. We have used secondary sequence modeling of P/rds previously to assist targeted mutagenesis studies and were surprised by the extent to which random coil elements dominate predictions for the protein’s C-terminus. shows the results of the application of two commonly used secondary structure prediction methods to the P/rds C-terminus. Modeling by both the GOR4 [28
] and JNET [29
] methods predicts more than half of the C-terminal sequence to be unstructured. In contrast, one strongly predicted helical element, centered at Leu320, is also suggested, and a previous FTIR evaluation of a 15 amino acid synthetic peptide encoding Val312-Leu326 supports the prediction of helical content in this region [17
]. An additional short region of helix is also predicted by each method; however, the locations assigned do not concur. Beyond the agreement of the helical region centered at Leu320, the most obvious commonality is the extent of random coil structure. Given this suggestion of limited secondary structure, we applied a Predictor of Natural Disordered Regions (PONDR) VL-XT analysis [30
], to further evaluate this region. This approach uses a series of neural network predictors that utilize amino acid sequence data to predict disorder for a given sequence; it defines disordered regions as entire proteins or regions of proteins which lack a fixed tertiary structure, essentially being partially or fully unfolded in situ
]. The prediction generated by the PONDR VL-XT program for the C-terminus is qualitatively similar to both the GOR4 and JNET results. As well, the bulk of predicted disorder is clearly confined to the C-terminus; whereas roughly 55% of the C-terminus is predicted to be disordered, only ~14% disorder is found for P/rds overall. Moreover, when PONDR VL-XT is applied to P/rds regions other than the C-terminus, it predicts only 3.6% disorder. Overall, both secondary structure and intrinsic disorder prediction methods suggest that, with the exception of a single helix, the P/rds C-terminus is anticipated to lack fixed structure.
Figure 1. Modeling of CTER structural elements. Above: linear representation of full-length P/rds. Sequences predicted to form transmembrane helices (shaded) and disordered regions (hatched) are indicated. Below: the 63 C-terminal amino acids of bovine P/rds are (more ...)
C-terminal structure in P/rds from rod OS membranes. We employed limited proteolysis to examine whether the disorder predicted is characteristic of the P/rds C-terminus in the context of its native membrane environment. shows a Western blot analysis of bovine rod OS membranes probed with MAbC6, a monoclonal antibody that binds an epitope at the C-terminus of P/rds. The rod OS membranes were burst hypotonically to expose the P/rds cytoplasmic C-termini, then were pretreated with one of two denaturing conditions. The membranes were washed free of denaturants and then were digested with Proteinase K for the times indicated. Membranes pretreated with 5 mM Tris, pH 7.5 are shown for comparison (control). We found that approximately 70% of the C-terminal epitope was destroyed over 30 min in the control reaction. Highly structured, globular domains typically display an increased accessibility to proteolytic digestion upon denaturant induced unfolding. In contrast, the relatively harsh pretreatments used here, including 4M urea and 100 mM sodium carbonate, pH 11, had no significant effect on the time course of proteolysis. Similar results were obtained when trypsin, an enzyme with greater sequence specificity was employed (not shown). Thus, strong denaturants do not increase accessibility of the C-terminus to proteases, suggesting that an extended conformation is present in the native protein. We also tested the effect of pretreatment with 10 mM DTT, a strong, membrane-permeable reductant that is reported to disrupt P/rds interactions with other photoreceptor proteins [31
], and found it also had no appreciable effect on the protease sensitivity of the P/rds C-terminus (). These findings demonstrate that the C-terminus is relatively accessible to limited proteolysis and are consistent with the extensive regions of disorder predicted by structure modeling.
Figure 2. Protease sensitivity of the P/rds C-terminus in photoreceptor rod OS membranes. (A) Hypotonically lysed rod OS membranes were pretreated with 4M urea, 100 mM NaCarbonate, pH 11, or left untreated prior to limited digestion with Proteinase K at 37°C (more ...)
Expression and purification of a soluble P/rds C-terminal domain (CTER). Structural determinations of integral membrane proteins continue to present numerous technical challenges, particularly with respect to characterization of regions of flexibility. We therefore designed a soluble version of the P/rds C-terminal domain as a cleavable GST fusion protein, based on a previous study [18
]. The new expression vector (designated PGEX6CTER) takes advantage of a PreScission protease cleavage site and offers the advantage of facile protease removal using a glutathione affinity matrix. The fusion protein itself, GST6-CTER, is highly expressed in E. coli strain BL21 and yields of approximately 30mgs per liter of LB culture were obtained routinely. presents a Coomassie blue stained Tricine gel containing fractions from the fusion protein purification procedure. The initial affinity purification from the bacterial lysate yielded a single major band of apparent MW ~32kDa, with a secondary band of ~25Da, likely a GST truncation product. Cleavage of GST6-CTER with PreScission protease yielded the expected fragments for GST alone ~25kDa and the predicted CTER domain ~7 kDa (). A second (negative) affinity purification to remove GST, uncleaved GST6-CTER, and the PreScission protease, results in a single major band of the predicted ~7 kDa molecular weight.
