BACKGROUND
tic <dig> is an integral membrane protein and a core component of the translocon at the inner envelope membrane of chloroplasts  that is involved in the import of nucleus-encoded proteins into the organelles  <cit> .
other proteins reported to be involved in protein translocation across the inner membrane include tic <dig>  tic <dig>  tic <dig> and tic <dig>  <cit> , and the more recently identified tic <dig>  tic <dig> and tic  <dig>  <cit> .
tic <dig>  tic <dig> and tic <dig> have been reported to play regulatory roles in protein import across the inner membrane  <cit> .
tic <dig> was originally identified in pisum sativum  using chemical cross-linking, which showed that it associates with preproteins transiting the inner membrane  <cit> .
there are four tic <dig> isoforms in arabidopsis thaliana   <cit> .
attic20-i is the dominant arabidopsis isoform , the most similar to the originally characterized isoform from pea  <cit> , and is the focus of the current study.
based on the in vitro cross-linking data, and more recent in vivo evidence and bioinformatics analysis, tic <dig> has been hypothesized to serve as a preprotein conducting channel of the tic complex.
in arabidopsis, knock-down of attic <dig> using anti-sense technology resulted in plants with a pale phenotype; the plastids of these plants were arrested at a pre-chloroplastic developmental state, and preprotein translocation was inhibited at the inner membrane  <cit> .
more recently, attic20-i knockout mutants  have been characterized with pale phenotypes that are consistent with the phenotype of the antisense plants  <cit> .
attic <dig> is also reported to be a core component of a  <dig> mda complex isolated from the inner envelope membrane of arabidopsis chloroplasts that is thought to comprise the channel-forming tic complex  <cit> , and electrophysiological experiments have shown that tic <dig> does have some capacity to function as an ion-permeable pore in reconstituted membranes  <cit> .
further evidence in support of tic <dig> having a role in membrane translocation comes from bioinformatic analyses, which show that it shares homology with cyanobacterial amino acyl transporters, as well as with the mitochondrial channel protein, tim <dig>  <cit> .
the similarity with tim <dig> extends to the predicted topology of the proteins.
originally, it was proposed that pstic <dig> contained  <dig> transmembrane α-helical domains  <cit> , but it now seems likely that it has  <dig> such domains  <cit> , as does tim <dig>  <cit> .
despite the evidence in support of a function for tic <dig> in protein conductance across the inner membrane, direct evidence for the mechanism by which tic <dig> might transport preproteins is lacking.
the preprotein channel of the toc complex, toc <dig>  is a β-barrel through which preproteins transit  <cit> .
tic <dig> is unlikely to transport preproteins in the same manner at toc <dig>  owing to its predicted α-helical conformation.
that tic <dig> is present in the  <dig> mda complex of the inner membrane together with the newly discovered tic components tic <dig>  tic <dig> and tic <dig>  <cit> , but in the absence of tic <dig>  which is another tic component that has been suggested to serve a channel function  <cit> , is additional evidence in support of a role for tic <dig> as a preprotein channel.
the role of the new tic components is unknown, but the study provides evidence that attic <dig> provides the preprotein conduit across the inner membrane  <cit> .
there is also evidence that tic <dig>  a protein related to tic <dig>  may serve a channel function, perhaps at specific stages of development  <cit> .
if tic <dig> does provide the pre-protein channel across the inner membrane, it may function in a manner similar to the related mitochondrial protein tim <dig>
one of the structural features of tim <dig> is an intrinsically disordered domain on its n-terminus that resides in the mitochondrial intermembrane space, and that has a role in precursor protein recognition  <cit> .
intrinsically disordered proteins with inducible structure are known to participate in protein-protein interactions  <cit> .
the members of the toc <dig> family of proteins contain large n-terminal domains  that have been demonstrated to be intrinsically disordered and are hypothesized to play a role in transit peptide recognition and binding  <cit> .
it is of interest whether the n-terminal extramembranous region of attic <dig> is also intrinsically disordered; if so, it may have a role in preprotein recognition, or in tic complex assembly.
the current study makes use of recombinant versions of attic <dig> produced in e. coli using an autoinducing expression system  <cit> .
recombinant full-length  attic <dig> and an n-terminally truncated version  were expressed in e. coli membranes at a high yield.
these proteins were folded with a high helical content when extracted in mild detergents and when reconstituted into lipid vesicles.
on the other hand, a synthetic peptide corresponding to the  <dig> n-terminal amino acids of attic <dig> displayed features of intrinsically disordered proteins .
evidence for direct self-association of attic <dig> was also observed in vitro for the first time.
collectively, these data shed light on the role of attic <dig> in the dynamics and physiology of the tic complex.
RESULTS
in silico analysis for attic20
tic <dig> has been predicted to have  <dig> tm helices  <cit> , and our in silico modelling using toppred, 3d distill servers and tmhmm are consistent with this prediction   <cit> .
the predictions are also consistent with attic <dig> having small n- and c-terminal soluble segments  <cit> , which we then examined for predicted intrinsic disorder using iupred  <cit> .
using the short disorder parameter, iupred predicted that the n-terminal segment has a strong tendency towards disorder .
based on this analysis, we devised two constructs for further work: one encoding a mature, transit peptide-lacking version of attic <dig> and an n-terminally truncated version  lacking the n-terminal amino acids of the mature protein that were predicted to form the intrinsically disordered segment .
additionally, we prepared a synthetic peptide corresponding to the extramembrane n-terminal domain that was predicted to be disordered.figure  <dig>
design of attic <dig>  its truncated mutant and extramembrane n-terminal peptide.
schematic representation of different forms of attic <dig> used in this study.
a cdna encoding pre-attic <dig>  was used to generate constructs encoding mature attic <dig> and attic20Δn <dig>  a truncated mutant lacking the  <dig> amino acid n-terminal domain , with c-terminal hexahistidine tags .
a 21-amino acid peptide corresponding to the ntd was also synthesized .
results of in silico analyses suggest that attic <dig> contains four transmembrane domains and that its n-terminal peptide is disordered.
iupred was used for disorder predictions of attic <dig>  transmembrane prediction for attic <dig> was based on  hydrophobicity plot and  tmhmm analysis.
refer to the methods for further detailed method and analysis.
optimization of attic <dig> and attic20Δn <dig> expression in bacterial membranes
until recently, production of recombinant versions of tic <dig> in e. coli had not been reported in the literature  <cit> .
kovacs-bogdan et al.
reported producing codon-optimized pstic <dig> in an e. coli cell-free lysate system, and attic <dig> in e. coli bl <dig> in inclusion bodies  <cit> .
in order to optimize the expression of recombinant attic <dig> and attic20Δn <dig> in the current study, a variety of expression conditions were compared.
production of recombinant attic <dig> in e. coli bl <dig> codonplus -ripl using iptg under various temperatures did not produce significant yields of protein, except in inclusion bodies .
to overcome this problem, auto-induction was used as a milder expression method  <cit> .
expression of the recombinant versions of attic <dig> in bacterial cells using the auto-induction method at rt resulted in enriched growth of the cultures.
auto-induction resulted in improved production of both recombinant attic <dig> and attic20Δn <dig>  both proteins were abundant in the inclusion body fraction, as revealed on coomassie-stained gels .
