNF: dinitrophenate
TFA: trifluoroacetate
PFF: pentafluorophenate
TfO: triflate
Pic: picrate
3.8
R2 = 0.98
3.6
R2 = 0.87
3.6
3.4
3.4
3.2
3.2
3
3
2.8
2.8
0.5
1
1.5
2
2.5
3
3.5
Partition Coefficient: LogP
10
11
12
13
14
15
16
RP-HPLC retention time (min.)
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EP = electrostatic potential of anion
Bartoli and Roelens, JACS (2002), 124, 8307-8315.
Garland R. Marshall, Washington University, St. Louis, MO
Peptide Analogs
Garland R. Marshall, Washington University, St. Louis, MO
Binding Affinity vs. Electronic Potential
of Substituent at 350
IKENLKDCGLF (native seq.)
R=
(7) IKENLKDCGLF(-p-NO2)-NH2
R=
3.0
(8) IKENLKDCGLF(-F5)-NH2
R=
2.5
(9) IKENLKDCGLY-NH2
R=
(10) IKENLKDCGLY(Me)-NH2
R=
(11) IKENLKDCGL(2-Nal)-NH2
R=
(12) IKENLKDCGLF(-p-t-butyl)-NH2
R=
(13) IKENLKDCGL(Cha)-NH2
R=
2
R = 0.85
2.0
1.5
1.0
0.5
(14) IKENLKDCGLF-NH2
Matt Anderson, thesis research
EC50 (mM)
Remove
carboxylate
charge as
carboxamide
and retest
effect of psubstituents
Structure of
Substituent
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R=
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
0.0
-1.0
0.0
1.0
2.0
3.0
Hammet sigma constant
! " = #( pKa - pK aH)/$
Bio5325 Protein Course, November 16, 2006
Rhodopsin
CHASING ANOTHER ARTIFACT?
QUESTION THREE: Are the studies on the α -peptide interacting
with R* relevant to the α -subunit in particular and to the Gprotein transducin in general?
DOGMA: Small segments of a protein do not necessarily interact
in the same way as when they are in context.
HYPOTHESIS: R*-bound α -peptide provides motif for interaction
of α -subunits of G-proteins with activated GPCRs.
GPCRs.
LITERATURE: SAR studies of α-peptide and mutational studies
of α -subunit give a consistent set of results.
Bio5325 Protein Course, November 16, 2006
α-Peptide Fused with Crystal Structure of Transducin
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
Protein
Expression
GαiΔCT
Rhodopsin-Gt Interface
Peptide Synthesis
EPR / NMR Probes
– Is the conformational
change of Gtα(340-350)
representative of the full
length G-protein?
Protein Ligation
– What is the structural
relationship between the
alpha peptide and
rhodopsin along the
signal-transduction
pathway?
NMR
EPR
Smith, SUNY Stony Brook
Hubbell, UCLA
Conformational Changes
Dynamics
Distance Constraints
Bio5325 Protein Course, November 16, 2006
Sakmar, TP. Curr Opin Cell Biol. 2002, 14, 189-195
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Intein-Mediated
Intein-Mediated Protein Ligation
Intein Methodology / Mechanism
HS
O
Intein
N-Extein
H2N
HS
H
N
N
H
O
C-Extein
N→S acyl transfer
Transesterification
H2 N
O
G!i1"CT
H
N
N
H
NH2
Succinimide formation
S →N shift
HS
O
N
H
G!i1"CT
contaminants
H2 N
G!i1"CT
-
N
H
SO 3
S
HS
peptide
H 2N
Intein CBD
N-S Acyl
Transfer
Native Chemical
Ligation
G!i1"CT
CNLKDCGLF
CO NH Cys
peptide
Semi-synthetic Protein
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Synthetic Peptides - NMR
SO3 Na+
HS
O
HS
H2 N
CBD
Synthetic peptide
+ MESNA
Intein CBD
O
O
Intein
Transthioesterification
Affinity
Purification
S
S
H2N
HS
H2 N
H. Paulus
Paulus.. Annu
Annu.. Rev. Biochem
Biochem.. 2000. 69
69:447-96.
