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Originally published In Press as doi:10.1074/jbc.M709032200 on November 28, 2007

J. Biol. Chem., Vol. 283, Issue 5, 2962-2972, February 1, 2008
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Functional Role for a Conserved Aspartate in the Spo0E Signature Motif Involved in the Dephosphorylation of the Bacillus subtilis Sporulation Regulator Spo0A*Formula

Alejandra R. Diaz{ddagger}, Sophie Stephenson{ddagger}, J. Michael Green{ddagger}, Vladimir M. Levdikov§, Anthony J. Wilkinson§, and Marta Perego{ddagger}1

From the {ddagger}Department of Molecular and Experimental Medicine, Division of Cellular Biology, The Scripps Research Institute, La Jolla, California 92037 and the §Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, United Kingdom

Received for publication, November 2, 2007 , and in revised form, November 28, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sporulation is a complex developmental system characterizing Gram-positive bacteria of the genus Bacillus and Clostridium. In Bacillus subtilis the phosphorelay signal transduction system regulates the initiation of sporulation by integrating a myriad of positive and negative signals through the action of histidine sensor kinases and aspartyl phosphate phosphatases. The Spo0E family of phosphatases dephosphorylates the Spo0A response regulator and transcription factor of the phosphorelay. In this study we analyzed the role of the Spo0E signature motif in protein activity. This family is characterized by a conserved signature motif centered around the sequence "SQELD." Alanine scanning mutagenesis was carried out on the T35IXXSQ ELDCLI46 residues of B. subtilis Spo0E and in vivo and in vitro activities were analyzed. The ability of the mutant proteins to interact with Spo0A~P was assayed by fluorescence resonance energy transfer spectroscopy. The results suggested that aspartate 43 has a critical role in Spo0E catalytic activity, whereas the other residues have a role in protein conformation and/or interaction with Spo0A. Residues Thr35 and Cys44 did not seem to have any critical functional or structural role. We propose that Asp43 of Spo0E may function in a manner similar to the one proposed for the catalytic mechanisms of nucleotidase members of the haloacid dehalogenase family. These proteins use an aspartyl nucleophile as their common catalytic strategy and the active site of haloacid dehalogenase proteins shares a common geometry and identity of conserved amino acids with the active site of response regulators ( Ridder, I. S., and Dijkstra, B. W. (1999) Biochem. J. 339, 223-226[CrossRef][Medline] [Order article via Infotrieve] ).


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sporulation is a complex development system that characterizes Gram-positive organisms of the genus Bacillus. The mechanism regulating spore formation has been studied in great detail in Bacillus subtilis, thanks to its amenability to genetic manipulation, but many aspects are applicable to other species or even Genus such as Bacillus anthracis and Clostridia (1, 2). The initial step of the sporulation process is regulated by the phosphorelay, a multicomponent signal transduction system of the two-component family (3, 4). In the phosphorelay, activating signals are integrated by 5 histidine sensor kinases (KinA, -B, -C, -D, and -E) whose autophosphorylation on a histidine residue is followed by a transfer of the phosphoryl group to an aspartate residue of the single domain response regulator intermediate Spo0F. From Spo0F, the phosphoryl group is relayed to the Spo0A response regulator through a phosphohistidine of the phosphotransferase Spo0B (Fig. 1) (5-8).

Modulation of the phosphorelay system is a critical aspect of Bacillus physiology because it is relevant to the persistence of the organism in the environment or, in the case of pathogenic organisms, in the host (9). Modulation of the system may occur at different levels by means of positive and negative effectors. Activating signals sensed by the kinases are essential but their identification has so far proved elusive. Nevertheless, the identification of negative regulators of the main sporulation kinase, KinA, and additional experimental evidence indicate that the cell cycle, metabolism, and environmental conditions influence the activity of the histidine kinases (10, 11). Fundamental to proper physiological signal transduction are negative regulatory mechanisms that allow the cell to rapidly respond to changes in environmental or growth conditions. In the sporulation phosphorelay, the Spo0F response regulator is subject to negative regulation by members of the Rap family of phosphatases (RapA, -B, -E, and -H) in response to signals that induce pathways alternative to sporulation, such as growth or competence to DNA transformation (12-14).

An additional negative regulatory mechanism is exerted specifically on the Spo0A protein by the three members of the Spo0E family of phosphatases (Spo0E, YisI, and YnzD). Members of this family were previously shown to be small proteins (ranging between 56 and 85 amino acids) that shared a consensus motif, SQELD, generally preceded by a hydrophobic residue (located two amino acids upstream) and followed by two hydrophobic residues (located one amino acid downstream). A threonine residue immediately preceding the first hydrophobic residue is also invariant in the B. subtilis Spo0E proteins and highly conserved in the Spo0E-like proteins of other Bacilli (Fig. 2A) (15).


Figure 1
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  		FIGURE 1.
Schematic representation of the phosphorelay signal transduction system for sporulation initiation in B. subtilis. Negative regulation exerted by the Rap or Spo0E proteins is indicated by the perpendiculars.

 
The structures of two Spo0E-like proteins of B. anthracis (BA1655 and BA5174) have been solved by NMR spectroscopy. Each is organized in two anti-parallel {alpha}-helices with the conserved signature motif SQELD (SRDLD in BA1655) located in the middle of helix {alpha}2. Notably the residues of the signature motif corresponding to glutamine 40 and aspartate 43 in B. subtilis Spo0E have their side chains projecting outwards from the protein surface with neither forming intramolecular interactions (16).

In this study, alanine mutagenesis was carried out on the signature motif of B. subtilis Spo0E to determine whether any of its residues had critical roles in the phosphatase activity. In addition to genetic and biochemical studies, these mutants were analyzed in vitro by fluorescence resonance energy transfer (FRET)2 spectroscopy to gain information on the interaction of Spo0E with Spo0A.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Growth Conditions—The B. subtilis strain JH12745 (trpC2, phe-1, spo0E::cat) (17) was used throughout this study. B. subtilis strains were grown in Schaeffer sporulation medium (18) supplemented with erythromycin (5 µg/ml).

Escherichia coli DH5{alpha} was used for plasmid construction and propagation. Strains were grown in Luria-Bertani medium supplemented with ampicillin (100 µg/ml) or kanamycin (30 µg/ml).