while the majority of the recombinant protein was directed to inclusion bodies , it could also be detected in the bacterial membrane fraction , albeit not as abundantly.
the protein could be extracted from the membrane fraction using mild detergent , and purified to near homogeneity, and in good yield , using ni-affinity chromatography .
the identity of the recombinant protein in the membrane faction was confirmed using a western blot probed with anti-histidine antibody .
the membrane content of the fraction from which attic <dig> and attic20Δn <dig> were extracted was confirmed using the bacterial membrane marker nadh oxidase .
collectively, these data show that the auto-induction method resulted in production of recombinant attic <dig> and attic20Δn <dig> in the bacterial membrane fraction, and that pure protein could be obtained in high yield using mild extraction and purification conditions.
the two purified recombinant proteins  are compared using semi-native page stained with coomassie blue in figure 2d.figure  <dig>
expression of attic <dig> and its truncated mutant  in bacterial membranes.
fractionation of e. coli bl <dig> codonplus -ripl cells expressing recombinant attic <dig> using the autoinduction method.
protein profiles of bacterial cell fractions  and attic <dig> purified from the membrane fraction  were compared using sds-page stained with coomassie blue.
<dig> μg of total protein was loaded in each lane.
the left lane contains a protein molecular weight  ladder .
western blot detection of recombinant attic <dig> in the bacterial membrane fraction of auto-induced  and uninduced  bacterial cells using mouse igg2b anti-histidine antibody, detected by chemiluminescence using a horseradish peroxidase-linked secondary antibody.
an assay for the membrane marker nadh oxidase was used to confirm the presence of membranes in the “membrane fraction” and to compare to the soluble protein fraction from figure 2a.
the nadh oxidase assay measures the conversion of nadh from its reduced to its oxidized form, indicated by a decrease in a <dig> over time.
average specific nadh oxidase activity  of the soluble and bacterial membrane fractions are shown.
comparison of purified attic <dig>  and its truncated mutant , resolved using semi-native page  stained with coomassie blue.
the molecular weights  of the protein ladder  are indicated.
refolding of attic <dig> and attic20Δn <dig> in detergents and liposomes
the secondary structure of purified attic <dig> extracted from bacterial membranes using four different detergents  at concentrations above their cmc value was compared.
the secondary structure of purified attic <dig> in each detergent was examined by far-uv cd spectroscopy, and the helical content for each was estimated .
among the four detergents tested, ldao and tx- <dig> induced the least helical content in attic <dig> .
in addition, the protein exhibited a low stability in these detergents, resulting in protein aggregation/precipitation at 4°c .
the far-uv cd spectra of attic <dig> in 1% chaps indicated the protein had 40% α-helical content, with local minimum ellipticities at  <dig> nm and  <dig> nm.
the protein, however, also displayed a low level of stability in this detergent.
the protein extracted and purified in  <dig> % zw 3- <dig> exhibited the highest α-helical content of the detergents tested, with local minimum ellipticities at  <dig> nm and  <dig> nm, and a positive maximum ellipticity near  <dig> nm .
deconvolution of the cd spectrum of the protein in  <dig> % zw3- <dig> revealed a 47% α-helical content .
in addition, attic <dig> was solubilized from the bacterial membrane fraction much more efficiently with zw 3- <dig>  as compared to the other detergents, which resulted in this detergent providing the highest yield .
collectively, the data suggested that attic <dig> remained folded in a native-like functional form when extracted from bacterial membranes using  <dig> % of the zw 3- <dig> detergent.
this condition was also used successfully to isolate and purify attic20Δn <dig> .
although lacking the 20-amino acid n-terminal domain, purified attic20Δn <dig> in  <dig> % zw 3- <dig> still exhibited a highly helical secondary structure, similar to the full-length version .
the far-uv cd spectra of attic20Δn <dig> also displayed minima at both  <dig> nm and  <dig> nm, which are characteristics of helical proteins.
deconvolution of the cd spectra of attic20Δn <dig> revealed a 43% helical content of the protein in  <dig> % zw 3- <dig> .
overall, our detergent screening revealed zw 3- <dig> to be a suitable detergent for extracting and purifying attic <dig> and attic20Δn <dig>  providing a stable protein-detergent mixed micelle environment for structural and functional studies.table  <dig>
secondary structure composition of attic <dig> and its truncated mutant in detergents and lipid vesicles
a
proteins
environment
α
β
turn
random
nrmsd
adeconvolution of cd spectra was performed using the cdsstr program on the dichroweb website .
the values represent the percentage of secondary structure composition.
nrmsd, normalized root mean square deviation, denotes the best fit between the calculated and experimental cd spectra.
*helical content of attic <dig> was predicted using the distill prediction program, as described in the methods.
cd analysis of attic <dig> and attic20Δn <dig> in detergents and liposomes.
far-uv cd spectra of purified attic <dig> and attic20Δn <dig> in  <dig> % zw 3- <dig> detergent.
the protein concentration was ~ <dig> μm, as determined by bradford protein assay.
far-uv cd of attic <dig> in detergent and popc lipid vesicles.
protein and lipid concentrations were ~  <dig> nm and  <dig>  mm, respectively.
all samples were measured in buffer containing  <dig> mm tris-hcl,  <dig> mm nacl, 1% glycerol, and ph  <dig> .
summary of deconvoluted data are shown in table  <dig>
the secondary structure of attic <dig> was also determined after reconstitution into liposomes .
far-uv cd spectra of reconstituted attic <dig> in popc liposomes showed an enhancement in molar ellipiticity.
a slight red shift of the local minimum at approximately  <dig> nm was also observed in the cd spectrum of attic <dig> following reconstitution into popc vesicles.
comparison of the cd spectra of attic <dig> in zw 3- <dig> to that of the protein in liposomes reveals that the two prominent local minima of the protein in detergent  have shifted towards a single minimum  and a negative shoulder at ~ <dig> nm in liposomes .
the deconvolution of the cd spectrum of attic <dig> in the popc liposomes revealed a helical content of ~74% , which is in good agreement with the helical content predicted from the amino acid sequence .
in conclusion, attic <dig>  extracted and purified from bacterial membranes using zw 3- <dig> detergent, exhibited a highly helical secondary structure following reconstitution into popc lipid vesicles.
associated state of attic <dig> monitored by cd spectroscopy and semi-native sds-page
attic <dig> extracted and purified from bacterial membranes using mild detergent or reconstituted into liposomes both displayed high helical contents as reveled by cd spectroscopy .
in liposomes, attic <dig> displayed a marked enhancement in helical content and its cd spectrum displayed the characteristics of α-helices, with the π → π* exciton split bands at ~ <dig> and  <dig> nm and the n → π* transition at ~ <dig> nm .
however, compared to a typical α-helical cd spectrum, the parallel band at ~ 208- <dig> nm exhibited a shoulder-like minimum ellipticity .
the θ208/ θ <dig> ratio of the attic <dig> spectrum in popc vesicles is less than  <dig> .
such low molar ellipiticity ratios between  <dig> and  <dig> nm have been used to signify coiled coil motifs or oligomerization of monomers and/or packing of helical domains within a monomer for other proteins  <cit> .
