 |
INTRODUCTION |
Heterotrimeric G proteins are activated by
seven-transmembrane-spanning receptors and relay signals to downstream
effectors, including cellular enzymes and ion channels. Upon agonist
binding, receptors become activated and in turn interact with G
proteins and catalyze GDP release from G protein
subunits. After
the release of GDP, the G
subunit, together with G
subunits,
remains in a tight complex with the receptor, which dissociates when
GTP binds to the empty G
subunit. Both the GTP-bound G
subunit
and the G
subunit complex are then capable of regulating a
variety of effectors on the intracellular face of the plasma membrane. The binding of G
and G
subunits is restored when the intrinsic GTPase activity in the G
subunit hydrolyzes the bound GTP to GDP.
In order to have a reliable method for studying the conformational
changes in G proteins, we developed a fluorescent monitor because
fluorescence is easily detectable and responsive to local environmental
change. Cysteines are highly reactive and can be labeled with a variety
of Cys-directed fluorescent groups. We determined the cysteines
that are accessible to sulfhydryl-specific fluorescence labeling on
G
subunits and characterized the fluorescence changes on G
when
different sites are labeled. We replaced those accessible cysteines
with serine to make a functionally cysteineless mutant in which to
place additional cysteines at sites where we would like to monitor
conformational changes or interaction with other proteins.
To study structural changes in G
t upon its activation,
we used the functional derivative of G
t,
G
t/G
i1 chimera (Chi6) in which residues
216-294 of G
t were replaced with the corresponding residues 220-298 from G
i1. Chi6 can be conveniently
expressed in Escherichia coli, and it was shown to have a
similar rate of rhodopsin-catalyzed GDP/GTP exchange as
G
t does, implying that its receptor and G
binding
properties are G
t-like (1). The crystal structure of
Chi6 in complex with G
1
1 has been
solved and revealed an identical geometry with wild type, native
G
t (2). High expression levels of this protein in
E. coli provided us milligram amounts of pure protein for
biochemical and fluorescent studies as well as ease in constructing
G
mutants.
Lucifer yellow, an environmentally sensitive fluorescent probe,
was selected because it is a good reporter of local changes. We
previously used this probe as a reporter of the binding of the
inhibitory subunit of cGMP phosphodiesterase to G
t
(3).
Ho and Fung (4) previously reported that by using
5,5'-dithiobis-(2-nitrobenzoic acid) titration and
N-ethylmaleimide modification, a total of five reactive
sulfhydryls in native G
t and nine reactive sulfhydryls
in the SDS-denatured G
t protein were found. Eight cysteines were found by cDNA sequencing of
t (5). In
1988, by using
125I-N-(3-indo-4-azidophenylpropionamido-S-(2-thiopyridyl)cysteine (125I-ACTP),1 a
cross-linking reagent, Dhanasekaran et al. (6) showed that Cys210 and Cys347 were the major reactive
cysteines. Here, we show that the major labeling sites for LY are
located on Cys210 and Cys347 in
G
t/G
i chimeras. Also, we show that single
labeling at either Cys210 or Cys347 can be used
to report the local conformational changes around the labeled sites. As
expected, Cys210 in the switch II region reports an
AlF4
-dependent
activating conformational change. Unexpectedly, there is also an
AlF4
-dependent
conformational change at Cys347, which may be important for
allosteric communication between receptor binding and GDP-binding sites
on the molecule.
 |
EXPERIMENTAL PROCEDURES |
Materials--
GTP, GTP
S, GDP, deoxyribonucleotides, and
imidazole were purchased from Boehringer-Mannheim. All restriction and
DNA modification enzymes were obtained from Boehringer-Mannheim and
Pharmacia Biotech Inc. Lucifer yellow vinyl sulfone was purchased from
Sigma. All other reagents were from sources described previously
(1).