Sporulation assays were carried out in 5 ml of Schaeffer medium at 37 °C for the times indicated in Table 1. Serial dilutions were plated in duplicate before and after treatment with 0.5 ml of CHCl3. Colony counts were averaged and the percentage of sporulation was calculated as the ratio between the survival and the viable counts.


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  		TABLE 1
Efficiency of sporulation of strain JH12745 overexpressing the wild type and mutant Spo0E proteins

 
Site-directed Mutagenesis of the spo0E Gene—Site-directed mutagenesis of the spo0E gene was carried out by Kunkel's method (19) using the Muta-Gene phagemid in vitro mutagenesis kit (Bio-Rad). The spo0E gene (181 bp of promoter region and 258 bp of coding sequence) was PCR amplified from chromosomal DNA of wild type strain JH642 using oligonucleotide primers OE5'SophPst and OE3'SophBam (supplemental Table 1S). The fragment was digested with PstI and BamHI, and ligated into similarly digested vector pTZ19U (Bio-Rad). The resulting plasmid, pTZ19U-0E, was used to generate the spo0E mutant genes according to the mutagenesis kit instructions (Bio-Rad). After verification by DNA sequencing, the wild type and mutated spo0E genes were then transferred as PstI-BamHI fragments to the multicopy pHT315 vector for genetic analyses (20).

Protein Expression and Purification—The wild type and mutant spo0E coding sequences were PCR amplified from the pTZ19U-0E plasmids using oligonucleotide primers Spo0E5'Nco and Spo0E3'Xho (supplemental materials Table 1S) and cloned in the pET28{alpha} expression vector (Novagen) thus generating a fusion to six histidine codons at the 3' end of the gene. The E. coli expression strain BL21(DE3) pLysS was freshly transformed with the pET28-spo0E wild type or mutant plasmids and single colonies were grown at 37 °C in LB medium containing kanamycin to an A600 of 0.6-0.8. The cultures were then induced with 1 mM isopropyl 1-thio-β-D-galactopyranoside and incubated for an additional 2 h. The cells were harvested by centrifugation and the pellets were frozen until use. The Spo0E proteins were purified essentially as previously described (21). The Spo0A protein (modified to carry a His6 tag at the carboxyl terminal end) and its phosphorylated form (Spo0A~P) were also purified as previously described (21).

Dephosphorylation Assay—Spo0A (5 µM) was phosphorylated in a reaction containing KinA (0.1 µM), Spo0F (2.5 µM), and Spo0B (0.1 µM) in the phosphorelay buffer (50 mM EPPS, pH 8.5, 20 mM MgCl2, 0.1 mM EDTA, 5% glycerol [{gamma}-32P]ATP (at 1.86 µCi/µl final concentration) and 900 µM ATP (22). The reaction was incubated for 45 min at room temperature. The Spo0E proteins were then added (5 µM) and aliquots were withdrawn at intervals as indicated in Fig. 6. The reactions were stopped by the addition of SDS-loading buffer and immediate freezing in a dry ice-ethanol bath. Samples were analyzed by 15% SDS-PAGE followed by PhosphorImager quantitation by the ImageQuant Software (Molecular Dynamics). Dephosphorylation assays using purified Spo0A~P were carried out as described previously (21).

Native Polyacrylamide Gel Electrophoresis—Tris-Tricine native PAGE was carried out according to Schagger and von Jagow (23). The gels were prepared with 3 M Tris-HCl buffer, pH 8.45, containing 5 mM dithiothreitol (DTT). The running buffer was 0.1 M Tris-HCl, 0.1 M Tricine, pH 8.25. Protein molecular weights were determined using molecular weight markers for non-denaturing gel electrophoresis (Sigma) as standards. Calculations were carried out as described in the supplier's instructions.

Size Exclusion Chromatography—A 200-ml Sephacryl S-100 column (120 x 1.5 cm) was equilibrated in buffer A (50 mM Tris-HCl, pH 8.5, 100 mM KCl). Approximately 10 mg of purified Spo0E protein in buffer A containing 5% glycerol was applied to the column. The elution was followed by reading at OD280 and the fractions were analyzed by SDS-PAGE. The molecular weights were determined based on the calibration curve obtained with the molecular weight standards.

Construction, Expression, and Activity of N-Spo0A Cys Mutants—Site-directed mutagenesis of the NH2 terminus of Spo0A from B. subtilis was performed with the QuikChange site-directed mutagenesis kit (Stratagene). Plasmid pET28{alpha} (Novagen) carrying the N-spo0A gene was used as template for the PCR. The N-spo0A gene, which encodes the amino-terminal domain of Spo0A (amino acids 1-128), was PCR amplified using the oligonucleotide primers Spo0A5'Nco and Spo0A3'Xho (supplemental Table 1S). Two single mutants, C7S and C42S, were created using oligonucleotide pairs Spo0AC7S-Spo0AC7Srev and Spo0AC42S-Spo0AC42Srev (supplemental Table 1S). The cysteine mutant proteins were expressed in E. coli strain BL21(DE3) grown at 37 °C in LB medium. The protein purification was carried out in 50 mM NaPO4 buffer, pH 8.0, 300 mM NaCl, 10 mM β-mercaptoethanol, 20 mM imidazole (Buffer A) and phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM. The cells were disrupted by sonication on ice and His6 tag N-Spo0A mutant proteins were purified by Ni-NTA resin column (Qiagen) chromatography as described for the wild type protein (21). The phosphorylation and dephosphorylation activity of N-Spo0AC7S or N-Spo0AC42S were tested as described above.


Figure 2
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  		FIGURE 2.
The Spo0E family of phosphatases. A, amino acid sequence alignment of members of the Spo0E family of B. subtilis (Bs) and B. anthracis (Ba) (13, 16). Alignment was obtained with the program Clustal W. Asterisks below denote invariant residues, whereas colons indicate conserved substitutions. The conserved signature motif is highlighted in gray. The extent of the {alpha} helices, based on the structure of BA5174 (16, 41), is shown above the alignment. B, alignment of the Spo0E conserved signature motif with similar sequences of FliY of B. subtilis (40, 42), and CheZ and FliM of E. coli (28). Identical or conserved residues are highlighted in gray. Numbers refer to the position of the first and last residue shown in the full-length protein (in parentheses).

