Title: RTX Calcium Binding Motifs Are Intrinsically Disordered in the Absence of Calcium
Abstract: The Repeat in Toxin (RTX) motif is a tandemly repeated calcium-binding nonapeptide sequence present in proteins that are secreted by the type I secretion system (T1SS) of Gram-negative bacteria. Here, we have characterized the structural and hydrodynamic properties of the RTX Repeat Domain (RD) of the CyaA toxin from Bordetella pertussis. This 701-amino acid long domain contains about 40 RTX motifs. We showed that, in the absence of calcium, RD was natively disordered, weakly stable, and highly hydrated. Calcium binding induced compaction and dehydration of RD, along with the formation of stable secondary and tertiary structures. The calcium-induced conformational switch between unfolded conformations of apo-RD and stable structures of holo-RD is likely to be a key property for the biological function of the CyaA toxin: in the low calcium environment of the bacterial cytosol, the intrinsically disordered character of the protein may facilitate its secretion through the secretion machinery. In the extracellular medium, calcium binding can then trigger the folding of the polypeptide into its functional state. The intrinsic disorder of RTX-containing proteins in the absence of calcium may thus be directly involved in the efficient secretion of proteins through T1SS. The Repeat in Toxin (RTX) motif is a tandemly repeated calcium-binding nonapeptide sequence present in proteins that are secreted by the type I secretion system (T1SS) of Gram-negative bacteria. Here, we have characterized the structural and hydrodynamic properties of the RTX Repeat Domain (RD) of the CyaA toxin from Bordetella pertussis. This 701-amino acid long domain contains about 40 RTX motifs. We showed that, in the absence of calcium, RD was natively disordered, weakly stable, and highly hydrated. Calcium binding induced compaction and dehydration of RD, along with the formation of stable secondary and tertiary structures. The calcium-induced conformational switch between unfolded conformations of apo-RD and stable structures of holo-RD is likely to be a key property for the biological function of the CyaA toxin: in the low calcium environment of the bacterial cytosol, the intrinsically disordered character of the protein may facilitate its secretion through the secretion machinery. In the extracellular medium, calcium binding can then trigger the folding of the polypeptide into its functional state. The intrinsic disorder of RTX-containing proteins in the absence of calcium may thus be directly involved in the efficient secretion of proteins through T1SS. The type I secretion system (T1SS) 3The abbreviations used are: T1SS, type one secretion system; ANS, 1-anilino-8-naphthalene sulfonate; CyaA, adenylate cyclase; HlyA, α-hemolysin; RTX, repeat in toxin; RD, RTX repeat domain; RALS, right angle light scattering; LALS, low angle light scattering; nOe, nuclear Overhauser effect; SEC, size exclusion chromatography; TDA, triple detector array; AUC, analytical ultracentrifugation; QELS, quasi-elastic light scattering. is one of the major export machineries that are used by Gram-negative bacteria to secrete proteins into their external medium (1Delepelaire P. Biochim. Biophys. Acta. 2004; 1694: 149-161Crossref PubMed Scopus (298) Google Scholar, 2Holland I.B. Schmitt L. Young J. Mol. Membr. Biol. 2005; 22: 29-39Crossref PubMed Scopus (194) Google Scholar). This machinery was originally described as a dedicated secretion apparatus of α-hemolysin (HlyA), an essential pore-forming toxin produced by diverse uropathogenic Escherichia coli strains (3Felmlee T. Pellett S. Lee E.Y. Welch R.A. J. Bacteriol. 1985; 163: 88-93Crossref PubMed Google Scholar, 4Felmlee T. Pellett S. Welch R.A. J. Bacteriol. 1985; 163: 94-105Crossref PubMed Google Scholar). Similar systems were soon identified in many other mammalian or plant pathogens and shown to secrete a wide variety of virulence factors endowed with many different biological activities such as pore-forming cytolytic capacities (like HlyA), adenylate cyclase, actin cross-linking, lipases, proteases, adhesion proteins, hemophores, etc. (1Delepelaire P. Biochim. Biophys. Acta. 2004; 1694: 149-161Crossref PubMed Scopus (298) Google Scholar, 2Holland I.B. Schmitt L. Young J. Mol. Membr. Biol. 2005; 22: 29-39Crossref PubMed Scopus (194) Google Scholar, 5Welch R.A. Curr. Top. Microbiol. Immunol. 