:447-96.
G!i1"CT
O
H2N
H2N
O
H2 N
Express in
E. coli
O
O
HS
Clone Gαi1 into Intein Vector
NH2
Garland R. Marshall, Washington University, St. Louis, MO
NMR Experiments: 13C-LGF
Ligated peptide (α
(α-subunit)
Unligated peptide
Leu344(uniform 13C), Gly348(2-13C), Phe350(ring 13C)
2 eq. AlF4-
1 eq. AlF4-
Leu C
Cα
αH peak shown; similar results for all labeled atoms
No AlF4-
Lori Anderson, thesis research
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Lori Anderson, thesis research
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
One can measure precise distances between free
electron of spin label and protons seen by transfer
NOE experiment due to change of relaxation time
(broadening of NMR signal).
Conceptually, a great experiment; unfortunately it
didn’
didn’t work for logistical reasons as tried with
rhodopsin/
rhodopsin/ α-peptide (technical difficulties).
*N
Nitroxide
α-peptide
Measure Relaxation Effect ofBio5325
Spin
Label
Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
Paramagnetic Broadening Effect
O
N
H 3C
CH3
CH3
S
S
N
CH3
O
CH3
35
CH3
H 3C
O
N
Proton Peak
Intensity
Decreases in
Presence of Spin
Label.
(2.) 3-Maleimido-PROXYL
(1.) methyl methanethiosulfonate
methanethiosulfonate-PROXYL
-PROXYL
O
C
H3C
N
H3C
OH
OH
O
CH3
CH2CH3
CH3
N
H
C
O
N
25
20
15
O
ICH2
CH2I
C
NH
(1/R6
O
O
(5.) Iodoacetamido salicylate
(4.) N- ethylmaleimide
(3.) Iodoacetamido
Iodoacetamido-PROXYL
-PROXYL
Protons in α -peptide estimated to
be greater than 20 Å from nitroxide
on Cys316
a
b
c
30
O
O
Distance (Å)
O
H 3C
H 3C
distance
between proton
and electron)
10
0
0.2
0.4
0.6
0.8
1
with spin
Intensity Ratio without
spin
Line widths and correlation times are 20Hz, 50ns (a); 20Hz,
40ns (b
(b) and 40Hz, 40ns (c
(c). ωh = 600 MHz
Reagents Used to Modify Cys140 and Cys316
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
-400
60
0
-200
-400
(a)
40
(b)
-600
20
Cys316
0
-200
Cys140+316
200
No labeling
200
80
Cys140+316
400
O
C
No Spin(C316)
100
600
No Spin(C140&316)
%
800
400
Cys140+316(no spin)
600
Cys140+316(no spin)
Binding of Gta(340-350)
Gta(340-350) to Cysteine Spin-labeled
Rhodopsin
OH
ICH2
C
NH
O
IAS →C316
(Iodoacetamido salicylate
salicylate))
CH2CH3
O
-600
OH
N
O
-800
3320
3340
3360
3380
3400
3320
3340
3360
3380
3400
EPR spectra for (a) labeled rhodopsin and (b) maleimide-Proxyl
0
none
(1)
(2)
(3)
IAS+(1) IAS+(2) NEM
IAS
• labeling Cys140 blocks peptide-binding
• labeling Cys316 doesn’
doesn’ t affect peptide-binding
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NEM → C140+316
(N-ethylmaleimide
(Nethylmaleimide))
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
Differential Effects of Labeling of Cys140 or Cys316 upon
Stabilization of MII by C-terminal Peptides of Gtα and Gtγ
Spin-labeling Sites on Rhodopsin
C140
maleimido-Proxyl
C316
iodoacetamidoProxyl
Gtα
Gt
α(340-350)
Bio5325 Protein Course, November 16, 2006
Labeling of C140 leads to 10-fold reduction in
affinity for Gt
Gtα
α C-terminal peptide and no effect on
Gtγγ C-terminal binding. Labeling of C316 leads to 3Gt
fold reduction in affinity for Gt
Gtγγ C-terminal peptide
and no effect on Gt
Gtα
α C-terminal binding.
