Disparate Impact of the S33V Mutation on Conformational Stability in

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Disparate Impact of the S33V Mutation on Conformational Stability in Rat β-Parvalbumin (Oncomodulin) and Chicken Parvalbumin 3† Anmin Tan, Lindsey A. Markus, and Michael T. Henzl* Department of Biochemistry, 117 Schweitzer Hall, UniVersity of Missouri, Columbia, Missouri 65211, United States ReceiVed: July 8, 2010; ReVised Manuscript ReceiVed: September 20, 2010

Rat β-parvalbumin (β-PV) and chicken parvalbumin 3 (CPV3) exhibit diminished Ca2+ affinity. Their sequences, 70% identical, are unusual in that serine replaces the consensus residue, valine, at position 33. Reasoning that the substitution of a compact, polar hydroxymethyl moiety for a bulky, apolar isopropyl group might contribute to the attenuated Ca2+ affinities, we have characterized the S33V variants of both proteins. The impact of the mutation in CPV3 differs decidedly from that in rat β. Whereas replacement of S33 by valine in CPV3 causes a substantial increase in the solvent-accessible apolar surface in the Ca2+-free protein, the mutation evidently decreases the exposed apolar surface area in rat β. Although the mutation has a minimal effect on divalent ion affinity in both proteins, the ∆∆H and -T∆∆S changes for Ca2+ binding in CPV3 S33V, but not rat β S33V, are consistent with increased burial of the apolar surface. The influence of the S33V substitution on conformational stability likewise differs for rat β-PV and CPV3. Whereas the stability of the former is virtually unperturbed by the sequence alteration, the latter is destabilized by 0.7 kcal/mol. Moreover, the mutation greatly exacerbates the tendency for CPV3 to aggregate. The concentration and scan rate dependence observed in DSC studies of CPV3 S33V denaturation suggest that unfolding proceeds through an intermediate state that is prone to aggregation. Consistent with this idea, reversible unfolding data, collected at very low protein concentration, likewise indicate that the thermal denaturation is not a two-state process. Introduction The EF-hand protein family is the largest class of intracellular Ca2+-binding proteins. The human genome, for example, encodes 242 family members. These proteins play central roles in Ca2+ signaling pathways,1-4 either as Ca2+-dependent regulators or cytosolic Ca2+ buffers. The EF-hand family is named for its distinctive Ca2+-binding motif, a strongly conserved central ion-binding loop flanked by short amphipathic helices. The spatial arrangement of these structural elements can be suggested with the thumb and first two fingers of the right hand.5 The similarity of their metal ion-binding sites notwithstanding, EF-hand proteins exhibit large variations in divalent ion affinity. We are studying the physical basis for these differences in members of the parvalbumin (PV) family.3,6,7 These small, vertebrate-specific proteins function primarily as cytosolic Ca2+ buffers. The PV tertiary structure includes six helices organized into two domains. The calcium-binding domain (CD-EF domain) encompasses the two EF-hand motifs, which are known as the CD and EF sites, in reference to the helical elements flanking the binding loops. The N-terminal AB domain, which includes residues 1-38, packs tightly against the hydrophobic aspect of the CD-EF domain. It is worth noting that the crystal structure of carp parvalbumin established the EF-hand paradigm.5 Parvalbumins can be assigned to R and β sublineages on the basis of isoelectric point (pI < 5 for β) and lineage-specific sequence differences.8,9 Mammals express one R-PV and one β-PV (aka oncomodulin).10 Birds express one R isoform and two β isoforms. Chicken parvalbumin 3 (CPV3) is one of two β-PV isoforms in the chicken; the other is called avian thymic †

Part of the “Robert A. Alberty Festschrift”. * To whom correspondence should be addressed. Phone: (573) 882-7485. Fax: (573) 884-4812. E-mail: [email protected].

Figure 1. Amino acid sequences of rat β-parvalbumin and CPV3C72S.

hormone (ATH). CPV3 and ATH were both originally discovered in the thymus gland11,12 and evidently play an endocrine role in the avian immune systems,13,14 influencing T-cell differentiation and proliferation. Both proteins have also been detected in other highly specialized tissues. ATH is expressed in the avian retina,15 and CPV3 is expressed in the hair cells of the avian auditory organ (or basilar papilla).16 Although the roles played by ATH and CPV3 in these alternative settings are presently unknown, the proteins are believed to function as specialized Ca2+ buffers. Interestingly, rat β-PV, identical to CPV3 at 75 of 108 residues (Figure 1), is highly expressed in the organ of Corti (the mammalian auditory organ),17 in the highly specialized sensory cells known as outer hair cells.18 Although widely viewed as interchangeable Ca2+ buffers, parvalbumins exhibit a range of Ca2+ affinities. Besides the fact that CPV3 and the mammalian β-PV are both expressed in the auditory organ of their respective species, both proteins also share attenuated divalent ion-binding affinity. In saline at pH 7.4, ATH and rat R-PV display overall ∆G°′ values for Ca2+ binding of -22.5 and -22.0 kcal/mol, respectively.19,20 By

10.1021/jp1063325  2010 American Chemical Society Published on Web 10/20/2010

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contrast, rat β-PV exhibits a value of -18.4 kcal/mol.20 CPV3 exhibits intermediate Ca2+ affinity, with a ∆G°′ value of -20.1 kcal/mol.19 In both proteins, the less favorable free-energy change results primarily from attenuated divalent ion binding at the CD site. Whereas the sequence of the rat β CD site harbors several notable departures from the parvalbumin consensus, the sequence of the CPV3 CD site is unexceptional. Presumably, the diminished affinity for Ca2+ is a consequence of nonconsensus sequence substitutions elsewhere in the molecule. As shown in Figure 1, the AB domains of CPV3 and rat β-PV are identical at 26 of 38 residues. Our attention was drawn to residue 33. Whereas this position is occupied by an apolar residue in other parvalbumin isoforms, typically valine, the mammalian β isoform and CPV3 both harbor serine. As part of an attempt to restore high Ca2+ affinity to CPV3, we examined the impact of replacing S33 by valine. Although the mutation has a modest impact on divalent ion affinity in both proteins, its impact on conformational stability is strongly context-dependent. Our findings are discussed herein. Experimental Methods Materials. CaCl2 · 2H2O, EGTA, Hepes, MgCl2 · 2H2O, NaCl, Na2EDTA · 2H2O, NaH2PO4, and NTA were purchased from Fisher Scientific Co. DEAE-Sepharose, Sephadex G-75, 8-anilino1-naphthalenesulfonate, lysozyme, urea (Fluka), DMPC, DPPC, and DSPC were obtained from Sigma-Aldrich. Ampicillin and LB broth were purchased from Research Products International Corporation. IPTG was from Gold Biotechnology. Protein Mutagenesis, Expression, And Purification. The S33V mutation was introduced into rat β-PV or CPV3 using the Quik-Change mutagenesis kit (Stratagene), employing oligonucleotides purchased from Integrated DNA Technologies (Coralville, IA). The fidelity of the resulting sequences was verified by automated DNA sequencing. The rat β-PV S33V variant was expressed constitutively in E. coli DH5R using the PLD2 expression vector, a derivative of Bluescript (Stratagene) described previously.21 The 1 L LBbroth cultures containing ampicillin (100 µg/mL) were inoculated with 5 mL of a stationary-phase starter culture. After 20 h at 37 °C, the cells were collected by centrifugation and resuspended in 20 mM Hepes, pH 7.4. After treatment with lysozyme (5 mg per g of cell paste), the suspension was extruded from a French pressure cell and then centrifuged (30 min, 27 000 × g, 4 °C). The clarified lysate was fractionated on DEAEagarose, employing a 0-0.6 M NaCl gradient, in 20 mM Hepes, pH 7.4. Fractions containing the S33V variant were identified by nondenaturing PAGE, combined, concentrated to 5 mL, and then fractionated on a Sephadex G-75 column (3.0 × 70 cm), in 0.15 M NaCl, 0.025 M Hepes, pH 7.4. A 1 L culture yields 75-100 mg of protein. The wild-type CPV3 sequence includes a solvent-exposed cysteine at position 72 and oligomerizes readily in the absence of reductant.22 To avoid the experimental complications associated with this behavior, we have replaced C72 with the parvalbumin consensus residue, serine. This substitution has no discernible impact on divalent ion affinity.12,19 The CPV3-C72S coding sequence, optimized for expression in E. coli, was obtained from Genscript USA Inc. (Piscataway, NJ) and cloned into pET11a (Novagen) using the Nde I and BamHI restriction sites. Following mutagenesis as described above, E. coli BL21(DE3) harboring the pET11-CPV3-C72S-S33V vector was cultured at 37 °C in LB broth containing ampicillin (100 µg/mL). IPTG was added (0.25 mM) when the culture turbidity

