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1 Departments of Molecular Biosciences and 2 Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66045, USA
Reprint requests to: William D. Picking, Division of Biology, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA; e-mail: picking{at}ku.edu; fax: (785) 864-5294.
(RECEIVED July 27, 2005; FINAL REVISION November 9, 2005; ACCEPTED December 9, 2005)
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Abstract |
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G°0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m-values obtained for the urea-induced unfolding of the proteins suggest that they undergo only a small change in solvent-accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein–protein interactions. Keywords: type III secretion; MxiH; Shigella; needle protein; protein stability; protein structure/folding; stability and mutagenesis; structure/function studies; other spectroscopies; circular dichroism; fluorescence
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051733506.
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Introduction |
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The needle of S. flexneri is composed of multiple copies of a single protein called MxiH (Allaoui et al. 1992; Hueck 1998; Blocker et al. 2001; Jouihri et al. 2003). The related gastrointestinal pathogen Salmonella typhimurium possesses a similar TTSS, which has an external needle composed of multiple copies of the protein PrgI (Kimbrough and Miller 2000; Kubori et al. 2000). The exposed portion of the TTSS of Shigella and Salmonella is possibly involved in sensing the approach of the pathogen to a target cell and triggering the secretion of the effector proteins that subvert normal cell processes (Kenjale et al. 2005). Thus, these needles and their component proteins are attractive candidates for antigens that might induce a protective immune response against these important pathogens. It is therefore important to fully understand the physical, biochemical, and biological properties of these potential vaccine candidates.
Recently, Blocker et al. (2003; Cordes et al. 2003) described the helical packing of the Shigella needle and found it to be similar to that of the Gram-negative bacterial flagellar filament, despite the fact that, at 83 amino acids, it is much smaller than flagellin. Like the polymerizing core of flagellin, MxiH is predicted to have a secondary structure composed largely of 
-helix with a minor 
-strand component and extensive turns (Kenjale et al. 2005). Furthermore, if the flagellin analogy is appropriate, it is likely that deleting a portion of the C terminus of the needle proteins from various TTSSs should allow them to be purified in monomeric form for biophysical and biochemical study (Yonekura et al. 2003).
The Shigella and Salmonella needle proteins have a high degree of sequence similarity (Fig. 1
) and are predicted to have similar secondary structures. In this study, we describe the purification of monomeric forms of these proteins after removal of their five C-terminal amino acid residues. In contrast, the full-length proteins appear to polymerize and aggregate when expressed in Escherichia coli (data not shown). The soluble 5 variants provide excellent candidates for structural studies. Thus, we provide the first biophysical characterization of the structure and stability of needle protein monomers from Shigella and Salmonella in this work. The reversibility of the folding/unfolding of the two proteins allowed us to use spectroscopic and calorimetric methods to also thermodynamically characterize their stability properties. In this form, they are also strong candidates for high-resolution structure determination.
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) show that they have a similar secondary structure with high helical content. Deconvolution and analysis of the spectra using Dichroweb (Whitmore and Wallace 2004) suggests that that PrgI contains more helix than MxiH (Table 1). At 10°C, the recombinantly expressed MxiH has a little >50% -helical content and ~20% -sheet, whereas PrgI has almost 70%-helix and ~10%-sheet. Additionally, MxiH appears to have more random and turn structure than its Salmonella homolog. At temperatures as low as 25°C, both proteins seem to lose a significant amount of secondary structure, especially with respect to helical content (Table 1). This indicates that both proteins may be relatively unstable (see below).
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) and UV absorbance spectroscopy (Figs. 4, 5). Analysis of the CD unfolding curves in Figure 3 yield an approximate Tm of 42°C and 37.5°C for MxiH and PrgI, respectively. Correspondingly, nearly indistinguishable H values of 22.3 and 23.9 kcal/mol are seen.
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, 5). The three Phe and the highest wavelength of the Trp peaks show little evidence for well-defined thermal transitions for both proteins. In contrast, Tyr peaks manifest typical thermal unfolding curves. Assuming a two-state unfolding model, the Tyr profiles yielded Tm and corresponding H values that are similar, within error limits, to those observed in the CD studies (Table 2).
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Urea-induced unfolding as monitored by CD
To further access the stability of these proteins, urea unfolding studies were performed using the intrinsic CD signal of the proteins at 222 nm to monitor changes in secondary structure as a function of urea concentration. The unfolding curves, as seen in Figure 6, show some degree of cooperativity (10°C) (Fig. 6A1,A2), which diminishes with increasing temperature (25°C) (Fig. 6B1,B2). The hyperbolic nature of the unfolding curves at 25°C, as opposed to the more sigmoidal curve observed in the lower temperature experiments, indicates a loss in cooperativity. From the urea unfolding data, the intrinsic free energy of unfolding (G°0,un) and their dependence on denaturant concentration (m-values) of the proteins were calculated using a nonlinear least-square analyses for a two-state unfolding transition. These results are reported in Table 3. The results show that MxiH is slightly more stable than PrgI and also has a higher m-value, indicating that more surface area of MxiH is exposed during unfolding than during PrgI unfolding.