3D Structure of Rhodopsin (orange) and Transducin (alpha
= grey, beta/gamma = blue), C-terminal domains of alpha
and gamma that interact with R* shown in yellow. Arrows
indicate proposed paths of interaction between R* and
GTP-binding site.
From Downs et al., Vision Research - online 2006
Garland R. Marshall, Washington University, St. Louis, MO
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Marshall et al., JACS 112:963 (1990)
Solid-State Magic-Angle Spinning (MAS) NMR
1. Can measure precise interatomic distances in solids or
lyophilized samples (up to 10 Å)
2. Requires isotopic labeling of biological sampling
3. Requires large sample size and/or long signal averaging
4. Rapid rotation at “ magic angle”
angle” removes dipole/dipole
couplings which are selectively reintroduced and
measured by pulse sequences synchronized with rotor
cycles.
Pulse sequence for a version of
rotational-echo, double-resonance
(REDOR) I3C NMR. Two equally
spaced, I5N 180° pulses per rotor
period result in the dephasing of
transverse carbon magnetization
produced by a cross-polarization
(CP) transfer from dipolar-coupled
protons. Carbon-nitrogen dipolar
coupling determines the extent of
dephasing. A I3C 180° pulse
replaces the I5N pulse in the
middle of the dephasing period and
refocuses isotropic I3C chemical
shift differences at the beginning of
data acquisition. After the CP
transfer, resonant decoupling
removes the protons from the
experiment. The illustration is for
four rotor periods.
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
REDOR = 4.07 Å
X-Ray = 4.128 Å
Marshall et al., JACS 112:963 (1990)
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Rienstra et al. PNAS
99:10260-10265 (2002)
“Measurement of
carbon–nitrogen internuclear
distances in
[U-13C,15N]f-MLF-OH by
frequency-selective REDOR (16).
(a) Structural model of f-MLF-OH
displaying the distances
measured in b–d. Experimental
REDOR SS0 curves (S0 and S
represent the reference and
dipolar dephasing experiments,
respectively) and simulations are
shown for Met(C)–Leu(N) (b),
Leu(C)–Leu(N) (c), and
Met(C)–Phe(N) (d), and they
correspond to internuclear
distances of 3.12 0.03 Å (b),
3.64 0.09 Å (c), and 4.12 0.15 Å
(d). The average rmsd error in
the calculated torsion angles was
3.5° for the full structure
calculation and 1.0° for the CNS
calculation.”
Garland R. Marshall, Washington University, St. Louis, MO
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Rienstra et al.
PNAS 99:1026010265 (2002)
Garland R. Marshall, Washington University, St. Louis, MO
“An illustration of a family of nearly identical structures
that is representative of the entire ensemble and is
consistent with the
SSNMR torsion angle measurements, 13C–15N
distances, and excluded-volume
constraints. The structure of the backbone is of
especially high quality (0.02 Å
rmsd). Since the formyl group was not labeled, it was
permitted to assume
both the cis and trans conformations in the calculation,
and it exhibits the
appearance of a carboxyl group in the figure. The
carboxyl terminus and the
Phe ring appear disordered because no torsion angle
methods currently exist
to constrain the terminal or Φ angle. The ring
conformation is largely
determined by excluded volume constraints, and it is
likely undergoing twofold
flips. “
Rienstra et al. PNAS 99:10260-10265 (2002)
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
Synthetic Peptides: EPR
EPR Spin-Labeling Studies
[Toac343]]-α
α-peptide
MTS-Cys
MTS-Cys
HN
S
HN
S
N
– 5 rotatable bonds
– Compromises accuracy
O
O
MTS-Cys
Toac/Tpig
Fmoc
HN
N
O
O
HN
N
O
NH
Toac
OH
O
– Incorporated as amino acids
– Toac:
Toac: α -helical conform.
