Distinguishing unfolding and functional conformational transitions of calmodulin using ultraviolet resonance Raman spectroscopy

Eric M. Jones,1 Gurusamy Balakrishnan,1 Thomas C. Squier,2 and Thomas G. Spiro1* 1

Department of Chemistry, University of Washington, Seattle, Washington 98195-1700 Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99354

2

Received 9 April 2014; Accepted 28 May 2014 DOI: 10.1002/pro.2495 Published online 3 June 2014 proteinscience.org

Abstract: Calmodulin (CaM) is a ubiquitous moderator protein for calcium signaling in all eukaryotic cells. This small calcium-binding protein exhibits a broad range of structural transitions, including domain opening and folding–unfolding, that allow it to recognize a wide variety of binding partners in vivo. While the static structures of CaM associated with its various binding activities are fairly well-known, it has been challenging to examine the dynamics of transition between these structures in real-time, due to a lack of suitable spectroscopic probes of CaM structure. In this article, we examine the potential of ultraviolet resonance Raman (UVRR) spectroscopy for clarifying the nature of structural transitions in CaM. We find that the UVRR spectral change (with 229 nm excitation) due to thermal unfolding of CaM is qualitatively different from that associated with opening of the C-terminal domain in response to Ca21 binding. This spectral difference is entirely due to differences in tertiary contacts at the interdomain tyrosine residue Tyr138, toward which other spectroscopic methods are not sensitive. We conclude that UVRR is ideally suited to identifying the different types of structural transitions in CaM and other proteins with conformation-sensitive tyrosine residues, opening a path to time-resolved studies of CaM dynamics using Raman spectroscopy. Keywords: calmodulin; Raman spectroscopy; conformational change; calcium binding

Introduction Calcium plays a vital role in cellular signaling in virtually all eukaryotes.1 Chief among the cellular

Conflict of Interest Statement The authors declare no competing financial interests. Grant sponsor: National Institutes of Health grant (to T.G.S.); Grant number: GM-25158. Grant sponsor: Pacific Northwest National Laboratory (PNNL) is operated for the Department of Energy by the Battelle Memorial Institute; Contract number: DE-AC06-76RL0 1830. *Correspondence to: Thomas G. Spiro; PO Box 351700, Seattle, WA 98195-1700; E-mail: [email protected]

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receptors of calcium is calmodulin (CaM), a highly conserved 16.7 kDa acidic protein, which binds up to four Ca21 ions (in four EF-hand domains, two each in the protein’s N-terminal and C-terminal lobes) and modulates the activity of numerous binding partners in a calcium-dependent manner.1–3 In response to regulated changes in cytosolic Ca21 concentration, CaM forms complexes with and redistributes among binding partners, ultimately modulating a variety of downstream cellular processes such as muscle contraction, metabolism, and synaptic transmission.1,3,4 Structural2,5–9 and kinetic binding studies10–12 have shed much light on the means by which CaM recognizes its various targets and discriminates between

C 2014 The Protein Society Published by Wiley-Blackwell. V

them on the basis of calcium occupancy. Far less is known, however, about how the different mechanisms of target-binding relate to the intrinsic dynamics of CaM, an unusually flexible and dynamic protein,13–17 and how these mechanisms depend on the inherent differences in the functional motions of the Nterminal and C-terminal domains. In the last decade, CaM has become a useful model system for general studies of protein dynamics by NMR and various computational methods.17–26 These studies suggest that CaM has a surprisingly broad repertoire of conformational dynamics for such a small protein, and that these dynamics may relate to the functional need for CaM to select among dozens of binding partners in response to changes in a single variable, calcium concentration. CaM is believed to sample distinct conformational spaces in the Ca21bound and Ca21-unbound states, with binding activities dependent on the range of accessible conformers in a particular Ca21-bound microstate.16,26 Moreover, it has been suggested that CaM undergoes structural transitions like that involved in calcium activation of the protein (a “closed–open” transition), and global unfolding, under physiological conditions, at least in the C-terminal lobe.15,16,27 However, it remains a particular challenge to directly examine these structural transitions in real time because the spectral markers (absorbance and fluorescence) usually used in the study of CaM cannot easily distinguish these different structural transitions. Thus, experimental studies of CaM dynamics using real-time methods have lagged behind computational work. Ultraviolet resonance Raman (UVRR) spectroscopy is a sensitive method for examining structural transitions in unlabeled proteins.28,29 Raman excitation in resonance with an electronic transition of a protein chromophore (such as aromatic sidechains of Tyr and Trp) provides site-selective interrogation of vibrational modes with minimal spectral overlap from nonresonant groups. Our laboratory has successfully used both static and time-resolved UVRR to examine unfolding and functional motions of such proteins as myoglobin, hemoglobin, cytochrome c, and model polypeptides.28,30–33 In the present study, we identify distinct UVRR spectral signatures for conformational transitions involved in the Ca21 activation and the unfolding of CaM. This work exploits the Raman signal of CaM’s two Tyr residues, in particular Tyr138, which is involved in the Ca21dependent structural transition of CaM.34,35 We find that the two classes of CaM structural change mentioned above give readily distinguishable UVRR signals, opening the way to real-time spectroscopic studies of CaM dynamics.