we therefore used semi-native page as a method to examine whether attic <dig> reconstituted into liposomes displayed characteristics of an oligomeric protein.
semi-native page revealed a mixture of oligomeric forms of attic <dig> at the lowest levels of sds tested .
monomers and dimers were most prevalent at all concentrations of sds.
the low but detectable amounts of trimers and tetramers were eliminated as the concentration of sds was increased, such that primarily monomer, and a very small amount of dimer, were visible at the highest concentration tested .
thus, the ability of attic <dig> to self-associate in liposomes has been observed in vitro for the first time.
it is important to note that reconstituted attic20Δn <dig> also showed evidence of self-assembly, as revealed by cd spectroscopy  and semi-native page .figure  <dig>
titration of attic <dig> in popc liposomes with sds.
far-uv cd spectra of reconstituted attic <dig> in popc liposomes titrated with sds .
shows the low  region of the spectra in greater detail , where higher sds concentrations enhanced negative ellipticities at both  <dig> and  <dig> nm.
shows the high  region  in greater detail, where increased sds concentrations resulted in only small changes in the molar ellipticity of attic <dig>   plot of rθ
vs.  reveals a cooperative dissociation of associated attic <dig> in the presence of sds.
the rθ was normalized and k1/ <dig>  was calculated using the hill fitting .
semi-native page  of attic <dig> and attic20Δn <dig> in popc liposomes in the presence of increasing concentrations of sds added to the sample buffer .
the gels were stained with coomassie brilliant blue.
monomeric , dimeric , trimeric  and tetrameric  forms of attic <dig> and attic20Δn <dig> are indicated.
relative proportion of the different associated states of attic <dig> and attic20Δn <dig> measured during the sds titration as determined by measuring band intensity on the semi-native page gels shown in c. band intensity was quantified using quantiy one  as explained in the methods.
the dissociation/unfolding pathway and stability of attic <dig> in liposomes was further analyzed by using cd spectroscopy to monitor the protein conformation during an sds titration of attic <dig> reconstituted into liposomes.
cd spectral analysis revealed two conformational states of the protein when sds was added .
at the lower concentration range of sds , far-uv cd spectral shape of reconstituted attic <dig> displayed a drastic change as sds was added to the proteoliposome mixture.
the negative ellipticities at  <dig> nm and  <dig> nm were both enhanced, and the negative shoulder at  <dig> blue-shifted towards  <dig> nm and gradually became a visible local minimum .
these results suggest a transition of reconstituted attic <dig> towards a less constrained conformation with fewer intermolecular interactions between helices and more independence for monomers.
further increasing the sds concentration above  <dig>  mm  did not produce significant changes in the far-uv cd spectral shape of attic20; the protein’s cd spectra remained unchanged with two negative maxima at  <dig> and  <dig> nm.
plotting the negative ellipticity ratio of these two maxima  as a function of sds concentration revealed a two-state dissociation of attic <dig> in popc with a dissociation constant k1/ <dig>  of  <dig>  ±  <dig>  mm sds .
the cd data are consistent with the semi-native page analysis, which showed that the addition of sds resulted in simultaneous loss of higher order oligomers  from the liposomes and concurrent enrichment of monomers .
the relative amounts of the different oligomeric states of attic <dig> were calculated based on protein band intensities on the semi-native page gel .
at the lowest concentration of sds used , approximately 65% of the protein was in the monomeric form, ~26% was dimeric, ~7% trimeric, and only ~2% was tetrameric .
the tetramers were no longer detectable when  <dig> mm sds was included in the sample buffer, and at concentrations of  <dig> mm sds and above, both attic <dig> and attic20Δn <dig> were almost exclusively found as a monomers, with only trace amounts of dimer detectable  at  <dig> mm sds .
collectively, the spectroscopic measurements and semi-native sds-page data suggested that both attic <dig> and its n-terminally truncated mutant were able to self-associate in lipid bilayers under our experimental conditions.
the oligomers dissociated readily in sds, suggesting that the interaction between monomers was weak.
additionally, the spectroscopic two-state dissociation of the protein from liposomes during sds titration, supported by the semi-native page data, implies that the monomeric and dimeric states of the protein predominate.
overall, the combination of experimental techniques used in this study provide evidence that attic <dig> has the propensity to form oligomers in a lipid environment.
the n-terminal domain of attic <dig> is intrinsically disordered
to examine the secondary structure of the extramembrane n-terminal domain of attic <dig>  cd spectroscopy of a corresponding synthetic peptide  was carried out at various temperatures, phs, and buffer conditions.
under non-denaturing conditions , the peptide exhibited a cd spectrum typical of disordered proteins, indicated by the presence of a deep minimum at ~  <dig> nm   <cit> .
it is noteworthy that the negative ellipticity at ~  <dig> nm could be attributed to the close proximity and interaction of trp residues present at the c-terminus of the peptide  <cit> .
this spectroscopic result supports our computational prediction that the n-terminal domain of attic <dig> is disordered under normal physiological conditions .figure  <dig>
a synthetic peptide corresponding to the n-terminal domain of attic <dig> has characteristics of an intrinsically disordered protein.
temperature-dependent far-uv cd spectra of n-terminal peptide of attic <dig>   ph-dependent far-uv cd spectra of n-terminal peptide of attic <dig>   far-uv cd spectra of n-terminal peptide of attic <dig> in the absence and presence of 10% and 50% tfe at 25°c.
peptide content was kept at a concentration of  <dig> μm in tris buffer for all measurements.
the peptide gained modest amounts of structure with increasing temperature, ph, and tfe concentration, which is consistent with the domain being intrinsically disordered.
the structural properties of the n-terminal domain of attic <dig> were further characterized through its changes at different temperatures, phs, and increasing concentrations of trifluoroethanol    <cit> .
comparing the far-uv cd spectra of the peptide at different temperatures revealed a modest gain of peptide structure as temperature increased, revealed by an enhancement of the negative ellipticity at ~  <dig> -  <dig> nm .
the influence of trp-trp interactions on the peptide's cd spectra might also be lessened at higher temperatures, as indicated by the slight blue shift of the negative ellipticities at ~  <dig> nm.
in addition, a reduction of the negative minimum at ~  <dig> nm revealed a decrease in random coil content of the peptide .
thus, an increase in temperature induced partial folding of the n-terminal domain of attic <dig>  in addition, a slight gain in the peptide structure was also observed at high ph  .
carrying a number of basic amino acid residues, the n-terminal peptide possesses a large positive charge at neutral ph .
under basic conditions, this net charge would be minimized, thus reducing the electrostatic repulsion between amino acid residues and inducing partial folding of the peptide  <cit> .
finally, the secondary structure stabilizer tfe enhanced the n-terminal peptide structure .
at 50% tfe, a notable increase in secondary structure was observed, indicated through the enhancement of local minima at ~  <dig> nm and ~  <dig> nm.
overall, the n-terminal domain of attic <dig> displayed an unstructured conformation with the propensity to gain structure depending on its environment, which is consistent with being characterized as an intrinsically disordered protein domain.
the ability to adopt different conformations may play a role in its physiological function.