 
Fluorescent Labeling—The Spo0A and Spo0E proteins were first incubated overnight in their storage buffer supplemented with 1 mM DTT. The DTT was removed prior to the incubation with the Alexa Fluor molecules by using a NAP5 column (Amersham Biosciences) equilibrated with 50 mM sodium phosphate buffer, pH 7 (for Spo0A) or pH 8 (for Spo0E). The proteins were eluted in the same sodium phosphate buffer and concentrated with a Microcon column (Amicon). To completely remove the DTT, the proteins were diluted 20-fold in sodium phosphate buffer and concentrated again. Labeling of N-Spo0A proteins or Spo0E proteins with the maleimide derivative of Alexa Fluor 488 and 555 (Molecular Probes), respectively, was carried out by reacting the proteins with a 3-fold molar excess of dye for 1 h at room temperature in the dark. Unreacted dye was removed by gel-filtration through Sephadex G-25. Labeled protein was separated from unlabeled material by an octyl-Sepharose CL-4B column equilibrated with sodium phosphate buffer (50 mM, pH 7 or 8) containing 1.5 M (NH4)SO4. The protein was then eluted using a step gradient of sodium phosphate buffer containing (NH4)SO4 at 1, 0.5, and 0 M. Labeled protein concentrations were estimated by absorption spectroscopy using the following extinction coefficients for the conjugated dyes: N-Spo0AC42S-Alexa488 or N-Spo0AC7S-Alexa488, 72,000 M-1 cm-1 at 496 nm; Spo0E-Alexa 555, 158,000 M-1 cm-1 at 556 nm.

FRET Measurements—FRET efficiencies were determined from steady-state fluorescence. Fluorescence emission spectra were taken on an SLM Aminco 8100 spectrofluorometer. Emission spectra of donor (N-Spo0AC42S-Alexa488 or N-Spo0AC7S-Alexa488), both in the presence and absence of the acceptor (Spo0E-Alexa 555), were recorded over the wavelength range of 470-520 nm with a spectral band-pass of 8 nm. Samples were temperature controlled at 20 °C. FRET efficiencies were calculated with the formula E = 1 - IDA/ID, in which I is the intensity of the signal in the presence of the donor only (D) or the donor and acceptor (DA).

Binding Assay—All measurements were carried out using 40 nM unphosphorylated or phosphorylated N-Spo0AC42S-Alexa488 or N-Spo0AC7S-Alexa488 in 1 x phosphorelay buffer, pH 8.5, unless otherwise stated. The concentrations of the Spo0E-Alexa 555 used are given in Fig. 7, E and F.

Analysis of Steady-state Fluorescence Titrations—We considered a simple model in which the interaction between N-Spo0A and Spo0E can be treated as a simple 1:1 association, with an apparent equilibrium constant given by: Kapp = [DA]/[D][A]. In this equation [DA] is the equilibrium concentration of the N-Spo0AC42S-Spo0E complex, whereas [D] and [A] are the equilibrium concentrations of free N-Spo0AC42S and Spo0E, respectively (24).

Antibody Production—A polyclonal antibody against Spo0E was obtained by standard procedures after immunization of a rabbit with the purified protein.

Docking Calculations—Protein docking calculations were performed with HADDOCK 2.0 (25) using standard protocols. The NMR structure of BA5174 (Protein Data Bank code 2C0S) and one of the four subunits of the asymmetric unit of the crystal structure of Spo0A~P (PDB code 1QMP) were docked. The Ca2+ ion in the active site of Spo0A~P structure was replaced with a Mg2+ ion and water molecules were omitted from the calculations. The topology parameters for the phosphorylated aspartate in the Spo0A~P structure were obtained using the Dundee PRODRG server (26). Six amino acid residues of Spo0A~P (Asp9, Asp10, Asn11, Asp55, Thr84, Lys106) and three amino acid residues of BA5174 (Ser35, Gln36, Asp39) were considered as potential sources of ambiguous interaction restraints in the docking calculations. The solvent accessibility of these residues was calculated, and those that are exposed at the surface were further selected for ambiguous interaction restraints (ACS). The solutions from the docking calculations were ranked according to their average interaction energies (sum of Eelec, Evdw, EACS) and their average buried surface area (25).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Phenotypic Analysis of Spo0E Mutants—With the objective of measuring the contribution of the conserved signature sequence of the Spo0E family of proteins to their phosphatase activity, we carried out alanine scanning mutagenesis of these residues in B. subtilis Spo0E. Residues Thr35, Ile36, Ser39, Gln40, Glu41, Leu42, Asp43, Cys44, Leu45, and Ile46 were singly mutated to alanine by means of the single-stranded site-directed mutagenesis method (19) (see "Experimental Procedures"). Each allele was cloned in the multicopy shuttle vector pHT315 (20) and transformed into the B. subtilis spo0E mutant strain JH12745 (17). Because overexpression of the wild type spo0E gene results in inhibition of sporulation, any alanine substitution affecting the activity of the Spo0E mutant proteins would result in a less drastic sporulation defect. Liquid sporulation assays were carried out to quantitate the in vivo activity of the Spo0E mutant proteins compared with the wild type. The results shown in Table 1 indicated that whereas alanine substitution at residues Thr35, Glu41, and Cys44 did not affect the activity of the Spo0E protein, the remaining mutant proteins were significantly affected in their ability to inhibit sporulation. These observations suggested a significant role in the structure and/or function of Spo0E for Ile36, Ser39, Gln40, Leu42, Asp43, Leu45, and Ile46.