2001; 257: 85-111PubMed Google Scholar). T1SS is a tripartite machinery consisting of: (i) an inner membrane ABC transporter that recognizes the protein substrate, (ii) a membrane fusion protein (MFP), and (iii) an outer membrane protein (OMP) (1Delepelaire P. Biochim. Biophys. Acta. 2004; 1694: 149-161Crossref PubMed Scopus (298) Google Scholar, 2Holland I.B. Schmitt L. Young J. Mol. Membr. Biol. 2005; 22: 29-39Crossref PubMed Scopus (194) Google Scholar, 5Welch R.A. Curr. Top. Microbiol. Immunol. 2001; 257: 85-111PubMed Google Scholar, 6Koronakis V. Sharff A. Koronakis E. Luisi B. Hughes C. Nature. 2000; 405: 914-919Crossref PubMed Scopus (881) Google Scholar). The proteins are usually recognized through a non-cleavable secretion signal, localized at the C terminus of the polypeptide chain and are exported from the cytosol to the extracellular medium without periplasmic intermediates (1Delepelaire P. Biochim. Biophys. Acta. 2004; 1694: 149-161Crossref PubMed Scopus (298) Google Scholar, 2Holland I.B. Schmitt L. Young J. Mol. Membr. Biol. 2005; 22: 29-39Crossref PubMed Scopus (194) Google Scholar, 3Felmlee T. Pellett S. Lee E.Y. Welch R.A. J. Bacteriol. 1985; 163: 88-93Crossref PubMed Google Scholar, 5Welch R.A. Curr. Top. Microbiol. Immunol. 2001; 257: 85-111PubMed Google Scholar). Although the exported proteins vary widely in terms of biochemical function as well as in size (from less than one hundred to several thousands amino acids), one of their prominent features is the presence of characteristic repeated motifs known as RTX (for Repeat in Toxin), localized immediately upstream to the C-terminal secretion signal (1Delepelaire P. Biochim. Biophys. Acta. 2004; 1694: 149-161Crossref PubMed Scopus (298) Google Scholar, 2Holland I.B. Schmitt L. Young J. Mol. Membr. Biol. 2005; 22: 29-39Crossref PubMed Scopus (194) Google Scholar, 5Welch R.A. Curr. Top. Microbiol. Immunol. 2001; 257: 85-111PubMed Google Scholar). RTX sequences are glycine- and aspartate-rich nonapeptides of the prototype GGXGXDXLX (X stands for any amino acid, L is occasionally substituted by V, I, F, or Y) that are present in variable number (from 5 to more than 50), generally in a tandem fashion. These motifs constitute a specific type of Ca2+ binding site that is essential for the function of the proteins. Indeed most cytolysins of the HlyA family are calcium-dependent and few of them (e.g. E. coli HlyA and the adenylate cyclase toxin (CyaA) from Bordetella pertussis) have been shown to bind calcium in solution (7Ludwig A. Jarchau T. Benz R. Goebel W. Mol. Gen. Genet. 1988; 214: 553-561Crossref PubMed Scopus (142) Google Scholar, 8Boehm D.F. Welch R.A. Snyder I.S. Infect. Immun. 1990; 58: 1959-1964Crossref PubMed Google Scholar, 9Sanchez-Magraner L. Viguera A.R. Garcia-Pacios M. Garcillan M.P. Arrondo J.L. de la Cruz F. Goni F.M. Ostolaza H. J. Biol. Chem. 2007; 282: 11827-11835Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 10Rose T. Sebo P. Bellalou J. Ladant D. J. Biol. Chem. 1995; 270: 26370-26376Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Crystallographic studies of two metalloproteases and a lipase revealed that these repeats, in the presence of calcium, fold into a parallel β-roll, in which the first six amino acids GGXGXD form a turn while the remaining three residues XLX fold into a short β-strand, with the leucine residue (or other nonpolar amino acids), making the hydrophobic core of the β-roll (11Baumann U. Wu S. Flaherty K.M. McKay D.B. EMBO J. 1993; 12: 3357-3364Crossref PubMed Scopus (430) Google Scholar, 12Meier R. Drepper T. Svensson V. Jaeger K.E. Baumann U. J. Biol. Chem. 2007; 282: 31477-31483Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The arrangement of consecutive turns and β-strands builds up a right-handed helix of parallel β-strands, one turn of this helix consisting of two consecutive RTX motifs. Calcium is bound between two adjacent turns by the conserved aspartic acids and by backbone carbonyls. Previous studies indicated that the RTX structures are important for the secretion of the corresponding substrates through the T1SS, without being per se part of the secretion signal (13Nicaud J.M. Mackman N. Gray L. Holland I.B. FEBS Lett. 1986; 204: 331-335Crossref PubMed Scopus (75) Google Scholar, 14Felmlee T. Welch R.