discussion
a novel method for expressing and purifying recombinant attic <dig> in a native-like folded form is reported in the current study.
in addition, the self-association of attic <dig> in mild detergent and lipid systems was observed in vitro for the first time, using a combination of biochemical and biophysical techniques, and evidence is presented that the extramembrane n-terminal domain has properties of an intrinsically disordered protein.
the evidence for oligomerization may be important when exploring the hypothesized function of the protein as a preprotein channel of the tic complex.
novel recombinant protein expression system for attic20
several studies, including ours, predict attic <dig> to contain four tm domains that span the chloroplast inner envelope membrane  <cit> .
the hydrophobic nature of the protein has made it difficult to produce recombinant tic <dig> until recently, when kovacs-bogdan et al.
reported obtaining attic <dig> in bacterial inclusion bodies  <cit> .
a disadvantage of extracting proteins from inclusion bodies is that many such proteins have been shown to be only partially folded  <cit> .
other disadvantages of this method include the heterogeneity of protein conformations that can be obtained when refolding proteins from inclusions bodies, and the limited number of detergents suitable for the solubilization and subsequent refolding of the protein  <cit> .
in the current study, using a mild auto-induction expression system  <cit> , together with nutrient-rich media, we were able to heterologously express attic <dig> in e. coli in such a way that some of the protein was directed to the bacterial membranes, even though the majority was still directed to inclusion bodies .
targeting to the bacterial membrane occurred without introduction of a signal sequence onto the n-terminus of attic <dig>  although we did not attempt to characterize the nature of the targeting, it is known that the signal recognition particle  targets multi-spanning transmembrane  domain proteins to the membrane in e. coli.
srp is able to recognize either cleavable n-terminal signal sequences or internal tm domains  for targeting proteins to bacterial membranes, and insertion can occur via the secyeg or yidc translocon  <cit> .
furthermore, srp is known to be recruited by the presence of tryptophan-enriched transmembrane helices, such as is found in the first predicted tm domain of attic <dig>  <cit> .
it therefore seems likely that attic <dig> was targeted to the bacterial membrane in an srp-dependent manner, with the first tm domain serving as a signal anchor sequence.
in summary, production of the recombinant protein relied on using the “codon plus” strain of e. coli, much like kovacs-bogdan et al.
<cit> , who used a codon-optimized cdna for expression of attic <dig> in inclusion bodies.
targeting of the protein to the bacterial membrane rather than to inclusion bodies was presumably a result of using the “mild” auto-induction method.
in addition, the method resulted in the purification of recombinant attic <dig> in detergents in relatively high yield .
both attic <dig> and its truncated version  were shown to retain high helical contents following their extraction from the membrane using  <dig> % of the mild detergent zw3- <dig> .
the helical content of the protein  following reconstitution into popc liposomes was very similar to that predicted from the primary sequence .
thus, the expression and reconstitution protocol described in this study is appropriate for preparing highly pure and native-like folded protein that can potentially be used for obtaining high-resolution structures of attic <dig>  the method might also be applied to other tic components, whose structural and functional features still remain elusive.
in the study reported by kovacs-bogdan et al.
<cit> , pstic <dig>  expressed in a cell-free system also exhibited a highly helical secondary structure  in the presence of  <dig> % brij- <dig> detergent .
the far-uv cd spectrum of pstic <dig> reported in that study was indicative of a monomeric conformation, featuring two minima at  <dig> and  <dig> nm and a positive maximum at  <dig> nm  <cit> .
similar cd spectral properties were also observed in the current study for dissociated attic <dig> following the titration of protein from popc liposomes with sds .
therefore, we conclude that expression of attic <dig> in bacterial membranes allows for the formation of oligomers that aren’t formed when the protein is expressed in a cell-free environment in the presence of detergent.
self-association of attic20
the recent report that tic <dig> comprises part of a  <dig> mda complex, together with new components tic <dig>  tic <dig> and tic <dig>  that forms preprotein-conducting channels independent of tic <dig>  has contributed to a debate about the stoichiometry of the components in the tic translocon  <cit> .
one issue in particular that has been raised is whether tic <dig> is present in sufficient quantities to serve as the primary channel forming protein  <cit> .
and if it is the primary channel forming protein, the mechanism by which it operates is still unknown.
determining the structure and oligomeric state of attic <dig> represents an important step toward answering these questions.
in this study, we report the observation that attic <dig> has the capacity to self-associate in lipid membranes .
this homo-oligomerization of attic <dig> was detected for protein extracted directly from bacterial membranes using mild detergent , and when reconstituted into liposomes .
the predominant associated form of attic <dig> was dimeric, although higher-order oligomers  were also observed at much lower levels .
under our experimental conditions, attic <dig> oligomeric forms in liposomes could be dissociated with relatively low concentrations of sds .
this relatively weak self-association of attic <dig> could be an indication that the oligomeric state of the protein is dynamic, with the ability to associate and dissociate readily in response to particular signals.
in the context of the tic complex at the chloroplast, one such signal could be the presence of preproteins, which has been shown to trigger tic complex assembly  <cit> .
the potential function of the different oligomers, or which one might be more prevalent in vivo, remains to be determined; however, kikuchi et al.
<cit>  hypothesize that the  <dig> mda complex isolated from chloroplast inner envelope membranes that interacts with translocating preproteins may contain  <dig> copies of attic <dig>  it is possible that an oligomer of attic <dig>  forms the core of the  <dig> mda complex and provides the conduit through which preproteins travel.
it is also possible that tic <dig> operates in a manner similar to tim <dig>  the mitochondrial translocase component with which it shares sequence and topological similarities, and that is the central component of the tim <dig> complex  <cit> .
interestingly, these mitochondrial transporters are dimeric in their functional state  <cit> , which is comparable to the results of this study, underscoring the existence of oligomeric forms of attic <dig> in equilibrium with its monomeric form in membranes.
evidence in support of the propensity of attic <dig> to form oligomers  was provided by the transmembrane helical dimer prediction program preddimer   <cit> .
while no dominant set of pairings emerges from this analysis, the results from the preddimer prediction suggest the possibility of different sets of distinct helix-helix alignments, allowing for multimeric forms of attic <dig> to adopt several conformations.figure  <dig>
possible helix dimerization of attic <dig>  a 3-d model of attic <dig> was generated using the distill program  <cit>  as described in the methods section.
each tm helix shown in the model was used in the analysis of predicted tm helical dimer conformations .
for each predicted dimer conformation, fscor is evaluated, representing the packing quality of helices.
a higher fscor reflects a high packing efficiency of the dimer  <cit> .
the detailed prediction method is described in the methods.
this work also revealed some of the structural features of attic <dig> .
the n-terminal peptide preceding the  <dig> predicted tm domains of the protein displayed characteristics that are consistent with instrinsically disordered proteins, exhibiting flexible conformations in different environments .
this conformational elasticity could be important for the physiological function of attic <dig>  such as peptide recognition or interaction with protein partners in the tic complex.
these protein partners potentially include the other members of the recently discovered  <dig> mda complex of the inner envelope membrane   <cit> .
interestingly, the truncated version of attic <dig>  lacking the n-terminal peptide, displayed conformations and showed evidence of self-assembly similar to the full-length protein in detergents and lipid bilayers .
thus, the ability to self-associate is independent of the n-terminus, and is presumably conferred by one or more of the transmembrane domains.
the n-terminal domain may participate in protein interactions with other components of the translocon complex.
of note, is that the extramembrane n-terminal domain of tim <dig> has also been reported to be intrinsically disordered; in that case, it is believed that the domain  is involved in preprotein binding  <cit> .