Biochemical Analysis of the Spo0E Mutants—Because the Spo0E protein inhibits sporulation when overexpressed by dephosphorylating the Spo0A~P response regulator, we purified the wild type and Spo0E mutant proteins to quantitate their activity in an in vitro assay. COOH-terminal His6 tag derivative proteins were purified from overexpressing E. coli strains as described under "Experimental Procedures" and their activity was tested against purified phosphorylated Spo0A protein. The time course of Spo0E-dependent dephosphorylation and autodephosphorylation of Spo0A~P were monitored and the results are shown in Fig. 3. In agreement with the in vivo analysis, the T35A and C44A mutant proteins were not affected in their dephosphorylation activity toward Spo0A~P. Also in agreement with the in vivo data were the observation that the Q40A mutant protein was slightly affected showing a 4-min delay in reducing the amount of Spo0A~P to its basal level. Under the assay conditions used, dephosphorylation of Spo0A~P by the E41A mutant protein was also delayed compared with the wild type protein, even though in vivo it seemed to have wild type activity. The I36A, I46A, and S39A proteins were ~50% less active than the wild type, whereas the L42A protein was slightly less active than the wild type. The D43A protein was essentially inactive in agreement with the lack of activity in vivo. The in vitro activity did not seem to correlate with the in vivo activity only for the Spo0E L45A mutant that, although affected in vivo, was as active as the wild type in vitro.

Western Blot Analysis—To determine whether the in vivo activities of the mutant proteins were affected by protein instability, immunoblot analysis of whole cell lysates of strains expressing the Spo0E mutant proteins were carried out. The results (shown by a representative gel in Fig. 4) indicated that alanine substitutions at residues Thr35, Ile36, Ser39, Gln40, Leu42, and Asp43 give rise to levels of proteins comparable with that in the wild type strain, thus excluding the possibility that any observed phenotype was the result of protein instability. Variability in protein concentrations within several independent analyses is most likely attributable to the low level of Spo0E protein present in the cells and the weakness of the anti-Spo0E antibody. On the contrary, strains expressing the L45A and I46A mutant proteins consistently contained almost undetectable levels of Spo0E. Thus, whereas the substitution of Leu45 with alanine impaired the stability of the protein in vivo but not in vitro, the I46A replacement gave rise to an unstable protein in vivo and an inactive one in vitro.

Oligomerization of Spo0E Wild Type and Mutant Proteins—To determine the oligomeric state of the Spo0E proteins, we carried out analyses by native gel electrophoresis, size exclusion chromatography, and mass spectrometry. Native gel electrophoresis was carried out at four different concentrations of polyacrylamide (12, 13, 14, and 15%) with Tris-Tricine buffer (see "Experimental Procedures") in the presence of 5 mM DTT to minimize the oxidation of the cysteine residue at position 44. The results revealed that the Spo0E wild type protein separated into three major bands whose molecular masses were calculated to be 22, 48, and 66 kDa (supplemental materials Fig. S1).

To obtain precise molecular weights of the Spo0E forms, analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry was carried out. The wild type protein sample showed three peaks with masses of 10,728, 21,454, and 32,218 daltons, consistent with monomer, dimer, and trimer forms (supplemental materials Fig. S2). These values are also consistent with the predicted molecular weight if removal of the initial methionine is taken into account. Taking into account the possibility of a molecular size overestimation associated with the non-denaturing gel electrophoresis method, the results are consistent with a possible organization of Spo0E as monomeric, dimeric, or trimeric forms with the former being the most abundant form (Fig. 5A).


Figure 3
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  		FIGURE 3.
Dephosphorylation of Spo0A~P by Spo0E proteins. Purified, wild type Spo0A~P(5 µM) was incubated in the absence or presence of 2 µM Spo0E proteins and aliquots were withdrawn at the indicated times. Samples were run on a 15% SDS-PAGE and analyzed by the ImageQuant software after exposure to a PhosphorImager plate (Amersham Biosciences). The remaining Spo0A~P was calculated as a percentage of the value measured at the zero time point.

 


Figure 4
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  		FIGURE 4.
Western blot analysis of B. subtilis strains expressing Spo0E wild type and mutant proteins. Total cell lysates of strain JH12745 ({Delta}spo0E, cat) expressing the wild type or mutant spo0E genes from plasmid pHT315 were run on 15% SDS-PAGE and the Spo0E proteins were detected with an anti-Spo0E polyclonal antibody. Lane 1, Spo0E wild type; lane 4, Spo0ET35A; lane 5, Spo0EI36A; lane 6, Spo0ES39A; lane 7, Spo0EQ40A; lane 8, Spo0EE41A; lane 9, Spo0EL42A; lane 10, Spo0ED43A; lane 11, Spo0EL45A; lane 12, Spo0EI46A. Lane 2 is the negative control strain JH12745 carrying the vector alone and lane 3 is 2 pg of purified Spo0EC44A protein as molecular weight standard. Cells were grown in Schaeffer medium to A525 = 2.1 and visualized by immunoblotting as described under "Experimental Procedures." The gel is representative of several independent experiments.

 
All the alanine mutant proteins generated in this study showed the same oligomerization pattern as wild type Spo0E with the exception of the C44A mutant protein whose major species was the monomer with only a minor presence of the dimeric form (Fig. 5A). These data, together with the in vivo and in vitro analyses, suggest that, because the alanine substitution of the cysteine residue does not impair the dephosphorylation of Spo0A, the monomeric form of Spo0E may be sufficient for activity although a dimer can be formed even in the absence of the cysteine. The trimer form may be an artifact due to oxidation of the cysteine residue in vitro.

To determine whether the oligomeric forms of the Spo0E protein were stable or interconverting, we purified the Spo0E wild type protein by gel filtration chromatography (supplemental materials Fig. S3). Standards of known molecular weights were used to estimate the size of the protein fractions. The subunit molecular weight of His tag-modified Spo0E is 10,856. By Sephacryl S-100 chromatography, the Spo0E wild type protein eluted in a first, broad peak of an estimated molecular mass of 42 kDa likely corresponding to a trimer. Two additional sharp peaks with estimated molecular masses of 28 and 14.8 kDa, corresponding to the dimer and monomer forms, respectively, also eluted from the column. When the three fractions were analyzed by native gel electrophoresis, the monomer form of the third fraction was essentially pure, whereas the dimer of the second fraction contained ~20% of the bigger oligomer but no monomeric protein, arguing against the possibility of a rapid monomer-dimer equilibrium. The first fraction contained ~30% of dimeric and 70% of trimeric protein (Fig. 5B).