– Tpig:
Tpig: doesn’
doesn’ t constrain
backbone
[Proxyl340]]-α
α-peptide
Tpig
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Lori Anderson, thesis research
Bio5325 Protein Course, November 16, 2006
Proxyl-VLEDLKSC(Me)
ToacLF + rod-outer-segment membranes
Proxyl-VLEDLKSC(Me)ToacLF
(top panel = dark-adapted; bottom panel = light-activated)
DEER EPR spectra, distance distribution on right
EPR – DEER experiment to measure spin/spin interaction
Lori Anderson (WU), Ned Van Eps and Wayne Hubbell (UCLA)
Bio5325 Protein Course, November 16, 2006
Lori Anderson (WU), Ned Van Eps and Wayne
Hubbell (UCLA)
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
Time-Resolved Binding of Spin-Labeled
Peptide to Photoactivated Rhodopsin
EPR Experiments
0.4
Spectral properties of nitroxide
Laser pulse (! = 500 nm)
1. Mobility of side chain
2. Solvent Accessibility
0.2
– hydrophobic/hydrophilic boundaries
3. Interspin distances up to 50 Å
What are the conformational changes of
specific residues in the α-subunit relative to
labeled positions in rhodopsin?
rhodopsin?
EPR data
Optical Data (! = 370 nm)
0.0
-20
0
20
40
60
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Release of Bound Toac Peptide
80
Time (ms)
Garland R. Marshall, Washington University, St. Louis, MO
Comparison of R*-bound conformations of Arg,
Arg, Ser
analog with α-peptide
τ = 49 min
Intensity
0
50 100 150 200 250
time (min)
R*+P
R*P
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
50 µM TOAC343V (by mass)
88 µM Rhodopsin
(Urea Washed ROS)
Dark
Light
Native: IKENLKDCGLF
VLED: VLEDLKSCGLF
HASV: VLEDLKSVGLF
EC50
0.53
0.010
0.0064
R2
0.97
0.82
0.91
Bio5325 Protein Course, November 16, 2006
High-affinity Toac343 binding
20 mM MES
100 mM NaCl
1 mM MgCl2
pH 6.5
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
Mapping Peptide/Rhodopsin Interactions
Summary of 250R1 Data
Bleached Rhodopsin 250R1 without peptide
Bleached Rhodopsin 250R1 with 4 mM high affinity
peptide (no TOAC)
Bleached Rhodopsin 250R1 with 4 mM high affinity
TOAC peptide
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Four-pulse DEER (Double Electron-Electron Resonance)
Jeschke, Chemphyschem 2002, 3, 927-932
Garland R. Marshall, Washington University, St. Louis, MO
DEER analysis of
Gα peptide
binding to
rhodopsin
3.6 nm
TOAC
PROXYL
1.9 nm
Dipolar evolution
function
0
0.2
0.4
0.6
Time, µs
Dipolar
evolution
function
Dipolar
Evolution
Function
Distance distribution
(up to 80 Angstroms)
0.8
1.0
Rhodopsin
+ peptide, dark
Rhodopsin
+ peptide, light
Fourier
Transform
Garland R. Marshall, Washington University, St. Louis, MO
Use of TrNOE Structures of Peptide Analogs to Probe
Plausible Sets of R* Intracellular Loop Conformers for
Binding Partner
A.
0
1
2
Distance, nm
3
4
1.9 nm
1.9 nm
Interspin
Bio5325distance
Protein Course, November 16, 2006
τ2
Distance
distribution
Fourier Transform
B.
