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Back | Show only these items: | Preliminary structural characterisation of the 33 kDa protein (PsbO) in solution studied by site-directed mutagenesis and NMR spectroscopy

1
Preliminary structural characterisation of the 33 kDa protein (PsbO) in solution studied by site-directed mutagenesis and NMR spectroscopy

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa1

Site-directed mutagenesis experiments combined with 1D and 2D NMR spectra provide a preliminary insight into the structure and dynamics of the 33 kDa protein (PsbO) from T. elongatus free in solution.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa1

The NMR spectra suggest that PsbO rather than forming a ‘molten globule’ state or being ‘natively unfolded’, contains both a well folded core and highly flexible regions.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

This core shows remarkable stability over a broad range of temperatures and pH values.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con2

Additional experiments with Cys residues introduced at different positions indicate sites of increased accessibility/flexibility which may be important for the docking to the PS2 core complex.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con3

Introduction

Photosystem 2 (PS2) is part of the thylakoid membrane of chloroplasts and cyanobacteria and performs one of the key reactions on our planet: the light-driven oxidation of water.^1,2

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac1

It consists of more than 20 polypeptides, most of them with no function identified yet.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

Especially, the role of the numerous small membrane spanning peptides is still unclear.³

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac3

The primary electron transfer reaction occurs at the heterodimeric core of PS2 formed by the subunits D1 and D2, which bind most of the redox active cofactors.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac4

Water oxidation to molecular oxygen is catalysed via the sequence of four one-electron oxidation steps (S-states) by the manganese containing water-oxidising complex (WOC).⁴

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

Recent advances in X-ray structure analysis of PS2 crystals^5,6 provide a detailed view of the structure and organisation of the WOC.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac6

At the lumen side of PS2, the WOC is shielded by several extrinsic proteins.⁷

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac7

One of them, the 33 kDa protein (PsbO), is of special interest because it is ubiquitous in all photosynthetic organisms performing water-splitting and its extraction results in a strong decrease of the oxygen evolving activity.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot1

Therefore, it is often referred to as manganese stabilizing protein (MSP).

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac8

However, it is obvious from the recently published PS2 X-ray structure⁶ that the PsbO subunit is not directly involved in binding of manganese as part of the WOC.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac9

Several lines of experiments using different kinds of spectroscopic techniques or biochemical and theoretical analyses have made suggestions about the PsbO structure and its behaviour free in solution.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac10

It was concluded from Fourier transform infrared (FTIR) spectroscopy^8–10 and circular dichroism (CD) spectroscopy^11,12 that the PsbO subunit in solution consists of a high content of β-sheet (30%) and random/loop structure (60%) whereas α-helical parts are only present in small amounts (10%).

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac11

These results were consistent with secondary structure prediction methods and folding models of the 33 kDa protein.^13–15

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac12

Some authors suggested an unusual folding behaviour for PsbO in solution and therefore postulated an ‘intrinsically disordered’, ‘natively unfolded’ or ‘molten globule’ protein structure.^10,16

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac13

Experiments with focus on the dynamic properties of the PsbO subunit led to the observation of pH-dependent and cofactor-induced structural changes in solution.^11,17,18

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac14

Recently, it was reported that reconstitution of the PS2-complex with PsbO is accompanied by changes of its secondary structure.¹⁹

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac15

Indeed, there are hints for structural changes of the bound PsbO subunit depending on the oxidation state of the manganese cluster.^20,21

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac16

Here we report preliminary results from NMR-spectroscopy on PsbO from Thermosynechococcus elongatus (T. elongatus) describing its structural and dynamic properties.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa1

The 1D and 2D NMR spectra suggest that PsbO free in solution adopts a defined structured core.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

The spectra also indicate that the PsbO from T. elongatus in solution contains a considerable amount of flexible regions.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

The PsbO protein shows a remarkable temperature, pH, and long term stability beeing stable for at least four weeks under the conditions tested in this study.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con2

Due to this extraordinary stability of PsbO, we characterised four cysteine mutants serving as local probes for structural and dynamic properties of PsbO.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa2

