(Received for publication, March 15, 1995; and in revised form, December 22, 1995)
From the
The fluorescent probe tetramethylrhodamine iodoacetamide was attached to cysteine residues substituted at various specific locations in full-length and deletion variants of the homodimeric Escherichia coli ribosomal protein L7/L12. Ground-state tetramethylrhodamine dimers form between the two subunits of L7/L12 depending upon the location of the probe. The formation of tetramethylrhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the different protein variants. Intersubunit tetramethylrhodamine dimers form when tetramethylrhodamine acetamide is attached to two different sites in the N-terminal domain of the L7/L12 dimer (residues 12 or 33), but not when attached to sites in the C-terminal domain (residues 63, 89, or 99). The tetramethylrhodamine dimers do form at sites in the C-terminal domain in L7/L12 variants that contain deletions of 11 or 18 residues within the putative flexible hinge that separates the N- and C-terminal domains. The tetramethylrhodamine dimers disappear rapidly (within 5 s) upon addition of excess unlabeled wild-type L7/L12. It appears that singly labeled L7/L12 dimers are formed by exchange with wild-type dimers. Binding of L7/L12:tetramethylrhodamine cysteine 33 or cysteine 12 dimers either to L7/L12-depleted ribosomal core particles, or to ribosomal protein L10 alone, results in disappearance of the 518-nm absorption band. This result implies a conformational change in the N-terminal domain of L7/L12 upon its binding to the ribosome, or to L10.
Tetramethylrhodamine iodoacetamide (TMRIA) is a
sulfhydryl-specific reagent used primarily for fluorescence
spectroscopy and microscopy. TMRIA is a derivative of rhodamine B which
is well known as a quantum counter (1) and as an active medium
for lasers(2) . It is commercially available from Molecular
Probes Inc. (Eugene, OR) as a mixture of the 5` and 6` isomers (Fig. 1). Recently the pure 5` isomer also became available from
Molecular Probes. Observations of an absorption band with a maximum at
518 nm associated with high concentrations of rhodamine B in aqueous
solutions were reported by Förster and
König(3) . They attributed this absorption
to a ground-state rhodamine B dimer with a dissociation constant (K
) of 667 µM. Selwyn and
Steinfeld (4) studied the aggregation equilibria of a number of
xanthene dyes and concluded that rhodamine B formed a dimer in aqueous
solutions with a dissociation constant of 680 µM. Arbeloa
and Ojeda (5) extended these studies and determined that
neutral and cationic rhodamine B forms ground-state dimers at high
concentrations in aqueous solutions (K
= 476 and 714 µM, respectively). Both
the Förster and Arbeloa studies reported a ratio of
1.3 for the extinction coefficients at 518 and 555 nm (518/555 ratio)
for the rhodamine B dimer and a ratio of 0.4 for the monomer.
Figure 1: Structure of 5`- (or 6`-)TMRIA.
Tetramethyl derivatives of rhodamine have also been reported to form
ground-state dimers. Ajtai et al.(6) reported K values for dimer dissociation of the 5`
or 6` isomers of TMRIA as 137 and 56 µM, respectively;
this study also included absorption spectra of the 5` and 6` isomers of
TMRIA indicating that at 500 µM the 518/555 ratios reached
are less than 1.0. Edmundson et al.(7) , however,
reported a 518/555 ratio for dimers of 5`- and
6`-carboxytetramethylrhodamine of about 1.3.
L7/L12 is a 12-kDa (120 residues) protein present in four copies (in the form of two dimers) in the 50 S subunit of Escherichia coli ribosomes, where it is essential for optimal ribosome function in protein biosynthesis(9, 10) . A high resolution crystal structure for monomeric C-terminal domain (residues 52-120) has been determined(11) . Native L7/L12 possesses no cysteine, but several site-specific cysteine substitution mutants of L7/L12 have been prepared and utilized in cross-linking(12, 13, 14, 15, 16, 17) and fluorescence studies(18, 19) . Both methods have been used to study the conformation, aggregation state, and ribosomal location of L7/L12. The fluorescence methods essentially provide an average view of the conformation of the protein population on the time scale of nanoseconds, whereas the cross-linking method determines whether two distinct locations can come into contact within a time on the order of minutes.