Results The UVRR spectral analysis of CaM largely depends on the signal of Tyr138, located at the interface of

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Figure 1. Structures of apo-CaM (1CFD, left) and holo-CaM (1CLL, right),36,37 showing the opening of the N-terminal and C-terminal lobes upon binding of four Ca21 ions (not shown), and formation of a hydrogen bond between Tyr138 and Glu82 in the open conformation. Tyr99 remains solventexposed in both structures, and therefore, contributes negligibly to UVRR difference signals.

the protein’s C-terminal lobe and the central linker domain. This residue is mostly shielded from solvent in the absence of Ca21, but is exposed and donates a hydrogen bond to Glu82 on the central linker when the protein is Ca21-bound, in response to the opening of the C-terminal lobe (Fig. 1). This “closed– open” transition has long been known to affect UV absorptivity and fluorescence emission from Tyr138,38,39 although unfolding and calcium binding give extremely similar signals using these methods. The only other Tyr residue in CaM, Tyr99, is fully solvent-exposed in both the presence and absence of bound Ca21, and therefore, contributes negligibly to spectral changes on activation or unfolding. We questioned whether UVRR spectroscopy would be useful for monitoring unfolding of CaM. Static UVRR spectra of 125 mM apo-CaM were collected at various temperatures. Previous work40–42 has shown that apo-CaM denatures in a broad transition centered at about 50 C; the N-terminal and Cterminal lobes melt at different temperatures.40,42 Figure 2(A) shows UVRR spectra of apo-CaM at 14 and 77 C, at which CaM is expected to be fully folded and fully unfolded, respectively, and the unfolded-minus-folded difference spectrum. The UVRR spectral change associated with thermal unfolding is evident mainly as a decrease in peak intensity [Fig. 2(A)] owing to a change in the environment of one or both Tyr residues. Since Tyr99 is solvent-exposed (in both Ca21-bound and Ca21-free structures), the UVRR intensity loss is most likely due to a conformational change at Tyr138, possibly due to exposure to water on unfolding. This weakening of the spectral intensity affects all bands; however, the transition can be conveniently monitored

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Figure 2. UVRR spectral signature of thermal unfolding of apo-CaM. A, static UVRR spectra (229 nm excitation) of apo-CaM at 14 and 77 C and the temperature-induced difference spectrum (77–14 C), indicative of unfolding (top). Bands marked with an asterisk are from the MOPS/EGTA buffer. The integrated intensity of the Y9a band at 1177 cm21 is plotted as a function of temperature in the bottom panel, fit to a two-state folded–unfolded transition (solid line). From this best fit line, the 50% folded temperature for the CaM C-domain was calculated to be 48 C; the thermodynamic parameters of unfolding have been calculated elsewhere.42 B, second singular vector (from SVD analysis) for thermal unfolding of apo-CaM monitored by UVRR (top) and intensity of this component as a function of temperature (bottom). Note difference in horizontal scale between upper panels of A and B.