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Use of TrNOE Structures of Peptide Analogs to Probe
Plausible Sets of R* Intracellular Loop Conformers for
Binding Partner
A. Gtα(340-350) and its analogs B. GTA2, C.
GTA11, D. GTA14, E. GTA19, and F. 1LVZ, are
shown in the common binding mode. The first
and last 3 Cα atoms in the loop structures were
superimposed to find the common binding pose.
IC1 is shown in red, IC2 in yellow, IC3 in green
and IC4 in blue. Gtα(340-350) and its analogs
are shown in magenta.
A. TrNOE structure of Gtα(340-350) - IKENLKDCGLF. The analogs
of Gtα(340-350) have a similar structure. B. Ensemble of
intracellular loop structures.
Bio5325 Protein Course, November 16, 2006
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Garland R. Marshall, Washington University, St. Louis, MO
WHY SHOULD YOU CARE?
Human Diseases Due to Mutations in G Protein-Coupled
Receptors
Disease
of Mutation
Receptor
Type and Function
(-) = Loss of Function
(+) = Gain of Function
--------------------------------------------------------------------------------------------------------------------Retinitis pigmentosa
rhodopsin
(-) apoptosis of rod cells
Retinitis pigmentosa
rhodopsin
(-) null mutations
Stationary Night Blindness
rhodopsin
(+) missense mutations
Color blindness
opsins
(-) X chromosome
rearrangements
Nephrogenic DI
V2-receptor
(-)
Isolated glucocorticoid deficiency
ACTH-receptor
(-)
Hyperfunctioning thyroid adenomas TSH-receptor
(+) missense
Familial precocious puberty
LH-receptor
(+) missense
Familial hypocalciuric hypercalcemia Ca+2 receptor
(-) missense
Neonatal severe hyperparathroidism
Ca+2 receptor
(-) missense
---------------------------------------------------------------------------------------------------------Bio5325 Protein Course, November 16, 2006
Acknowledgments
Garland R. Marshall, Washington University, St. Louis, MO
O
O
N
O
H
N
O
O
O
O
O
N
HN
Gly Analog (1)
H
N
N
N
N
H
Meta II Stabilization at 1 mM Concentration
of Peptide
N
H
Ala Analog (2)
O
N
N
N
N
HN
λ (nm)
O
Bio5325 Protein Course, November 16, 2006
Garland R. Marshall, Washington University, St. Louis, MO
Graduate Students - Rieko Arimoto (BME), Lori Anderson (Bioorganic), Eric
Welsh (Biophysics), Matt Anderson (Bioorganic)
Prof. Gregory Nikiforovich (WUMS) – Rhodopsin Modeling
Dr. Christine M. Taylor (WUMS) - Modeling of Rh
Rh*
* Loops with TrNOE peptides
Prof. Wei-jun Zhang (WUMS) – Chemical Ligation of TM Segments
Dr. Wei Sha (WUMS) – π–Cation and Cyclic Peptide Studies
Dr. Yaniv Barda (WUMS) – Constrained Analog Synthesis
Prof. Oleg G. Kisselev (St. Louis University) - TrNOE Studies
Prof. Tom Baranski (WUMS) – Molecular Biology of GPCRs
Prof. Janusz Zabrocki ( Polytechnika
Polytechnika,, Lodz
Lodz,, Poland) – Tetrazole Analogs
Prof. David Cistola (WUMS) - TrNOE Studies of Peptide Analogs
Prof. Steve O. Smith (SUNY Stony Brook) – MAS NMR Studies
Prof. Wayne L. Hubbell and Dr. Ned Van Eps (UCLA) – ESR Studies
NIH for Financial Support (EY12113 and GM53630)
Bio5325 Protein Course, November 16, 2006
THE END of the BEGINNING*
*Assuming funding, of course
Bio5325 Protein Course, November 16, 2006