Tan et al. (600 nm) reached 0.6. Following an additional 20 h of incubation, the culture was harvested by centrifugation. The lysis and purification steps parallel those outlined above; details are described elsewhere.19 Each liter yielded 50-75 mg of protein, with purity exceeding 98%. Because rat β-PV and CPV3 both lack tryptophan, the A274/ A290 ratio provides an indication of homogeneity. In each case, the purity of the preparations exceeded 98%. Concentrations were estimated spectrophotometrically, with extinction coefficients determined from parallel absorbance and interference measurements in a Beckman XL-I analytical ultracentrifuge. The following values (in units of M-1 cm-1) were employed: wildtype rat β-PV, 2890; rat β S33V, 2900; wild-type CPV3, 1560; and CPV3 S33V, 1600. Fluorescence Spectroscopy. The ANS emission spectrum was examined at 25 °C, in the absence and presence of the proteins of interest, using an SLM-Aminco 8100 fluorometer, modified for photon counting. Data were collected at 1 nm intervals between 380 and 600 nm, averaging for 1 s at each point, with excitation at 365 nm. A nominal 4 nm bandpass was employed for both excitation and emission channels. A 1.0 mM ANS solution was prepared in 0.15 M NaCl, 0.025 M Hepes, pH 7.4 (Hepes-buffered saline, HBS) and standardized spectrophotometrically (ε ) 4950 M-1 cm-1).23 This stock solution was diluted to 10 µM in HBS containing 50 mM EDTA. After collecting the spectrum of ANS solution alone, additions of Ca2+-free protein were made to yield final concentrations of 25, 50, 75, and 100 µM protein. Data were collected at 25 °C in a 1.0 cm quartz cuvette. The emission from Y26 was used to monitor thermally induced unfolding of CPV3 S33V. The temperature of the 5.0 µM sample, contained in a sealed 1.0 cm quartz cuvette, was regulated by means of a Peltier-equipped sample holder. Fluorescence was excited at 274 nm and monitored at 305 nm using an 8 nm bandpass for excitation and emission. Readings were made at 1.0 degree intervals, permitting the sample to equilibrate for 3 min at each temperature. Data were averaged for 10 s. Replicate data sets were analyzed simultaneously using the following two-state unfolding model

y) (yf + mfT) + (yu + muT){exp[(-∆H/RT)(1 - T/Tm)]} 1 + {exp[(-∆H/RT)(1 - T/Tm)]} (1) In eq 1, ∆H is the denaturational enthalpy at the transition midpoint, R is the gas constant, T is the absolute temperature, Tm is the transition midpoint, yf and mf are the intercept and slope of the pretransition baseline, and yu and mu are the intercept and slope of the post-transition baseline.24 ∆H and Tm were treated as global fitting parameters, whereas yf, mf, yu, and mu were treated as local parameters. Light Scattering. Light scattering was measured in the SLMAminco fluorometer. Excitation and emission monochromators were set to 450 nm, and the scattered light was monitored as a function of temperature. Data were acquired at 5.0 degree intervals, averaging the scattered signal for 10 s. If the size of the particle is small relative to the wavelength of the scattered radiation, then the Rayleigh ratio, Rθ, is related to the molecular weight of the scatterer, M, by the equation

S33V Variants of β-PV and CPV3

KC 1 ) + 2BC + ... Rθ M

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(2)

K is a collection of constants

K)

2π2n2o(dn/dC)2 Nλ4

(3)

in which no is the refractive index of the solvent, dn/dC is the specific refractive index increment, N is Avogadro’s number, and λ is the wavelength of the scattered light. The Rayleigh ratio is an instrument-independent measure of the scattering intensity

Rθ )

iθ r2 Io (1 + cos2 θ)

(4)

where iθ is the scattering intensity, Io is the intensity of the unpolarized incident light, r is the distance from the sample to the detector, and θ is the angle between the transmitted beam and the direction of observation.25 In eq 2, B is the second virial coefficient, a measure of solution nonideality. In the analysis of the temperature-dependent light scattering of CPV3 S33V, we assumed that K was temperature-independent and that the scattering particles behaved ideally (i.e., B ) 0). With these assumptions, the ratio of the scattering intensities at 25 °C and some other temperature should approximate the ratio of weightaveraged particle molecular weights at the two temperatures. Chemical Denaturation. Samples (2.0 mL, 5.0 µM) of Ca2+free protein in PBS/EDTA were titrated with 10.0 M urea while monitoring ellipticity at 222 nm in an Aviv 62DS circular dichroism spectrometer. Denaturant was added with an automated titrator (Hamilton Microlab 500) operated in the constantvolume mode. Following each titrant injection, the sample was allowed to equilibrate with stirring for 120 s prior to data collection (30 s). Data from three or four titrations, corrected for dilution, were treated globally with the following two-state unfolding model based on the linear extrapolation method26 y) (yn + mn[urea]) + (yu + mu[urea]) exp[-(∆Go - m[urea])/RT] 1 + exp[-(∆Go - m[urea])/RT]

(5) where yn and mn represent the intercept and slope of the pretransition baseline, yu and mu represent the intercept and slope of the post-transition baseline, ∆Go is the extrapolated conformational stability in the absence of urea, m describes the sensitivity of the conformational stability to [urea], R is the gas constant, and T is the absolute temperature.27 ∆Go and m were global fitting parameters, identical for all of the experiments; yn, mn, yu, and mu were local fitting parameters, allowed to vary between experiments. To prepare the titrant solution, 60.07 g of urea was transferred to a 100 mL volumetric flask, with 10.0 mL of 10X PBS/EDTA solution and sufficient water to affect dissolution. After equilibration to room temperature, the solution was diluted to 100 mL and the pH readjusted to 7.40. The solution was divided into aliquots, to avoid repeated freeze/thaw cycles, and stored at -20 °C. The molar urea concentration (M) was standardized

by refractometry, using the relationship M ) 117.66(∆η) + 29.753(∆η)2 + 185.56(∆η)3, where ∆η is the difference between the refractive index of the urea solution and that of the buffer.28 The predicted and measured concentrations agreed within 1%. Analytical Ultracentrifugation. Analytical ultracentrifugation measurements were performed in a Beckman XL-I analytical ultracentrifuge. Sedimentation velocity data were collected at 45 000 rpm and 20 °C in dual-sector charcoal-Epon centerpieces. Data collection was initiated immediately following rotor acceleration. The radial solute distributions were monitored at 274 nm. Ca2+-free CPV3 S33V, obtained by extensive dialysis against PBS/EDTA, was also examined by sedimentation equilibrium analysis at 35 °C, employing rotor speeds of 20 000, 30 000, and 40 000 rpm. The use of six-sector charcoal-Epon centerpieces permitted simultaneous examination of three separate loading concentrations. Absorbance was monitored at 274 nm, averaging 10 readings at each radial position. Radial solute distributions were collected at 1 h intervals at each speed until successive data sets were indistinguishable. The resulting nine radial distributions (three loading concentrations at each of three rotor speeds) were subjected to global nonlinear least-squares minimization in Origin v. 7.5 (OriginLab). The data were satisfactorily accommodated by an equation describing the radial distribution of a single ideal species

[

a ) ao exp

]

Mω2(1 - VjF) 2 (r - r2o) + bl 2RT

(6)

where a is the absorbance at the radial position r, ao is the absorbance at an arbitrary reference position ro, M is the molecular weight, ω is the angular velocity, Vj is the partial specific volume, F is the solution density, R is the gas constant, T is the absolute temperature, and bl is a baseline offset to account for optical mismatch between the sample and solvent sectors. The partial specific volume was set to 0.72 cm3/g, based on the amino acid composition of the protein,29 and the solvent density was measured with an Anton-Paar DMA 5000 densimeter. Isothermal Titration Calorimetry. Isothermal titration calorimetry experiments were performed with a MicroCal VP-ITC in HBS at 25 °C. The protein samples were titrated with Ca2+ in the presence and absence of competitive chelators (EDTA, EGTA, NTA), with Ca2+ in the presence of Mg2+, and with Mg2+ in the presence and absence of EDTA. Each titration protocol included a 2 µL preinjection, the heat from which was ignored during the fitting process. Raw ITC data were integrated with software supplied with the instrument and compiled into a master file. This composite data set was subjected to least-squares minimization, to obtain estimates of the binding constants and enthalpies for both Ca2+ and Mg2+. The data treatment and error analysis are described in detail elsewhere.20,30,31 Prior to analysis, residual divalent metal ions were removed from the proteins by passage over EDTA-agarose.32 The resulting material contained less than 0.02 mol equiv of Ca2+, measured by atomic absorption spectrometry. The preparation of the chelating matrix and details of its use are described elsewhere.31,33 Thermal Denaturation of CPV3 S33V with CD Detection. A 5.0 µM sample of the protein in PBS/EDTA, in a sealed 1.0 cm cuvette, was heated from 5 to 80 °C while monitoring the ellipticity at 222 nm in an Aviv 62DS spectrometer. Replicate data sets were analyzed with the two-state unfolding model described earlier (eq 1).