CONCLUSIONS
in conclusion, the novel protein expression system reported in this study resulted in the protein being targeted to bacterial membranes, which is a more biologically relevant system than inclusion bodies, and led to the finding that the protein can exist as a monomer and higher-order associated forms.
the existence of associated forms of attic <dig> in membranes may be important for understanding its hypothesized function as a preprotein transporter.
the observed dimeric, trimeric and tetrameric forms, together with the low dissociation constant  of the protein in liposomes implies a relatively weak interaction between the monomers, which in turn suggests a dynamic nature of the equilibrium between the monomeric and associated forms.
this may be an indication that changes in the protein's local environment, which could include presence/absence of preproteins, could trigger changes in the multimeric state of the protein.
the ability of tic <dig> to switch between its monomeric and associated forms is consistent with previous observations that tic complex assembly is triggered by the presence of preproteins, and that the  <dig> mda complex likely contains more than one copy of attic <dig>  and will be important when testing its hypothesized function as a preprotein channel protein of the inner envelope membrane of chloroplasts.

 tic is an integral membrane protein and a core component of the translocon at the inner envelope membrane of chloroplasts that is involved in the import of nucleus-encoded proteins into the organelles. other proteins reported to be involved in protein translocation across the inner membrane include tic tic tic and tic, and the more recently identified tic tic and tic. tic tic and tic have been reported to play regulatory roles in protein import across the inner membrane. tic was originally identified in pisum sativum using chemical cross-linking, which showed that it associates with preproteins transiting the inner membrane. there are four tic isoforms in arabidopsis thaliana. attic20-i is the dominant arabidopsis isoform, the most similar to the originally characterized isoform from pea, and is the focus of the current study. based on the in vitro cross-linking data, and more recent in vivo evidence and bioinformatics analysis, tic has been hypothesized to serve as a preprotein conducting channel of the tic complex. in arabidopsis, knock-down of attic using anti-sense technology resulted in plants with a pale phenotype; the plastids of these plants were arrested at a pre-chloroplastic developmental state, and preprotein translocation was inhibited at the inner membrane. more recently, attic20-i knockout mutants have been characterized with pale phenotypes that are consistent with the phenotype of the antisense plants. attic is also reported to be a core component of a mda complex isolated from the inner envelope membrane of arabidopsis chloroplasts that is thought to comprise the channel-forming tic complex, and electrophysiological experiments have shown that tic does have some capacity to function as an ion-permeable pore in reconstituted membranes. further evidence in support of tic having a role in membrane translocation comes from bioinformatic analyses, which show that it shares homology with cyanobacterial amino acyl transporters, as well as with the mitochondrial channel protein, tim. the similarity with tim extends to the predicted topology of the proteins. originally, it was proposed that pstic contained transmembrane α-helical domains, but it now seems likely that it has such domains, as does tim. despite the evidence in support of a function for tic in protein conductance across the inner membrane, direct evidence for the mechanism by which tic might transport preproteins is lacking. the preprotein channel of the toc complex, toc is a β-barrel through which preproteins transit. tic is unlikely to transport preproteins in the same manner at toc owing to its predicted α-helical conformation. that tic is present in the mda complex of the inner membrane together with the newly discovered tic components tic tic and tic, but in the absence of tic which is another tic component that has been suggested to serve a channel function, is additional evidence in support of a role for tic as a preprotein channel. the role of the new tic components is unknown, but the study provides evidence that attic provides the preprotein conduit across the inner membrane. there is also evidence that tic a protein related to tic may serve a channel function, perhaps at specific stages of development. if tic does provide the pre-protein channel across the inner membrane, it may function in a manner similar to the related mitochondrial protein tim one of the structural features of tim is an intrinsically disordered domain on its n-terminus that resides in the mitochondrial intermembrane space, and that has a role in precursor protein recognition. intrinsically disordered proteins with inducible structure are known to participate in protein-protein interactions. the members of the toc family of proteins contain large n-terminal domains that have been demonstrated to be intrinsically disordered and are hypothesized to play a role in transit peptide recognition and binding. it is of interest whether the n-terminal extramembranous region of attic is also intrinsically disordered; if so, it may have a role in preprotein recognition, or in tic complex assembly. the current study makes use of recombinant versions of attic produced in e. coli using an autoinducing expression system. recombinant full-length attic and an n-terminally truncated version were expressed in e. coli membranes at a high yield. these proteins were folded with a high helical content when extracted in mild detergents and when reconstituted into lipid vesicles. on the other hand, a synthetic peptide corresponding to the n-terminal amino acids of attic displayed features of intrinsically disordered proteins. evidence for direct self-association of attic was also observed in vitro for the first time. collectively, these data shed light on the role of attic in the dynamics and physiology of the tic complex.  in silico analysis for attic20 tic has been predicted to have tm helices, and our in silico modelling using toppred, 3d distill servers and tmhmm are consistent with this prediction. the predictions are also consistent with attic having small n- and c-terminal soluble segments, which we then examined for predicted intrinsic disorder using iupred. using the short disorder parameter, iupred predicted that the n-terminal segment has a strong tendency towards disorder. based on this analysis, we devised two constructs for further work: one encoding a mature, transit peptide-lacking version of attic and an n-terminally truncated version lacking the n-terminal amino acids of the mature protein that were predicted to form the intrinsically disordered segment. additionally, we prepared a synthetic peptide corresponding to the extramembrane n-terminal domain that was predicted to be disordered.figure design of attic its truncated mutant and extramembrane n-terminal peptide. schematic representation of different forms of attic used in this study. a cdna encoding pre-attic was used to generate constructs encoding mature attic and attic20Δn a truncated mutant lacking the amino acid n-terminal domain, with c-terminal hexahistidine tags. a 21-amino acid peptide corresponding to the ntd was also synthesized. results of in silico analyses suggest that attic contains four transmembrane domains and that its n-terminal peptide is disordered. iupred was used for disorder predictions of attic transmembrane prediction for attic was based on hydrophobicity plot and tmhmm analysis. refer to the methods for further detailed method and analysis. optimization of attic and attic20Δn expression in bacterial membranes until recently, production of recombinant versions of tic in e. coli had not been reported in the literature. kovacs-bogdan et al. reported producing codon-optimized pstic in an e. coli cell-free lysate system, and attic in e. coli bl in inclusion bodies. in order to optimize the expression of recombinant attic and attic20Δn in the current study, a variety of expression conditions were compared. production of recombinant attic in e. coli bl codonplus -ripl using iptg under various temperatures did not produce significant yields of protein, except in inclusion bodies. to overcome this problem, auto-induction was used as a milder expression method. expression of the recombinant versions of attic in bacterial cells using the auto-induction method at rt resulted in enriched growth of the cultures. auto-induction resulted in improved production of both recombinant attic and attic20Δn both proteins were abundant in the inclusion body fraction, as revealed on coomassie-stained gels. while the majority of the recombinant protein was directed to inclusion bodies, it could also be detected in the bacterial membrane fraction, albeit not as abundantly. the