In Vitro Activity of the Spo0E Oligomers—To assess whether the oligomeric forms of Spo0E separated by gel filtration were active, we tested their ability to dephosphorylate Spo0A~P. The results shown in Fig. 6 indicated that a rate of dephosphorylation comparable with the wild type Spo0E was reached by the purified monomeric form as well as the dimeric form, whereas the third fraction (mixture of dimer and trimer) was significantly less active. This suggests that the monomer and dimer forms of Spo0E are equally active, the trimer may be an inactive form, most likely the result of a purification artifact.

FRET Analysis of Spo0A/Spo0E Interaction—We took advantage of the FRET spectroscopy technique to obtain direct information on the molecular interaction between Spo0A and Spo0E. Our mutagenesis data implicated the "SQELD" signature motif in Spo0E-catalyzed dephosphorylation of Spo0A~P. By FRET we aimed at determining whether alanine substitutions of this sequence affected the interaction of Spo0E with its substrate. The presence in Spo0E of a cysteine residue near the signature motif allowed us to label the wild type and mutant proteins with the fluorescent dye Alexa Fluor 555. All FRET studies were carried out with the monomeric forms of the Spo0E proteins purified by size exclusion chromatography. The amino-terminal domain of Spo0A was used instead of the full-length protein to avoid possible interference on the Spo0A/Spo0E interaction by the Spo0A carboxyl-terminal domain. Because N-Spo0A contains two cysteine residues (Cys7 and Cys42) we mutated each one to a serine residue and tested the activity of each mutant protein in vitro. Both proteins were as active as the wild type N-Spo0A protein in being phosphorylated by the phosphorelay reactions and in being dephosphorylated by wild type Spo0E (data not shown).


Figure 5
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  		FIGURE 5.
Native gel analysis of Spo0E wild type and mutant proteins. Proteins were run on non-denaturing Tris-Tricine 15% polyacrylamide gels and stained with Coomassie stain. A, native gel of Spo0E wild type and alanine mutant proteins (8 µg) after purification by Ni-NTA chromatography. B, the Spo0E wild type protein was analyzed before (WT) and after separation by gel filtration chromatography. The separated monomer (M), dimer (D), and mixture of dimer and trimer (D/T) are shown. {alpha}LA, {alpha} lactalbumin; CA, carbonic anhydrase; CEA, chicken egg albumin; BSA, bovine serum albumin.

 


Figure 6
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  		FIGURE 6.
Dephosphorylation rates of Spo0A~P by Spo0E proteins fractionated by size exclusion chromatography. The Spo0E wild type protein after purification by Ni-NTA chromatography and before gel filtration (WT, {square}) or the fractioned monomer ({blacktriangleup}), dimer ({diamond}), or mixture of dimer/trimer (*) proteins at 5 µM were incubated with purified Spo0A~P(5 µM) and time points were withdrawn at the indicated times. The zero time point of the control reaction containing Spo0A~P with only Spo0E storage buffer ({diamondsuit}) was used as the 100% value to calculate the percentage of remaining Spo0A~P.

 
The phosphorylated N-Spo0AC7S and N-Spo0AC42S proteins were labeled with the Alexa Fluor 488 donor fluorophore and the emission spectrum of a 40 nM sample was recorded using an excitation wavelength of 470 nm in the presence and absence of wild type Spo0E labeled with the acceptor fluorophore (Fig. 7A). The fluorescence intensity of the Spo0AC42S-phosphorylated donor decreased significantly after the addition of the Spo0E acceptor, indicating that the probes were sufficiently close (<105 Å) for fluorescence energy transfer to occur. The transfer efficiency, E, was calculated to be 0.29 ± 0.03.

When the phosphorylated Spo0AC7S protein was used as the fluorescence donor, no fluorescence energy transfer was detected in the presence of the Spo0E acceptor indicating either that Cys42 of Spo0A and the Cys44 of Spo0E were not within the distance range for energy transfer to occur (>105 Å) (data not shown) or that the fluorophore affected protein conformation.

Notably, no fluorescence energy transfer was detected between the fluorescently labeled but unphosphorylated N-Spo0AC42S and the Spo0E protein indicating that phosphorylation of the response regulator is a requirement for effective interaction (Fig. 7B).

FRET Analysis of Spo0A Interaction with the Spo0E Alanine Mutants—To determine whether the Spo0E proteins with alanine substitutions analyzed in vivo and in vitro were affected in their ability to interact with Spo0A, we labeled each mutant (with the exception of C44A) with the Alexa 555 acceptor fluorophore and measured the fluorescence intensity after addition to the phosphorylated N-Spo0AC42S-Alexa 488 donor. As shown in Fig. 7, C and D, we detected FRET when the donor was in the presence of the labeled Spo0ET35A and Spo0ED43A mutant proteins. The efficiency of FRET was in the order of 0.14 ± 0.04 and 0.17 ± 0.05, respectively. These results suggest that the alanine substitutions at residue Thr35 or Asp43 did not significantly affect the interaction detectable by FRET. However, whereas the T35A mutant was essentially as active as the wild type in vivo and in vitro, the activity of the D43A protein was severely affected in both assays (Table 1 and Fig. 3E).


Figure 7
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  		FIGURE 7.
FRET analysis of Spo0A~P/Spo0E interaction measured by steady-state fluorescence. Emission spectra of 40 nM Spo0AC42S~P labeled with Alexa Fluor 488 at residue Cys7 was recorded after the addition of 40 nM Spo0Ewt labeled with Alexa Fluor 555 at the Cys44 residue; spectra obtained with phosphorylated Spo0AC42S are in A, whereas the ones obtained with unphosphorylated response regulator are in B. Emission spectra of labeled Spo0AC42S~P and labeled mutant Spo0ET35A and D43A are in panels C and D, respectively. Emission spectra of labeled Spo0AC42S~P titrated with different concentrations of Spo0Ewt are reported in panel E (for clarity only the data relative to 6 of 12 concentrations of Spo0E are shown). The decrease in fluorescence upon addition of increasing concentrations of Spo0E was monitored with excitation and emission wavelengths set to 470 and 520 nm, respectively. The fitted curve (obtained using the equation described under "Experimental Procedures" and the data from the titrations analysis) corresponding to the apparent binding constant (Kapp) is shown in F. The schematic was obtained using the GraphPad Prism program and is representative of three independent experiments used to calculate the average Kapp and Kd.