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5269-5273Crossref PubMed Scopus (100) Google Scholar, 15Letoffe S. Wandersman C. J. Bacteriol. 1992; 174: 4920-4927Crossref PubMed Google Scholar). It has been suggested that the RTX motifs may serve as a flexible hinge between the secretion signal, and the upstream domains of the substrate. Besides, it is generally assumed that the newly synthesized polypeptide chain has to adopt a loosely folded conformation compatible with the passage through the narrow channel of the T1SS (6Koronakis V. Sharff A. Koronakis E. Luisi B. Hughes C. Nature. 2000; 405: 914-919Crossref PubMed Scopus (881) Google Scholar). Given the low calcium concentration within the bacterial cytosol, it has been hypothesized that, inside bacteria, the RTX sequences could adopt an unfolded conformation favorable for the secretion of the protein substrate (1Delepelaire P. Biochim. Biophys. Acta. 2004; 1694: 149-161Crossref PubMed Scopus (298) Google Scholar, 2Holland I.B. Schmitt L. Young J. Mol. Membr. Biol. 2005; 22: 29-39Crossref PubMed Scopus (194) Google Scholar, 5Welch R.A. Curr. Top. Microbiol. Immunol. 2001; 257: 85-111PubMed Google Scholar). To provide direct experimental evidences for this model, we have examined the conformational flexibility of the RTX motifs from the adenylate cyclase toxin (CyaA) of B. pertussis, the causative agent of whooping cough (16Ladant D. Ullmann A. Trends Microbiol. 1999; 7: 172-176Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 17Vojtova J. Kamanova J. Sebo P. Curr. Opin. Microbiol. 2006; 9: 69-75Crossref PubMed Scopus (145) Google Scholar). CyaA, one of the major virulence factors of B. pertussis, is secreted by a dedicated T1SS, made of the CyaB, CyaD, and CyaE proteins, and is able to invade eukaryotic target cells, where upon activation by calmodulin, it produces supraphysiological levels of cAMP. CyaA is a 1706-amino acid long protein that contains in its C-terminal moiety (i.e. residues 1006-1706) about 40 RTX motifs organized in five successive blocks, each consisting of 8-10 RTX motifs, separated by linkers of variable length (from 23-49 residues). Here, we have characterized the conformational, thermodynamic, and hydrodynamic properties of the apo (Ca2+-free) and holo (Ca2+-bound) forms of the CyaA RTX domain (RD) by various biophysical approaches. We showed that at low calcium concentrations, similar to those prevailing in the bacterial cytosol, RD presents the characteristic features of an intrinsically disordered protein (18Dunker A.K. Lawson J.D. Brown C.J. Williams R.M. Romero P. Oh J.S. Oldfield C.J. Campen A.M. Ratliff C.M. Hipps K.W. Ausio J. Nissen M.S. Reeves R. Kang C. Kissinger C.R. Bailey R.W. Griswold M.D. Chiu W. Garner E.C. Obradovic Z. J. Mol. Graph. Model. 2001; 19: 26-59Crossref PubMed Scopus (1910) Google Scholar, 19Fink A.L. Curr. Opin. Struct. Biol. 2005; 15: 35-41Crossref PubMed Scopus (613) Google Scholar, 20Dyson H.J. Wright P.E. Nat. Rev. Mol. Cell. Biol. 2005; 6: 197-208Crossref PubMed Scopus (3117) Google Scholar), while in the presence of millimolar calcium concentrations (like those found in the extracellular medium), the protein exhibits a stable and compact β-sheet conformation. The biological relevance of these findings for the secretion by the T1SS of CyaA and, more generally, of RTX-containing protein substrates will be discussed. Materials—Hepes-d18 (D18, 98%, DLM-3786-0) was purchased from Cambridge Isotope Laboratories. D2O (D215B), NaOD, and DCl were from Euriso-top (C.E.A. Saclay, Gif-Sur-Yvette, France). Experiments were done in 5 mm Hepes, 150 mm NaCl, pH 7.3 (buffer A), at 37 °C, unless stated otherwise. Protein Preparation—The RD of CyaA corresponds to residues 1006-1706 of CyaA. The RD protein was overproduced in E. coli and purified by Ca2+-dependent phenyl-Sepharose chromatography as described previously (10Rose T. Sebo P. Bellalou J. Ladant D. J. Biol. Chem. 1995; 270: 26370-26376Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). After