protein could be extracted from the membrane fraction using mild detergent, and purified to near homogeneity, and in good yield, using ni-affinity chromatography. the identity of the recombinant protein in the membrane faction was confirmed using a western blot probed with anti-histidine antibody. the membrane content of the fraction from which attic and attic20Δn were extracted was confirmed using the bacterial membrane marker nadh oxidase. collectively, these data show that the auto-induction method resulted in production of recombinant attic and attic20Δn in the bacterial membrane fraction, and that pure protein could be obtained in high yield using mild extraction and purification conditions. the two purified recombinant proteins are compared using semi-native page stained with coomassie blue in figure 2d.figure expression of attic and its truncated mutant in bacterial membranes. fractionation of e. coli bl codonplus -ripl cells expressing recombinant attic using the autoinduction method. protein profiles of bacterial cell fractions and attic purified from the membrane fraction were compared using sds-page stained with coomassie blue. μg of total protein was loaded in each lane. the left lane contains a protein molecular weight ladder. western blot detection of recombinant attic in the bacterial membrane fraction of auto-induced and uninduced bacterial cells using mouse igg2b anti-histidine antibody, detected by chemiluminescence using a horseradish peroxidase-linked secondary antibody. an assay for the membrane marker nadh oxidase was used to confirm the presence of membranes in the “membrane fraction” and to compare to the soluble protein fraction from figure 2a. the nadh oxidase assay measures the conversion of nadh from its reduced to its oxidized form, indicated by a decrease in a over time. average specific nadh oxidase activity of the soluble and bacterial membrane fractions are shown. comparison of purified attic and its truncated mutant, resolved using semi-native page stained with coomassie blue. the molecular weights of the protein ladder are indicated. refolding of attic and attic20Δn in detergents and liposomes the secondary structure of purified attic extracted from bacterial membranes using four different detergents at concentrations above their cmc value was compared. the secondary structure of purified attic in each detergent was examined by far-uv cd spectroscopy, and the helical content for each was estimated. among the four detergents tested, ldao and tx- induced the least helical content in attic. in addition, the protein exhibited a low stability in these detergents, resulting in protein aggregation/precipitation at 4°c. the far-uv cd spectra of attic in 1% chaps indicated the protein had 40% α-helical content, with local minimum ellipticities at nm and nm. the protein, however, also displayed a low level of stability in this detergent. the protein extracted and purified in % zw 3- exhibited the highest α-helical content of the detergents tested, with local minimum ellipticities at nm and nm, and a positive maximum ellipticity near nm. deconvolution of the cd spectrum of the protein in % zw3- revealed a 47% α-helical content. in addition, attic was solubilized from the bacterial membrane fraction much more efficiently with zw 3- as compared to the other detergents, which resulted in this detergent providing the highest yield. collectively, the data suggested that attic remained folded in a native-like functional form when extracted from bacterial membranes using % of the zw 3- detergent. this condition was also used successfully to isolate and purify attic20Δn. although lacking the 20-amino acid n-terminal domain, purified attic20Δn in % zw 3- still exhibited a highly helical secondary structure, similar to the full-length version. the far-uv cd spectra of attic20Δn also displayed minima at both nm and nm, which are characteristics of helical proteins. deconvolution of the cd spectra of attic20Δn revealed a 43% helical content of the protein in % zw 3-. overall, our detergent screening revealed zw 3- to be a suitable detergent for extracting and purifying attic and attic20Δn providing a stable protein-detergent mixed micelle environment for structural and functional studies.table secondary structure composition of attic and its truncated mutant in detergents and lipid vesicles a proteins environment α β turn random nrmsd adeconvolution of cd spectra was performed using the cdsstr program on the dichroweb website. the values represent the percentage of secondary structure composition. nrmsd, normalized root mean square deviation, denotes the best fit between the calculated and experimental cd spectra. *helical content of attic was predicted using the distill prediction program, as described in the methods. cd analysis of attic and attic20Δn in detergents and liposomes. far-uv cd spectra of purified attic and attic20Δn in % zw 3- detergent. the protein concentration was ~ μm, as determined by bradford protein assay. far-uv cd of attic in detergent and popc lipid vesicles. protein and lipid concentrations were ~  nm and mm, respectively. all samples were measured in buffer containing mm tris-hcl, mm nacl, 1% glycerol, and ph. summary of deconvoluted data are shown in table  the secondary structure of attic was also determined after reconstitution into liposomes. far-uv cd spectra of reconstituted attic in popc liposomes showed an enhancement in molar ellipiticity. a slight red shift of the local minimum at approximately nm was also observed in the cd spectrum of attic following reconstitution into popc vesicles. comparison of the cd spectra of attic in zw 3- to that of the protein in liposomes reveals that the two prominent local minima of the protein in detergent have shifted towards a single minimum and a negative shoulder at ~ nm in liposomes. the deconvolution of the cd spectrum of attic in the popc liposomes revealed a helical content of ~74%, which is in good agreement with the helical content predicted from the amino acid sequence. in conclusion, attic extracted and purified from bacterial membranes using zw 3- detergent, exhibited a highly helical secondary structure following reconstitution into popc lipid vesicles. associated state of attic monitored by cd spectroscopy and semi-native sds-page attic extracted and purified from bacterial membranes using mild detergent or reconstituted into liposomes both displayed high helical contents as reveled by cd spectroscopy. in liposomes, attic displayed a marked enhancement in helical content and its cd spectrum displayed the characteristics of α-helices, with the π → π* exciton split bands at ~ and nm and the n → π* transition at ~ nm. however, compared to a typical α-helical cd spectrum, the parallel band at ~ 208- nm exhibited a shoulder-like minimum ellipticity. the θ208/ θ ratio of the attic spectrum in popc vesicles is less than. such low molar ellipiticity ratios between and nm have been used to signify coiled coil motifs or oligomerization of monomers and/or packing of helical domains within a monomer for other proteins. we therefore used semi-native page as a method to examine whether attic reconstituted into liposomes displayed characteristics of an oligomeric protein. semi-native page revealed a mixture of oligomeric forms of attic at the lowest levels of sds tested. monomers and dimers were most prevalent at all concentrations of sds. the low but detectable amounts of trimers and tetramers were eliminated as the concentration of sds was increased, such that primarily monomer, and a very small amount of dimer, were visible at the highest concentration tested. thus, the ability of attic to self-associate in liposomes has been observed in vitro for the first time. it is important to note that reconstituted attic20Δn also showed evidence of self-assembly, as revealed by cd spectroscopy and semi-native page.figure titration of attic in popc liposomes with sds. far-uv cd spectra of reconstituted attic in popc liposomes titrated with sds. shows the low region of the spectra in greater detail, where higher sds concentrations enhanced negative ellipticities at both and nm. shows the high region in greater detail, where increased sds concentrations resulted in only small changes in the molar ellipticity of attic plot of rθ vs. reveals a cooperative dissociation of associated attic in the presence of sds. the rθ was normalized and k1/ was calculated using the hill fitting. semi-native page of attic and attic20Δn in popc liposomes in the presence of increasing concentrations of sds added to the sample buffer. the gels were stained with coomassie brilliant blue. monomeric, dimeric, trimeric and tetrameric forms of attic and attic20Δn are indicated. relative proportion of the different associated states of attic and attic20Δn measured during the sds titration as determined by measuring band intensity on the semi-native page gels shown in c. band intensity was quantified using quantiy one as explained in the methods. the dissociation/unfolding