 
All the remaining Spo0E alanine mutants (Ile36, Ser39, Gln40, Leu42, Leu45, and Ile46) did not generate any energy transfer with N-Spo0A most likely because the proteins were not interacting or their interaction was too rapid to detect and thus the distance or orientation between the fluorophores on Cys44 of Spo0E and Cys7 of Spo0A was not optimal for energy transfer to occur. Two of the mutant proteins, Q40A and E41A, were delayed in dephosphorylation activity in vitro, which may account for the lack of detectable energy transfer from Spo0A. Similarly, the initial rates of I36A, S39A, L42A, and I46A as well as their overall efficiency both in vivo and in vitro, were reduced compared with the wild type protein, consistent with lack of efficient interaction required for FRET measurements. These observations suggest that conformational changes occurred as a result of the amino acid substitutions in all mutant proteins except the T35A and D43A. The reason why the L45A mutant, which is affected in vivo but not in vitro, did not interact with Spo0A sufficiently enough to generate transfer of energy is probably most strongly related to structural changes.


Figure 8
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  		FIGURE 8.
Structural model of Spo0A/Spo0E interaction by the HADDOCK program. A, overview of the BA5174-Spo0A complex. Polar intermolecular interactions are shown. In green is shown the starting BA5174 structure prior to docking. This structure has been superposed onto the modeled BA5174. A degree of bending on the second helix is apparent. Side chains of residues contributing to the hydrophobic core of BA5174 are shown in yellow space-filling format. B, polar interaction network in the active site region of the BA5174-Spo0A complex.

 
Determination of the Apparent N-Spo0A:Spo0E Binding Constant—To determine the apparent affinity (Kapp) of N-Spo0AC42S for Spo0E, the decrease in fluorescence intensity was monitored after the addition of acceptor-labeled molecules to the donor fluorophore-labeled protein. The N-Spo0AC42S-Alexa 488 (40 nM) was titrated with different concentrations of Spo0E-Alexa 555 after normalizing the signal intensity with unlabeled Spo0E protein. The donor signal due to FRET was calculated by taking the FDA/FD ratio and then normalizing the result. We analyzed these titrations according to the apparent 1:1 binding model as described under "Experimental Procedures."

The efficiency of energy transfer (E), calculated as described under "Experimental Procedures, " increased hyperbolically with increasing concentrations of Spo0E, with an apparent binding constant Kapp of 3.3 ± 0.3 x 107 M-1 and a Kd of 3.0 ± 0.27 x 10-8 M. These values suggest a tight interaction between Spo0E and the phosphorylated form of Spo0A.

Docking Calculations of the BA5174 Aspartic Acid Phosphatase to the Spo0A Sporulation Regulator—To predict the mode of interaction of Spo0E and Spo0A~P, molecular modeling of the complex formed between the Spo0E orthologue from B. anthracis BA5174, and the receiver domain of Spo0A~P from Bacillus stearothermophilus was performed using the docking program HADDOCK 2.0 (25). In this protein-protein docking procedure, we included interaction information derived from the alanine scanning mutagenesis of Spo0E that highlighted the importance of conserved residues in the Spo0E signature motif for interactions with Spo0A. The active residues used in the definition of the ambiguous interaction restraints for docking consisted of Ser35, Gln36, and Asp39 from BA5174 (corresponding to Ser39, Gln40, and Asp43, respectively, of Spo0E) and Asp9, Asp10, Asn11, Asp55, Thr84, and Lys106 from Spo0A.

BA5174 and Spo0A~P in the highest scoring modeled complex maintained their overall folds. The juxtaposition of the BA5174 and Spo0A~P molecules is reminiscent of that of (i) CheZ and CheY (PDB code 1KMI) and (ii) Spo0B and Spo0F (PDB code 2FTK) in their complexes (supplemental Fig. S4) (27, 28). Protein association is accompanied by formation of a polar interaction between the Asp39 carboxylate of BA5174 and the side chain amide of Asn11. The Asp39 carboxylate forms a ligand of the Mg2+ ion that additionally coordinates the phosphoryl group of Asp56 and the carboxylates of Asp9 and Asp10 (Fig. 8). In the HADDOCK generated model, Gln36 of BA5174 forms a hydrogen bond with Lys106 of Spo0A, which in turn forms an ionic interaction with the Asp56 phosphoryl group. Flanking the active sites of the respective molecules in the complex are interactions between the His29 imidazole of BA5174 and the main chain carbonyl of Phe108 of Spo0A and between the side chains of Asn43 of BA5174 and Asp10 of Spo0A (Fig. 8A).

The functional importance of Leu38 for BA5174 activity is reflected in the model by its contribution to the hydrophobic core of the two-helix BA5174 structure. The essential function of Ser35 for BA5174 activity is less obvious from the structure because this residue is not in the putative interface. One explanation would invoke helix bending. The capacity of the Ser35 hydroxyl to hydrogen bond to the main chain carbonyl oxygen of Val31 could compensate for loss of intrahelical main chain hydrogen bonding were the helix to bend (29). Such an influence on the helix conformation may facilitate the extension of the Gln36 and Asp39 side chains into the active site cleft of Spo0A.

The modeling studies of the complex reconcile structural data on the respective partner proteins and data derived from functional analysis. The model is, nevertheless, hypothetical and subject to refinement as further experimental data emerge.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this work, we analyzed the role of the conserved signature motif of Spo0E-like proteins by means of alanine-scanning mutagenesis followed by genetic and biochemical analyses as well as FRET. We have identified residues in the signature motif of the B. subtilis Spo0E phosphatase (T35IXXSQELDCLI46) that are involved in specific interaction with the Spo0A~P response regulator or in the phosphatase catalytic activity. Other residues appear to be necessary for protein structure and stability.


Figure 9
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  		FIGURE 9.
Comparison of the active site of Spo0A~P and dNT-2, a mitochondrial deoxyribonucleotidase of the HAD family (37). Nitrogen atoms are blue, oxygen atoms are red, and carbon atoms are gray. The phosphorus is in yellow, the Ca+ bound to Spo0A~Pisin green, whereas the Mg2+ in dNT-2 is in cyan. Hydrogen bonds are shown as gray lines. The figure was made with University of California, San Francisco, Chimera (43) using the coordinates 1DZ3 (Spo0A) (44) and 1MH9 (dNT-2) (37).

