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Juxtamembrane tryptophans possess distinct roles in defining the OmpX barrel-membrane boundary and packing-facilitated protein-lipid stability/strength/association
Defining the span of the transmembrane region, a key requirement to ensure correct folding, stability and function of bacterial outer membrane β-barrels, is achieved by the amphipathic property of tryptophan. We demonstrate two unique and distinctive roles of the interface Trp76 and Trp140 of outer membrane protein X (OmpX), and map their positional relevance in the refolding process, barrel formation, and the resulting stability in dodecylphosphocholine (DPC) micelles. The solvent-exposed Trp76 acts as a rigid nucleating point for barrel refolding and micelle interaction, while Trp140 defines the global thermal and chemical stability of the refolded barrel through direct protein-DPC association.
The Outer membrane protein X (OmpX) of Escherichia coli is a structurally well characterised eight stranded antiparallel β barrel (2). It belongs to the family of Outer membrane proteins (OMPs) of gram negative bacteria which help in attachment and invasion but OmpX of E. coli is not reported to perform any such function till now. Although the exact function of E. coli OmpX is not well defined, it is thought to promote ability of bacterial cells to be internalized and resist bactericidal activity of human serum (3).
Biological membranes are complex entities with diverse composition and fluidity, anchoring key integral membrane proteins with assorted functions. Governing factors that mediate protein-lipid interactions form the basis of existence and stability of membrane proteins in their lipid milieu. The aromatic girdle in membrane proteins, preferentially localised at the membrane interface, is speculated to play an important role in governing interactions critical for membrane protein folding and subsequent anchoring (REF). Among the aromatic amino acid side chains, the indole presents unique properties of possessing the largest non-polar surface area, along with hydrogen bonding capability of the indole NH moiety. In addition, being the most polarizable amino acid makes tryptophan the most ideal amphiphilic residue for stabilizing the interfacial region of a membrane protein (1).
OmpX consists of two tryptophans at unique and distinct positions (REF). According to the reported structure W76 is supposed to be an exposed tryptophan present at the interface where as W140 is expected to be a buried one. Tryptophans are biologically expensive amino acids and hence we assume them to play specific role in folding and function of OmpX. To understand the role of each tryptophan, single tryptophan mutants of OmpX were generated where either of the tryptophan was mutated to phenylalanine.
In this study, we have tried to understand the positional relevance of each tryptophan in OmpX both from global and local perspective. Thermal and chemical denaturation studies were performed for both the mutants and the obtained thermodynamic parameters were compared within themselves as well as with the wild type. Fluorescence life time and anisotropy based measurements were carried out in order to comment upon the local environment and rigidity or flexibility of the fluorophore. We observed that the mutant devoid of W140 was least stable and W140 even being a buried tryptophan, was conformationally flexible whereas W76 was rigid. In addition, acrylamide and iodide quenching experiments were also performed to know the accessibility of tryptophan at two different positions.
2. Materials and methods
2.1 Cloning and Expression
OmpX WT gene (without the signal sequence) was a king gift from Prof. Kurt Wüthrich at the Scripps Research Institute. C.A. OmpX has two tryptophans at 76th and 140th position respectively. Two single tryptophan mutants were generated using site directed mutagenesis as reported earlier where in either of the tryptophan was mutated to phenylalanine. The mutants thus generated are referred as W76F and W140F in this study. The WT and mutants were expressed as inclusion bodies in E. coli C41 cells and purified using anion exchange chromatography under denaturing conditions.
2.2 In vitro protein folding
Purified protein fractions were dialysed against water to remove urea and lyophilised to obtain powder. Refolding was carried out using rapid dilution strategy as reported previously (Ref). Protein dissolved in 8M urea was rapidly diluted 20 fold against refolding solution containing 50, 100 or 250mM DPC (n dodecyl phosphocholine) and 50mM Tris pH 9.5.
2.3. Thermal denaturation experiments
Folded OmpX was thermally melted from 5°C to 95°C and recovered back to 5°C as reported previously (ref) employing tryptophan’s fluorescence as a probe on Fluoromax-4 spectrofluorometer equipped with peltier from Horiba Jobin-Yvon, France. The tryptophans were excited at 295nm and emission scan was recorded from 310-400nm at regular temperature interval of 1°C. Protein concentration of 30µM was maintained constant with varying concentrations of DPC as 10, 20 and 50mM to look for LPR dependency. The data thus obtained was blank subtracted and corrected for dark counts.
2.4 Equilibrium unfolding
Refolded protein was subjected to chemical denaturation using GdnHCl as a denaturant. The unfolding was monitored as fluorescence on SpectraMax M5 microplate reader at 25°C (Molecular Devices, U.S.A) using 96-well flat bottom black plates (SPL Life Sciences Co., Ltd, Korea).With 295nm as λex, emission was scanned from 320-400 nm at every 1nm in a 200µl reaction having 10, 20 and 50mM DPC and protein concentration of 30µM. All the data was blank subtracted and thermodynamic parameters were obtained by averaging data from 2-3 independent experiments. The two - state equation used for fitting the fraction unfolded graphs is as mentioned below:
2.6 Fluorescence life time and Anisotropy measurements.
Fluorescence life time of tryptophan in the case of all three proteins was estimated on FluoroLog spectroflorometer (Horiba Jobin-Yvon, France) using TCSPC (Time correlated single photon counting).The excitation wavelength used was 295nm where as emission wavelength of 340nm and 355nm were used for folded and unfolded samples respectively. Instrument Response Function (IRF) calculated using anthracene was found to be Í 830ps. Amplitude fraction (αi) and corresponding life time (τi) was calculated by fitting the deconvoluted data to three exponential function using DAS6 v6.4 software (Horiba Scientific, France). The average life time was calculated using
Anisotropy was measured with increasing temperature on Fluoromax-4 spectrofluorometer equipped with peltier from Horiba Jobin-Yvon, France. The excitation and emission wavelengths were set at 295 and 340nm. Each data point was obtained from average of 5 trials with maximum 2 % target error. The integration time was set as 5 seconds and G factor as 1.8. Temperature independent anisotropy measurements were recorded at 25°C.
2.7 Acrylamide and Iodide quenching
Refolded protein was subjected to quenching in presence of acrylamide (ref) and iodide from 0M to 0.5M. In the case of iodide quenching, equivalent amounts of KCl was also added to ignore the effect of salt concentration on the refolded sample. The reactions were performed in the dark with incubation of 5 minutes at 25°C before recording.
2.8 Equilibrium Folding Experiments from Urea
Unfolded protein samples containing 8M urea and 50m DPC in 50mM Tris pH 9.5 was subsequently diluted to achieve minimum concentrations of urea for folding the protein keeping the constant DPC concentration of 10mM throughout the urea gradient. The reaction was monitored as increasing amount of fluorescence as the protein folds on SpectraMax M5 microplate reader (Molecular Devices, U.S.A). Folding for each mutant was monitored till 216 hours to achieve maximum refolding at 25°C. Fraction unfolded sigmoidal curves thus obtained were fitted to a two state equation to derive âˆ†G, m-value and Cm.
3. Results and discussion
1) Fluorescence and Anisotropy Melting
To better understand the importance of the two tryptophans, single tryptophan mutants were generated where in each mutant, one of the tryptophan was substituted with phenylalanine. Hence mutants thus generated were named as: W76F where interfacial tryptophan is absent and W140F, where transmembrane tryptophan is not present. Upon thermal denaturation of W140F from 5°C to 95°C, there was no loss in the fluorescence intensity observed and the protein recovered completely when cooled back to 5°C. On the other hand anisotropy decreased dramatically at around 80°C but came back to the starting point when recovered back to 5°C. Insignificant loss in fluorescence intensity can be attributed to the fact that 76th tryptophan present in W140F is already present exposed to the solvent to start off and thus shows no change in fluorescence intensity profile. Decrease in anisotropy indicates that the tryptophan present in this mutant is a rigid one and thus at higher temperatures, its rigidity is lost transiently but recovers back on decreasing the temperature. On the other hand, W76F which has a buried tryptophan at 140th position shows a unique profile on melting and recovery. There is a sudden increase in fluorescence intensity upon heating till around 80°C after which it drops when further heated till 95°C. Upon cooling, fluorescence intensity increases again till 60°C and decreases further till it reaches to the original position. We hypothesise this trend to the presence of a quencher near W140.As the refolded protein is heated the quencher moves away and thus the fluorescence intensity increases but after increasing the temperature even further beyond 80°C, the protein starts unfolding and thus intensity drops down. On cooling the protein starts folding back but at a particular temperature of 60°C, quencher is again very close to the fluorophore and thus the intensity decreases again. Anisotropy on the other hand is more or less constant throughout the heating cooling cycle, forcing us to believe that although buried inside the lipid micelles, W140 is relatively flexible in the local pocket and resist any further significant movement upon heating. OmpX WT shows the cumulative effect of both the tryptophans as compared to the single tryptophan mutants both in terms of fluorescence intensity and anisotropy melting cooling profiles.
2) Life time Measurements, anisotropy and quenching experiments
W76F having a transmembrane tryptophan shows highest life time of 4.6ns where as W140F having the interfacial tryptophan has a lowest life time of 2.24ns. OmpX WT shows average life time of both the tryptophans which is around 3.4 ns. These results suggested that W140 is present inside the transmembrane region and is fully covered inside the lipid micelles. On the other hand, high Ksv value and comparatively low anisotropy values contradicted the results obtained from life time measurements. As acrylamide is a non polar molecule, we thought that it is somehow not able to access the exposed fluorophore and thus used a different quencher Iodide to verify the assumption. High anisotropy values for W140F suggested W76 to be a rigid fluorophore but low Ksv values in acrylamide quenching indicated its low accessibility for acrylamide. However, iodine being a polar molecule was able to access the exposed tryptophan leading to high Ksv values for W140F as compared to W76F.
This also led us to conclude that protein lipid systems are rather complex heterogeneous systems and thus one can expect to see varied results for quenching depending upon the nature of the environment of flouorophore and the type of quencher employed.
3. Results/Results and discussion/Discussion
3.1. Text here
3.2. Text here
Appendix A: Supplementary Data
Supplementary data associated with the article can be found in the online version.
For use within text:
Fig. 1. (A) Figure legend. (Insert figure above this figure legend)