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Differential Scanning Calorimetry. Differential scanning calorimetry was performed with a Nano-DSC (Calorimetry Sciences Corporation), equipped with 0.32 mL cylindrical hastalloy cells. Temperature calibration was confirmed using samples of DMPC, DPPC, and DSPC. The accuracy of the differential power measurements was verified with internally generated electrical calibration pulses. Prior to analysis, protein samples were dialyzed extensively against PBS/EDTA, which was then employed as the reference buffer. Sample and reference solutions were degassed under vacuum for 5 min immediately before loading. Reversibility was examined by rescanning each sample. If the sample exhibited an endotherm upon rescan comparable in area to the original scan, the unfolding process was assumed to be reversible. Data were then collected at several protein concentrations and subjected to global analysis to extract estimates of Tm, ∆Hcal, ∆HvH, and ∆Cp, as described previously.34 A baseline, obtained with sample and reference cells filled with buffer, was subtracted from the protein data prior to analysis. Although the thermal unfolding of wild-type CPV3 is reversible at low to moderate protein concentrations, the posttranslational baseline is not well-defined, necessitating truncation of the data sets at 65 °C. To improve the parameter estimates, particularly ∆Cp, the DSC data were analyzed simultaneously with urea denaturation experiments conducted at 20, 25, 30, and 35 °C. The use of chemical denaturation data in the determination of ∆Cp was originally suggested by Pace and Laurents.35 The fitting strategy that was employed is comparable to that of McCrary et al.36 Equation 5 was used to treat the urea denaturation data. However, rather than treating ∆Go as a fitting parameter, its value is calculated with the Gibbs-Helmholtz equation

(

∆Go ) ∆HvH

)

T 1+ ∆Cp[(T - Tm) - T ln(T/Tm)] Tm (7)

Thus, least-squares minimization yields values of Tm, ∆HvH, and ∆Cp consistent with both the chemical and thermal denaturation data. To estimate the area of an irreversible unfolding transition, a fourth-order polynomial was fit to the data immediately preceding and immediately following the transition. The area of the transition between the heat capacity curve and the baseline was integrated numerically. Results Divalent Ion Binding. Figure 2A and B compares raw calorimetry data from titrations of wild-type CPV3 and the CPV3 S33V with Ca2+. Corresponding data for rat β-PV and rat β S33V are displayed in panels C and D. The S33V substitution has a greater impact on the appearance of the CPV3 data than it has on the rat PV-β data. The divalent ion-binding properties of the rat β-PV and CPV3 S33V variants were measured by global ITC analysis, as described in Experimental Methods. Aliquots of the Ca2+-free proteins were titrated with Ca2+ in the absence and presence of competing chelators or Mg2+ and with Mg2+ in the absence or presence of EDTA. Estimates of the binding parameters for both Ca2+ and Mg2+ were obtained by global least-squares analysis of the resulting data. The integrated data and the best leastsquares fit to the data are displayed in Figures 3 and 4 for β-PV

Tan et al.

Figure 2. Raw ITC data. (A) Titration of 74 µM CPV3 with 1.03 mM Ca2+. (B) Titration of 63 µM CPV3 S33V with 1.10 mM Ca2+. (C) Titration of 62 µM rat β-PV with 1.04 mM Ca2+. (D) Titration of 58 µM rat β S33V with 1.10 mM Ca2+.

S33V and CPV3 S33V, respectively. The optimal parameter values and experimental uncertainties are listed in Table 1. The thermodynamic parameters associated with Ca2+ and Mg2+ binding in the wild-type and S33V variants are presented in Table 2. In both wild-type rat β-PV and CPV3, the EF and CD sites are occupied sequentially in titrations with Ca2+ and Mg2+. We assume that this order of binding is preserved in the variant proteins. Ca2+ Binding. In CPV3, replacement of S33 with valine yields a small, but perceptible, improvement in the free-energy change associated with Ca2+ binding to the EF site. The ∆∆G value (-0.27 kcal/mol) results from relatively large and compensating changes in enthalpy (∆∆H ) 1.93 kcal/mol) and entropy (-T∆∆S ) -2.22 kcal/mol). In the case of rat β-PV, the ∆∆G value (-0.13 kcal/mol) is realized through a minor improvement in binding enthalpy. Whereas the free-energy change associated with binding to the EF site is slightly improved, binding to the CD site is less favorable in both proteins. Once again, the underlying energetic basis for the change in binding free energy differs in the two proteins. For rat β S33V, the ∆∆G value of 0.31 kcal/mol primarily reflects a less favorable entropic contribution (-T∆∆S ) 0.25 kcal/mol). By contrast, the 0.22 kcal/mol value observed for the S33V mutation in the CPV3 background is the result of offsetting changes of comparable magnitude in enthalpy (∆∆H ) -0.48 kcal/mol) and entropy (-T∆∆S ) 0.72 kcal/mol). Due to the compensating changes in free energy for the CD and EF site binding events, the overall changes in ∆G°′ resulting from the S33V mutation are miniscule for both proteins (-0.18 kcal/mol for rat β-PV and -0.05 kcal/mol for CPV3) and approach the experimental uncertainties.


Figure 3. Global ITC analysis of divalent ion binding by rat β-PV S33V. (A) 1.10 mM Ca2+ versus 57 µM protein (red); 1.10 mM Ca2+ versus 29 µM protein (green); 1.10 mM Ca2+ versus 56 µM protein, 0.1 mM NTA (blue); 1.10 mM Ca2+ versus 57 µM protein, 1.0 mM NTA (magenta). (B) 1.99 mM Mg2+ versus 54 µM protein (red); 1.99 mM Mg2+ versus 58 µM protein, 0.12 mM EDTA (green). (C) 1.1 mM Ca2+ versus 58 µM protein, 1.0 mM Mg2+ (red); 1.1 mM Ca2+ versus 56 µM protein, 5.0 mM Mg2+ (green); 1.1 mM Ca2+ versus 58 µM protein, 10.0 mM Mg2+ (blue). (D) 1.1 mM Ca2+ versus 58 µM protein, 72 µM EDTA (red); 1.1 mM Ca2+ versus 58 µM protein, 58 µM EGTA (green).

Mg2+ Binding. In rat β, the mutation affords a minor improvement in Mg2+ binding at the EF site (∆∆G ) -0.15 kcal/mol) due to slightly more favorable entropic (-T∆∆S )


Figure 4. Global ITC analysis of divalent ion binding by CPV3 S33V. (A) 1.10 mM Ca2+ versus 56 µM protein (red); 1.10 mM Ca2+ versus 28 µM protein (green); 1.10 mM Ca2+ versus 55 µM protein, 0.1 mM NTA (magenta); 1.10 mM Ca2+ versus 55 µM protein, 1.0 mM NTA (blue). (B) 1.99 mM Mg2+ versus 62 µM protein (red); 1.99 mM Mg2+ versus 60 µM protein, 0.12 mM EDTA (green). (C) 1.1 mM Ca2+ versus 57 µM protein, 1.0 mM Mg2+ (red); 1.1 mM Ca2+ versus 52 µM protein, 5.0 mM Mg2+ (green); 1.1 mM Ca2+ versus 52 µM protein, 10.0 mM Mg2+ (blue). (D) 1.1 mM Ca2+ versus 56 µM protein, 72 µM EDTA (green); 1.1 mM Ca2+ versus 54 µM protein, 65 µM EGTA (red).

-0.09 kcal/mol) and enthalpic (∆∆H ) -0.05 kcal/mol) contributions. As observed for Ca2+, this minor affinity increase is nearly offset by the diminished affinity of the second Mg2+binding event. The opposing enthalpy and entropy changes associated with this second event are somewhat larger, ∆∆H ) 0.75 kcal/mol and -T∆∆S ) -0.53 kcal/mol. As a result of