Construction of Mutants and Chimera Expression and
Purification--
The construction of Chi6 was described previously
(1). The construction of all mutants were based on Chi6 (1), composed of G
t cDNA except amino acids 216 to 294, which were
from G
i cDNA. Mutants were all constructed by using
the QuikChangeTM site-directed mutagenesis kit from
Stratagene Ltd. Strategy of oligo design for polymerase chain
reaction-based mutagenesis as suggested by the QuikChangeTM
kit instruction manual. The NH2-terminal sequence of Chi6
is derived from G
t expression construct (1) and preceded
by MA(His)6A. All chimera proteins were expressed and
purified by nickel affinity chromatography followed by anion exchange
chromatography using Protein Pak Q15 HR resin (Millipore) as described
previously (1). The concentrations of chimeras in the eluates were
determined spectrophotometrically and proteins were stored in buffer A
(50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM MgCl2, 50 µM GDP, 0.1 mM phenylmethylsulfonyl fluoride and 5 mM
-mercaptoethanol). Samples were aliquoted and kept at
80 °C for
several months with no loss of functional activity. The final yield of
all chimeras was between 1 and 3 mg/liter bacterial culture with an
average of 90% purity.
Expression of P
in E. coli--
The P
expression and
purification was performed as described (7). The P
was additionally
purified by reversed-phase chromatography on HPLC Vydac C-4 column
(size of 0.46 × 20 cm) using a gradient of acetonitrite.
Acetronitrite was removed from P
samples by Speed-Vac
re-concentrating procedure. The final yield of the P
ranged from 15 to 30 mg/liter of more than 95% pure protein.
Lucifer Yellow Labeling--
Labeling of G
with LY was
performed immediately after the samples were purified. Protein samples
(in buffer A without
-mercaptoethanol and phenylmethylsulfonyl
fluoride) and LY were mixed in a molar ratio of G
protein:LY (1:5)
followed by incubation on ice for 30 min. Labeling reactions were
stopped by addition of 4 mM
-mercaptoethanol. The excess
LY was removed by overnight dialysis and the labeled samples were again
purified and resolved by HPLC using protein Pak Q15 HR column (capacity
of 2 ml).
Stoichiometry Determination--
All samples were scanned from
260 to 550 nm with 8452A Diode Array Spectrophotometer (Hewlett
Packard). The stoichiometry (n) of LY labeling was
calculated as a ratio of the concentration of LY and the concentration
of the labeled protein in the HPLC eluted samples. The relationship
applied is,
|
(Eq. 1)
|
where CLY is the concentration of LY and
CChi6-LY is the concentration of labeled Chi6
protein (or the other labeled G
chimeras). The concentration of LY
(CLY) was determined using,
|
(Eq. 2)
|
where A430,LY is the absorbance of LY at
430 nm and
430,LY is the molar extinction
coefficient of LY at 430 nm which is 12,400. Since LY also absorbs at
280 nm (
280,LY = 24,000) the concentration of total
labeled protein (CChi6-LY) was determined by a
subtraction of LY absorbance at 280 nm from the total absorbance at 280 nm using the relationship,
|
(Eq. 3)
|
where A280,Chi6 is the absorbance of
Chi6-LY at 280 nm and
280,Chi6 is the molar extinction
coefficient of Chi6 at 280 nm which is 43,100. The value of
280,Chi6 was calculated by applying the
relationship,
|
(Eq. 4)
|
where
280,Trp, is the molar extinction
coefficient of Trp at 280 nm (5,300),
280,Tyr is the
molar extinction coefficient of Tyr at 280 nm (1, 400) while the molar
extinct coefficient of GDP at 280 nm,
280,GDP is 9,000. This relationship used to determine
280,Chi6 is based on
the Trp, Tyr, and GDP content (Chi6 has 3 Trp, 13 Tyr, and 1 GDP). For
the concentration of Chi6, the following relationship is used,
|
(Eq. 5)
|
Fluorescence Assays--
To monitor
AlF4
-dependent
conformational change of G
t and chimeras, tryptophan
fluorescence, before and after addition of AlF4
(simultaneously addition of
10 mM sodium fluoride and 50 µM
AlCl3 to the test sample), was determined with excitation
at 280 nm and emission at 340 nm. For LY-labeled samples, an additional measurement for
AlF4
-dependent
fluorescence change of LY was performed with excitation at 430 nm and
emission at 520 nm. All the experiments were performed on an AMINCO
Bowman® Series 2 Luminescence Spectrometer running with
OS/2 Warp 4.0.