pathway and stability of attic in liposomes was further analyzed by using cd spectroscopy to monitor the protein conformation during an sds titration of attic reconstituted into liposomes. cd spectral analysis revealed two conformational states of the protein when sds was added. at the lower concentration range of sds, far-uv cd spectral shape of reconstituted attic displayed a drastic change as sds was added to the proteoliposome mixture. the negative ellipticities at nm and nm were both enhanced, and the negative shoulder at blue-shifted towards nm and gradually became a visible local minimum. these results suggest a transition of reconstituted attic towards a less constrained conformation with fewer intermolecular interactions between helices and more independence for monomers. further increasing the sds concentration above mm did not produce significant changes in the far-uv cd spectral shape of attic20; the protein’s cd spectra remained unchanged with two negative maxima at and nm. plotting the negative ellipticity ratio of these two maxima as a function of sds concentration revealed a two-state dissociation of attic in popc with a dissociation constant k1/ of  ±  mm sds. the cd data are consistent with the semi-native page analysis, which showed that the addition of sds resulted in simultaneous loss of higher order oligomers from the liposomes and concurrent enrichment of monomers. the relative amounts of the different oligomeric states of attic were calculated based on protein band intensities on the semi-native page gel. at the lowest concentration of sds used, approximately 65% of the protein was in the monomeric form, ~26% was dimeric, ~7% trimeric, and only ~2% was tetrameric. the tetramers were no longer detectable when mm sds was included in the sample buffer, and at concentrations of mm sds and above, both attic and attic20Δn were almost exclusively found as a monomers, with only trace amounts of dimer detectable at mm sds. collectively, the spectroscopic measurements and semi-native sds-page data suggested that both attic and its n-terminally truncated mutant were able to self-associate in lipid bilayers under our experimental conditions. the oligomers dissociated readily in sds, suggesting that the interaction between monomers was weak. additionally, the spectroscopic two-state dissociation of the protein from liposomes during sds titration, supported by the semi-native page data, implies that the monomeric and dimeric states of the protein predominate. overall, the combination of experimental techniques used in this study provide evidence that attic has the propensity to form oligomers in a lipid environment. the n-terminal domain of attic is intrinsically disordered to examine the secondary structure of the extramembrane n-terminal domain of attic cd spectroscopy of a corresponding synthetic peptide was carried out at various temperatures, phs, and buffer conditions. under non-denaturing conditions, the peptide exhibited a cd spectrum typical of disordered proteins, indicated by the presence of a deep minimum at ~  nm. it is noteworthy that the negative ellipticity at ~  nm could be attributed to the close proximity and interaction of trp residues present at the c-terminus of the peptide. this spectroscopic result supports our computational prediction that the n-terminal domain of attic is disordered under normal physiological conditions.figure a synthetic peptide corresponding to the n-terminal domain of attic has characteristics of an intrinsically disordered protein. temperature-dependent far-uv cd spectra of n-terminal peptide of attic ph-dependent far-uv cd spectra of n-terminal peptide of attic far-uv cd spectra of n-terminal peptide of attic in the absence and presence of 10% and 50% tfe at 25°c. peptide content was kept at a concentration of μm in tris buffer for all measurements. the peptide gained modest amounts of structure with increasing temperature, ph, and tfe concentration, which is consistent with the domain being intrinsically disordered. the structural properties of the n-terminal domain of attic were further characterized through its changes at different temperatures, phs, and increasing concentrations of trifluoroethanol. comparing the far-uv cd spectra of the peptide at different temperatures revealed a modest gain of peptide structure as temperature increased, revealed by an enhancement of the negative ellipticity at ~  - nm. the influence of trp-trp interactions on the peptide's cd spectra might also be lessened at higher temperatures, as indicated by the slight blue shift of the negative ellipticities at ~  nm. in addition, a reduction of the negative minimum at ~  nm revealed a decrease in random coil content of the peptide. thus, an increase in temperature induced partial folding of the n-terminal domain of attic in addition, a slight gain in the peptide structure was also observed at high ph. carrying a number of basic amino acid residues, the n-terminal peptide possesses a large positive charge at neutral ph. under basic conditions, this net charge would be minimized, thus reducing the electrostatic repulsion between amino acid residues and inducing partial folding of the peptide. finally, the secondary structure stabilizer tfe enhanced the n-terminal peptide structure. at 50% tfe, a notable increase in secondary structure was observed, indicated through the enhancement of local minima at ~  nm and ~  nm. overall, the n-terminal domain of attic displayed an unstructured conformation with the propensity to gain structure depending on its environment, which is consistent with being characterized as an intrinsically disordered protein domain. the ability to adopt different conformations may play a role in its physiological function. discussion a novel method for expressing and purifying recombinant attic in a native-like folded form is reported in the current study. in addition, the self-association of attic in mild detergent and lipid systems was observed in vitro for the first time, using a combination of biochemical and biophysical techniques, and evidence is presented that the extramembrane n-terminal domain has properties of an intrinsically disordered protein. the evidence for oligomerization may be important when exploring the hypothesized function of the protein as a preprotein channel of the tic complex. novel recombinant protein expression system for attic20 several studies, including ours, predict attic to contain four tm domains that span the chloroplast inner envelope membrane. the hydrophobic nature of the protein has made it difficult to produce recombinant tic until recently, when kovacs-bogdan et al. reported obtaining attic in bacterial inclusion bodies. a disadvantage of extracting proteins from inclusion bodies is that many such proteins have been shown to be only partially folded. other disadvantages of this method include the heterogeneity of protein conformations that can be obtained when refolding proteins from inclusions bodies, and the limited number of detergents suitable for the solubilization and subsequent refolding of the protein. in the current study, using a mild auto-induction expression system, together with nutrient-rich media, we were able to heterologously express attic in e. coli in such a way that some of the protein was directed to the bacterial membranes, even though the majority was still directed to inclusion bodies. targeting to the bacterial membrane occurred without introduction of a signal sequence onto the n-terminus of attic although we did not attempt to characterize the nature of the targeting, it is known that the signal recognition particle targets multi-spanning transmembrane domain proteins to the membrane in e. coli. srp is able to recognize either cleavable n-terminal signal sequences or internal tm domains for targeting proteins to bacterial membranes, and insertion can occur via the secyeg or yidc translocon. furthermore, srp is known to be recruited by the presence of tryptophan-enriched transmembrane helices, such as is found in the first predicted tm domain of attic. it therefore seems likely that attic was targeted to the bacterial membrane in an srp-dependent manner, with the first tm domain serving as a signal anchor sequence. in summary, production of the recombinant protein relied on using the “codon plus” strain of e. coli, much like kovacs-bogdan et al., who used a codon-optimized cdna for expression of attic in inclusion bodies. targeting of the protein to the bacterial membrane rather than to inclusion bodies was presumably a result of using the “mild” auto-induction method. in addition, the method resulted in the purification of recombinant attic in detergents in relatively high yield. both attic and its truncated version were shown to retain high helical contents following their extraction from the membrane using % of the mild detergent zw3-. the helical content of the protein following reconstitution into popc liposomes was very similar to that predicted from the primary sequence. thus, the expression