 
The recently determined NMR structures of two members of the Spo0E family from B. anthracis, BA1655 and BA5174 (16), have shown that each protein is composed of two anti-parallel {alpha}-helices flanked by flexible regions at the NH2 and COOH termini. The signature motif SQELD, which is actually SRDLD in BA1655, is situated in the middle of helix {alpha}2 with the residues corresponding to Gln40 and Asp43 of Spo0E projecting their side chains into the solvent and therefore potential mediators of protein-protein interactions. The side chain of the residue corresponding to Glu41 also projects outward from the molecule but it forms a salt bridge with the residue corresponding to Lys17 in helix {alpha}1 in 50% of the ensemble of NMR structures. Our alanine mutagenesis studies indicated that although the Q40A and E41A substitutions similarly slowed Spo0E-catalyzed dephosphorylation of Spo0A~P in vitro, Q40A had some effect in vivo, whereas E41A had no impact in vivo. In contrast, the D43A substitution completely inactivated the protein both in vivo and in vitro. Because all three proteins were expressed at essentially the same level as the wild type protein and each behaved similarly to the wild type protein in native gel electrophoresis analyses, we interpret the results to mean that Gln40 and Asp43 have a role in Spo0A~P binding and/or catalysis of the dephosphorylation reaction. The FRET analysis suggested that the interaction with the substrate was affected in the Q40A and E41A mutant proteins, this interaction was not affected by the D43A substitution. Thus Asp43 appears to be critical for catalysis of the dephosphorylation reaction, whereas the Gln40 and perhaps Glu41 are more important in protein:protein recognition and binding.

The results obtained with the D43A substitution in Spo0E are reminiscent of the effect of alanine substitution of Gln147 of CheZ, which dephosphorylates the chemotaxis response regulator in E. coli, CheY (28). CheY and Spo0A share a common three-dimensional structure and active site stereo-chemistry (supplemental materials Fig. S4) (30, 31). The active site contains the conserved phosphorylatable aspartate located at the COOH-terminal end of β-strand 3 in an acidic pocket that contains two other conserved aspartate residues, a conserved lysine, and a conserved threonine residue. The Q147A mutant of CheZ binds CheY~P, albeit with moderate affinity, but it is deficient in phosphatase activity. The co-crystal structure of CheZ and Formula (a phosphoryl group mimicked in proteins phosphorylated on aspartate)-activated CheY showed that the side chain of Gln147 is inserted into the active site of CheY where it forms a coordinate bond to the Mg2+ ion and makes van der Waals interactions with both Phe14 of CheY and the Formula species. Based on these observations, a role for Gln147 in catalysis was proposed in which it contributes to the positioning of the water molecule for nucleophilic attack (28). Molecular modeling of the Spo0E-Spo0A~P interaction in the program HADDOCK (Fig. 8) suggests that the Asp39 residue of BA5174 (Asp43 is Spo0E) may coordinate the Mg2+ ion, thus extending the analogy to the Gln147 residue of CheZ. In this view, the Asp43 residue of Spo0E could have a similar catalytic role in the dephosphorylation of Spo0A~P.

Interestingly, the basic geometry of the response regulator active site, including the identity of the conserved amino acids (3 aspartates, lysine, and threonine) is shared by the HAD (haloacid dehalogenase) superfamily of proteins, which includes a range of dehalogenases, nucleotidases, phosphatases, and phosphomutases (Fig. 9) (32, 33). A shared characteristic of HAD superfamily enzyme action is a nucleophilic aspartate residue. Structural studies using Formula, have provided evidence for a phosphoaspartate intermediate in members of the HAD family that catalyze dephosphorylation or phosphotransfer reactions (MjPSP, β-phosphoglucomutase, P-type ATPases, 5' nucleotidases such as dNT-2) (34-37). In this subgroup of the family, a fourth aspartate residue is present in the active site (Asp43, Asp51, or Asp13 in dNT-2, pyrimidine 5' nucleotidase P5N-1, and MjPSP, respectively). In dNT-2, for example, the additional aspartate is Asp43 (Fig. 9) with Asp41 corresponding to the phosphorylatable Asp55 of Spo0A and with Asp175 and Asp176 arranged similarly to Asp9 and Asp10 in this response regulator. This additional aspartate may have a bifunctional role in the reaction acting as a general acid for the protonation of the leaving group of the substrate and a general base for the activation of the nucleophilic water molecule (37). Given the critical catalytic role of Asp43 in Spo0E, it is tempting to propose a functional role for this residue similar to the one assigned to the Asp43 or Asp51 residues of dNT-2 and P5N-1, respectively.

We have previously identified four residues in Spo0A from B. subtilis peripheral to the active site but critical for its dephosphorylation by Spo0E. These residues, Asn12, Pro60, Leu62, and Phe88 are conserved in Spo0A proteins from different species (B. anthracis, Bacillus thuringiensis, B. cereus, and B. strearothermophilus), are surface exposed, and are located on the β1-{alpha}1, β3-{alpha}3, and β4-{alpha}4 loops of Spo0A surrounding the active site (21). In the BA5174/Spo0A~P model constructed using the HADDOCK program, these loops are at the molecular interface. Asn12 (Asn11 in B. stearothermophilus Spo0A~P) is involved in interactions with Asp39 (Asp43 of Spo0E), Pro59 (Pro60) contributes to the protein:protein interface, and the other two residues are adjacent to it. The model places the key catalytic residues corresponding to Gln40 and Asp43 in close proximity to the phosphorylated aspartate residue of the response regulator. The model is tentative, however, because water molecules were not included in the docking calculations and the presence of a metal ion adds additional complexity to the interface. The model cannot therefore be used to discriminate between alternative catalytic mechanisms in which water and metal coordination are integral elements. Instead it is consistent with mechanisms that invoke catalytic functions for either Gln36 (Gln40 in Spo0E), which could adopt a conformation that stabilizes a water molecule for nucleophilic attack, or Asp39 (Asp43 in Spo0E), which could act as a general base in the reaction. In either view, aspartyl phosphate phosphatases have evolved to complete the active site of their response regulator substrate so as to augment the rate of their autodephosphorylation reactions. In doing so a further regulatable check point in two-component signaling is introduced.