Limited Proteolytic Analysis--
Protein samples were treated
with trypsin with a weight ratio of 16.6 (protein to trypsin) for 0, 5, 15, and 30 min before the reactions were stopped by TLCK (8, 9). After
running in polyacrylamide gel electrophoresis gels, the LY-labeled
fragments were observed by illuminating the gel with UV light.
Surface Exposed Area Calculation--
Solvent-accessible surface
area was calculated using the method of Eisenberg et al.
(10). The program used for this analysis was Access running in Silicon
Graphic System IRIX 6.2.
Data Analysis--
All curve fitting and kinetic analysis were
performed by using Prism version 2.01 for Windows 95 from GraphPad.
 |
RESULTS |
Determination of Solvent-accessible Surface Areas of Cysteines in
G
t--
There are eight cysteines in G
t and
according to the crystal structure (11), only four of them are surface
exposed: namely Cys62, Cys210,
Cys321, and Cys347 (Fig.
1). To obtain a quantitative criterion of
surface exposure, we calculated the solvent-accessible surface area by
the method of Eisenberg and McLachlan (10) using the coordinates of
G
t-GDP (11) and G
t-GTP
S (12). The
solvent-accessible surface areas of all eight cysteines in Chi6 are
summarized in Table I. Among these
cysteines, Cys210 has greater surface exposed area in its
GDP binding form than Cys62, Cys321, and
Cys282. Three other cysteine residues (135, 216, and 250)
are completely buried inside the protein according to this calculation.
Cys210 becomes less accessible in the G
-GTP
S binding
form. Since the COOH terminus of G
is disordered and not visible in
the crystal structure, it is likely that Cys347 is highly
accessible.

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Fig. 1.
Locations of all cysteines in Chi6
protein. Crystal structure of G t-GDP (11) is shown.
All cysteines (except Cys347) are space-filled
(yellow), helixes are red, -sheets are
cyan, while the loops are silver. GDP buried in
the central part of the molecule is purple.
Cys62, Cys210, and Cys321 (labeled
with red text) are more surface-exposed than other cysteines.
Cys347 is not shown because the last 7 amino acid residues
are disordered in the crystal structure. Graphic was generated using
"WebLab ViewerLite" V3.10 (Molecule Simulation Inc).
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Determination of LY Labeling Sites on Chi6--
To determine
accessible cysteines in Chi6, we first labeled Chi6 with LY under mild
conditions and identified the labeling sites. After the excess LY was
removed, the labeled samples were purified by anion-exchange HPLC. The
presence of LY adds negative charges to the protein and, thus, allows
separation of variously labeled species by anion exchange
chromatography (Fig. 2). Chi6 showed
three major labeled peaks as well as one unlabeled peak which
co-migrates with the original protein. Each peak in the HPLC elution
was collected separately and the stoichiometry of labeling was
determined. Proteins from labeled peaks 1 and 2 each contains a single
fluorescence group per molecule, while protein in labeled peak 3 contains two fluorescent groups (Table
II).

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Fig. 2.
HPLC purification of the LY-labeled Chi6 and
molecular structure of LY bound to cysteine. A, labeled G
(Chi6) samples were purified by HPLC using protein Pak Q 15 column. The
stoichiometry of LY to protein in labeled peak 1 as well as labeled
peak 2 in Chi6 was 1:1, whereas it was 2:1 in labeled peak 3. B, molecular structure of LY bound to cysteine. Retention
time for each fraction is indicated on the top of the peak.
