A two-stranded a-helical coiled coil was prepared having a Cys(4) metal-binding site within its hydrophobic interior. The addition of Cd2+ results in the incorporation of 2 equiv of metal ion, which is accompanied by a conformational change of the peptide, as observed by circular dichroism (CID) spectroscopy. Isothermal titration calorimetry (ITC) shows that the addition of Cd2+ is accompanied by two thermodynamic events. A comparison of the time dependence of the ITC behavior with those of the UV absorption and CID behavior allows the assignment of these events to a preliminary endothermic metal-binding step followed by a slower exothermic conformational change

Mukherjee, Madhumita

Ogawa, Michael Y.

Zhu, Xianchun

English

Bowling Green State University: ScholarWorks@BGSU

Bowling Green State University ScholarWorks@BGSU Chemistry Faculty Publications Chemistry 6-2008 Cd2+-induced Conformational Change Of A Synthetic Metallopeptide: Slow Metal Binding Followed By A Slower Conformational Change Madhumita Mukherjee Xianchun Zhu Michael Y. Ogawa Bowling Green State University, mogawa@bgsu.edu Follow this and additional works at: https://scholarworks.bgsu.edu/chem_pub  Part of the Chemistry Commons Repository Citation Mukherjee, Madhumita; Zhu, Xianchun; and Ogawa, Michael Y., "Cd2+-induced Conformational Change Of A Synthetic Metallopeptide: Slow Metal Binding Followed By A Slower Conformational Change" (2008). Chemistry Faculty Publications. 175. https://scholarworks.bgsu.edu/chem_pub/175 This Article is brought to you for free and open access by the Chemistry at ScholarWorks@BGSU. It has been accepted for inclusion in Chemistry Faculty Publications by an authorized administrator of ScholarWorks@BGSU. Cd2+-Induced Conformational Change of a Synthetic Metallopeptide:Slow Metal Binding Followed by a Slower Conformational ChangeMadhumita Mukherjee, Xianchun Zhu, and Michael Y. Ogawa*Department of Chemistry and Center for Photochemical Sciences, Bowling Green State UniVersity,Bowling Green, Ohio 43403Received December 5, 2007A two-stranded R-helical coiled coil was prepared having a Cys4metal-binding site within its hydrophobic interior. The addition ofCd2+ results in the incorporation of 2 equiv of metal ion, which isaccompanied by a conformational change of the peptide, asobserved by circular dichroism (CD) spectroscopy. Isothermaltitration calorimetry (ITC) shows that the addition of Cd2+ isaccompanied by two thermodynamic events. A comparison of thetime dependence of the ITC behavior with those of the UVabsorption and CD behavior allows the assignment of these eventsto a preliminary endothermic metal-binding step followed by aslower exothermic conformational change.The three-dimensional structures of proteins are oftenaffected by the binding of transition-metal ions.1 In somecases, metal-induced protein-folding events are essential forthe proper functioning of metalloproteins, as seen for thezinc finger proteins, metallothioneins, and metalloregulatoryproteins such as MntR.2–4 In other cases, the binding of metalions may cause protein misfolding, which may result in thegeneration of pathological events. Such may be the case whenCu2+, Fe2+, or Zn2+ bind to the amyloid  (A) peptide,resulting in the generation of the amyloid plaques associatedwith Alzheimer’s disease.5 For these reasons, metal-inducedprotein-folding processes have been the focus of recentstudies through the use of theoretical methods6 and well-defined model systems.7–10Previous work by our group examined the metal-bindingproperties of a synthetic 32-residue polypeptide calledC16C19-GGY.11,12 It was found that this peptide undergoesa significant conformational change from a monomericrandom coil to a metal-bridged coiled coil upon its bindingof various d10 metal ions.13 Interestingly, the oligomerizationstate of the resulting holoproteins was found to be metal-ion-dependent because the Cd2+ adduct was shown to existas a two-stranded coiled coil containing a single metal ionand the Cu+ species was found to assemble into a four-stranded