Materials and methods

Sample preparation

The recombinant 33 kDa protein was expressed in E. coli from an expression plasmid containing the full-length sequence of the T. elongatus psbO gene resulting in the export of PsbO into the periplasmic space.²²

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

Point mutations were constructed by oligonucleotide-directed mutagenesis.²³

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met1

The periplasmic cell-fraction highly enriched with 33 kDa protein was isolated,²⁴ dialysed against 20 mM Tris pH 8.0, 0.5 M mannitol, and loaded onto a ResourceQ column (Amersham-Pharmacia).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

The protein was eluted with a linear gradient from 0 to 0.5 M NaCl.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

By this method, we obtained highly purified PsbO (up to 20 mg L⁻¹ culture).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs1

The ¹⁵N-labeled samples were prepared from minimal medium with ¹⁵N NH₄Cl as the nitrogen source.²⁵

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met2

Unless otherwise stated, PsbO protein samples for NMR experiments were prepared in 20 mM Na-phosphate buffer, pH 3.6, supplemented with 10% D₂O.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp4

To prove the integrity of the recombinant protein, reconstitution experiments were performed with PS2 particles prepared according to .^{ref. 26}

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met3

These particles were washed with 1 M CaCl₂ to remove the extrinsic proteins before adding the recombinant PsbO protein²⁷.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

NMR spectroscopy

NMR experiments were carried out at different pH and at different temperatures on a Bruker DRX600 spectrometer equipped with a 5 mm BBI probe and a z-axis gradient unit.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp6

Water suppression for 1D and 2D experiments was achieved by the watergate pulse scheme.²⁸

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met4

The 1D ¹H NMR spectrum was recorded with a time domain of 32 k complex points and a sweep width of 12376.24 Hz.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met5

The 2D ¹H-¹⁵N HSQC spectrum was recorded with a time domain of 2 k complex data points with 256 complex increments collected, and a sweep width of 12019.23 Hz in the ¹H and 2128.68 Hz in the ¹⁵N dimension.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met5

Modified versions of the experiment proposed previously were used to determine ¹⁵N{¹H}-NOE at 600.13 MHz proton frequency.^29–36

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met6

The ¹⁵N{¹H}-NOE experiment was recorded using a 4.9 s saturation period and an additional recycle delay of 5 s.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met5

The ¹⁵N{¹H}-NOE was recorded in an interleaved manner to reduce influences from possible instabilities under the chosen experimental conditions.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met5

Data analysis

NMR spectra were processed and analysed with XWINNMR (Bruker, Rheinstetten, Germany), NMRPipe, NMRDraw, and NMRView.³⁷

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

NOE values were calculated from the ratio of the peak heights in the experiment with and without proton saturation.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

Uncertainties for NOE values were determined from triple recording of the whole relaxation experiment.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod2

Albeit no assignments are available, the NOE values were quantified based on the fraction of resolved peaks with NOE values less than 0.5 versus the total number of resolved resonances observed in the experiment.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod3

Results and discussion

Recombinant wild-type PsbO (WT-PsbO)

In this report, we have used NMR-spectroscopy to elucidate preliminary structural and dynamic properties of the 33 kDa protein from the donor side of PS2.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa1

In order to investigate functional aspects of this protein, we have also carried out site-directed mutagenesis experiments.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa3

The PsbO protein from the thermophilic cyanobacterium T. elongatus was overexpressed in E. coli. Its ability to restore the water splitting activity of CaCl₂-washed PS2 was shown by reconstitution experiments.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met7

Routinely, about 50% of the original oxygen-evolving activity was obtained with the reconstituted PS2 (data not shown).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs2

Fig. 1 shows the 1D ¹H NMR spectrum of wild-type PsbO free in solution.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs3

The dispersion of aliphatic and amide proton resonances clearly indicates that the protein is, at least in part, comprised of a well folded core domain.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res1

In order to test the influence of different experimental conditions on the stability of WT-PsbO we recorded several 1D ¹H NMR spectra at different pH values and at different temperatures.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa4