hydrophobic interaction chromatography, RD was further purified by size exclusion chromatography on Sephacryl S300 (20 mm Hepes, pH 7.3, 150 mm NaCl) and by ion exchange chromatography (IEC) on Q Sepharose. Elution was done with 20 mm Hepes, pH 7.3, 500 mm NaCl at room temperature. The IEC elution buffer was finally exchanged against 5 mm Hepes, pH 7.3 on a G25SF column. The protein solution was stored at -20 °C or dialyzed against 10 mm NH4HCO3 and lyophilized. We checked by CD, fluorescence, and trypsin partial digestion that the lyophilization process did not affect the calcium-induced conformational changes of RD. Protein batches were analyzed by SDS-PAGE, N-terminal sequencing, and by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS model PCS 4000, Ciphergen). A molar mass of 72,621 g·mol-1, and a molar extinction coefficient of 72000 m-1·cm-1 were computed from the amino acid sequence of RD. Circular Dichroism Spectroscopy—CD spectra were recorded on an Aviv circular dichroism spectrometer model 215, equipped with a water-cooled Peltier unit. CD measurements were carried out in a 1-mm path length Suprasil cell (110.QS, Hellma) at a scan rate of 0.5 nm/sec (step: 0.5 nm and integration time: 1 s) with a time constant of 100 ms and a bandwidth of 1 nm. Four scans were averaged to obtain each spectrum. The spectrum of buffer A was subtracted to all spectra. The CD units used are the mean residue ellipticity (MRE), expressed in degrees square centimeter per decimol of residue ((deg·cm2)/(dmol·res)) and calculated from the relationship in Equation 1, [θ]F=(100θm)/(CIN),(Eq. 1) where θm is the measured ellipticity in degrees, C is the concentration in moles per liter, l is the path length of the cell in centimeter, and N is the number of residues. The value 100 arises from the conversion of the concentration in mole per liter to decimole per cubic centimeter. Fluorescence Spectroscopy—Thermal-induced denaturation of RD at various calcium concentrations was followed by fluorescence spectroscopy. Measurements were performed with an FP-6200 spectrofluorimeter (Jasco) in a Peltier-thermostatted cell holder, using a 1-cm path length quartz cell (111-QS) with constant stirring. The tryptophan emission spectra of RD at a concentration of 1 μm at different calcium concentrations (from 0 to 2 mm) were recorded from 300 to 400 nm (excitation at 292 nm at a scan rate of 125 nm·min-1) as a function of temperature, ranging from 10 to 98 °C, with a temperature increment of 1 °C between each spectrum. A bandwidth of 5 nm was used for both excitation and emission beams. The ratio of fluorescence intensities (rFI) at 360 nm and 320 nm (rFI 360/320) was used to determine the fraction of unfolded protein fU using the relation: fU = (rFIT - rFI10)/(rFI98 - rFI10) where rFI10, rFI98, and rFIT, are the rFI at 10 °C, 98 °C, and temperature T, respectively. The fraction of native protein fN that populated the native state, N, is given by Equation 2, fN=NN+U=(1+(UN))−1=(1+KT)−1(Eq. 2) where U is the concentration of RD in the thermal-unfolded state and KT the equilibrium constant at the given temperature: KT = U/N. The equilibrium constant KT is related to the free energy ΔG according to K = exp(-ΔG/RT), and in the vicinity of the half-melting point Tm, the fraction of native RD, fN, is related to the Van't Hoff enthalpy ΔHvH according to Equation 3.fN=(1+exp(ΔHvHR×(1Tm−1T)))−1(Eq. 3) Fittings were done with Kaleidagraph (Synergy Software, Reading, PA). Nuclear Magnetic Resonance Spectroscopy—Purified RD in 20 mm Hepes, 2 mm EDTA was buffer-exchanged against 10 mm ammonium bicarbonate (NH4HCO3) on prepacked G25SF desalting columns at room temperature and freeze-dried. Two cycles of resuspension of the lyophilized protein in D2O and freeze-drying were performed to exchange amide protons and to reduce H2O concentration. Buffers and guanidine hydrochloride (GdnHCl) were D2O-exchanged twice by repeated freeze-drying and resuspension. Apo-RD samples were prepared by dissolving lyophilized RD in 5 mm Hepes-d18, 150 mm NaCl, pH 7.5, prepared in D2O (99.99%), supplemented or not with 5.3 m GdnHCl. Holo-RD samples were prepared from apo-RD samples by addition of 2 mm CaCl2. Protein concentrations ranged between 55 and 75 μm. NMR experiments were conducted at 37 °C on an Inova 600 (Varian Inc., Palo Alto) spectrometer with a 14.1 Tesla magnetic field, and equipped with a cryoprobe. Spectra were recorded, processed and analyzed using Vnmr 6.1C (Varian). Water signal was suppressed by low-power irradiation during the 2 s recovery delay or using the jump-return (21Plateau P. Guéron H. J. Am. Chem. Soc. 1982; 104: 7310-7311Crossref Scopus (1148) Google Scholar) or double pulse-field gradient stimulated-echo (22Dalvit C. Shapiro G. Böhlen J.