coiled coil having a tetranuclear copper center.11–13The rich conformational properties of this system musttherefore arise from a complicated interplay occurringbetween the coordination chemistry of the metal ions andthe peptide’s ability to self-associate into ordered three-dimensional structures. To more easily study the nature ofthis relationship, we sought to design a related system inwhich simpler metal-induced conformational behavior canbe observed. Here, we report the metal-binding propertiesof the peptide AQ-C16C19, which exists as a two-strandedR-helical coiled coil in the absence of metal ions andundergoes a small, but noticeable, conformational changewhen two Cd2+ ions bind to a preformed Cys4 metal-bindingsite within its hydrophobic interior. In particular, the additionof Cd2+ to AQ-C16C19 is seen to produce two stepwisethermodynamic events that are assigned to a preliminaryendothermic metal-binding step followed by a slowerexothermic conformational change.The peptide AQ-C16C19 has the sequence Ac-Q(IAA-LEQK)2(CAACEQK)(IAALEQK)GGY-amide, which issimilar to that previously used to prepare a stable two-stranded R-helical coiled coil14 but modified to containcysteine residues at two of its hydrophobic positions.High-performance size-exclusion chromatography (HPSEC)shows that the purified peptide exists as a dimer (MW )6.8 kDa), and circular dichroism (CD) spectroscopy showsthat it is R-helical in nature, as evidenced by minima at208 and 222 nm (Figure 1). The observed ellipticity ratio* To whom correspondence should be addressed. E-mail: mogawa@bgsu.edu.(1) Wilson, C. J.; Apiyo, D.; Wittung-Stafshede, P. Q. ReV. Biophys. 2004,37, 285.(2) Berg, J. M. J. Biol. Chem. 1990, 265, 6513.(3) Golynskiy, M. V.; Davis, T. C.; Helmann, J. D.; Cohen, S. M.Biochemistry 2005, 44, 3380.(4) Duncan, K. E. R.; Stillman, M. J. JIB 2006, 100, 2101.(5) Jun, S. M.; Saxena, S. Angew. Chem., Int. Ed. 2007, 46, 3959.(6) Dudev, T.; Lim, C. J. Am. Chem. Soc. 2007, 192, 12497.(7) Ghosh, D.; Pecoraro, V. L. Inorg. Chem. 2004, 43, 7902.(8) Cerasoli, E.; Sharpe, B. K.; Woolfson, D. N. J. Am. Chem. Soc. 2005,127, 15008.(9) Harris, K. L.; Lim, S.; Franklin, S. J. Inorg. Chem. 2006, 45, 10002.(10) Reddi, A. R.; Gibney, B. R. Biochemistry 2007, 46, 3745.(11) Kharenko, O. A.; Ogawa, M. Y. J. Inorg. Biochem. 2004, 98, 1971.(12) Kharenko, O. A.; Kennedy, D. C.; Demeler, B.; Maroney, M. J.;Ogawa, M. Y. J. Am. Chem. Soc. 2005, 127, 7678.(13) Hong, J.; Kharenko, O. A.; Ogawa, M. Y. Inorg. Chem. 2006, 45,9974.(14) Tsurkan, M. V.; Ogawa, M. Y. Biomacromolecules 2007, 8, 3908.Inorg. Chem. 2008, 47, 4430-44324430 Inorganic Chemistry, Vol. 47, No. 11, 2008 10.1021/ic702370k CCC: $40.75  2008 American Chemical SocietyPublished on Web 04/29/2008of θ222/θ208 ≈ 1.1 is greater than that seen for monomerichelices for which θ222/θ208 ) 0.86 and is consistent withthe assignment of a coiled-coil structure.15 The CDspectrum of the peptide is concentration-dependent, andthe change in θ222 was analyzed in terms of a two-statemonomer-dimer equilibrium16 (data not shown) to yieldKd ) 6.5 ( 0.6 µM and a maximum ellipticity of θmax )-33 400 ( 200. Together, these results show that theapopeptide exists as a two-stranded coiled coil and canbecome 97% helical at high concentrations.16The metal-binding properties of AQ-C16C19 were studiedby UV and CD spectroscopy as well as ITC. Figure 2 (inset)shows that the addition of Cd2+ to the peptide solution resultsin the appearance of a new absorption band at 240 nm, whichis assigned to a Cys-SH to Cd2+ ligand-to-metal charge-transfer transition. The increase in absorbance with Cd2+addition was analyzed in terms of the equilibrium1n[Cdn · Peptide]h1n[Peptide]+Cdfor whichKd )[Peptide]free1/n[Cd]free[CdnPeptide]1/n(1)R)Kd[Peptide]total{ S1- S}1 ⁄ n + n(S) (2)where R ) [Cd]total/[Peptide]total, S ) (A - A0)/(A∞ - A0) or(θ - θ0)/(θ∞ - θ0) and [Peptide] refers to the molarconcentration of the peptide dimer (Figure 2). The absorptiontitration data can be accurately described by the bindingisotherm (eq 2) to yield