Aggregated t-CuP binds to poly-L-glutamate through supramolecular interactions, revealing itself to be an extremely sensitive probe for the major conformations of the polymeric scaffold

de Luca, G.

Pasternack, Robert F.

Romeo, A.

Scolaro, L. M.

English

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Swarthmore College Works Chemistry & Biochemistry Faculty Works Chemistry & Biochemistry 1-21-2010 Conformations Of A Model Protein Revealed By An Aggregating Cu?? Porphyrin: Sensing The Difference G. de Luca A. Romeo L. M. Scolaro Robert F. Pasternack Swarthmore College, rpaster1@swarthmore.edu This work is brought to you for free and open access by . It has been accepted for inclusion in Chemistry & Biochemistry Faculty Works by an authorized administrator of Works. For more information, please contact myworks@swarthmore.edu. Follow this and additional works at: https://works.swarthmore.edu/fac-chemistry  Part of the Inorganic Chemistry Commons Let us know how access to these works benefits you  Recommended Citation G. de Luca, A. Romeo, L. M. Scolaro, and Robert F. Pasternack. (2010). "Conformations Of A Model Protein Revealed By An Aggregating Cu?? Porphyrin: Sensing The Difference". Chemical Communications. Volume 46, Issue 3. 389-391. DOI: 10.1039/b918433c https://works.swarthmore.edu/fac-chemistry/114 Conformations of a model protein revealed by an aggregatingCuII porphyrin: sensing the diﬀerencewzGiovanna De Luca,*ab Andrea Romeo,ab Luigi Monsu` Scolaro*a and Robert F. PasternackbReceived (in Austin, TX, USA) 8th September 2009, Accepted 28th October 2009First published as an Advance Article on the web 19th November 2009DOI: 10.1039/b918433cAggregated t-CuP binds to poly-L-glutamate through supra-molecular interactions, revealing itself to be an extremely sensitiveprobe for the major conformations of the polymeric scaﬀold.Structural motifs play a central role in the proper functioningof biomolecules and considerable eﬀort has been focused onthe search for molecular probes capable of reporting onspeciﬁc conformations of nucleic acids and proteins.1 Porphyrinsare appealing candidates for this purpose because of theirspectroscopic features which are markedly aﬀected by self-aggregation and by the speciﬁc microenvironment.2 Here wereport on the chiroptical properties of an aggregating cationicmetalloporphyrin, Cu(II) 5,15-bis(N-methylpyridinium-4-yl)-10,15-bis(diphenylporphine) (t-CuP, Fig. 1a), on polyglutamate,as a model scaﬀold, and demonstrate that this porphyrin is apromising candidate as a structural probe of protein conformation.The copper(II) derivative was selected because of its stabilityover an extended pH range, its lack of pH-dependent andredox behavior (which would complicate the interpretation ofexperimental results), and its tendency to form extendedassemblies.3–5 The analogous tetracationic Cu–porphyrin,which shows little tendency to aggregate, does not displaysensitive, speciﬁc spectroscopic features required of a structuralprobe, losing its induced CD signal long before polyglutamatecompletely switches from helix to coil.6The spectral features of t-CuP in the absence of poly-L-Glu(PLG) at 5 mM, no buﬀer or salt added, are shown in Fig. 1(solid lines). The extinction spectrum (Fig. 1a) presents arather broad B-band at 418 nm, accompanied by two weakerQ-bands at lower energy. As expected for an achiral moleculethe corresponding CD spectrum (Fig. 1b) is silent, and the lowintensity of scattered light (Fig. 1c) rules out the presence oflarge aggregates in solution.7 However, increasing temperatureleads to signiﬁcant changes in the UV/Vis extinction spectrum(Fig. S1z) suggesting that this porphyrin is an aggregate(probably a dimer) at room temperature, even at lowconcentration. Such small aggregates (dimers or oligomers)have been reported in low ionic strength (IS) aqueoussolutions of the parent metal-free porphyrin.8 Addition ofPLG (FW B 17 kD, 100 mM Glu residues, 2 mM