-M. Parella T. Magn. Reson. in Chem. 1999; 37: 7-14Crossref Scopus (37) Google Scholar) schemes. Proton spectra were acquired with 4096 complex points and a sweep width of 12 ppm. Transverse relaxation times (T2) were obtained from standard spin-echo experiments that used short (≤4 ms) variable τ relaxation delays (π/2-τ-π/2) to safely neglect J-coupling evolution. Bulk T2 values were estimated by fitting the data of the aliphatic region (below 3.2 ppm) to a single exponential decay. One-dimensional saturation transfer experiments were performed by selective saturation (1.7 ppm) of the aromatic region using trains of 90° gaussian pulses centered at 7.1 ppm. Thirty-two scans were accumulated for each saturation time, which varied between 0 and 1.7 s. An identical set of experiments with off-resonance saturation was used as reference. The nuclear Overhauser effect (nOe) between aromatic and aliphatic protons was calculated from the intensities of the aliphatic region (upfield of 2.5 ppm) of the on- (IS) and off- (IR) resonance experiments as follows: nOe = [IS-IR]/IR. Intrinsic Viscosity and Molecular Mass Measurements with SEC-TDA—Size exclusion chromatography (SEC) experiments were done on a Superdex 200 column (GE Healthcare) controlled by a GPCmax module and connected on-line to a triple detector array (TDA) model 302 (Viscotek Ltd., Houston, Basingstoke, UK). The oven of the TDA contained (i) a static light scattering cell with two photodiode detectors, at 7° for low angle (LALS) and at 90° for right angle laser light scattering (RALS), (ii) a deflection refractometer, (iii) a photometer, and (iv) a differential viscometer. The general procedures described by Viscotek were followed. All solutions were filtered on 0.2-μm filters and allowed to equilibrate at 10 °C. SEC was performed at 10 °C, and detection in the TDA oven was done at 20 °C. All experimental sequences contained injections of bovine serum albumin (2 mg/ml, various volumes) and apo-RD or holo-RD (at least four injections of different volumes). Bovine serum albumin injections were used for TDA internal constants calibration. The refractive index increments, dn/dc, were experimentally determined and were similar for both states (dn/dc: 0.184). Buffer A (5 mm Hepes, 150 mm NaCl, pH 7.3) was used for apo-RD and buffer A supplemented with 2 mm CaCl2 for holo-RD. All data were acquired using the Omnisec software. Protein concentration was determined using both the photometer and the deflection refractometer. The RALS and LALS data, in combination with the concentration, provided the molecular mass M. Intrinsic viscosity [η] was calculated using the differential viscometer, which is made of a balanced four-capillary bridge, an original adaptation of the electrical Wheatstone bridge to fluid materials (23Harding S.E. Prog. Biophys. Mol. Biol. 1997; 68: 207-262Crossref PubMed Scopus (307) Google Scholar). Both molecular mass and intrinsic viscosity were calculated with the Omnisec software. Analytical Ultracentrifugation—Sedimentation equilibrium and velocity experiments were performed at 20 °C on a Beckman XL-A analytical ultracentrifuge (Beckman Coulter) equipped with an AN60-Ti rotor. The samples were centrifuged at 13,000 × g for 10 min prior to experiments. Detection of the protein concentration as a function of radial position and time was performed by optical density measurements at a wavelength of 280 nm. Buffer A supplemented with either 2 mm CaCl2 or 2 mm EDTA was used for holo-RD or apo-RD, respectively. The buffer viscosity η and density ρ, calculated with Sednterp 1.09 were η = 1.016 cP and 1.00456 g·ml-1. For sedimentation equilibrium experiments, protein samples (40 μl, 2.5 μm) were loaded in a 