and reconstitution protocol described in this study is appropriate for preparing highly pure and native-like folded protein that can potentially be used for obtaining high-resolution structures of attic the method might also be applied to other tic components, whose structural and functional features still remain elusive. in the study reported by kovacs-bogdan et al., pstic expressed in a cell-free system also exhibited a highly helical secondary structure in the presence of % brij- detergent. the far-uv cd spectrum of pstic reported in that study was indicative of a monomeric conformation, featuring two minima at and nm and a positive maximum at nm. similar cd spectral properties were also observed in the current study for dissociated attic following the titration of protein from popc liposomes with sds. therefore, we conclude that expression of attic in bacterial membranes allows for the formation of oligomers that aren’t formed when the protein is expressed in a cell-free environment in the presence of detergent. self-association of attic20 the recent report that tic comprises part of a mda complex, together with new components tic tic and tic that forms preprotein-conducting channels independent of tic has contributed to a debate about the stoichiometry of the components in the tic translocon. one issue in particular that has been raised is whether tic is present in sufficient quantities to serve as the primary channel forming protein. and if it is the primary channel forming protein, the mechanism by which it operates is still unknown. determining the structure and oligomeric state of attic represents an important step toward answering these questions. in this study, we report the observation that attic has the capacity to self-associate in lipid membranes. this homo-oligomerization of attic was detected for protein extracted directly from bacterial membranes using mild detergent, and when reconstituted into liposomes. the predominant associated form of attic was dimeric, although higher-order oligomers were also observed at much lower levels. under our experimental conditions, attic oligomeric forms in liposomes could be dissociated with relatively low concentrations of sds. this relatively weak self-association of attic could be an indication that the oligomeric state of the protein is dynamic, with the ability to associate and dissociate readily in response to particular signals. in the context of the tic complex at the chloroplast, one such signal could be the presence of preproteins, which has been shown to trigger tic complex assembly. the potential function of the different oligomers, or which one might be more prevalent in vivo, remains to be determined; however, kikuchi et al. hypothesize that the mda complex isolated from chloroplast inner envelope membranes that interacts with translocating preproteins may contain copies of attic it is possible that an oligomer of attic forms the core of the mda complex and provides the conduit through which preproteins travel. it is also possible that tic operates in a manner similar to tim the mitochondrial translocase component with which it shares sequence and topological similarities, and that is the central component of the tim complex. interestingly, these mitochondrial transporters are dimeric in their functional state, which is comparable to the results of this study, underscoring the existence of oligomeric forms of attic in equilibrium with its monomeric form in membranes. evidence in support of the propensity of attic to form oligomers was provided by the transmembrane helical dimer prediction program preddimer. while no dominant set of pairings emerges from this analysis, the results from the preddimer prediction suggest the possibility of different sets of distinct helix-helix alignments, allowing for multimeric forms of attic to adopt several conformations.figure possible helix dimerization of attic a 3-d model of attic was generated using the distill program as described in the methods section. each tm helix shown in the model was used in the analysis of predicted tm helical dimer conformations. for each predicted dimer conformation, fscor is evaluated, representing the packing quality of helices. a higher fscor reflects a high packing efficiency of the dimer. the detailed prediction method is described in the methods. this work also revealed some of the structural features of attic. the n-terminal peptide preceding the predicted tm domains of the protein displayed characteristics that are consistent with instrinsically disordered proteins, exhibiting flexible conformations in different environments. this conformational elasticity could be important for the physiological function of attic such as peptide recognition or interaction with protein partners in the tic complex. these protein partners potentially include the other members of the recently discovered mda complex of the inner envelope membrane. interestingly, the truncated version of attic lacking the n-terminal peptide, displayed conformations and showed evidence of self-assembly similar to the full-length protein in detergents and lipid bilayers. thus, the ability to self-associate is independent of the n-terminus, and is presumably conferred by one or more of the transmembrane domains. the n-terminal domain may participate in protein interactions with other components of the translocon complex. of note, is that the extramembrane n-terminal domain of tim has also been reported to be intrinsically disordered; in that case, it is believed that the domain is involved in preprotein binding.  in conclusion, the novel protein expression system reported in this study resulted in the protein being targeted to bacterial membranes, which is a more biologically relevant system than inclusion bodies, and led to the finding that the protein can exist as a monomer and higher-order associated forms. the existence of associated forms of attic in membranes may be important for understanding its hypothesized function as a preprotein transporter. the observed dimeric, trimeric and tetrameric forms, together with the low dissociation constant of the protein in liposomes implies a relatively weak interaction between the monomers, which in turn suggests a dynamic nature of the equilibrium between the monomeric and associated forms. this may be an indication that changes in the protein's local environment, which could include presence/absence of preproteins, could trigger changes in the multimeric state of the protein. the ability of tic to switch between its monomeric and associated forms is consistent with previous observations that tic complex assembly is triggered by the presence of preproteins, and that the mda complex likely contains more than one copy of attic and will be important when testing its hypothesized function as a preprotein channel protein of the inner envelope membrane of chloroplasts. 
as a component of the tic complex, it is believed to form a preprotein-conducting channel across the inner membrane. this method resulted in the recombinant protein being directed to the bacterial membrane without the addition of a bacterial targeting sequence. we report a method for producing large amounts of recombinant attic using a codon-optimized strain of e. coli coupled with an autoinduction method of protein expression. the arabidopsis thaliana protein attic is a key component of the protein import machinery at the inner envelope membrane of chloroplasts. furthermore, we present evidence that the extramembranous n-terminus of the mature protein displays characteristics that are consistent with it being an intrinsically disordered protein domain.
 the arabidopsis thaliana protein attic is a key component of the protein import machinery at the inner envelope membrane of chloroplasts. as a component of the tic complex, it is believed to form a preprotein-conducting channel across the inner membrane.  we report a method for producing large amounts of recombinant attic using a codon-optimized strain of e. coli coupled with an autoinduction method of protein expression. this method resulted in the recombinant protein being directed to the bacterial membrane without the addition of a bacterial targeting sequence. using biochemical and biophysical approaches, we were able to demonstrate that attic homo-oligomerizes in vitro when solubilized in detergents or reconstituted into liposomes. furthermore, we present evidence that the extramembranous n-terminus of the mature protein displays characteristics that are consistent with it being an intrinsically disordered protein domain.  our work strengthens the hypothesis that attic functions similarly to other small α-helical integral membrane proteins, such as tim that are involved in protein transport across membranes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords tic20tic complexprotein self-assemblycircular dichroismprotein foldingstructure-function relationshipprotein reconstitutionchloroplast membrane proteinsissue-copyright-statement© the author 2014 