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TABLE 1: Divalent Ion-Binding Parametersa quantity

rat β-PVb

rat β-PV S33 V

CPV3c

CPV3 S33V

2+

∆H1 k1 ∆H2 k2 ∆H1 k1 ∆H2 k2

-4.10 (0.13) 2.45 (0.53) × 107 -3.46 (0.13) 1.43 (0.45) × 106

Ca Binding -4.30 (0.13) 3.08 (0.77) × 107 -3.40 (0.17) 8.42 (2.36) × 105

-5.46 (0.40) 4.50 (0.93) × 107 -0.13 (0.11) 2.43 (0.47) × 107

-3.53 (0.16) 7.19 (1.70) × 107 -0.61 (0.16) 1.67 (0.44) × 107

3.01 (0.11) 9.40 (0.91) × 103 4.16 (0.35) 1.64 (0.33) × 102

Mg2+ Binding 2.96 (0.21) 1.22 (0.28) × 104 4.91 (0.73) 1.13 (0.37) × 102

1.53 (0.39) 4.98 (1.38) × 104 1.62 (0.53) 2.12 (0.54) × 104

1.67 (0.26) 6.34 (2.32) × 104 1.68 (0.07) 2.43 (0.73) × 104

a Measured at 25 °C, in 0.15 M NaCl, 0.025 M Hepes, pH 7.4. Enthalpies are expressed in units of kcal/mol; the microscopic binding constants are expressed in units of M-1. Numbers in parentheses are the 95% confidence intervals. b From Henzl et al.20 c From Henzl and Agah.19

the compensatory changes in ∆∆G associated with the two binding events, the overall change in Mg2+ affinity resulting from the S33V mutation (∆∆G ) 0.07 kcal/mol) is minimal and within the experimental uncertainty. In CPV3, the S33V mutation causes modest improvements in binding free energy for both Mg2+-binding events (∆∆Gtot ) -0.24 kcal/mol). In each case, the improvement reflects a slightly more favorable entropic contribution. ANS Fluorescence Studies. The quantum yield of 8-anilino1-naphthalenesulfonate (ANS) is highly sensitive to solvent polarity, and the molecule has long been used to probe for solvent-accessible apolar surface area in proteins.37 Strongly quenched in aqueous solution, ANS emission increases dramatically with access to apolar surface. To determine whether replacement of S33 with valine provokes a perceptible conformational change in rat β-PV and CPV3, we compared the ANSbinding behavior of the S33V variants and the wild-type proteins. Aliquots of Ca2+-free protein were added to 10 µM solutions of the fluorescent probe. Following each addition, the emission spectrum was acquired between 380 and 600 nm, with excitation at 365 nm. Fluorescence intensity at 497 nm is plotted versus protein concentration in Figure 5A for rat β-PV (O) and rat β S33V (b) and in Figure 5B for CPV3 (O) and CPV3 S33V (b).

Figure 5. ANS fluorescence. (A) Impact of the addition of Ca2+-free rat β-PV (O) or rat β S33V (b) to a 10 µM solution of ANS in 0.15 M NaCl, 0.025 M Hepes, 0.05 M EDTA, pH 7.4, 25 °C. (B) Corresponding data for Ca2+-free rat CPV3 (O) or CPV3 S33V (b).

Interestingly, the S33V mutation has sharply contrasting effects on protein conformation in the two protein backgrounds. The much steeper rise in ANS emission observed upon addition

TABLE 2: Energetics of Divalent Ion Bindinga quantity

rat β-PV

rat β-PV S33 V

CPV3

CPV3 S33V

2+

a

∆G1 ∆H1 -T∆S1 ∆G2 ∆H2 -T∆S2 ∆Gtot ∆Htot -T∆Stot

-10.08 ( 0.11 -4.10 ( 0.13 -5.98 ( 0.17 -8.39 ( 0.16 -3.46 ( 0.13 -4.93 ( 0.21 -18.47 ( 0.19 -7.56 ( 0.18 -10.91 ( 0.26

Ca Binding -10.21 ( 0.13 -4.30 ( 0.13 -5.91 ( 0.18 -8.08 ( 0.15 -3.40 ( 0.17 -4.68 ( 0.23 -18.29 ( 0.20 -7.70 ( 0.21 -10.59 ( 0.29

-10.44 ( 0.11 -5.46 ( 0.40 -4.9 ( 0.41 -10.07 ( 0.10 -0.13 ( 0.11 -9.94 ( 0.15 -20.51 ( 0.15 -5.59 ( 0.41 -14.92 ( 0.44

-10.71 ( 0.12 -3.53 ( 0.16 -7.18 ( 0.20 -9.85 ( 0.14 -0.61 ( 0.16 -9.24 ( 0.21 -20.56 ( 0.18 -4.14 ( 0.23 -16.3 ( 0.29

∆G1 ∆H1 -T∆S1 ∆G2 ∆H2 -T∆S2 ∆Gtot ∆Htot -T∆Stot

-5.42 ( 0.06 3.01 ( 0.11 -8.43 ( 0.13 -3.02 ( 0.11 4.16 ( 0.35 -7.18 ( 0.37 -8.44 ( 0.13 7.20 ( 0.37 -15.6 ( 0.39

Mg2+ Binding -5.57 ( 0.12 2.96 ( 0.21 -8.52 ( 0.24 -2.80 ( 0.16 4.91 ( 0.73 -7.71 ( 0.75 -8.37 ( 0.20 7.87 ( 0.76 -16.24 ( 0.79

-6.40 ( 0.15 1.53 ( 0.39 -7.93 ( 0.42 -5.89 ( 0.14 1.62 ( 0.53 -7.51 ( 0.55 -12.29 ( 0.21 3.15 ( 0.66 -15.44 ( 0.69

-6.55 ( 0.18 1.67 ( 0.26 -8.22 ( 0.32 -5.98 ( 0.16 1.68 ( 0.07 -7.66 ( 0.17 -12.53 ( 0.24 3.35 ( 0.27 -15.88 ( 0.36

Energies are expressed in kcal mol-1. All data were collected at 25 °C, in 0.15 M NaCl, 0.025 M Hepes, pH 7.4.



Figure 6. Urea denaturation. (A) Replicate titrations of wild-type rat β-PV (5.0 µM) with 10.0 M urea, at 25 °C, in PBS, containing 0.5 mM EDTA. Unfolding was monitored by circular dichroism at 230 nm. (B) Corresponding titrations of rat β S33V. (C) Corresponding titrations of wild-type CPV3. (D) Corresponding data for CPV3 S33V. The solid lines reflect the best least-squares fit to eq 5. Data have been offset vertically for clarity.

TABLE 3: Urea Denaturation Parametersa protein

∆Go (kcal mol-1)

m (kcal mol-1 M-1)

rat β-PV rat β-PV S33 V CPV3 CPV3 S33V

4.03 ( 0.07 4.08 ( 0.08 4.81 ( 0.06 4.09 ( 0.06

1.42 ( 0.02 1.40 ( 0.03 1.46 ( 0.02 1.42 ( 0.02

a All data acquired at 25 °C, in 0.15 M NaCl, 0.010 M NaPi, 0.005 M EDTA, pH 7.40.

of CPV3 S33V, a 2.6-fold increase in slope relative to the wildtype protein, implies that replacement of S33 with valine has enlarged the area of the solvent-exposed apolar surface in CPV3. By contrast, the much shallower increase in ANS emission observed upon addition of rat β S33V, a 2.7-fold reduction in slope relative to the wild-type protein, suggests that the S33V substitution has decreased the solvent-accessible apolar surface area in rat β-PV. Chemical Denaturation of the S33V Variant Proteins. Urea denaturation experiments were performed to assess the intrinsic conformational stabilities of the S33V variants. The proteins were titrated with urea at 25 °C in the presence of 0.5 mM EDTA. Data for wild-type rat β-PV are displayed in Figure 6A; data for the rat β S33V variant are displayed in Figure 6B. Figure 6C and D presents corresponding analyses for wild-type CPV3 and CPV3 S33V, respectively. All of the unfolding transitions were reversible. The solid lines represent the best global least-squares fit to the data. Table 3 lists the conformational free energies in the absence of urea (∆Go) and the m value, a measure of the sensitivity of the unfolding reaction to [urea], for each protein. The ∆Go of 4.03 kcal mol-1 obtained for wild-type rat β agrees well with the previously reported value.34 Upon replacement of S33 by valine, the ∆Go increases to 4.08 kcal mol-1.

Figure 7. Thermal denaturation of the S33V variants of rat β-PV and CPV3. (A) DSC analysis of rat β S33V at concentrations of 1.7, 3.4, 5.1, and 6.8 mg/mL in PBS/EDTA. The solid red line denotes the best least-squares fit to the data, indicated by the black line. (B) Urea denaturation of 5.0 µM rat β S33V at 20 (O), 25 (0), 30 (4), and 35 (]) °C. These data were subjected to simultaneous least-squares minimization, together with the DSC data shown in panel A. The solid red lines represent the optimal global fit to the data. (C) DSC analysis of CPV3 S33V in PBS/EDTA, at concentrations of 7.1 (black), 5.3 (red), 3.6 (green), and 1.8 (blue) mg/mL. (D) Thermal denaturation of 5.0 µM CPV3 S33V in PBS, 0.5 mM EDTA, pH 7.4, monitored by CD at 230 nm. For clarity, only every other data point is displayed. The red lines indicate the best global fit to the three data sets. (E) Thermal denaturation of 5.0 µM CPV3 S33V in PBS, 0.5 mM EDTA, pH 7.4, monitored by fluorescence. Excitation at 274 nm; emission at 305 nm. The red line denotes the best simultaneous fit of the two data sets to a two-state unfolding model. (F) CD spectra of 5.0 µM CPV3 S33V before (black) and after (red) CD-monitored thermal denaturation.