Time-resolved fluorescence and energy transfer
studies (18, 19) ()in our laboratories have
indicated that the two C-terminal domains of L7/L12 possess
considerable mobility and are, on average, widely separated from one
another, even though they can be trapped in high yield by cross-linking
as covalently linked disulfide dimers(17) . By contrast, these
fluorescence studies indicated that the two N-terminal domains
(residues 1-33) that are responsible for the
dimerization(20) , are, on average, closer together. In the
present work, the extent of formation of ground-state rhodamine dimers
between TMRA bound to the various cysteine locations in full-length or
deletion variants of L7/L12, both free and reconstituted into
ribosomes, was investigated to extend the findings from cross-linking
and fluorescence studies on the conformations of the two domains of the
protein.
Figure 2: Schematic diagram of the domain structure of E. coli L7/L12 dimer. The C-terminal and N-terminal domains are indicated along with the sites of the various cysteine substitutions and the hinge deletion.
518/555
ratios of 1.30 ± 0.03 were obtained for the TMRA labeled C-33 or
C-12 proteins and this value was taken to represent 100% labeling. The
bases of this assumption were the
[C]iodoacetamide studies described above and the
number of reports in the literature (see Introduction) that the ratio
of the two principle absorption bands in such ground-state dimers is
approximately 1.3. Addition of excess (15-fold) unlabeled wild-type
L7/L12 led to virtually complete disappearance of the TMRA dimer
absorption (as evidenced by the resulting 518/555 ratio of 0.41).
Subsequent to subunit exchange the absorption at 555 nm was measured
and an extinction coefficient for the TMRA monomer bound to the C-12 or
C-33 positions was estimated to be 72,000 M
cm
± 2,000, assuming 100% labeling.
This value may be compared to the extinction coefficient of 76,000 M
cm
given in the
Molecular Probes catalogue for the isomeric mixture of TMRIA reacted
with
-mercaptoethanol. We note that Corrie and Craik (23) determined an extinction coefficient of 96,900 ±
5,300 M
cm
at 549 nm for
the 5` isomer of TMRIA attached to the thionucleotide ATP
S. The
extinction coefficient of TMRA attached to cysteines located within the
putative structureless loops (11) of the L7/L12 C-terminal
domains (C-63, C-89, and C-99) is the same in buffer or 4 M guanidine hydrochloride (data not shown), indicating negligible
effects of secondary or tertiary structure on this property. Assuming
that the extinction coefficient of TMRA attached to the C-terminal
residues is also approximately 72,000 M
cm
at 555 nm (see above), we can estimate that
the labeling ratio obtained for the three C-terminal positions
approaches 100%.
Figure 3:
Extinction coefficients for TMRA bound to
L7/L12. Values for the dimeric (A) C-33 and (B) C-89
L7/L12 variants are indicated. The extinction coefficients were based
on a value of 72,000 M cm
at 555 nm for monomeric TMRA and a 518/555 ratio of 1.3 for
dimeric TMRA (see ``Materials and
Methods'').
The absorption spectra for the TMRA labeled C-63 and
C-99 conjugates of L7/L12 were essentially identical to that of the
C-89 variant (Fig. 3). The spectra of all of the C-terminal
domain, full-length variants, however, differed significantly from the
absorption spectra corresponding to the TMRA labeled C-12 and C-33
variants. Specifically, they exhibited a single prominent absorption
band with a maximum at 555 nm and had 518/555 ratios of 0.40 ±
0.01. These spectral characteristics indicated the absence of rhodamine
interaction for the probes located at these sites in the C-terminal
domain in the full-length protein. However, the behavior of the probes
located in the same sites changed in deletion variants of L7/L12
missing either 11 (C-89:42-52) or 18 residues
(C-63:
35-52) in the hinge linking the C-terminal domain to
the N-terminal domain. Specifically, the 518/555 ratios for the TMRA
labeled deletion variants were 1.30 ± 0.03, indicating that TMRA
dimers form in the CTD sites when the hinge residues were deleted.
Figure 4: Dissociation of L7/L12 dimers. Effect of dilution of TMRA labeled C-12 (solid circles), C-33 (open circles), and C-89 (open triangles) L7/L12 on the 518/555 ratio of TMRA absorption. Data represent averaged values from three experiments.