Figure 3. Expression and purification of a soluble form of the P/rds C-terminus analyzed by SDS-PAGE. (A) IPTG induction of a pGEX6CTER transformed BL21 culture resulted in the appearance of a new protein band at ~33 kDa. GST affinity purification yielded (more ...) Self association of CTER.
Previous studies have demonstrated that full-length P/rds from ROS membranes is detergent solubilized as a non-covalent tetramer under reducing conditions [32
]. Moreover, specific determinants within the large extracellular/intradiskal loop region, EC2, are used to guide protein folding and subunit assembly [12
]. Although no evidence has yet been presented that C-terminal defects can alter protein stoichiometry, we thought it worthwhile to directly test the possibility that this domain may self-associate - particularly since a 15 amino acid synthetic peptide corresponding to sequence within this region is reported to form dimers and tetramers [17
]. We asked whether we could detect a similar self-association of CTER using a hydrodynamic assay. Low pressure size exclusion chromatography (SEC) was performed in a moderate ionic strength buffer system at neutral pH, using protein concentrations from 0.1 to 1.5 mg/ml on a column of Sephacryl S-100HR. shows a typical SEC elution profile of purified CTER as measured by A280
. A single symmetrical peak, with an elution time of 118.1 min is observed - corresponding to an apparent MW of approximately 25kDa. The left inset figure shows that an identical profile was observed when TCA-precipitated SEC fractions were analyzed by Tricine-buffered SDS-PAGE in conjunction with Coomassie blue staining. The CTER elution position was unaffected by initial protein concentration (0.05 to 1.5 mg/ml), low ionic strength, or phospholipid vesicles (not shown). Collection, concentration and re-injection of the CTER peak resulted in an essentially identical elution profile (not shown). These results suggested that the purified CTER was present as a single non-equilibrating species of ~25 kDa apparent MW. Given its predicted monomer mass of 7.2 kDa, the SEC data suggested that CTER could self-associate as a non-equilibrating trimer or tetramer. This was an intriguing outcome, given our previous findings that: 1) tetrameric P/rds subunit assembly is not affected by large insertional mutations in its C-terminus [12
], and 2) strong denaturants do not increase accessibility of the C-terminus to proteases (). However, since molecular mass determinations by SEC assume globularity and can be affected by binding interactions with chromatographic media [26
], we decided to consider an alternative interpretation to self-association and test the possibility that CTER is non-globular.
Figure 4. CTER apparent MW under non-denaturing conditions was assessed by SEC. Purified CTER was chromatographed in 50mM TRIS-HCl, 150mM NaCl, 1 mM TCEP, pH 7.5, on a calibrated column of Sephacryl S-100HR at 0.2 ml/min. The protein eluted as a single major peak (more ...)
Analytical ultracentrifugation (AUC) was employed to differentiate between self-association and non-globularity and rigorously define CTER stoichiometry. CTER sedimentation velocity was assessed for protein concentrations ranging from 0.1 to 1.0 mg/ml and the data were analyzed using a model of continuous distribution, c(s), of sedimentation coefficients [25
]. The results are presented in a scaled and offset form in . Peak positions and ratios remain essentially constant; these findings indicate that CTER does not participate in a reversible mass-action equilibrium at the concentrations measured. The weight-average s-values (Sw
) presented in were obtained by a second-moment integration of the sedimentation profiles. The similar values confirm that Sw
remains constant over the range of protein concentration examined. A model of non-interacting discrete species as applied to the 1 mg/ml velocity sedimentation data suggest that CTER is present as a single major species, with < 10% of a higher MW contaminant present.
Figure 5. Analytical ultracentrifugation of purified CTER by velocity sedimentation. CTER was prepared in 50mM Tris-HCl, 150mM NaCl, 1 mM TCEP, pH 7.5 at concentrations of 0.1, 0.3, and 1.0 mg/ml. After reaching temperature equilibrium, the samples were centrifuged (more ...)
Weight-average S-values for samples of purified CTER at concentrations from 0.1 to 1.0 mg/ml obtained from second order integration of the respective velocity sedimentation profiles. No change is observed over the concentration range examined.
CTER was subsequently subjected to sedimentation equilibrium analysis at three protein concentrations ranging from 0.11 to 1.0 mg/ml. Data sets (six in total) collected for each concentration at both 28k and 40k rpm were analyzed with nonlinear least-squares techniques using the WinNonlin program. As was expected from sedimentation velocity experiments, fits using only a single ideal species were poor, and large systematic errors indicated an incorrect model. Various association models were tried, and the best fit was obtained assuming two non-interacting discrete species. The results from this fit, using the known value for CTER of 7.21 kDa is shown in . The deviations are essentially random and close to the noise level inherent in the optical system. The values of the fitted K’s indicate a higher order MW contaminant present in the CTER sample at approximately 5-10%. In sum, the combined AUC measurements indicate that the purified CTER is monomeric and that the sample also contains <10% of a higher molecular weight contaminant. The rigorous determination that CTER is monomeric (by AUC) combined with a substantially retarded hydrodynamic elution position (observed by SEC) suggests that CTER possesses an extended conformation and may have a weak affinity for the SEC matrix.