1.2-mm thick six channels epon centerpiece. Apo-RD samples were centrifuged successively 3 h at rotor speeds of 10,000 rpm, 2 h at 12,000 rpm, and then 2 h at 18,000 rpm. Holo-RD samples were centrifuged 3 h at 18,000 rpm, 2 h at 22,000 rpm, and 2 h at 26,000 rpm. Data were recorded for each speed after controlling that the sedimentation/diffusion equilibrium had been effectively reached. The baseline was measured at 42,000 rpm after 2 h. Radial distributions were analyzed by global fitting of the three speeds using the one species model of the Ultrascan 9.5 software. Partial specific volumes were obtained by fixing the molecular mass to the computed mass of the monomer. For sedimentation velocity experiments, the protein samples (300 μl at 2.5, 7, 15, and 25 μm) were loaded in 1.2-mm thick epon double sector cells and spun at 35 000 rpm. Sedimentation velocity profiles were monitored at 3-min intervals. Data were analyzed with the Sedfit 11.3 software using a continuous size distribution c(s) model with invariant diffusion coefficient D (24Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3152) Google Scholar). Quasi-elastic Light Scattering—QELS experiments were performed on a DynaPro MS800 (Wyatt), using the Protein Solution Dynamics software version 6.2.05. The laser power was 100%, and its incident light wavelength was λQELS: 824.7 nm. Buffers and samples were filtrated on 0.2-μm filters prior to acquisition. Protein concentration was routinely 5 μm. A microcuvette of dimensions 3 × 8.5 mm (105.251 QS) was loaded with 100 μl of RD. Samples were thermally equilibrated for 10 min in the cell compartment at 20 °C. Acquisition time was 10 s, with an interval time of 1 s. At least 30 acquisitions were averaged to produce a data collection. A set of three independent data collections was obtained for each experimental condition. The data were processed with SEDFIT 9.2 and analyzed using: (i) Continuous Hydrodynamic Radius Distribution and (ii) Stokes Radii models. The maximum entropy and the Tikhonov-Phillips 2nd derivative regularization procedures were used with a confidence level (F-ratio) of 0.55. The hydrodynamic radius, RH, was calculated from the Stokes-Einstein equation RH = (kBT)/6πηsDt, where kB is the Boltzmann's constant, T the temperature, and ηs the viscosity of the solvent. The frictional ratio from quasi-elastic light scattering is given by f/f0 = RH/RS, where RS stand for the radius of an anhydrous sphere of the same mass as RD. Protein Shape and Hydration—The viscosity increment v (also called the Simha-Einstein hydrodynamic function) is related to the axial ratio a/b of an ellipsoid of revolution and can be calculated from the Einstein's viscosity relation: M[η] = νVHNA, where VH is the hydrodynamic volume defined by VH = 4πRH/3 and NA the Avogadro number. Hydration is calculated from the intrinsic viscosity relation described below. The intrinsic viscosity in a defined solvent depends on the shape, the hydration, the molecular volume of the protein, and electroviscous effects. Its expression is the product of the viscosity increment ν and the swollen volume Vs according to [η]=vVs=v(v¯+δ/ρ), from which the hydration parameter is extracted: δ=(([η]/v)−v¯)ρ. The swollen volume, Vs, is the sum of the partial specific volume v¯ of the protein (volume occupied by one gram of protein) and the time-averaged apparent hydration δ of the protein (volume of water per gram of protein). The hydration parameter of the protein includes: (i) the water molecules bound to the protein and (ii) the water molecules dragged by the diffusion of the protein. The molecular mass M was measured by SELDI and static light scattering. The partial specific volume v¯ was determined by equilibrium AUC and the translational diffusion coefficient Dt by velocity AUC and QELS (see above). The viscosity increment provides the axial ratio a/b, using the inversion formulae of the hydrodynamic function v using the parameters for polynomial fit described by Harding and Cölfen (25Harding S.E. Colfen H. Anal. Biochem. 