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Table II
Summary of percentage of LY labeling in various chimera proteins and
AlF4 -dependent fluorescence change for
all the labeled peaks. The ratio indicated after protein is the
stoichiometry of LY:protein labeling with LY
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|
To identify the labeling site(s) for each peak, limited tryptic
digestion and mutagenesis studies were performed. The potential tryptic
cleavage sites in Chi6 protein are lysine 18, arginine 204, and
arginine 310 (8, 9) as illustrated at the bottom of Fig.
3. Protein from labeled peak 3 of Chi6
was treated with trypsin for 0, 5, 15, and 30 min before the reactions
were stopped by TLCK. The LY-labeled fragments on the gel were observed
with UV illumination. As shown in Fig. 3, the double labeled peak 3 showed fluorescent fragments of 38, 19, 15, and 5 kDa, which suggested the labeled cysteines were within the region composed of amino acids
205-350, which contain Cys210, Cys321, and
Cys347. To identify the labeled site(s), a mutant (Chi6a)
was constructed in which Cys210 was changed to serine. As
shown in Table II, after been labeled with LY, Chi6a-LY
(Cys347-LY) migrated on Mono-Q column as a single labeled
peak with a retention time of 13 min similar to labeled peak 2 in
Chi6-LY (Table II). After this peak was treated with trypsin, the size of the LY-labeled fragments were 38, 19, and 5 kDa, which suggested the
labeled site was either Cys347 or Cys321 (Fig.
3).

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Fig. 3.
Proteolytic analysis of labeled peak of Chi6
and Chi6a-LY. The proteins represented by labeled peak 3 of
Chi6-LY (upper left panel) and labeled peak of Chi6a-LY
(upper right panel) were treated with trypsin (weight ratio
protein:trypsin = 16.6:1). Trypsin fragments were resolved by
polyacrylamide gel electrophoresis and each gel contains protein
samples with trypsin treatment time of 0, 5, 15, and 30 min before the
reactions were stopped by TLCK. A UV transilluminator was used to
observe the LY-labeled fragments on the gel. Simplified scheme for
limited proteolytic digestion pattern of Chi6 is shown on the
lower panel. The cleavage sites in Chi6 protein for trypsin
are shown with a hollow arrow (under Lys18,
Arg204, and Arg310). See "Experimental
Procedures" for more details.
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|
To further clarify the possible labeling sites suggested in tryptic
digestion experiments, two more mutants were constructed: Chi6b, with
Cys347 mutated to serine, and Chi6ab, which has both
Cys210 and Cys347 mutated to serine. As shown
in Table II, after labeling with LY, Chi6b showed a single labeled peak
with a retention time of 12 min, which corresponded to peak 1 for Chi6,
whereas in the double mutant (Chi6ab), all major labeled peaks
disappeared. We therefore conclude that the protein eluted in labeled
peak 1 was labeled at Cys210 while protein eluted in peak 2 was labeled at Cys347. Thus, cysteine 210 and cysteine 347 are the major accessible sites for LY labeling. A comparison of the
areas of the peaks shows that the efficiency of labeling at
Cys347 was 6.5 times higher than that of
Cys210. However, for mutants with one cysteine available
for labeling (Chi6a and Chi6b), the efficiency of LY labeling at
Cys210 and Cys347 was comparable, which
suggested that the labeling of Cys347 in Chi6 prevented the
labeling of Cys210.
Functional Assays for LY-labeled and Unlabeled Chimera
Proteins--
To determine whether the mutant proteins folded
properly, had GDP bound, and could undergo GTP-dependent
conformational changes, we measured the intrinsic fluorescence change
of Trp207 after addition of
AlF4
. All the mutant proteins
were able to undergo conformational change upon binding to
AlF4
(Table II). After LY
labeling, the
AlF4
-dependent
intrinsic fluorescence change of Cys347-LY in Chi6a protein
was 50% but Cys210-LY in Chi6b protein showed
significantly less (1.2%) fluorescence change (Fig.