Thus, the mutation has a negligible impact on the conformational stability in the rat β-PV. By contrast, the S33V mutation reduces the stability of CPV3 from ∆Go is 4.81 to 4.09 kcal mol-1. Thermal Denaturation of the S33V Variant Proteins. The thermal stabilities of the S33V variants were initially examined by scanning calorimetry. Data for rat β S33V are displayed in Figure 7A. Simultaneous analysis of DSC data, plus urea denaturation data (Figure 7B) collected at 20, 25, 30, and 35 °C, yielded a Tm of 49.8 °C, ∆HvH of 67.1 kcal mol-1, ∆Hcal of 66.5 kcal mol-1, and estimated ∆Cp of 1.58 kcal mol-1 K-1. These values are comparable to those obtained previously for wild-type rat β-PV, Tm ) 49.3 °C, ∆HvH ) 67.9 kcal mol-1, ∆Hcal ) 72.9 kcal mol-1, and ∆Cp ) 1.60 kcal mol-1 K-1. Corresponding data for CPV3 S33V are displayed in Figure 7C. The observed behavior is highly concentration-dependent. With increasing protein concentration, the primary transition shifts to lower temperature. The apparent transition temperatures were 45.7, 44.8, 43.4, and 43.0 °C at 1.8, 3.5, 5.3, and 7.1 mg/ mL, respectively. A secondary transition is observed near 80

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Figure 9. Light scattering by CPV3 S33V as a function of temperature. A 5.4 mg/mL sample of CPV3 S33V was irradiated at 450 nm. The scattered radiation was monitored at an angle of 90° in an SLM 8100 fluorometer as the sample was heated from 25 to 60 °C.

Figure 8. Conformational stability of wild-type CPV3. (A) DSC data were collected at protein concentrations of 1.95, 1.58, and 1.01 mg/ mL. (B) Urea denaturation of 5.0 µM CPV3 at 20 (O), 25 (0), 30 (4), and 35 (]) °C. The data in these two panels were subjected to simultaneous least-squares minimization. The solid red lines represent the optimal fit to the composite data set.

°C. The intensity of this broad endotherm increases with increased sample concentration, and the maximum shifts to higher temperature, increasing from 80.1 °C at a protein concentration of 3.5 mg/mL to 81.6 °C at 5.3 mg/mL and to 84.7 °C at 7.1 mg/mL. The concentration-dependent behavior and the substantially decreased endotherm observed upon reheating (vide infra) suggest that the transition is not reversible under these conditions. However, if the thermal denaturation of CPV3 S33V is conducted at much lower protein concentration, reversible behavior is observed. Figure 7D displays the thermal denaturation of 5.0 µM (0.06 mg/mL) CPV3 S33V, monitored by circular dichroism at 222 nm. The best global fit of the three replicate data sets to eq 1, denoted by the solid lines, yields estimates for the melting temperature and denaturational enthalpy of 48.9 ( 0.1 °C and 57.8 ( 0.9 kcal/mol, respectively. The far-UV CD spectrum obtained immediately upon cooling the sample to room temperature is virtually identical to the spectrum collected prior the analysis (Figure 7F), an indication that the thermally denatured protein is capable of facile refolding at low concentration. Although wild-type CPV3 also exhibits concentration-dependent unfolding behavior, at concentrations of 2.5 mg/mL or lower, the Tm is concentration-independent, and the unfolding is completely reversible. An evaluation of CPV3 conformational stability by global analysis of DSC and urea denaturation data is displayed in Figure 8. The analysis yielded a Tm of 50.7 °C, ∆HvH ) 77.8 kcal/mol, ∆Hcal ) 77.9 kcal/mol, and ∆Cp ) 1.30 kcal mol-1 K-1. The van’t Hoff enthalpy for denaturation describes the temperature dependence of the equilibrium constant for unfolding, increasing with the sharpness of the transition. The virtual identity of the van’t Hoff and calorimetric enthalpies indicates that thermal unfolding of wild-type CPV3 proceeds from the native to the denatured state with negligible population of intermediate states. The much lower ∆HvH value obtained for

CPV3 S33V, 57.8 kcal/mol versus 77.8 kcal/mol, suggested that thermal unfolding of the variant is not, in fact, a two-state process. To test this idea, we monitored the thermal unfolding of CPV3 S33V by fluorescence. The resulting data are displayed in Figure 7E. The best simultaneous fit to replicate data sets, assuming two-state behavior, afforded a Tm of 48.3 ( 0.1 °C and ∆HvH of 63.0 ( 0.6 kcal/mol. These values differ significantly from the estimates obtained by CD (48.9 °C, 57.8 kcal/mol). The fact that distinct spectroscopic probes yield disparate estimates for the thermodynamic parameters provides additional evidence that the denaturation process is not twostate and proceeds through a partially unfolded intermediate. The DSC behavior exhibited by CPV3 S33V suggested that the denatured protein was aggregating. To test this hypothesis, we examined the intensity of Rayleigh scattering from a sample of CPV3 S33V as a function of temperature. A 450 µM sample of the protein was heated from 25 to 60 °C by means of the Peltier-controlled sample holder in our SLM 8100 fluorometer. The intensity of the scattering signal is plotted versus temperature in Figure 9. The major increase in scattering observed between 40 and 50 °C indicates that the protein is in fact aggregating. As described in Experimental Methods, the ratio of the scattering intensities at 25 and 55 °C should roughly equal the ratio of the weight-averaged molecular weights for the two temperatures. The calculated ratio is approximately 7.6. Sedimentation equilibrium data (not shown) return a molecular weight close to the sequence-derived molecular weight of 11 986. Thus, the light-scattering data suggest that, at the higher temperature, the weight-averaged molecular weight of the denatured preparation is approximately 91 000. Sedimentation velocity data (vide infra) provide further evidence that CPV3 S33V aggregates irreversibly during thermal denaturation at high concentration. The impact of the DSC scan rate on the thermal denaturation behavior is displayed in Figure 10. Samples of the protein were heated at rates of 2.0, 1.0, 0.5, 0.25, and 0.125 deg min-1. The apparent Tm, 45.1 °C, is independent of scan rate at or below 1.0 deg min-1. At 2.0 deg min-1, however, the maximum is shifted downward, to 43.2 °C. If the samples are cooled and rescanned, a transition is observed on the rescan, albeit markedly reduced in amplitude. Figure 11A shows the behavior observed at a scan rate of 2.0 deg min-1. The area of the first transition observed on the rescan is just 30% that of the original scan. The peak maximum is shifted upward by approximately 0.6 deg. Interestingly, the area



Figure 10. Scan rate dependence of CPV3 thermal denaturation at heating rates of 0.125 (magenta), 0.25 (blue), 0.50 (green), 1.0 (red), and 2.0 (black) degrees per minute. Due to the length of time required for data acquisition at the two slowest heating rates, narrower temperature ranges were employed for the two slowest heating rates. Figure 12. Sedimentation velocity analysis of denatured ensemble. Samples of CPV3 S33V that had been subjected to thermal denaturation by DSC were removed when the calorimeter temperature reached 65 °C, loaded into sedimentation velocity cells, and centrifuged at 45 000 rpm. Data for the initial 10 scans are displayed. The sample in panel A was heated in the calorimeter at a rate of 2.0 deg min-1. The sample in panel B was heated at a rate of 0.125 deg min-1.

Figure 11. Reproducibility of DSC behavior as a function of scan rate. (A) Data were acquired at a scan rate of 2.0 deg min-1. (B) Data acquired at 0.125 deg min-1. In both cases, the black line corresponds to the original scan, and the red line corresponds to the rescan.

of the second transition, near 85 °C, is comparable to that of the original scan. That second transition is sharper and shifted upward by approximately 1.4 deg. The corresponding behavior observed at 0.125 deg min-1 is displayed in Figure 11B. Due to the much longer time required for the experiment, data acquisition was terminated at lower temperature. At this slower scan rate, the area of the rescan is 40% of the original. It may be significant that the transition is broadened on the rescan and shifted to higher temperature. To determine whether the properties of the denatured ensemble were sensitive to scan rate, samples were heated in the calorimeter (at either 2.0 or 0.125 deg min-1), removed when the instrument temperature reached 65 °C, immediately cooled to room temperature, and subjected to sedimentation velocity analysis. Figure 12A and B shows the first 10 scans acquired on material denatured in the calorimeter at 2.0 and 0.125 deg min-1, respectively. The material denatured at the higher scan rate exhibits much greater evidence of aggregation. At least three populations of molecules are evident. A very rapidly sedimenting population of particles has nearly traversed the length of cell in the time required for the rotor acceleration and acquisition of the initial scan. It should be noted that the very high “absorbance” readings are a consequence of intense scattering