Table II. Parameters obtained from fitting sedimentation equilibrium data obtained at concentrations of CTER from 0.11 to 1.0 mg/ml at 28,000 and 40,000 rpm. Fits using a single ideal species were poor, and the heterogeneous model used here indicates that CTER (more ...) Tryptophan solvent exposure.
We wished to test further the hypothesis that CTER possesses an extended conformation; this model predicts that the two tryptophan residues present should be readily accessible to solvent. We measured intrinsic tryptophan fluorescence of CTER under a variety of solution conditions and found this to be so. (solid line) shows an emission spectrum of CTER, using an excitation wavelength of 295 nm. The characteristic broad peak at 348 nm suggested to us that the two tryptophans contained within this domain were rather solvent exposed, as had been predicted by the SEC and AUC results. Variation of ionic strength and divalent ion concentration had no appreciable effect on these results (not shown). The idea that CTER possesses an extended conformation was supported further by the finding that the addition of a strong denaturant, 6M GuCl, altered the emission spectrum peak only marginally (, dashed line). Protein denaturation and increase of tryptophan solvent exposure is typically accompanied by a significant red shift; however in this instance, the small shift observed likely results solely from an altered solvent environment. Finally, we assessed tryptophan accessibility by adding increasing amounts of the commonly used fluorescence quencher, acrylamide. As seen in , acrylamide quenching of CTER intrinsic tryptophan fluorescence is highly effective and shows a steep dependence similar to that seen for random coil peptides [34
]. The combined data clearly indicate a high solvent accessibility for both of the tryptophan residues in CTER, and these observations are most consistent with an extended protein conformation.
Figure 6. Hydrophobic residue exposure to solvent as assessed by intrinsic tryptophan fluorescence. Intrinsic tryptophan fluorescence was measured in response to 295nm excitation of a 5 μg/ml solution of CTER in 50 mM Tris-HCL, 150 mM NaCl, 1 mM TCEP, pH (more ...) Far UV Circular dichroism.
Since findings from SEC, AUC and intrinsic tryptophan fluorescence all pointed to an extended conformation and lack of globular structure, far UV CD was employed to help assay the degree of structure present in CTER. A previous investigation using far UV CD concluded that CTER displays considerable secondary structure in aqueous solution, but did not address whether tertiary structure is present [18
]. shows averaged far UV-CD spectra measured for CTER at 10°C (solid line) and 20°C (dotted line). They are similar and characterized by roughly 10% α-helix, in agreement with prior measurements. As expected from SEC, AUC, intrinsic tryptophan fluorescence and previous CD measurements, the spectra also indicate strong contributions from random coil elements (estimated here at ~36%). Far UV CD was subsequently applied to ask whether CTER displays significant tertiary structure. A thermal denaturation of CTER (from 10°C to 60°C) was performed while monitoring CD at 220 nm, a routine diagnostic for loss of α-helical and tertiary structure. Contrary to expectations for a folded protein, illustrates a small increase in CTER α-helix with elevated temperature. The combined results suggest that CTER largely lacks fixed tertiary structure and possesses limited secondary structure.
Figure 7. Far UV circular dichroism spectroscopy of purified CTER. (A) CD spectra of 0.31 mg/ml CTER taken in 50 mM Tris-HCl, 150 mM NaCl, 1mM TCEP, pH 7.5 at 10°C (solid line) and 20°C (dotted line) as described in Methods. The spectra are similar (more ...)
Since evidence suggests that the high macromolecule concentrations in cellular environments can promote compact, vs. extended protein conformations [35
], we examined whether macromolecular crowding effects could induce additional structure in CTER. As well, given the fact that the native P/rds C-terminus is membrane anchored, exists in a membrane-rich environment, and can interact with liposomes [3
], we tested the effect of a membrane mimetic environment on CTER structure. The presence of 320 mg/ml dextran (40,000), a commonly used macromolecular crowding agent [35
], did not have a appreciable effect on the far UV CD spectrum of CTER; an increase in helical content of roughly 3% was estimated. In contrast, increasing concentrations of trifluoroethanol (TFE), a cosolvent that is documented to stabilize functionally important protein structures [36
], had a significant effect on increasing α-helical content in CTER. shows that signals at 220 nm rise with TFE concentration. CTER helical content is estimated to increase more than threefold (10% to 32%), in the presence of 50% TFE. Overall these results suggest that CTER structure, like that in other disordered domains is not affected by macromolecular crowding effects [22
], but is appreciably increased in the presence of a hydrophobic environment.