4, Table II).

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Fig. 4.
Kinetics of the
AlF4 -dependent
fluorescence change monitored at 340 nm (Trp) and 520 nm (LY). The
fluorescence of Trp (open circle) and LY (open
triangle) in Chi6b-LY (Cys210-LY) (A) and
Chi6a-LY (Cys347-LY) (B) was monitored (66 nM protein) during activation by
AlF4 (10 mM sodium
fluoride and 50 µM AlCl3) at the time
indicated by arrow and expressed as the percent of initial
fluorescence change. C, the fluorescence of LY at
Cys347 was expressed as percentage of the maximum
fluorescence change upon addition of
AlF4 . The best fit obtained by using
equation Ft = F0 + F1(1-expact Kactt)
for tryptophan (dashed line) and LY (solid line)
are shown. Ft is fluorescent emission at time
t, while F0 is the initial
fluorescence before addition of AlF4 .
F1 is maximal change in fluorescence, and
Kact is the rate constant for Chi6 activation.
Initial fluorescence was set to 0 and maximal fluorescent change is
expressed as 100%. Both fluorescence change in LY and Trp can be
reversed by addition of 10 mM of EDTA to chelate
AlF4 and this reverse process is EDTA
concentration dependent (data not shown).
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|
LY Is a Fluorescent Reporter for Conformational Change in Switch
II--
There was only a 1.2%
AlF4
-dependent
intrinsic Trp fluorescence change in Cys210-LY G
(Table
II). To determine whether there was an
AlF4
-dependent
conformational change in Cys210-LY, we measured the LY
fluorescence change in Cys210-LY (Chi6b), which is within
the switch II region, after addition of
AlF4
. The fluorescence of
Cys210-LY increased 220% in response to
AlF4
(Fig. 4, Table II). This
fluorescence change can be reversed by 10 mM EDTA (data not shown).
Comparison of crystal structures of GDP and GTP
S bound
G
t revealed decreased accessibility of
Cys210 in the activated form. Therefore, the accessibility
of cysteine residues to thiol-specific reagents (like LY) should
decrease upon activation. Chi6b was labeled with LY in the presence or absence of AlF4
and a decrease of
77.8% of LY labeling efficiency for Chi6b was observed in
AlF4
bound Chi6b (Fig.
5). Thus, in the active conformation,
Cys210 is less accessible for LY labeling.

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Fig. 5.
LY labeling efficiency of Cys210
and Cys347 in the presence or absence of
AlF4 . Equal amounts of
chimera proteins from the same preparation were taken and divided into
control and AlF4 preactivated group.
Before LY labeling, the latter group was activated by addition of
AlF4 (10 mM sodium
fluoride and 50 µM AlCl3) for 10 min and
followed by measurements of
AlF4 -dependent
Trp207 intrinsic fluorescence change in both groups.
Labeling efficiency for two chimera proteins, Chi6a and Chi6b, were
shown in the figure. Total amount of labeled protein in control group
was set to 100%. The labeling efficiency of LY to Chi6a
(Cys347-LY) was reduced by 58.6% in preactivation group
while a 77.8% reduction was observed in preactivated Chi6b
(Cys210-LY).
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To further test the existence of a switch II conformational change in
Cys210-LY, the ability of the mutant to interact with the
downstream effector enzyme, cGMP phosphodiesterase, was tested.
G
t-GTP binds to the inhibitory
subunit (P
) with
higher affinity than G
t-GDP (1). The interaction of P
with Cys210-LY G
in the presence or absence of
AlF4
was monitored by LY
fluorescence. There was only a small fluorescent change upon addition
of increasing concentrations of P
in the absence of
AlF4
, while in the presence of
AlF4
there was a P
concentration-dependent increase in fluorescence (Fig.