of the incident radiation by the high-molecular-weight particles. There is a second rapidly sedimenting population that has largely cleared the cell by scan 10. The remaining material exhibits sedimentation behavior consistent with a monomeric parvalbumin species. The plateau absorbance for this latter species is 0.38. In the 1.2 cm path length of the sedimentation velocity centerpiece, the 0.65 mM sample should display an absorbance of 1.25. Thus, after heating to 65 °C in the calorimeter, approximately 30% of the sample is present as monomer and 70% as higher-molecular-weight aggregates. When the material denatured in the calorimeter at 0.125 deg min-1 is treated similarly, the observed behavior is qualitatively very different. Although there is a population of rapidly sedimenting particles, the vast majority of material remains monomeric, as judged by the fact that the residual plateau absorbance following the 10th scan is near 1.25. This observation suggests that the thermal unfolding should exhibit greater reversibility at the lower scan rate. Nevertheless, as noted above, the transition area observed on the rescan at the lower scan rate is only marginally greater than that observed at 2.0 deg min-1. Our present inability to offer a satisfactory explanation for the discrepancy is unsatisfying. However, it is clear that the apparent reversibility of the thermal unfolding event is a complex function of the initial scan conditions, including protein concentration and scan rate. Discussion The S33V substitution evidently has different structural consequences for the two proteins. Addition of Ca2+-free CPV3 S33V to a solution of the hydrophobic surface probe, ANS, produces a substantially larger increase in ANS emission than addition of a corresponding amount of wild-type CPV3. This observation implies that the unliganded variant exposes substantially more apolar surface than the wild-type protein. Conceivably, replacement of S33 with valine provokes a reorientation of the B-helix comparable to that observed in Ca2+free ATH. In the latter, removal of Ca2+ causes the coordinated

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displacement and rotation of helix B, resulting in exposure of apolar side-chains in the B-helix and creation of a hydrophobic pocket.38 By contrast, addition of the unliganded S33V variant of rat β-PV to an ANS solution produces a substantially smaller increase in probe emission than the corresponding addition of wild-type rat β. The implication is that the area of the solventaccessible apolar surface is reduced in the Ca2+-free protein by introduction of the S33V mutation. The binding/dissociation of Ca2+ from rat β-PV is accompanied by substantial reorganization of the hydrophobic core.39 The energetic cost of this structural remodeling may contribute to the attenuated divalent ion affinity displayed by the protein (vide infra). Apparently, the replacement of S33 with valine alters the details of this structural rearrangement. In light of the putative structural changes provoked by the S33V mutation, the muted impact on divalent ion affinity is interesting. In rat β, a minor increase in EF site Ca2+ affinity, coupled with a slightly greater decrease in CD site affinity, produces a very minor net loss in overall Ca2+ affinity. The effect of the mutation on Mg2+ affinity is virtually identical. In CPV3, the mutation likewise affords a modest increase in the Ca2+ affinity of the EF site and a modest decrease in the CD site affinity. However, because the magnitudes of the perturbations are comparable, the overall Ca2+ affinity is unchanged. Overall Mg2+ affinity is slightly improved. In CPV3, the negligible alteration of the ∆G for Ca2+ binding belies substantial, offsetting changes in ∆H and -T∆S. The S33V mutation yields a ∆∆H for the EF site of +1.93 kcal mol-1 and a corresponding -T∆∆S term of -2.20 kcal mol-1. Recall that the ANS-binding data for CPV3 S33V indicate that the unliganded variant protein exposes more apolar surface than wild-type CPV3 and, presumably, buries correspondingly more surface upon Ca2+ binding. Burial of the apolar surface is enthalpically unfavorable and entropically favorable. Thus, the perturbations of the EF site binding enthalpy and entropy are consistent with the proposed change in the solvent-accessible apolar surface area. The fact that the ∆∆H and -T∆∆S terms associated with the binding event at the CD site are substantially smaller (-0.48 and 0.70 kcal/mol, respectively) suggests that the Ca2+-provoked conformational change is largely complete with binding of the first ion. Significantly, the S33V mutation does not have a comparable impact on the underlying Mg2+-binding energetics. The enthalpic and entropic contributions for the EF site are essentially unchanged by the mutation. This finding implies that the conformational change provoked by Mg2+ binding to CPV3 differs fundamentally from that produced by binding of Ca2+. Whereas the -z glutamyl side-chain in an EF-hand motif coordinates Ca2+ in a bidentate fashion, it coordinates Mg2+ as a monodentate ligand.40 Consequently, the structural alterations in parvalbumin triggered by Mg2+ binding will not necessarily be identical to those that accompany Ca2+ binding. In pike parvalbumin (pI 4.1), the observed differences are minor and largely restricted to residues adjacent to the ion-binding loops.41 However, there is compelling circumstantial evidence that the Ca2+-bound and Mg2+-bound conformations of ATH may differ profoundly.42 ANS-binding data, far-UV CD data, and sedimentation velocity data all suggest that the conformation of the Mg2+-bound protein more closely resembles the Ca2+-free form of the protein. Earlier, a parallel was drawn between the ANSbinding behavior of unliganded CPV3 S33V and that of Ca2+free ATH, which prompted speculation that Ca2+ removal might provoke a comparable reorientation of the B-helix in CPV3

Tan et al. S33V. Perhaps the resemblance between CPV3 S33V and ATH extends to the Mg2+-bound conformation. In contrast to the CPV3 system, the unliganded S33V variant of rat β-PV has less solvent-accessible apolar surface than the wild-type protein. Consequently, the binding of Ca2+ to the variant should be accompanied by reduced burial of the apolar surface. Although the negative ∆∆H and positive -T∆∆S values would be consistent with decreased sequestration of the apolar surface, their magnitudes are small. It is apparent that the nature of the conformational change that accompanies Ca2+ binding to rat β-PV S33V differs fundamentally from that of CPV3 S33V. The S33V mutation has a markedly different impact on conformational stability in rat β-PV and CPV3. In urea denaturation experiments on the former, ∆Go increases slightly from 4.03 to 4.08 kcal/mol, and the m value is decreases slightly from 1.42 to 1.40 kcal mol-1 M-1. DSC analysis confirms that the mutation has a minimal impact on stability of rat β. By contrast, the conformational stability of CPV3 is reduced by 0.72 kcal/mol, from 4.81 to 4.09 kcal/mol. The m value is reduced from 1.46 to 1.42 kcal mol-1 M-1. Beyond its impact on the native-state stabilities of rat β-PV and CPV3, the S33V mutation also influences the unfolded proteins differently. Judging from the DSC behavior of rat β S33V, replacement of S33 by valine has little effect on denatured rat β-PV. The unfolding transition remains approximately twostate, and the thermal transition is entirely reversible. By contrast, when CPV3 S33V is examined by DSC, the observed behavior is strongly concentration-dependent. At a scan rate of 1 deg min-1, the maximum in the heat capacity curve decreases from 45.6 °C at a protein concentration of 1.8 mg/mL to 43.0 °C at 7.1 mg/mL. With the decrease in the apparent melting transition, a second broad endothermic transition appears near 80 °C. The maximum in this second transition shifts to higher temperature with increased protein concentration, increasing from 80.1 °C at a protein concentration of 3.5 mg/mL to 81.6 °C at 5.3 mg/mL and to 84.7 °C at 7.1 mg/mL. The concentration-dependent behavior suggested that the CPV3 S33V aggregates upon unfolding. That hypothesis was confirmed by light-scattering measurements and sedimentation velocity data. This finding prompted speculation that the thermal unfolding behavior might be reversible at sufficiently low protein concentration. Although sensitivity considerations precluded examination of the issue by DSC, spectroscopic methods (farUV CD, fluorescence), allowed the thermal denaturation of CPV3 S33V to be monitored at 5 µM (0.06 mg/mL). At this concentration, the protein unfolds reversibly. After cooling to room temperature, the far-UV CD spectrum of the heated sample is indistinguishable from the starting material between 250 and 195 nm. Thus, at sufficiently low concentration, thermal denaturation is thermodynamically reversible, and the protein refolds facilely upon cooling. The fact that the protein was capable of reversible thermal unfolding coupled with the DSC evidence for concentrationdependent aggregation suggested that the reversible unfolding transition was accompanied by, or closely followed by, aggregation of the denatured molecules. The implication was that thermal denaturation, at high protein concentration, was kinetically controlled. Because the scan rate dependence of the apparent melting temperature can provide an estimate for the activation energy of the kinetically limiting step,43,44 we examined the denaturation of CPV3 S33V in the scanning calorimeter as a function of scan rate.