by measuring the area under the relatively isolated Y9a band near 1177 cm21 [Fig. 2(A), bottom]. As measured by the intensity of this peak, unfolding is weakly cooperative and is centered at 48 C, consistent with earlier work on the C-terminal domain of CaM.42 While the data of Figure 2(A) provide a useful view of the unfolding process in CaM, the number of environmental variables that influence the intensity of a single band makes it desirable to monitor unfolding (a global process) using a global data analysis method that considers contributions from the entire UVRR spectrum. For this purpose, we have used singular value decomposition (SVD) of the temperature-dependent UVRR spectra.43 The first SVD basis spectrum (singular vector) for apo-CaM unfolding reflects primarily the spectral intensity, and the contribution of this component to the overall spectrum fluctuates quasi-randomly with temperature, due to variations in laser power over the course of the experiment (data not shown). Figure 2(B) shows the second singular vector for apo-CaM unfolding. This global basis spectrum shows a weakening and slight red shift of the Y9a mode and blue shifts of the Y8b and Y8a modes, together with a modest loss of intensity. The similarity between this basis spectrum and the static unfolded-minus-folded difference spectrum [Fig. 2(A), bottom] is remarkable. The SVD spectrum component exhibits an essentially noncooperative increase in contribution to the total spectrum [Fig. 2(B), bottom], indicating that this component varies more continuously than the isolated Y9a band. One source of this variation

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may be the weakly enhanced amide III and amide S modes, which vary with the secondary structure content of proteins.44–46 The broad, shallow minimum at 1339 cm21 and maximum at 1240 cm21 (amide III region) are consistent with conversion of ahelices to disordered structure, as would be expected upon unfolding of CaM. Likewise, the increase in intensity at 1395 cm21 (amide S band) is suggestive of the loss of helical structure, in which the amide S mode is poorly enhanced.44 The overlap of unfolding amide signals from the N-terminal and Cterminal lobes with the Tyr signal of the latter may be responsible for the apparent lack of cooperativity in the transition of the second SVD component; the C-terminal and N-terminal lobes of apo-CaM unfold in broad transitions centered around 46 and 60 C, respectively.42 In contrast to the apo-protein, Ca21-saturated CaM is thermally stable up to over 90 C,47 with a DGunfold of 4.7 and 6.5 kcal/mol for the N-domains and C-domains, respectively, in urea denaturation experiments at 20 C; these values were slightly lower when guanidinium chloride was used as a denaturant.42 Thus, no thermal transition was expected in the UVRR spectra of holo-CaM collected at temperatures up to 80 C, the limit of our experimental apparatus. Consistent with this hypothesis, the temperature-dependent UVRR spectra of Ca21CaM show very little temperature dependence [Fig. 3(A)]. The difference spectrum between static spectra collected at 76 and 15 C must be multiplied by five for any features to become evident [Fig. 3(A), top]; the difference spectrum shows a rather uniform

Calmodulin Transitions by UV Raman Spectroscopy

Figure 3. UVRR reveals minimal structural change on heating of Ca21-CaM. A, static UVRR spectra (229 nm excitation) of Ca21-saturated CaM at15 and 76 C and the temperature-induced difference spectrum (76–15 C, top). Bands marked with an asterisk are from the MOPS/EGTA buffer. The integrated intensity of the Y9a band at 1173 cm21 is plotted in the bottom panel. B, Second singular vector for the heating of Ca21-CaM monitored by UVRR (top), with the intensity of this spectral component plotted as a function of temperature (bottom). Note difference in horizontal scale between upper panels of A and B.

decrease of spectral intensity. When the integrated area of the Y9a band (CAH plus CAOH bend)48 is plotted as a function of temperature [Fig. 3(A), bottom], a general, noncooperative decrease in intensity is observed. This decrease is consistent with the general temperature-dependent decrease in the inherent Tyr UVRR cross section, also seen in Tyr solutions.30,49 SVD analysis of these data shows that the second singular vector varied approximately linearly with temperature, and indicated a slight red-shift of the Y9a and blue-shift of the Y8a bands, consistent with a temperature-dependent weakening of the hydrogen bond between Tyr138 and Glu82 (the first basis spectrum in the SVD analysis, not shown, reflected spectral intensity and varied randomly due

to fluctuations in the excitation laser power, as for the apo protein). Thus, UVRR confirms earlier results that show CaM is thermally stable when saturated with calcium, and that little conformational change is present at equilibrium when Ca21-CaM is heated. We next explored the spectral changes upon activation of CaM by calcium binding, which is known to induce formation of the aforementioned hydrogen bond between Tyr138 and Glu82. Endpoint UVRR spectra of apo-CaM and Ca21-CaM, and their difference spectrum, are shown in Figure 4(A; top). The most prominent spectral difference induced by Ca21 binding is a weakening and shift in the Y9a band at 1176 cm21, together with weakening and

Figure 4. Calcium-induced changes in CaM UVRR spectra. A, UVRR spectra (229 nm excitation) of 125 mM CaM in the presence of 30 mM and 3.3 mM total Ca21, with the Ca21 2 apo difference spectrum, showing the red shift and decrease in intensity of the Y9a peak (top). The position of the Y9a band is plotted versus [Ca21] in the bottom panel. B, Second singular vector for the Ca21 titration of apo-CaM, monitored by UVRR (top) and intensity of this component as a function of [Ca21] (bottom). The buffer contained 100 mM MOPS pH 7.4, 100 mM NaClO4, 2.5 mM EGTA, and varying concentrations of CaCl2 up to the indicated final [Ca21].