6). Thus, the mutant that fluorescently
labeled G
undergoes an activating conformational change that causes
an increased affinity for PDE
, and association with PDE
further
increases the fluorescence of Cys210-LY.

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Fig. 6.
Binding of Chi6b-LY protein to P subunit
in the presence or absence of
AlF4 . The relative
increase in LY fluorescence change (% of initial) was measured after
addition of increasing concentrations of P to 50 nM
Chi6b-LY (square) or
AlF4 -preactivated Chi6b-LY
(triangle). The kinetic parameters calculated from the best
fit to the four parameter logistic (sigmoidal) equation were:
Kd = 100 ± 11 nM,
FMAX 99%, and Hill slope 1.2.
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|
Since the switch II region of Chi6b-LY undergoes a conformational
change upon binding to AlF4
, and
this conformational change can be monitored by LY, we therefore concluded that LY labeled at Cys210 is a sensitive
fluorescent reporter for the conformation of the switch II region.
The COOH Terminus of G
Undergoes a Conformational Change Upon
AlF4
-dependent
Activation--
The COOH terminus of G
t is not ordered
in crystal structure (2, 11-13). To assess its conformation and
whether it changes conformation during activation, we labeled
Cys347 with LY in Chi6a (Cys347-LY). As shown
in Table II and Fig. 4B, an 91% LY fluorescence increase
was observed upon addition of
AlF4
in Cys347-LY
protein. This observation suggests that upon binding of
AlF4
, the COOH terminus of G
undergoes a conformational change. To understand and confirm the
existence of this COOH-terminal conformational change, we determined
the accessibility of Cys347 for LY labeling in the presence
and absence of AlF4
. In the
AlF4
-activated Chi6a, a decrease
of 58.6% of LY labeling efficiency to Chi6a was observed (Fig. 5)
suggesting a decreased accessibility of Cys347 in the
activated protein.
To determine whether this fluorescence change had a similar rate as the
AlF4
-dependent
fluorescence change at Trp207, the kinetics of the
AlF4
-dependent Trp and LY
fluorescence at Cys347-LY were measured. The rates of the
intrinsic fluorescence change of Trp207 and LY fluorescence
at Cys347-LY proteins were very similar, with a
Kact of 0.462 and 0.364, respectively (Fig.
4C). This result suggested that upon binding of
AlF4
, the COOH terminus of G
undergoes a fast conformational change that follows the slow
AlF4
-dependent
conformational change in the switch II region. Alternatively, the
change in conformation of the switch II region could lead to an
increased contact with the COOH terminus that resulted in the increase
of LY fluorescence.
 |
DISCUSSION |
Knowledge of the functional structure of heterotrimeric G proteins
has been greatly advanced with the solution of the crystal structure of
all their subunits (2, 11-18). A comparison of the three-dimensional
structure of the
subunits in their GDP, GTP
S, and transition
state analogue forms has revealed the molecular principles of
nucleotide binding, hydrolysis, and the nature of the conformation
changes upon protein activation. The COOH-terminal region of
G
t, known to be important for receptor interaction (19-24), was not ordered in crystal structures (2, 11-13). Thus, other biochemical or biophysical methods are needed to further define
the structural features of this region and understand its role in
mediating receptor interaction and receptor induction of GDP release.
In this report, we have developed an approach of sensitive real-time
monitoring of the structural changes in G
using targeted fluorescent
labeling of single surface-exposed cysteine residues. Our studies have
identified the labeling sites of LY on
G
t/G
i1 chimera (Chi6), characterized each
labeled protein and, most importantly, shown the existence of a
previously unsuspected conformational change in the COOH terminus of
G
upon activation.
The existence of two negative charges in LY and the mild conditions
used for labeling Chi6 with LY allowed us to resolve differently modified proteins by anion-exchange chromatography (Fig. 2). The labeling sites on G
identified in this report, Cys210
and Cys347, confirms the studies of Dhanasekaran et
al. (6) who found that Cys347 is highly accessible and
Cys210 is partially accessible to 125I-ACTP,
when this cross-linking reagent was used to label
G
t.