S33V Variants of β-PV and CPV3 For aggregating systems, the transition temperature typically increases with increased scan rate. However, the apparent Tm for CPV3 S33V was 45.1 °C at a scan rate of 1.0 deg min-1 and 43.2 °C at 2.0 deg min-1, that is, the transition temperature decreases with scan rate. Although there was no further change in the primary transition temperature with further decreases in scan rate, the intensity and position of the second transition were strongly dependent on scan rate throughout the entire range. The intensity of the second transition increased markedly with the scan rate increase, and the peak maximum was shifted to lower temperature. Sedimentation velocity data collected at 0.125 and 2.0 deg min-1 indicate that both the extent of aggregation and the size of the aggregates increase with scan rate. Our analysis suggests that at 2.0 deg min-1, roughly 70% of the original sample has formed high-molecular-weight oligomers by 65 °C, the temperature at which the material was removed from the calorimeter for the sedimentation study. Consistent with that estimate, the endotherm observed on the rescan at 2.0 deg min-1 had 30% of the original area. At the lower scan rate, a much smaller fraction of the sample was present as high-molecular-weight aggregates. Somewhat unexpectedly, however, the apparent reversibility did not show a corresponding improvement at the lower scan speed. At 0.125 deg min-1, the area of the transition measured for the rescan was still just 40% of the original. The finding that the extent of the aggregation is dependent on both concentration and scan speed may offer some insight into the details of the unfolding/aggregation process. If we were witnessing two-state unfolding followed by aggregation of the denatured molecules, then the transition temperature should have increase with scan rate. The fact that the end point, as defined by the size of the aggregates and the extent of aggregation, is sensitive to both concentration and scan rate implies that the unfolding must proceed through an intermediate state, which can either completely unfold or form aggregates. Raising the concentration of the intermediate state, either by raising the sample concentration or the scan speed, will favor the aggregation over complete unfolding of the protein monomers. The spectroscopic thermal unfolding data offer additional evidence that denaturation proceeds through a partially unfolded intermediate state. At the very low concentrations (5 µM, 60 µg/mL) employed for these analyses, the unfolding is entirely reversible, and there is no suggestion of aggregation. However, the fact that two independent spectroscopic probes, Y26 fluorescence emission and the far-UV CD signal, do not yield the same thermodynamic parameters further suggests that the transition is not a two-state process. The alteration in the overall ∆G°′ for divalent ion binding resulting from the S33V mutation reflects changes in the stabilities of the apo- and Ca2+-bound states. Figure 13 depicts the impact of the sequence alteration on the Gibbs free energies for the Ca2+-free and Ca2+-bound states of the two proteins. This treatment assumes that the free energy of the denatured state, arbitrarily set to zero, is unchanged by the presence of the divalent cation. In the case of rat β-PV, the S33V substitution causes small opposing changes in the stabilities of the unliganded and Ca2+-loaded protein, which produces a modest reduction in overall Ca2+ affinity. By contrast, the same mutation in CPV3 has a virtually identical effect on the free energies of the unliganded and bound states, so that overall Ca2+ affinity is unchanged. The disparate impact of the mutation suggests that the interaction between the B-helix, where residue 33 resides, and the remainder of the protein architecture is fundamentally different in the two proteins.


Figure 13. Free-energy diagram depicting the impact of the S33V mutation on the Gibbs free energies of the unfolded (U), unliganded native (N), and Ca2+-bound (Ca2N) states of rat β-PV and CPV3. (A) The S33V variant of rat β-PV and wild-type rat β-PV. (B) Wild-type CPV3 and the S33V variant of CPV3.

Although generally regarded as interchangeable Ca2+ buffer proteins, parvalbumins display broad variations in divalent ion affinity. At present, the relevant determinants of affinity are poorly understood. The solution structure of Ca2+-free rat β-PV offers, perhaps, some insight into the basis for the highly attenuated divalent ion affinity of that protein.39 Removal of Ca2+ triggers a substantial reorganization of the hydrophobic core that is not observed in high-affinity isoforms, such as rat R-PV45 and avian thymic hormone.38 Notably, F66 and F70 withdraw from the protein core, whereas F49 adopts an interior position. The side-chains of I46, I50, and L85 also experience displacements. Significantly, the side-chains of I49 and L50 associate with L85 in the apoprotein, and that interaction must be abolished when Ca2+ binds. Divalent ion-binding properties comparable to those of CPV3 can be produced in rat β by coupling rat β f CPV3 mutations within the CD site at residues 49, 50, 57, 58, 59, and 60 with the L85F mutation.34,46 However, the factors responsible for attenuation of the divalent ion in CPV3, relative to high-affinity parvalbumin isoforms, remain unknown. We have previously suggested that the N-terminal AB domain might figure prominently in this issue. In studies conducted some years ago with the isolated peptides, the CD-EF metal ionbinding module from rat β-PV exhibited substantially greater affinity for the AB peptide from rat R than for the homologous rat β AB peptide.47 Consistent with that finding, the heterologous RAB/βCD-EF complex bound Ca2+ substantially more tightly than the homologous β/β complex. Moreover, in addition to the structural alterations previously mentioned, the interface between the AB and CD-EF domains in rat β-PV undergoes remodeling upon binding/dissociation of Ca2+. Considered together, these observations suggested that the N-terminal AB domain might modulate parvalbumin divalent ion-binding properties and originally motivated our interest in the S33V mutation. The presence of serine at residue 33 in the AB domain is unusual among parvalbumin isoforms; valine is far more common. It seemed likely that replacement of the compact, polar hydroxymethyl group of S33 in rat β-PV and CPV3 with a

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bulky, apolar isopropyl group would produce a substantial increase in Ca2+ affinity. The minimal impact of the S33V substitution on divalent ion-binding properties suggests that the role of the AB domain in divalent ion binding is complex. The recent structural analysis of ATH in the unliganded and Ca2+-bound states lends support to this idea. As previously mentioned, in that protein, removal of Ca2+ provokes a rotation and displacement of the B-helix. However, ATH is a high-affinity parvalbumin isoform, with Ca2+ affinity comparable to that of rat R-PV, implying that the reorientation of helix B is energetically neutral. With the exception of that alteration, the conformations of the Ca2+-free and Ca2+-bound forms are virtually indistinguishable. Evidently, the role of the AB domain is context-dependent, determined both by the residues in the domain and the residues in the CD-EF metal ionbinding module with which it interacts. Conclusions Rat β-PV and CPV3 exhibit attenuated Ca2+ affinity. Because their sequences are distinguished from the parvalbumin consensus by the presence of serine, rather than valine, at position 33, we examined the effect of replacing S33 by valine in the two protein backgrounds. The substitution was found to have a minuscule impact on overall divalent ion affinity in both rat β and CPV3. Otherwise, the consequences of the sequence alteration differed profoundly in the two systems. In the unliganded CPV3 molecule, the S33V mutation provokes a marked conformational change, substantially increasing the area of the solvent-accessible apolar surface. Consistent with that finding, the observed thermodynamic parameters suggest that, relative to wild-type CPV3, Ca2+ binding in the S33V variant is accompanied by increased burial of the apolar surface. The S33V mutation reduces the conformational stability of the unliganded protein by 0.7 kcal/mol and exacerbates the tendency for the protein to aggregate upon unfolding. By contrast, substitution of valine for S33 in rat β-PV paradoxically reduces the solvent-accessible apolar surface area. Moreover, the mutation has a negligible impact on the stability of the unliganded protein, and the denatured rat β S33V variant exhibits no discernible tendency to aggregate. Acknowledgment. This work was supported by NSF Award MCB0543476 and by an award from the University of Missouri Research Board. Abbreviations ANS, 8-anilinonaphthalene-1-sulfonate; CD, circular dichroism; CD site, the metal ion-binding site in parvalbumin flanked by the C and D helical segments; CPV3, chicken parvalbumin 3; EF site, the metal ion-binding site flanked by the E and F helical segments; DMPC, dimyristoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; DSC, differential scanning calorimetry; DSPC, distearoylphosphatidylcholine; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(βaminoethyl ether)-N,N,N′,N′-tetraacetic acid; HBS, Hepesbuffered saline; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IPTG, isopropyl-β-D-thiogalactopyranoside; ITC, isothermal titration calorimetry; LB, Luria-Bertani, NaPi, sodium phosphate; NTA, nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PBS/EDTA, phosphate-buffered saline containing 0.0005 M EDTA; PV, parvalbumin.