Fig. 5shows the effect of addition of increasing quantities of wild-type L7/L12 dimers (up to a 15-fold molar excess) on the 518/555 ratio of TMRA labeled C-33 dimers (1 µM). Addition of equimolar unlabeled wild-type L7/L12 results in a decrease in the 518/555 ratio to 0.81. This result may be compared to the ratio of 0.72 expected for an equilibrium not altered by the TMRA association. The decrease in extinction coefficient at 518 nm of TMRA attached to C-33, subsequent to addition of 10-fold excess wild-type L7/L12, was completed in less than 5 s at 20° C. Wild type L7/L12 that had either been cross-linked with glutaraldehyde to render it incapable of dissociation and exchange, or treated to produce methionine sulfoxides with the resultant formation of L7/L12 monomers (24) incapable of reassociation, had no significant effect on the 518/555 ratio (Fig. 5). The retention time of the C-33 TMRA conjugate on the SE-HPLC column corresponded to that observed for unlabeled dimer and was identical in the presence and absence of 10-fold excess wild-type L7/L12 demonstrating the absence of either monomers or higher aggregates(21) .
Figure 5: Subunit exchange among L7/L12 dimers. Effect of addition of unlabeled wild-type L7/L12 (open circles), methionine oxidized L7/L12 (open triangles) or glutaraldehyde cross-linked L7/L12 to TMRA labeled C-33. Data represent averaged values from three or more experiments.
The formation of TMRA ground-state dimers in L7/L12 cysteine
substituted variants conjugated with the sulfhydryl specific probe was
used to investigate conformational aspects of the C-terminal and
N-terminal domains of L7/L12 dimer in solution. The formation of the
new 518-nm absorbance peak characteristic of the rhodamine-rhodamine
interaction and the ratio of the intensities of the new 518-nm band to
the pre-existing 555-nm band served as a convenient way of quantifying
the spectroscopic data. Probes situated at two locations in the
N-terminal domain both displayed the 518-nm band, whereas probes in the
C-terminal domain did not. This observation is consistent with mobility
of the CTD's inferred from H
NMR(25, 26) , ESR(27) , and electron
microscopy (28) studies and with energy transfer studies on
fluorescein and AEDANS attached to the C-63 and C-89 positions which
indicated that, on average, the C-63 to C-63 or the C-89 to C-89
intersubunit distances are >80 Å(19) .
Despite the absence of close proximity of the two C-terminal
domains, covalent disulfide cross-linked complexes have been obtained
in high yield for the C-63, C-89(19) , and C-99 (
)positions suggesting that, on the time scale of the
cross-linking experiments, these domains are mobile and can approach
close enough to permit disulfide bond formation. When the hinge region
linking the two domains is substantially shortened in deletion variants
(C-63:
35-52 and C-89:
42-52), TMRA dimer formation
takes place for probes in the C-terminal domain. This observation
implies that the hinge facilitates the mobility and separation of the
two C-terminal domains in the full-length protein.
TMRA attached to N-terminal sites 12 and 33, both within the known dimerization domain, formed rhodamine dimers in the free proteins with absorption properties closely resembling those of dimers formed by free dye at high concentrations (518/555 ratios of 1.3). This result is consistent with the parallel non-staggered model of the L7/L12 dimer as depicted in Fig. 2. It also suggests that both the 5` and 6` isomers of the probe were able to form dimers; however, it is also possible that one isomer in the mixture of 5` and 6` reacts preferentially with the target cysteine. Ajtai et al.(6) suggested that the TMRIA from Molecular Probes contains mainly the 6` isomer and also suggest differential reactivity of the two isomers.
The 518-nm absorption band of TMRA labeled C-12 or C-33 dimers disappeared rapidly (within 5 s) upon addition of excess unlabeled wild-type L7/L12. These results indicate that subunit exchange can occur among the L7/L12 dimer population and that the 518-nm absorption band of the TMRA dimers provide a convenient spectroscopic handle to monitor this process. These results are consistent with similar observations on rapid subunit exchange in other dimeric and tetrameric systems(29, 30, 31, 32) .