1995; 228: 131-142Crossref PubMed Scopus (50) Google Scholar). The semi axes a and b, with a>b, describe the shape of the ellipsoid of revolution. The values of a and b semi axes for prolate (a, b, b) and oblate (a, a, b) conformations were determined using Ultrascan. Calcium-induced Conformational Changes of RD—The RD protein (amino acids 1006-1706 of CyaA; molecular mass of 72.6 kDa) was overexpressed as a soluble polypeptide in E. coli and purified to homogeneity (10Rose T. Sebo P. Bellalou J. Ladant D. J. Biol. Chem. 1995; 270: 26370-26376Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 26Bauche C. Chenal A. Knapp O. Bodenreider C. Benz R. Chaffotte A. Ladant D. J. Biol. Chem. 2006; 281: 16914-16926Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The far-UV CD spectrum of the apo-state of RD (apo-RD, in the absence of calcium) was typical of a predominantly unfolded protein, as shown by the strong negative π0-π* band around 200 nm (Fig. 1). Yet, the weak negative n′-π* band, appearing as a shoulder around 220 nm, indicated the presence of some residual secondary structure elements in apo-RD. A very similar spectrum was observed for the protein equilibrated in the presence of 5 m GdnHCl. Upon addition of 2 mm calcium to apo-RD, secondary structures were formed, as revealed by the concomitant intensity decrease of the π0-π* band and the increase of the n′-π* band in the holo-RD spectrum, as previously reported (10Rose T. Sebo P. Bellalou J. Ladant D. J. Biol. Chem. 1995; 270: 26370-26376Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 26Bauche C. Chenal A. Knapp O. Bodenreider C. Benz R. Chaffotte A. Ladant D. J. Biol. Chem. 2006; 281: 16914-16926Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The structures of holo-RD were readily denatured upon addition of 5 m GdnHCl, leading to a spectrum similar to that of the apo-state. Proton nuclear magnetic resonance (NMR) was used to further characterize the calcium-induced conformational changes of RD. The one-dimensional spectrum of apo-RD in the presence of the denaturant GdnHCl (5.3 m) showed no evident secondary chemical shifts, with all peaks appearing at the expected frequencies for an unfolded protein, the so-called "random-coil" chemical shifts (Fig. 2A). The low chemical-shift dispersion was consistent with a protein with no stable secondary and tertiary structures. The spectrum of the apo-form under native conditions also showed very poor chemical shift dispersion, and resembled that of the protein denatured in GdnHCl, but with somewhat narrower lines. Noticeably, the bands of the apo-RD spectrum were strikingly narrow for a 701-residue long protein, suggesting that apo-RD had a highly dynamic structure in which most of its side-chains could freely reorient in solution. These observations suggested that apo-RD was mainly natively disordered. Yet, its NMR spectrum contained few signals that were well dispersed and that appeared at chemical shifts characteristic of a well-ordered conformation with stable tertiary structure (see Fig. 2B). The apo-RD spectrum indeed displayed some down-field shifted Hα resonances (≥4.8 ppm) indicative of β-sheet structures, upfield shifted signals in the methyl region (≤0.79 ppm), as well as several dispersed peaks in the aromatic region (7.7-6.5 ppm) at non-random coil frequencies indicating the presence of stable hydrophobic interactions. From integration of the downfield part of the Hα region and comparison with the whole Hα envelope integration (excluding the residual water signal at 4.62 ppm), we could roughly estimate that apo-RD contained at least ∼7% of β-sheets. Similarly, integration of the upfield signals in the methyl region (≤0.79 ppm) of the apo-RD spectrum suggested that ca. 17 methyls (i.e. 6% of the 286 Ile/Leu/Val methyls) were implicated in a stable hydrophobic environment that likely involved aromatic residues. The NMR spectrum of calcium-bound RD was drastically different. First, it exhibited an increase of the envelope of upfield-shifted Hα protons, providing direct evidence that holo-RD was more structured, with a β-sheet content (calculated as above) of at least 30-35%. Second, the signals became very broad as expected for a compact protein of 72.6 kDa. The line broadening of the 1H spectrum reflected a major change in the dynamic behavior of the protein upon calcium binding. Indeed, the value of the bulk transverse relaxation time T2 (which depends on the overa