Tan et al. References and Notes (1) Kretsinger, R. H. Structure and evolution of calcium-modulated proteins. CRC Crit. ReV. Biochem. 1980, 8, 119–174. (2) Kawasaki, H.; Kretsinger, R. H. Calcium-binding proteins 1: EFhands. Protein Profile 1995, 2, 297–490. (3) Celio, M. R.; Pauls, T.; Schwaller, B. Guidebook to the CalciumBinding Proteins; Oxford University Press: New York, 1996. (4) Kawasaki, H.; Nakayama, S.; Kretsinger, R. H. Classification and evolution of EF-hand proteins. BioMetals 1998, 11, 277–295. (5) Kretsinger, R. H.; Nockolds, C. E. Carp muscle calcium-binding protein. II. Structure determination and general description. J. Biol. Chem. 1973, 248, 3313–3326. (6) Pauls, T. L.; Cox, J. A.; Berchtold, M. W. The Ca2+-binding proteins parvalbumin and oncomodulin and their genes: New structural and functional findings. Biochim. Biophys. Acta 1996, 1306, 39–54. (7) Schwaller, B. The continuing disappearance of “pure” Ca2+ buffers. Cell. Mol. Life Sci. 2009, 66, 275–300. (8) Goodman, M.; Pechere, J. F. The evolution of muscular parvalbumins investigated by the maximum parsimony method. J. Mol. EVol. 1977, 9, 131–158. (9) Moncrief, N. D.; Kretsinger, R. H.; Goodman, M. Evolution of EF-hand calcium-modulated proteins. I. Relationships based on amino acid sequences. J. Mol. EVol. 1990, 30, 522–562. (10) Fohr, U. G.; Weber, B. R.; Muntener, M.; Staudenmann, W.; Hughes, G. J.; Frutiger, S.; Banville, D.; Schafer, B. W.; Heizmann, C. W. Human R and β parvalbumins. Structure and tissue-specific expression. Eur. J. Biochem. 1993, 215, 719–727. (11) Barger, B.; Pace, J. L.; Ragland, W. L. Purification and partial characterization of an avian thymic hormone. Thymus 1991, 17, 181–197. (12) Hapak, R. C.; Zhao, H.; Boschi, J. M.; Henzl, M. T. Novel avian thymic parvalbumin displays high degree of sequence homology to oncomodulin. J. Biol. Chem. 1994, 269, 5288–5296. (13) Novak, R.; Brewer, J. M.; Ragland, W. L. Immunophenotype of splenic lymphocytes with receptors for avian thymic hormone. FASEB J. 1996, 10, A1048. (14) Novak, R.; Henzl, M. T.; Ragland, W. L. Receptor cells for the CPV3 parvalbumin of chicken thymus in spleen and caecal tonsils. J. Allergy Clin. Immunol. 1997, 99, S202. (15) Rada, J. A.; Denis, C. S. Analysis of gene expression during the recovery from experimental myopia. Mol. Biol. Cell 2003, 14, 367a. (16) Heller, S.; Bell, A. M.; Denis, C. S.; Choe, Y.; Hudspeth, A. J. Parvalbumin 3 is an abundant Ca2+ buffer in hair cells. J. Assoc. Res. Otolaryngol. 2002, 3, 488–498. (17) Henzl, M. T.; Shibasaki, O.; Comegys, T. H.; Thalmann, I.; Thalmann, R. Oncomodulin is abundant in the organ of Corti. Hearing Res. 1997, 106, 105–111. (18) Sakaguchi, N.; Henzl, M. T.; Thalmann, I.; Thalmann, R.; Schulte, B. A. Oncomodulin is expressed exclusively by outer hair cells in the organ of Corti. J. Histochem. Cytochem. 1998, 46, 29–39. (19) Henzl, M. T.; Agah, S. Divalent ion-binding properties of the two avian β-parvalbumins. Proteins 2006, 62, 270–278. (20) Henzl, M. T.; Larson, J. D.; Agah, S. Influence of Monovalent Cation Identity on Parvalbumin Divalent Ion-Binding Properties. Biochemistry 2004, 43, 2747–2763. (21) Henzl, M. T.; Hapak, R. C.; Likos, J. J. Interconversion of the ligand arrays in the CD and EF sites of oncomodulin. Influence on Ca2+-binding affinity. Biochemistry 1998, 37, 9101–9111. (22) Hapak, R. C.; Zhao, H.; Henzl, M. T. Oligomerization of an avian thymic parvalbumin: Chemical evidence for a Ca2+-specific conformation. FEBS Lett. 1994, 349, 295–300. (23) Weber, G.; Young, L. Fragmentation of bovine serum albumin by pepsin. I. The origin of the acid expansion of the albumin molecule. J. Biol. Chem. 1964, 239, 1415–1423. (24) Pace, C. N.; Hebert, E. J.; Shaw, K. L.; Schell, D.; Both, V.; Krajcikova, D.; Sevcik, J.; Wilson, K. S.; Dauter, Z.; Hartley, R. W.; Grimsley, G. R. Conformational stability and thermodynamics of folding of ribonucleases Sa, Sa2 and Sa3. J. Mol. Biol. 1998, 279, 271–286. (25) van Holde, K. E.; Johnson, W. C.; Ho, P. S. Principles of Physical Biochemistry, 2nd ed.; Pearson Prentice Hall: Upper Saddle River, NJ, 2006. (26) Pace, C. N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 1986, 131, 266–280. (27) Santoro, M. M.; Bolen, D. W. Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl R-chymotrypsin using different denaturants. Biochemistry 1988, 27, 8063–8068. (28) Warren, J. R.; Gordon, J. A. On the refractive indices of aqueous solutions of urea. J. Phys. Chem. 1996, 70, 297–300. (29) Perkins, S. J. Protein volumes and hydration effects: The calculations of partial specific volumes, neutron scattering matchpoints and 280nm absorption coefficients for proteins and glycoproteins from amino acid sequences. Eur. J. Biochem. 1986, 157, 169–180.

S33V Variants of β-PV and CPV3 (30) Henzl, M. T.; Larson, J. D.; Agah, S. Estimation of parvalbumin Ca2+- and Mg2+-binding constants by global least-squares analysis of isothermal titration calorimetry data. Anal. Biochem. 2003, 319, 216–233. (31) Henzl, M. T. Characterization of parvalbumin and polcalcin divalent ion binding by isothermal titration calorimetry. Methods Enzymol. 2009, 455, 259–296. (32) Haner, M.; Henzl, M. T.; Raissouni, B.; Birnbaum, E. R. Synthesis of a new chelating gel: Removal of Ca2+ ions from parvalbumin. Anal. Biochem. 1984, 138, 229–234. (33) Henzl, M. T.; Agah, S.; Larson, J. D. Characterization of the metal ion-binding domains from rat R- and β-parvalbumins. Biochemistry 2003, 42, 3594–3607. (34) Henzl, M. T.; Ndubuka, K. Low-affinity signature of the rat β-parvalbumin CD site. Evidence for remote determinants. Biochemistry 2007, 46, 23–35. (35) Pace, C. N.; Laurents, D. V. A new method for determining the heat capacity change for protein folding. Biochemistry 1989, 28, 2520– 2525. (36) McCrary, B. S.; Edmondson, S. P.; Shriver, J. W. Hyperthermophile protein folding thermodynamics: Differential scanning calorimetry and chemical denaturation of Sac7d. J. Mol. Biol. 1996, 264, 784–805. (37) Daniel, E.; Weber, G. Cooperative effects in binding by bovine serum albumin. I. The binding of 1-aniline-8-naphthalenesulfonate. Fluorometric titrations. Biochemistry 1966, 5, 1893–1900. (38) Schuermann, J. P.; Tan, A.; Tanner, J. J.; Henzl, M. T. Structure of avian thymic hormone, a high-affinity avian β-parvalbumin, in the Ca2+free and Ca2+-bound states. J. Mol. Biol. 2010, 397, 991–1002.

J. Phys. Chem. B, Vol. 114, No. 49, 2010 16183 (39) Henzl, M. T.; Tanner, J. J. Solution structure of Ca2+-free rat β-parvalbumin (oncomodulin). Protein Sci. 2007, 16, 1914–1926. (40) Strynadka, N. C. J.; James, M. N. G. Crystal structures of the helixloop-helix calcium-binding proteins. Annu. ReV. Biochem. 1989, 58, 951–998. (41) Declercq, J. P.; Tinant, B.; Parello, J.; Rambaud, J. Ionic interactions with parvalbumins. Crystal structure determination of pike 4.10 parvalbumin in four different ionic environments. J. Mol. Biol. 1991, 220, 1017–1039. (42) Tan, A.; Henzl, M. T. Evidence for a Ca2+-Specific Conformational Change in Avian Thymic Hormone, a High-Affinity β-Parvalbumin. Biochemistry 2009, 48, 3936–3945. (43) Sanchez-Ruiz, J. M.; Lopez-Lacomba, J. L.; Cortijo, M.; Mateo, P. L. Differential scanning calorimetry of the irreversible thermal denaturation of thermolysin. Biochemistry 1988, 27, 1648–1652. (44) Freire, E.; Van Osdol, W. W.; Mayorga, O. L.; Sanchez-Ruiz, J. M. Calorimetrically determined dynamics of complex unfolding transitions in proteins. Annu. ReV. Biophys. Biophys. Chem. 1990, 19, 159–188. (45) Henzl, M. T.; Tanner, J. J. Solution structure of the Ca2+-free rat R-parvalbumin. Protein Sci. 2007, 17, 431–438. (46) Henzl, M. T.; Davis, M. E.; Tan, A. Leucine-85 is an Important Determinant of Divalent Ion Affinity in Rat β-Parvalbumin (Oncomodulin). Biochemistry 2008, 47, 13635–13646. (47) Henzl, M. T.; Agah, S.; Larson, J. D. Association of the AB and CD-EF domains from rat R- and β-parvalbumin. Biochemistry 2004, 43, 10906–10917.

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