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) show only very subtle differences with 
max values of 349.5 and 347.5 nm, respectively. These values indicate extensive exposure of the proteins’s single Trp residue to the solvent. This is consistent with the position of the 290 nm absorption peak (peak 6 in Figs. 4, 5). Both techniques suggest that the MxiH indole is slightly more exposed than the corresponding residue in PrgI.
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) shows a linear change in Trp fluorescence with increased urea concentration, with the slope of the curves at 0.04 and 0.07 M–1 for MxiH and PrgI, respectively. Model studies with the tryptophan derivative, N-acetyltryptophanamide (NATA), find that there is a linear dependence of indole fluorescence on urea concentration with a slope of 0.07M–1 (Eftink 1994). Thus, our results suggest that the urea-induced changes seen here are simply due to direct effects of urea on the MxiH and the PrgI indole side chains. Fluorescence based thermal unfolding studies show a curvilinear dependence of Trp fluorescence on temperature (Fig. 9). Furthermore, the emission peak position does not change with temperature, supporting the conclusion that the Trp environment does not significantly change.
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H of 27.9 kcal/mol, in excellent agreement with the CD and absorbance results (Fig. 10). Surprisingly, for reasons that are unknown at this time, we were unable to obtain a well-defined DSC endotherm for MxiH, even at high protein concentrations.
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-helix (>50%) with less than a quarter of the structure in -sheet (see Table 1), which is consistent with their predicted secondary structure. Thermally induced unfolding studies demonstrate that both proteins are rather unstable as monomeric proteins with comparable thermodynamic parameters. The thermally induced unfolding monitored by CD shows that both proteins reversibly unfold without aggregating. The midpoints of the thermal transitions of 42°C and 37.5°C for MxiH and PrgI, respectively, are relatively low for globular proteins with such an important function as involvement in the invasive process of the bacteria. Several considerations temper the significance of these observations. The transitions actually begin at much lower temperatures than the Tms with evidence for the initiation of the thermally induced changes at the lowest temperatures examined (i.e., 10°C). Furthermore, urea appears to induce the change at relatively low concentrations of the chaotrope.
Two non–mutually exclusive explanations seem most consistent with the obtained results. First, the C-terminal truncations that permit soluble, monomeric forms of the proteins to be produced may cause their destabilization, as could the presence of the C-terminal His tag. Second, the assembly of the proteins into their native helically packed complexes (Blocker et al. 2003) may result in much more stable structures. It should be noted that when full-length MxiH is generated in E. coli BL21(DE3) with the short His6 tag, it is sequestered into inclusion bodies. These inclusions can, however, be solubilized with 6 M urea, and the full-length MxiH-His6 purified by nickel-chelation chromatography. When the urea is then removed by dialysis, the portion of the protein remaining soluble can be subjected to gel filtration to give two forms of MxiH-His6. One is a very high molecular weight form that is composed of unfolded aggregates (based on CD spectroscopy and light scattering), and the other is fully folded, monomeric MxiH-His6. This monomeric form of MxiH also has a relatively low Tm (42.16° ± 1.6°C), indicating that the low Tm of MxiH5 is an intrinsic property of the MxiH monomer. Also, unfolding of the full-length MxiH-His6 was not completely reversible, indicating that it is not a particularly attractive candidate for vaccine use. Unfortunately, the studies described here cannot yet be done using intact needles. Although we can purify small amounts of needles, they are prepared from intact bacteria with intact type III secretion apparatuses and are thus contaminated with other proteins. Furthermore, these needles, once sheared from the bacterial surface, appear to be somewhat labile and tend to depolymerize into smaller forms (including monomers) within a relatively short period of time at low concentrations. It is important to note, however, that work by Jouihri et al. (2003) shows that the presence of a His6 on MxiH does not negatively impact its ability to form fully functional needles in Shigella flexneri, suggesting that the His tag does not greatly impact these studies on MxiH structure and stability.