The
experiments revealed differences in the two NTD sites implying
different orientations of the two polypeptide chains in the C-12 and
C-33 regions. Specifically, the dissociation constant for the TMRA
labeled C-12 dimer/monomer equilibrium is near 300 nM (judged
by the midpoint of the decrease in the 518/555 ratio upon dilution), in
good agreement with results of fluorescence polarization studies on the
dissociation of C-12 variants labeled with either one or two
fluorophores per L7/L12 dimer(19) . ()These
fluorescence polarization studies also indicated a dissociation
constant for singly and doubly labeled C-33 variants near 30
nM, a value consistent with the small change observed in the
518/555 ratio for TMRA labeled C-33 over the dilution range accessible
by absorption measurements (Fig. 4). These results and the
518/555 ratio observed for equimolar, subunit exchanged mixtures of
unlabeled wild-type L7/L12 and L7/L12:TMRA-C-33 suggest that TMRA dimer
formation does not significantly perturb the L7/L12 dimer/monomer
equilibrium. Conformational alterations of different magnitude in the
regions surrounding residues 12 or 33 may be necessary for the
TMRA-TMRA interaction, and the free energy of the TMRA dimer formation
may compensate for the free energy loss associated with such
alterations. Alternatively, microscopic details of the environment
around the TMRA moieties, such as the dielectric constant and
solvation, may differ sufficiently from the bulk solvent to alter the
free energy of formation of a ground-state complex, and may be
different for the probes at the two locations. Different orientations
for the TMRA molecules in the ground-state dimer have been proposed (5, 6, 7) but our results do not address this
issue. The reasons for the difference in the dimer/monomer equilibria
for the C-12 and C-33 variants is not presently clear.
The
dissociation of the TMRA dimers, located at both residues C-12 or C-33,
upon binding of L7/L12 to L10, suggests that a conformational change in
the N-terminal domain of L7/L12 accompanies the binding. Residue 12 is
near the N-terminal end of the domain, while residue 33 is near the
junction with the putative hinge region. This observation is consistent
with the finding that dimers of L7/L12 oxidatively cross-linked in
solution by disulfide bonds between the two C-33 or C-12 residues fail
to bind to ribosomal core particles (17) which
suggests that cross-linking prevents conformational changes at both
sites in the N-terminal domain required for or accompanying binding to
L10. Protein L10 is the major ribosomal component that anchors both
L7/L12 dimers to the ribosome, and it is not surprising that ribosomal
core particles lacking only L7/L12, but retaining L10, have the same
effect on TMRA dimers as does free L10. The effect must pertain to both
L7/L12 dimers even though they bind to non-equivalent sites in L10.
These observations are summarized in schematic form in Fig. 6.
The left hand image in Fig. 6A summarizes the locations
of all five rhodamine conjugates, and indicates the formation of
rhodamine dimers in the two N-terminal locations, but not in either of
the three C-terminal locations, the mobility and separation of which
are indicated by arrows. The right-hand images in Fig. 6B show the conformational perturbation and
disruption of the two N-terminal sites accompanying binding to the
ribosomal core particle or to purified L10.
Figure 6: Schematic diagram summarizing the formation and loss of TMRA dimers. Results are summarized for full-length L7/L12 dimers, both free and bound to L10 or 70 S ribosomal core particles. The arrows indicate mobility of the C-terminal domains.
Fig. 7shows the
effect of the deletion of residues in the hinge region on TMRA dimer
formation at two of the sites in the C-terminal domain. Extensive
studies have not yet been completed in which each of the three CTD
sites have been combined with both the longer and shorter deletions.
TMRA dimers form at sites 63 and 89, with the longer and shorter
deletions, respectively. The result suggests that the loss of
flexibility resulting from the hinge deletions facilitates stable
interaction between the probes. The magnitude of the 518-nm band in
these experiments indicates that the entire population undergoes this
interaction, despite the fact that residues 63 and 89 are located in
exposed loops facing in different directions, according to the x-ray
structure of C-terminal domain monomers(11) . The ability of
cysteine residues in these two locations (17) and also at
residue 99 to form zero-length disulfide cross-links has
been shown previously. These results suggested flexibility in the
orientation as well as the proximity of the two C-terminal domains. The
present results suggest that, even when the hinge is shortened,
sufficient flexibility remains to bring either the two 63 loops, or the
two 89 loops into proximity. The dynamics of the C-terminal domain and
its interaction with the N-terminal domain and L10 are being studied by
energy transfer and time-resolved fluorescence methods.
Figure 7: Schematic diagram indicating TMRA dimer formation in hinge-deleted L7/L12 variants.