buﬀer)strongly aﬀects all the porphyrin spectroscopic features, thechanges being pH dependent. At pH 4.7 (Fig. 1, dashed lines),where the peptide adopts an a-helical conformation,9 thet-CuP extinction spectrum (Fig. 1a) displays a hypsochromicand broadened B-band at 410 nm. An induced conservativesignal having a positive Cotton eﬀect appears in the CDspectrum under these conditions (Fig. 1b)3,6,10 and a modestincrease in intensity is observed in the resonance light scattering(RLS) spectrum (Fig. 1c) of the bound dimeric porphyrin(Fig. 1d, left). The appearance of an RLS signal in thesesamples implies some electronic coupling among the boundt-CuP dimers.In the presence of PLG (100 mM) at pH 7.2, where theprotein is in a random coil conformation,9 t-CuP spectralfeatures (Fig. 1, dotted lines) are very diﬀerent from thoseobserved at low pH. The porphyrin extinction spectrum(Fig. 1a) displays a split B-band having broad componentsFig. 1 Extinction (a), CD (b) and RLS (c) spectra of t-CuP in thepresence of PLG as a-helix (pH = 4.7; dashed lines) or random coil(pH=7.2; dotted lines). ([t-CuP]= 5 mM; [Glu]= 100 mM,FWB 17 kD;[buﬀer] = 2 mM). Solid curves: spectra of t-CuP (no protein).(d) Model for the interaction between PLG and t-CuP: (left) dimerson an a-helix; (right) aggregates on a random coil.aDipartimento di Chimica Inorganica, Chimica Analitica e ChimicaFisica and C.I.R.C.M.S.B., University of Messina, Salita Sperone 31,Vill. S. Agata, 98166 Messina, Italy. E-mail: delucag@unime.itbDepartment of Chemistry and Biochemistry, Swarthmore College,500 College Avenue, Swarthmore, PA 19081, USAw This paper is dedicated in memory of Prof. Raﬀaello Romeo.z Electronic supplementary information (ESI) available: Experimentaldetails; spectral changes on increasing T for t-CuP; spectra of t-CuP inMilliQ water or buﬀers; CD spectra for t-CuP/PGA on increasing pH;RLS spectra corresponding to Fig. 4. See DOI: 10.1039/b918433cThis journal is c The Royal Society of Chemistry 2010 Chem. Commun., 2010, 46, 389–391 | 389COMMUNICATION www.rsc.org/chemcomm | ChemCommPublished on 19 November 2009. Downloaded by Swarthmore College on 28/04/2015 14:29:23. View Article Online / Journal Homepage / Table of Contents for this issuecentered at 381 nm and 440 nm (minor and major,respectively) which, together with a strong signal in the RLSspectrum centered near the longer wavelength (Fig. 1c), pointto the presence of large J-aggregates.11 The CD spectrum(Fig. 1b) shows a rather complex induced signal, with a pairof bisignate features centered at the two extinction maximaand having opposite phasing. The formation of large t-CuPassemblies, normally observed under high IS conditions,12 ishere seen at 2 mM buﬀer, no salt added, by the interaction ofthe porphyrin with the completely deprotonated PLG strands(Fig. 1d, right).The presence of a large ICD signal in the region of t-CuPextinction when the dye interacts with PLG at pH 7.2 may atﬁrst seem surprising. The degree of chiral information of PLGin a random coil conformation is much less than that of thea-helices, and for systems thus far studied, dyes interactingwith this protein at pH B 7 have shown markedly reducedICD signals as a consequence of the transition from helix tocoil6,13 (unless other species act as stabilizers and allow apreviously induced dye organization to retain its chiralmemory).6,10,14 Only a few reports3,15 deal with a diﬀerentsignal phasing in the presence of the two diﬀerent polypeptideconformations, and these usually employ high dye loads thatmight inﬂuence the structure of the ﬁnal adducts. Here, themajor CD feature (centered around 440 nm) displayed by theporphyrin aggregates upon interaction with the random-coiledprotein presents an opposite (negative) Cotton eﬀect ascompared to that of t-CuP dimers bound to a-helices. Thisobservation may reﬂect the diﬀerent orientation of the porphyrinwith respect to the protein scaﬀold and also agrees with recentresults16 