values of n ) 1.6 ( 0.02 and Kd )18 ( 0.4 µM for the metal-binding process.17 The AQ-C16C19 peptide thus binds two Cd2+ ions to cysteineresidues located within its hydrophobic interior. Interestingly,the 113Cd NMR spectrum of the holopeptide consists of asingle resonance at 459 ppm, indicating that the two Cd2+ions either are in magnetically equivalent sites or are in rapidexchange. This chemical shift is similar to that reported forcadmium-substituted horse liver alcohol dehydrogenase inwhich the catalytic Cd2+ ion was thought to have a Cys2-His ligand set also containing a labile water molecule.18Figure 1 shows that the addition of Cd2+ to the peptideproduces a noticeable conformational change as evidencedby CD spectroscopy. Increasingly more positive values ofθ222 are observed that saturate at higher concentrations ofmetal ion, indicating a change in helicity from 72% to 59%.The spectra further show isodichroic points at 207 and 249nm, which indicates the existence of a two-state conforma-tional change upon the addition of Cd2+. Interestingly, theHPSEC behavior of the metallopeptide remains unchangedfrom that of the apopeptide, which shows that it remains adimer. The inset to Figure 1 shows that the CD titrationbehavior can be accurately described by eq 2, which yieldsvalues of n ) 1.7 ( 0.1 and Kd ) 15 ( 2 µM for the metal-binding process. These results are in agreement with thoseobserved from the absorption data discussed above.Isothermal titration calorimetry (ITC) was used to studythe thermodynamic events associated with the binding ofCd2+ to the AQ-C16C19 dimer. In these experiments, carewas taken to account for the effects of the buffer on theobserved enthalpy changes by use of an acetate buffer (pH5.5)19 and dilution experiments, which showed that theseeffects were negligible. Sufficient time (600 s) betweensuccessive injections ensured completion of the reaction ineach step. Figure 3A shows that each addition of metal ionis accompanied by an initial endothermic response followedby a smaller exothermic one. This behavior is reminiscentof that previously reported for the addition of Cu2+ toapoazurin in which metal binding was followed by aconformational change of the protein.20 However, in thatsystem, it was found that the initial binding event wasexothermic followed by an endothermic conformationalchange.(15) Hodges, R. S. Biochem. Cell Biol. 1996, 74, 133.(16) Wendt, H.; Berger, C.; Baici, A.; Thomas, R. M.; Bosshard, H. R.Biochemistry 1995, 34, 4097.(17) Lombardi, A.; Marasco, D.; Maglio, O.; Di Costanzo, L.; Nasti, F.;Pavone, V. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 11922.(18) Bobsein, B. R.; Myers, R. J. J. Am. Chem. Soc. 1980, 102, 2454.(19) Velazquez-Campoy, A.; Luque, I.; Todd, M. J.; Milutinovich, M.; Kiso,Y.; Freire, E. Protein Sci. 2000, 9, 1801.(20) Blasie, C. A.; Berg, J. M. J. Am. Chem. Soc. 2003, 125, 6866.Figure 1. CD spectrum of the AQ-C16C19 peptide (89.8 µM) with successiveadditions (anaerobic) of CdII in 20 mM acetate buffer (pH 5.4; temperature303 K). Inset: Plot of fractional saturation of sites vs mole ratio ([Cd]/[peptide]).The solid line shows the best fit of data points to eq 2. Figure 2. Plot of fractional saturation of sites vs mole ratio [Cd]/[peptide]obtained from the spectrophotometric titration of AQ-C16C19 peptide withsuccessive additions (anaerobic) of CdII. The data points are the average oftwo titrations with 128 and 119 µM peptide solutions in a 50 mM acetatebuffer (pH 5.4; temperature 303 K). The solid line shows the best fit ofdata points to eq 2. Inset: Appearance of a new absorption band upon Cd2+addition.COMMUNICATIONInorganic Chemistry, Vol. 47, No. 11, 2008 4431Examination of Figure 3B shows that the two thermody-namic events observed upon the addition of Cd2+ to AQ-C16C19 occur on very different timescales. Thus, to test thehypothesis that fast metal binding is accompanied by a slowerconformational change, the time profiles of the ITC signalswere compared with those of the UV absorption and CDspectra. Figure 4 (layers A1 and A2) shows