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overlapping shifts of the Y8a and Y8b peaks around 1600–1620 cm21. The latter two modes are wellknown markers of hydrogen bonding by Tyr;50 the Y9a mode, though not considered especially sensitive to H-bonding per se, contains much AOH bending character, and therefore, is sensitive to interactions which may change the orientation of the hydroxyl group relative to the phenyl ring plane.51 To examine the concentration dependence of this calcium-induced spectral change, apo-CaM (in the presence of excess calcium chelator EGTA) was titrated with aqueous calcium chloride at a constant temperature of 23 C, and spectra were collected at each calcium concentration. The spectra, shown as a function of estimated free [Ca21], are shown in Figure 4(A; bottom). In this figure, free calcium concentration was estimated (Materials and Methods), because fluorometric measurement of free [Ca21] in the presence of CaM is problematic in our experimental setup. These spectra show an almost stepwise shift in the Y9a and Y7a (CAO stretching)48 peaks; the shift in the Y9a peak is graphed as a function of estimated free [Ca21] in Figure 4(A; bottom). This step is centered at about 0.7 mM free Ca21; however, this is an overestimate, because our method of estimating free [Ca21] does not consider calcium binding by CaM itself, which is present at 125 mM. Thus, it is not possible to estimate the calcium occupancy of CaM needed to produce the stepwise shift in the signal of Tyr138 based on the present data. Nonetheless, we estimate that this transition occurs at roughly two Ca21 per CaM molecule, because previous data suggest that Ca21 binds at the two C-terminal binding sites of CaM first,52,53 producing a conformational change that stabilizes the central linker resulting in activation of the N-terminal lobe.36,54,55 A similar sharp transition, at the same calcium concentration, is evident in the SVD traces from the UVRR analysis of the Ca21 titration [Fig. 4(B)]. The basis spectrum for this change is remarkably similar to the static Ca21-minus-apo difference spectrum of Figure 4(A), showing the opposing shifts of the Y7a and Y9a bands, together with a weakening and shifting of the Y8a and 8b (both ring CAC stretching)48 modes. This transition is very cooperative, with a midpoint again at about 0.7 mM free Ca21 [Fig. 4(B), bottom]. It is, therefore, reasonable to suggest that the spectral changes indicated by Figure 4(A,B) reflect a Ca21-induced rearrangement of EF-hand domain 4, resulting in donation of a hydrogen bond by Tyr138 and concomitant opening of the region around this residue to solvent. Importantly, this spectral change is markedly different from that associated with CaM unfolding (Fig. 2), which is largely limited to a difference in spectral intensity affecting all bands. Thus, calcium activation and thermal denaturation of CaM give highly distinct

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UVRR spectral changes, allowing these two types of structural transition (activation and unfolding) to be distinguished based entirely on the resonance Raman signal of Tyr138.