MxiH and PrgI both have their Phe and Trp peaks located toward either their N or C terminus. In contrast, the Tyr residues are somewhat more dispersed throughout the proteins’s sequences (Fig. 1). The second derivative UV absorbance studies, as well as the intrinsic fluorescence results, are consistent, with neither the Phe nor the Trp side chains significantly changing their environments when the structure is perturbed by either temperature (Kueltzo et al. 2003a) or urea (Eftink 1994). The changes that are seen are entirely explicable in terms of the intrinsic effects of temperature and urea on their spectral properties. When combined with the observation that the changes in environment of Tyr residues give evidence of a distinct transition (also supported by the DSC results, at least in the case of PrgI), this suggests a model in which this analog of the fiber protein contains a structural core with less stable N and C termini. This would be consistent with the N and C termini being involved in polymerization of MxiH and PrgI into needle structures.
CD and absorbance unfolding experiments yield similar results in terms of Tm values. This indicates that both methods detect global unfolding events in the proteins despite the fact that one selectively monitors secondary structure change, whereas the other monitors tertiary structure alterations. Thus, no evidence for molten globule- like intermediates is seen in this case.
The urea unfolding studies support the observation that these recombinant proteins are relatively unstable. Their intrinsic free energy of unfolding is well below what is commonly seen for a globular protein, although these values are not outside the values occasionally encountered for proteins of their size. Nevertheless, the G values of <2 kcal/mol clearly classify them as unstable proteins. The resultant m-values can be correlated to the change in solvent-accessible surface area (ASA) as the proteins unfold (Fleming et al. 2005). For a protein of ~80 amino acid residues, the calculated ASA is ~6533 Å2, as obtained from Equation 9 below (Myers et al. 1995):
 | (9) |
where #res is the number of amino acid residues that take part in ASA. The m-values obtained from our urea-induced unfolding studies, however, predict from Equation 10 ASA values of 4109 Å2 and 3655 Å2 for MxiH and PrgI, respectively.
 | (10) |
The calculated ASA values summarized in Table 4 correspond to ~54 and 49 amino acid residues undergoing changes in the solvent-accessible surface area. This is compared to the 80 amino acid residues present in each of the proteins. Therefore, the relatively low m-values suggest that the proteins undergo significantly less change in solvent-accessible surface area than expected if complete unfolding were occurring or if the proteins are initially partially unfolded.
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Materials and methods |
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5 or PrgI5 with a short His6 affinity tag at the C terminus.
The expression plasmids containing MxiH5 or PrgI5 were transformed into E. coli BL21(DE3) for high-level protein production (Picking et al. 1996). After induction of protein expression, the bacteria were harvested by centrifugation (8000g), resuspended in affinity binding buffer (20 mM Tris at pH 7.9, 0.5 M NaCl, 5 mM imidazole) and the bacteria disrupted by sonication. Insoluble debris was removed by centrifugation (20 min at 20,000g), and the soluble fraction was used for affinity purification via the C-terminal His tag by nickel-chelation chromatography as previously described (Picking et al. 1996) using 20 mM Tris (pH 7.9), 0.5 M NaCl, 1 M imidazole for elution. Purified proteins were dialyzed against 10 mM NaPO4 (pH 7.2), 150 mM NaCl (PBS) with a resultant purity >95% in all cases as determined by SDS-PAGE with Coomassie staining.
Turbidity and second derivative UV absorption analysis
Protein concentration determination and second derivative UV spectroscopy were performed with an Agilent 8453 UV-visible spectrophotometer (Kueltzo et al. 2003b). Protein concentrations were determined using an extinction coefficient of 9970 M–1cm–1 and 11,460 M–1cm–1 calculated at 280 nm for MxiH and PrgI, respectively (Gill and von Hippel 1989). In temperature perturbation studies, spectra were acquired over a temperature range of 10°–90°C at 2.5° intervals within a 1-cm path length cell with continuous stirring. Each spectrum was collected with an integration time of 25 sec after a 5-min equilibration time. Using Chemstation software (Agilent), spectra were converted to second derivatives using a nine-point data filter and a fifth-degree Savitzky-Golay polynomial, and subsequently fitted to a cubic function with 99-point data interpolation. The resolution of the final spectra was ±0.01 nm. Data were imported into Microcal Origin from which aromatic peak positions were determined and then plotted as a function of temperature (Kueltzo et al. 2003a) as shown in Figures 8 and 9. The tyrosine peaks in these figures (peaks 4 and 5) were analyzed as discussed below. The optical density of the solution was monitored at 350 nm with respect to temperature to monitor protein aggregation. Protein concentrations ranged from 50 to 80 µM and variations of concentration within this range did not influence thermal stability.