All mutants were properly folded and functional as judged by several
criteria: first, all mutants had an increased Trp fluorescence upon
addition of AlF4
indicating that they
all have GDP bound and undergo conformational changes. Second, all
mutants activated with AlF4
had
similar affinity to P
when compared with Chi6 (1), which was in the
range of 1 µM. Third, the cleavage patterns of tryptic digestion were identical in Chi6 and Chi6a and therefore it can be
concluded the mutant chimeras were properly folded during expression.
Our data clearly show that LY at Cys210 is a reporter of
conformational change of the switch II region. The fluorescence of LY attached to Cys210 (Chi6a-LY) increases significantly
(220%) in the presence of AlF4
.
However, the intrinsic Trp fluorescence of Cys210-LY
(Chi6b-LY) under similar conditions underwent only 1.2% change, which
is much less than 50% change in Trp fluorescence for the unlabeled
mutant. But Chi6b-LY protein does indeed undergo switch II region
conformational change upon binding of
AlF4
as judged by acquisition of high
affinity for P
. An explanation for a decreased Trp fluorescence
change in Cys210-LY may be that the fluorescent group at
Cys210 increases local hydrophobicity in the environment of
Trp207 that causes an increase in Trp fluorescence in the
GDP-bound state of G
. Upon addition of
AlF4
, switch II moves and adopts the
conformation of active G
subunit, as Trp207 does.
However, the increased hydrophobicity of Trp207 in the new
environment is not much more than that created by the LY group, so that
it does not result in a regular change of intrinsic fluorescence.
The solvent-accessible surface area of Cys210 decreased
68.6% in the activated form of G
t. This calculation
matches well with our finding that the labeling efficiency at
Cys210 dropped 77.8% in the presence of
AlF4
. Thus, Cys210 of
Chi6b becomes partially protected from LY labeling in the presence of
AlF4
, reflecting these changes in
accessibility. The method is thus clearly useful for monitoring known
conformational changes.
The conformational changes seen at the COOH terminus of G
are the
first indications that the GTP-dependent or
activation-dependent conformational switch may extend to this
region of the molecule. Several facts support the existence of those
environmental changes around the COOH terminus. First, the fluorescence
of LY at Cys347-LY (Chi6a-LY) increases by more than 91%
in the presence of AlF4
and can be
reversed by 10 mM EDTA. There is no nonspecific interaction between LY and AlF4
since in the
presence of free LY, no fluorescence change was observed by addition of
sodium fluoride and AlCl3 (data not shown). Second, the
efficiency of Cys347 labeling with LY for Chi6a is
significantly reduced with addition of
AlF4
, suggesting that surface exposure
and reactivity of this residue is decreased as a result of protein
activation. To test whether a fluorescent tag on a cysteine in a part
of the molecule that is known to have no conformation change upon
activation, Chi6ab, with both reactive cysteines changed to serines,
was mutated to replace Val301 with cysteine. There was no
AlF4
-dependent fluorescent
change in the resultant mutant, Chi6ab-Cys301-LY (data not
shown). Thus the fluorescence change is not seen in regions known not
to change conformation. Third, these effects are very similar to those
we observed for Cys210 in Chi6b, which is an indicator of
the known conformational change of switch II region. Thus, we can
conclude that it is likely that a change in conformation does exist at
or near the COOH terminus of G
upon activation or more precisely in
the GDP-AlF4
form. The comparison of
the kinetics of fluorescence changes monitored by LY to the fluorescent
changes monitored by Trp207 for Chi6a-LY upon addition of
AlF4
shows that they have similar time
course (Fig. 4C). This analysis implies that the structural
change at the COOH terminus is rate limited by the relatively slow
movement of the switch II region upon binding of
AlF4
. Dhanasekaran et al.