No significant changes in the 1D ¹H NMR spectra were observed for spectra recorded at pH 3.6, 6.5, and 8.0.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res2

Obviously, PsbO retains its fold and is not denatured by lowering the pH from 6.5 to 3.6.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con4

Therefore, we recorded several 1D NMR spectra at 298 K, 310 K, 313 K, and 318 K with a buffer pH adjusted to 3.6.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met5

The line width of these 1D spectra did not change dramatically suggesting that PsbO structure is stable at higher temperatures.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con5

In conclusion, the free thermophilic PsbO in solution is stable at pH 3.6 and 310 K as judged from the signal dispersion in the NMR spectra.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con6

No significant protein degradation was observed even after four weeks.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs4

Fig. 2 shows a 2D ¹H-¹⁵N HSQC spectrum of PsbO isotopically enriched with ¹⁵N at pH 3.6 and 310 K. The 2D ¹H-¹⁵N HSQC spectrum is characterised by a substantial amount of well dipersed resonances indicative for secondary structure elements (especially β-sheet) present in PsbO.^38–40

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs5

However, the 1D ¹H NMR and the 2D ¹H-¹⁵N HSQC spectra also show intense signals at 1–2 ppm and at 8.5 ppm characteristic for unstructured polypeptide chains.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs6

This is consistent with the X-ray structure of PsbO that contains 30% β-sheet and 5% α-helix with several loops involved in binding to the PS2-complex.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

Remarkably, 65% of PsbO do not show any secondary structure elements in the X-ray structure of the PS2 complex.⁶

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac17

This is further corroborated by the ¹⁵N{¹H} NOE of wild-type PsbO.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res4

Out of 109 resolved and dispersed resonances in the ¹⁵N{¹H} NOE experiment, approximately 29 show values less than 0.5 suggesting that these residues are part of unstructured elements of PsbO (data not shown).

Type: Result | Advantage: None | Novelty: None | ConceptID: Res5

Peaks that cluster around 8.5 ppm have been omitted in this preliminary analysis due to spectral overlap.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met5

Based on the ¹⁵N{¹H} NOE, at least an estimated 30% to 40% of all residues of WT-PsbO show increased flexibility on the ps to ns time scale (data not shown).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs7

Nevertheless, this is only in part consistent with the analysis of the B-factors obtained from the X-ray structure where the loops do not show elevated values compared to the core of PsbO.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac18

From this preliminary comparison, it appears that flexible regions of free PsbO are motional restricted when bound to the PS2 complex.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con7

Overall, the 2D ¹H-¹⁵N HSQC spectrum of wild-type PsbO is characteristic for a folded protein which also contains a considerable amount of unstructured parts.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con8

However, the presence of a considerable amount of flexible regions in the free form of PsbO does not explain why PsbO from T. elongatus exhibits a higher thermostability than PsbO from spinach, especially as the content of Pro residues is virtually indentical in both organisms (5.6% for spinach compared to 5.3% for T. elongatus).⁴³

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac19

In conclusion, the structured core should be one of the major reasons for the thermostability of the thermophilic 33 kDa protein where a variety of factors may contribute to this stability as shown for other thermophilic proteins⁴¹.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con9

Site-directed mutagenesis of PsbO

In order to investigate the accessibility and mobility of surface-exposed structural protein elements, we created four mutants containing an additional cystein each at a different position in the amino acid chain of PsbO.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa3

These mutants were analysed by SDS-PAGE in the presence and absence of DTT.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met8

Fig. 3 shows that two of them, N58C and E180C, form dimers whereas T94C and Q196C are monomeric under oxidizing conditions.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs8

The mutations T94C and Q196C are located in the folded β-barrel core of PsbO while the mutations E180C and N58C are part of the extended loops.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac20

Obviously, the folded core region sterically prevents the formation of a dimer while the Cys residues in the loops are easily accessible for interaction.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac21

From these results we conclude, that the regions around residues Asn58 and Glu180 of PsbO are either flexible and/or solvent exposed to allow the formation of a disulfide bond between two PsbO molecules.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con10