Circular dichroism spectroscopy
Far-UV CD spectra and thermal unfolding monitored at 222 nm were performed using a JASCO J720 spectropolarimeter (JASCO Inc.). Far-UV spectra were recorded from 260 to 190 nm at a scan rate of 15–20 nm/min using a 0.01-cm path length cell. Spectra were acquired in triplicate and averaged. Thermally induced unfolding curves were acquired using a 0.1-cm path length cell with a temperature ramping rate of 15°C/h. The change in secondary structure as a function of temperature was monitored at 222 nm. The protein concentration in all cases was between 20 and 50 µM. The unfolding transitions were analyzed as discussed below.
Urea-induced unfolding
Urea concentration was determined based on the refractive index of the solution using a Bausch & Lomb refractometer and calculated using the method of Pace (1986). Urea titration experiments were conducted with an Aviv spectrophotometer (model 202SF). The sample temperature was controlled by a Peltier device. The protein solution was contained in a quartz cuvette with a 1-cm path length. The titrant was dispensed into the cuvette from a syringe connected to a Hamilton automatic titrator with narrow diameter tubing. The syringe contained both the titrant and a protein concentration identical to that in the cuvette. The concentration of protein in both the syringe and cuvette was kept the same to maintain constant protein concentration throughout the experiment. In addition, a constant volume was maintained in the cuvette. This was accomplished by a program that commands the titrator to withdraw an equal volume of solution from the cuvette that is equal to that added in the next titration step. The urea-induced equilibrium unfolding transitions were monitored at 222 nm at 10°C and 25°C in PBS at pH 7.0. During our titration experiments, 5-min intervals were allowed between the times denaturant was added and the time data acquisition began. We found no change in the CD signal within this period. Moreover, this is a relatively small protein and therefore is not expected to have complex or slow folding/unfolding kinetics.
Differential scanning calorimetry
DSC experiments were performed with a VP-DSC (MicroCal Inc.) , using a heating rate of 1°C/min. About 0.2 mM protein solutions were used with the final protein dialysis buffer (PBS at pH 7.0) used as the reference solution. Before scanning the samples, numerous water and buffer thermograms were obtained to establish a thermal history for the instrument. Between measurement of samples, soap solution was thermally scanned followed by water and then buffer. For each of the proteins, rescanning was performed to measure reversibility. The buffer/buffer thermogram was subtracted from that of the buffer/protein, and the data were normalized for protein concentration before processing. The H and Cp values were extracted by fitting to a two-state model using the fitting program supplied with the instrument.
Data analysis for equilibrium unfolding
The following equations describe a two-state unfolding between a native, N, and unfolded, U, state. The relationship between the equilibrium constant, Kun, for the unfolding process, the partition function, Q, and the mole fractions of the native, XN, and the unfolded, XU, protein is given in Equations 3 and 4.
 | (1) |
 | (2) |
 | (3) |
 | (4) |
The observed signal, S, in either the CD or the fluorescence experiments is given by Equation 5 below:
 | (5) |
where Si is the contribution of species i (with mole fraction Xi) to the signal, and P
Si/ P is the dependence of the signal on perturbant, P, at temperature, T, in thermal-induced unfolding and urea concentration, [d], in urea-induced unfolding studies. A linear free energy relationship for the urea-induced unfolding transitions is assumed as shown in Equation 6.
 | (6) |
 | (7) |
where G°0,un is the free energy of unfolding in the absence of perturbant, and m is the dependence of the free energy on denaturant concentration. Equations 1–6 were nonlinearly fit to the data using Igor Pro software. Thermally induced unfolding data were analyzed using the Gibbs-Hemholtz equation (Santoro and Bolen 1992) as shown in Equation 8, in combination with Equations 1–5.
 | (8) |
Fluorescence spectroscopy
Fluorescence emission spectra were acquired using a PTI QM-1 spectrofluorometer equipped with Felix software. Samples were excited at 295 nm (>95% Trp emission), and the emission spectra were collected between 305 nm and 400 nm at a scan rate of 30 nm/min using a 1-cm path length cell. In thermal unfolding studies, spectra were obtained at 2.5° intervals, beginning at 10°C up to 80°C. Each spectrum is an average of two scans. Depending upon protein concentration, slit widths were set between 4 nm and 6 nm. Additional fluorescence spectra were obtained using a JASCO FP-6500 spectrofluorometer (Jasco Inc.). Protein concentrations ranged from 50 µM to 80 µM, and within this range there was no concentration-dependent change in protein stability.
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Acknowledgments |
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References |
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Blocker, A., Jouihri, N., Larquet, E., Gounon, P., Ebel, F., Parsot, C., Sansonetti, P., and Allaoui, A. 2001. Structure and composition of the Shigella flexneri "needle complex," a part of its type III secretion. Mol. Microbiol. 39: 652–663.[CrossRef][Medline]
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