The residues in BA1655 and BA5174 that correspond to the Spo0E residues Leu42, Leu45, and Ile46 contribute to the close packing of the two helices of the protein. Thus the reduced activity and instability in vivo of the L45A and I46A mutant proteins may be explained by loss of structural integrity. The Ser35 side chain hydroxyl of BA1655 and BA5174 also forms a hydrogen bond with the main chain carbonyl oxygen of Val31 (Thr35 in Spo0E) raising the possibility that the corresponding S39A mutation, which also has drastic effects in vivo, affects the conformation and thus the activity of the protein. A strong effect in vivo and reduced activity in vitro was observed with the I36A mutant protein. The side chains of the corresponding Ile32 residue of BA5174 (monomer) or Leu32 of BA1655 (dimer) project outward from the face of the {alpha}2 helix; this face of the helix is distal to helix {alpha}1 in the monomer and also from the interface with the other molecule in the dimer, arguing against a structural role for this residue. Furthermore, the Spo0E I36A mutant protein did not interact with Spo0A~P in the FRET assay, suggesting a possible role for Ile36 in the interaction with Spo0A~P. In support of this idea, the corresponding Ile28 residue of BA5174 makes van der Waals contacts with residues 106-108 of Spo0A~P in the modeled complex.

The use of the FRET technique has provided useful information on the possible mechanism of Spo0E interaction with Spo0A. The results have shown that an interaction sufficiently close to generate energy transfer occurs only with the phosphorylated form of Spo0A; as inferred from the apparent affinity constant (Kapp 3.3 x 107M-1 and Kd 3 x 10-8M) the binding is relatively tight. This is reminiscent of the stable complex formed by the RapA phosphatase with the phosphorylated form of Spo0F that is detectable by native gel electrophoresis, whereas the unphosphorylated response regulator does not interact with the phosphatase, at least not strongly enough to be detected by the native gel assay (38).

The results of the FRET analysis also indicated that the T35A and D43A substitutions had little or no effect on the protein conformation and the spatial relationship of Spo0E with Spo0A. In contrast, all the other alanine substitutions resulted in loss of FRET signal. The absence of signal is likely to reflect loss or instability of the interaction between the Spo0E mutants (Ile36, Ser39, Gln40, Leu42, Leu45, and Ile46) and the Spo0A~P receiver domain.

Our in vitro data indicated that the active Spo0E protein can be in either the monomer or dimer form; in contrast, for the structurally characterized B. anthracis orthologues, BA5174 was a monomer, whereas BA1655 was a dimer. The dimer formed by BA1655 resembles the four-helix bundle of the Spo0B phosphotransferase with the distinction that the former is in head-to-tail apposition, whereas the latter is in head-to-head orientation (43). A long four-helix bundle composed of two {alpha} helices from each subunit in the dimer is also the structural feature of CheZ that dephosphorylates the chemotaxis response regulator CheY (44). These observations suggest the possibility that the Spo0E-like proteins, independently of their quaternary conformation, may interact with their target Spo0A~P in a manner similar to Spo0B interaction with Spo0F or CheZ interaction with CheY. The molecular modeling carried out with the HADDOCK program supports this suggestion (supplemental materials Fig. S4). The response regulator surface involved in this interaction is defined by helix {alpha}1 and all five β-{alpha} loops surrounding the active site. In agreement with this assumption, the previously mentioned mutations in Spo0A that resulted in resistance to Spo0E dephosphorylation mapped in the β-{alpha} loops (β-{alpha} loops 1, 3, and 4) (21).

The possibility that Spo0E may interact with a different surface of Spo0A was raised by the observation that the signature motif SQELD strongly resembles the sequence of the NH2-terminal peptide of FliM shown to interact with the chemotactic regulator CheY (Fig. 2B). The crystal structure of the BeF3-activated CheY bound to a 16-residue N-terminal peptide of FliM showed that the peptide binds to the {alpha}4-β5-{alpha}5 face of the response regulator with few, if any, interactions with residues in the β-{alpha} loops. Part of the N16-FliM sequence "LSQNEIDALL" is strikingly similar to a sequence in the COOH terminus of CheZ, which is also shown to interact with CheY at the {alpha}4-β5-{alpha}5 face of the molecule (supplemental materials Fig. S4) (28, 39). A similarly conserved sequence is present in the NH2-terminal region of FliY (40), which is also a phosphatase of CheY in B. subtilis.

The SQELD motif in the Spo0E-like proteins occurs in a helical region of the structure. As a result, the additional residue, inserted between Gln and Glu, in CheZ, FliM, and FliY would be expected to have a significant effect on the spatial arrangement of the associated side chains, arguing against structural and functional analogies. Similarly, the fact that no mutants were identified in the {alpha}4-β5-{alpha}5 face of Spo0A that resulted in a Spo0E-resistant response regulator, argue in favor of our view of a Spo0A:Spo0E binding interface most likely similar to the one in the Spo0F-Spo0B or the CheY-CheZ complexes involving the β-{alpha} loops.


   FOOTNOTES
 
* This work was supported in part by NIGMS, National Institutes of Health Grant GM55594 (to M. P.), the Wellcome Trust, and European Union Grant SPINE2-Complexes C-T-2006-031220 (to A. J. W.). DNA sequencing reactions and oligonucleotide synthesis was supported in part by the Stein Beneficial Trust. This is manuscript number 18946 from The Scripps Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and Figs. S1-S4. Back

1 To whom correspondence should be addressed: 10550 N. Torrey Pines Rd., MEM-116, La Jolla, CA 92037. Tel.: 858-784-7912; Fax: 858-784-7966; E-mail: mperego{at}scripps.edu.

2 The abbreviations used are: FRET, fluorescence resonance energy transfer; EPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid; HAD, haloacid dehalogenase; DTT, dithiothreitol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Ni-NTA, nickel-nitrilotriacetic acid. Back


   ACKNOWLEDGMENTS
 
We thank Stephanie Pond and J. C. Van der Schans in the laboratory of Dr. David Millar, The Scripps Research Institute, for help in developing the FRET analysis.



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 ABSTRACT
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 RESULTS
 DISCUSSION
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