Fig. 4 shows 1D ¹H NMR spectra of the oxidised and the reduced form of mutant N58C and E180C.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs9

Interestingly, in our experiments the signal dispersion and the line width in the 1D NMR spectra of the reduced mutant proteins does not dramatically differ from the spectra recorded in the absence of DTT.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs10

Typically, the line width for several selected aliphatic resonances is approximately 24–27 Hz for WT-PsbO, 28–29 Hz for oxidised N58C and 21–22 Hz for reduced N58C, and approximately 24 Hz for oxidised E180C as well as for reduced E180C.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs11

These values indicate that the line width does not significantly change upon reduction of the oxidised mutants of PsbO as a more dramatic change would be expected from the decrease in molecular weight (52 kDa compared to 26 kDa).

Type: Result | Advantage: None | Novelty: None | ConceptID: Res6

This suggests that both PsbO domains of the disulfide linked dimer are folded and tumble, at least to a significant extent, independently free in solution.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con11

From this we conclude that the loops carrying the point mutation exhibit an increased degree of flexibility in the apo-form of PsbO.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con12

Especially, the reduced and the oxidised form of the PsbO E180C mutant show a signal dispersion and line widths in the 1D NMR spectrum that are virtually identical and comparable to the spectra of WT-PsbO.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs12

It is interesting to note that especially the longer loop (carrying the mutation E180C) which is composed of 40 residues does not show any secondary structure elements in the X-ray structure of the PS2 complex from T. elongatus.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac22

This loop does not show elevated B-factors in comparison to the core of the protein which could be explained by its various contacts to other subunits of the PS2 complex, such as D2, CP47, and the 12 kDa-Protein (PsbU).

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac23

Comparing the preliminary results presented in this study with the B-factors for loop regions and the core of PsbO from the X-ray structure, we propose that originally flexible loops of free PsbO, e.g. the longer loop, are structurally better defined after complexation with PS2.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con13

Furthermore, our analysis of the cysteine mutants indicates that the presence of the disulfide bond C19–C44 in PsbO from T. elongatus might not be critical for a stable fold.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con14

It is also in accordance with the observation that removal of the cysteins of PsbO by mutagenesis neither impaired its binding to PS2 nor influenced its water-splitting activity.⁴²

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con14

Further, Fig. 1 shows that the line width of the 1D ¹H NMR spectrum of WT-PsbO is characteristic for a monomeric state of the protein in solution.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs13

This is corroborated by the results from HPLC size exclusion experiments that show a monodispersed peak corresponding to an approximate molecular weight of 20 kDa for the recombinant PsbO (data not shown).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs14

This suggests that WT-PsbO is monomeric in solution.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con15

In summary, we have shown by NMR-spectroscopy that apo-PsbO contains folded structural elements as concluded from the signal dispersion of 1D and 2D NMR spectra.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con16

Our data suggest that PsbO, rather than forming a ‘molten globule’ state or being ‘natively unfolded’, contains both a well folded core and highly flexible regions.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

In addition, the ¹⁵N{¹H}-NOE experiment provides evidence for a significant number of residues that show a higher degree of flexibility (approx.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res7

30–40%).

Type: Result | Advantage: None | Novelty: None | ConceptID: Res7

Spectra of the oxidised and reduced forms of PsbO N58C and PsbO E180C suggest that loops responsible for the interaction with the PS2 complex are flexible and/or solvent exposed when free in solution.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con8

100

Comparing our preliminary results with the B-factors for loop regions and the core of PsbO from the X-ray structure, there is evidence for the compelling hypothesis that originally flexible loops of free PsbO get motional restricted upon binding to PS2.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con7

101

This is further corroborated by the changes of apparent hyperfine splitting and line width of EPR spectra recorded with spin labeled PsbO E180C free in solution and after reconstitution of the PS2 complex (Nowaczyk, unpublished data).

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con7

102

Based on these results we propose that changes in the flexibility between the free and bound form of PsbO, particularly of the loop regions, are responsible for an efficient binding of PsbO to the PS2 core and might play a crucial role in modulating the binding affinity.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con17