(1988) also proposed that conformational changes could be transmitted
between domains containing Cys347 and Cys210 in
G
t because the photoactivation of the phenylazide moiety of 125I-ACTP labeled at Cys347 caused an
insertion to the 12-kDa fragment (residues
Arg204-Arg310), which contains
Cys210. Our data suggest that LY-bound Cys347
is in a more hydrophobic environment in
GDP-AlF4
than in the GDP-bound G
.
Another observation is the Cys347 is the major labeling
site for Chi6 and the labeling of Cys347 reduces the
labeling efficiency of Cys210, which confirms the result
suggested in 125I-ACTP labeling study (6). This leads us to
speculate that the COOH terminus of G
does not interact with the
2/
4 loop in G
-GDP form, but in the active conformation the
2/
4 loop and COOH terminus of G
move closer.
What is the nature of this COOH-terminal conformational change?
According to the crystal structure of G
-GTP
S (12), in two out of
three molecules in the asymmetric unit, the final eight residues are
disordered and not visible while in a third molecule, the COOH-terminal
residues 343-349 of G
t can be seen. The residues 343-349 make van der Waals contacts with residues 212-215 of the
2/
4 loop. It is not clear whether this is a crystal packing artifact or an indication of one possible orientation of the COOH terminus.
In G
t, Asn343 is the last residue that is
consistently seen. It is ~10 Å from the
2/
4 loop which is part
of Switch II. Two possibilities exist: 1) the conformation of the
COOH-terminal region changes upon activation, 2) the COOH terminus
itself does not undergo a conformational change, but its environment
changes. For example, the disordered COOH-terminal region including
Cys347 is close to the
2/
4 loop and in the active
conformation a binding pocket for the COOH terminus opens up near the
switch II region such that Cys347 becomes more buried. In
either case, the protection from fluorescent labeling by preactivation,
and the activation-dependent fluorescent change indicates
an important communication between the GDP binding pocket and the COOH
terminus of the protein.
The COOH-terminal conformational change that was detected in this study
is of significant interest because of the implications for receptor
interaction mechanisms. The COOH terminus of G
is known to be a key
determinant of the fidelity of receptor activation (19, 23). Since
known receptor-binding regions on G
are distant from the GDP-binding
site, it is likely that an allosteric mechanism triggers GDP release
(24). It appears from a number of studies that mutations at the COOH
terminus of G
proteins can both regulate specific receptor
interaction and affect GDP affinity (24-28). COOH-terminal peptides
from G
can both block receptor-G protein interaction and stabilize
the active conformation of G protein-coupled receptors (21, 29-32).
These data suggest that the COOH terminus may be a key relay for
communication between the activated receptor and the GDP binding
pocket. The activation-dependent conformational change
reported here thus suggests specific regulation of receptor affinity by
the status of the guanine nucleotide bound in the binding pocket. It is
known that when receptor, ligand, and G protein are bound, GDP release
leads to a high affinity "ternary complex" which only slowly
dissociates without guanine nucleotide binding (33-37). It is possible
that an activation-dependent conformational change at the
COOH terminus of G
might lead to a lowered receptor affinity and
dissociation from the ternary complex. Currently, it is thought that
dissociation of activated G protein from receptor is secondary to the
GTP-dependent dissociation of G
from G
. This
concept has not been rigorously tested, however. Future studies will
test the notion that the COOH-terminal conformational change after GTP
binding is a component of G protein dissociation from an activated receptor.
Site-specific Cys-directed fluorescent groups could be reporters for
conformation changes in various regions of G
. For example, they
could probe conformational changes in other regions of the G
subunit
like the NH2 terminus, which was not resolved in crystal structures of either the free G
GDP form (11) or the GTP
S form
(12, 14). Also, they could be used to monitor protein-protein interaction, such as G
interaction with receptors, G
, and
effectors. Future studies will explore these possibilities.