that both theinitial endothermic event and the UV absorption changesoccur in about 1 min, and layers B1 and B2 show that thatthe changes in the CD intensity and the secondary exothermicevent both occur on a ca. 50-fold longer timescale. Incomparison, the binding of Hg2+ to a weakly associatingpeptide was earlier shown to occur on the millisecondtimescale when performed under conditions similar to thoseused in Figure 4.7 The endothermic process seen in the ITCdata is thus assigned to a metal-binding event. This assign-ment is consistent with the observation of entropy-controlledZn2+ binding to a tetracysteine-containing peptide at low pH,where the cysteine residues are protonated.10 Endothermicheat changes accompanying metal-protein interactions havealso been reported for Cu2+ binding to the Cys site in bovineserum albumin,21 and in a control experiment, we observeonly endothermic changes when Cd2+ was added to a solutionof L-cysteine at pH 5.4. As a further test of this assignment,the endothermic peaks in the ITC titration data wereseparately analyzed according to one set of sites bindingmodel. As shown in Figure 5, a reasonable fit to the datawas obtained in which n ) 2.4 ( 0.04 and Kd ) 10 ( 2µM, which are in agreement with the results of the UV andCD titrations discussed above. The analysis also yields valuesof ∆G ) -6.9 kcal/mol and ∆H ) 1.6 kcal/mol to give ∆S) +28.1 cal/mol/K. This shows that the metal-bindingprocess is entropy-controlled, which is likely due to disrup-tion of the metal ion hydration sphere and structured watersurrounding the nonpolar residues of the peptide scaffoldupon metal binding. It is noted that the nature of conforma-tional change giving rise to the exothermic event remainsunclear at this time.In summary, the above study demonstrates the comple-mentary use of UV, CD, and ITC to elucidate metal bindingto peptides and proteins. The Cys-containing de novodesigned peptide undergoes a metal-induced helical transitionafter two Cd ions bind to the peptide dimer. The dataillustrate that these changes are entropically driven, withdehydration of both the peptide scaffold and metal ionexerting considerable influence on the reaction enthalpies,entropies, and free energy.Acknowledgment. This work was supported by NSFGrant CHE-0455441 and ACS-PRF Grant 40790-AC. Theauthors thank Prof. Max Funk at the University of Toledofor assistance in obtaining the ITC data.Supporting Information Available: 113Cd NMR protocol. Thismaterial isavailablefreeofchargevia theInternetathttp://pubs.acs.org.IC702370K(21) Zhang, Y.; Akilesh, S.; Wilcox, D. E. Inorg. Chem. 2000, 39, 3057.Figure 3. ITC titration of 218.5 µM peptide with 3.7 mM CdII in a 200mM acetate buffer, pH 5.4, 303 K: (A) raw titration data; (B) magnifiedraw data showing two stepwise events upon addition of CdII to the peptide.Figure 4. Comparison of the kinetics of CdII addition to peptide by UV,CD, and ITC. Layers A1 and A2: Comparison of the endothermic trace ofITC ([peptide] ) 218.5 µM; [Cd] ) 28 µM) with UV titration ([peptide]) 220 µM; [Cd] ) 26 µM. Layers B1 and B2: Comparison of theexothermic trace of ITC ([peptide] ) 217 µM; [Cd] ) 260 µM) with CDtitration in a 200 mM acetate buffer, pH 5.4. The CD titration is the averageof four titrations ([peptide] ) 205.5 µM; [Cd] ) 245 µM, [peptide] ) 120µM; [Cd] ) 183 µM, [peptide] ) 133 µM; [Cd] ) 157 µM. ITC traceswere translated to begin at time equals zero.Figure 5. Fits of the endothermic heat changes to a one set of sites modelfor the ITC titration of 218.5 µM peptide with 3.7 mM CdII in a 200 mMacetate buffer, pH 5.4, 303 K. The solid line shows the best fit.COMMUNICATION4432 Inorganic Chemistry, Vol. 47, No. 11, 2008

Cd2+-induced Conformational Change Of A Synthetic Metallopeptide: Slow Metal Binding Followed By A Slower Conformational Change

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Cd2+-induced Conformational Change Of A Synthetic Metallopeptide: Slow Metal Binding Followed By A Slower Conformational Change

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