Discussion It has long been established that calcium binding by CaM results in an opening of the two lobes of the protein, exposing hydrophobic pockets where amphipathic helices of target proteins can associate.5,37,56,57 Conversely, binding of target peptides has been shown to affect the binding of calcium by CaM, influencing the equilibrium between conformations that favor binding of one target over others.3,4,58 This balance between calcium and target binding influences the direction in which calciumdependent signals flow in the cell under a given stimulus; CaM acts as a switch at decision points in Ca21-dependent signaling processes.1,3,4 Recent studies, many of them computational, have explored the manners in which target and calcium binding influence the dynamics of CaM. These findings have informed a new understanding of CaM’s role as a cellular mediator. Specifically, Gsponer et al.26 showed that calcium binding to CaM restricts the accessible range of accessible conformations, allowing only certain types of target binding to occur. Similarly, another computational study27 found that the C-terminal lobe of apo-CaM (to which Ca21 binds first, and in which Tyr138 is located) samples opened, closed, and unfolded conformations, each of which would presumably exhibit distinct binding activities (or lack thereof). This latter result is supported by NMR dynamics studies of C-terminal CaM mutants.15,16 It is challenging to study these dynamic transitions by real-time experimental methods, however, owing to the nonspecific nature of most spectroscopic probes of CaM. Temperature-jump studies of apoCaM and holo-CaM have found microsecond relaxations associated with exposure of hydrophobic regions or a change in the environment of Tyr residues;59,60 however, these studies cannot distinguish “closed–open” transitions (like that seen upon Ca21 activation of CaM) from unfolding of the protein, due to the nonspecific spectroscopic probes used. Thus, it is unclear precisely which transitions (or what combination of transitions) was observed in these studies. The exquisite structural sensitivity of UVRR spectral markers, and the ability to selectively probe Tyr sidechains, makes UVRR ideally suited to defining the conformation and dynamic transitions of the C-domain of CaM. The observation that thermal denaturation of the protein (Fig. 2) and calcium-associated activation (Fig. 4) give entirely different spectral signatures shows that UVRR can distinguish the different types of conformational change undergone by the C-terminal lobe of CaM. In

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this context, UVRR is taking advantage of the different environmental changes that occur during closed–open and folded–unfolded transitions. Unfolding of CaM simply exposes Tyr138 to solvent, producing an overall loss of resonance enhancement due to the blue shift of the La electronic transition in a higher-dielectric environment.50,61 The closedopen transition, however, is associated with hydrogen bond donation by Tyr138 to Glu82. Thus, shifts in the hydrogen-bond sensitive Y9a, Y8a, and Y8b modes occur concomitant with this conformational change. As a result, the closed–open and folded– unfolded transitions, proposed to coexist in equilibrium in apo-CaM27 and possibly in partly Ca21-occupied CaM15,16 have unique, separable spectral signatures in UVRR spectroscopy, opening the possibility of real-time spectroscopic exploration of the dynamics of these transitions, as has been reviewed recently,62 in both apo-CaM and holo-CaM. It should be noted additionally that the closed– open transition also induces substantial ordering of Phe sidechains, via CH–p interactions, in the hydrophobic cores of both lobes of CaM37,56 (Tyr is only present in the C-terminal lobe). This ordering in principle suggests the possibility of monitoring CaM dynamics at both N-terminal and C-terminal via the UVRR signals of Phe residues, which are resonanceenhanced at shorter wavelengths.63,64 Indeed, Phe fluorescence has been used to successfully monitor calcium binding to the N-terminal domain of CaM.65,66 Preliminary experiments using 197 nm excitation, however, failed to uncover any significant change in the Phe UVRR signal associated with Ca21 binding, perhaps due to the relatively large number of Phe residues in CaM (data not shown). Nonetheless, alternate spectral probes (e.g., siteselective incorporation of Trp)52 may provide a means for separable monitoring of conformational change and dynamics in the N-terminal and C-terminal lobes of CaM.

Materials and Methods Buffers, salts, and other chemicals were from J.T. Baker and were of reagent grade. Vertebrate CaM was expressed, purified, and characterized as described previously.67,68 UV resonance Raman spectra were acquired using a laser apparatus described previously,69 with spectra collected in a 135 backscattering geometry from a flowing stream of sample solution. 229 nm excitation was chosen to selectively probe the Raman signals of Tyr sidechains (Trp is also resonance-enhanced at 229 nm, but CaM contains no Trp residues). The calcium concentration in all solutions was determined before and after each experiment using inductively coupled plasma optical emission spectroscopy, and the protein concentration was measured by UV absorptivity at 277 nm (e277 5 3300 M21cm21).70 All experiments were

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conducted at 23 C in the presence of 100 mM sodium 3-(N-morpholino)propanesulfonate (MOPS) pH 7.4, 100 mM sodium perchlorate (as a Raman intensity standard), and 2.5 mM ethylene glycolbis(2-aminoethylether)-N-N-N0 -N0 -tetraacetic acid (EGTA), with varying concentrations of added CaCl2. Free calcium concentration was estimated using the MaxChelator tool (http://maxchelator.stanford.edu), solving for free [Ca21] at known pH, ionic strength, and EGTA concentration.71–73 SVD of datasets was performed in Matlab; other data analysis was performed using Origin.

Acknowledgments The authors thank Yijia Xiong for assistance with the expression and purification of calmodulin.

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