showing that at alkaline pH PLG folds as a poly-proline II (PPII) left-handed helix. Very likely, this ability oft-CuP to sense the chirality of the less organized random coil isaﬀorded by the dye binding to the biopolymer strands in ahighly aggregated state, leading to an ampliﬁcation of thechiral information of the template (‘‘antenna eﬀect’’).17The transition between a-helix and random coil has beenstudied through changes in the CD spectrum of the protein inthe UV region.18 Fig. 2a shows that in a batch titration thetrend of ellipticity (at 222 nm) vs. pH for neat PLG (100 mM;data shown in black) is not modiﬁed markedly by the presenceof the porphyrin (data shown in red; [dye]/[Glu] = 1/20); thepresence of t-CuP does not perturb the conformational transi-tion of the peptide. The helix to coil transition in the presenceof the dye can also be monitored through changes in thespectral features of t-CuP. On increasing the pH, all spectro-scopic techniques consistently reveal the conversion fromt-CuP dimers on an a-helix to aggregates on a random coil(Fig. 2b). However, the inﬂection points displayed by thesignals belonging to the porphyrin are shifted to a somewhathigher pH than that exhibited by the protein (indicated by thedashed vertical line in Fig. 2). This diﬀerence suggests that thetwo binding modes of t-CuP to PLG are not energeticallyequivalent, with porphyrin dimers bound to a-helices as thefavored situation. As a consequence, the sensitivity to thepresence of a-helical protein increases if the signal chosen as aprobe belongs to the porphyrin: when the curve of the proteinCD signal vs. pH has already reached the plateau in the highpH region, the signals of the t-CuP have achieved only half oftheir dynamic range available to detect residual amounts ofa-helices still present in solution.Another important feature of the sensing capabilities oft-CuP becomes apparent in the presence of short PLG chains(FW B 1.5–3 kD) at around pH 4.5. It is known that thepropensity of this peptide to fold as a-helices decreasesdramatically for short strands, in line with the cooperativityof the coil to helix transition.18,19 For a short PLG, thecoexistence of helix and coil conformations has been detectedeven at low pH by comparing CD signals with those displayedby longer biopolymers in the UV spectral region.19 Whent-CuP (5 mM) is added to a 20 mM solution of short PLGat pH 4.7, the phase of the CD signal in the visible region(Fig. 3, solid line) is reminiscent of that observed in thepresence of random-coiled peptides (Fig. 1b, dotted line). Thisobservation is explained in terms both of the distribution ofthe peptide lengths and the face-on arrangement proposed forthe interaction between the porphyrin dimers and the a-helicalprotein. Estimates of the distance between the two positivecharges of t-CuP (B15.5 A˚) and the structural characteristicsof the a-helix (pitch: 5.4 A˚; residues per turn: 3.6) suggest that,for a dimer to bind to the helix with a parallel orientation ofthe dye planes to the helix axes, at least 10 contiguous Gluresidues have to adopt this conformation. However, at pH 4.7only a fraction of the short PLG is helical19 and when thelength distribution of this peptide is also taken into account itbecomes clear that the protein fraction suitable to allow theporphyrins to bind to helices as described above is evensmaller. In agreement with this hypothesis, when 5 mMt-CuP is added to a more concentrated (2 mM) solution ofshort PLG, at pH 4.7, the proﬁle of the ICD signal in thevisible spectral region (Fig. 3, dashed line) reverts to thatattributed to dimers bound to helices: the overall increase inprotein concentration causes a consequent increase of thefraction of strands having the appropriate length and helicityto allow the dimers to bind the protein in a parallel fashion.The porphyrin response to the diﬀerent conformations ofPLG has also been tested under conditions usually leading toaggregated t-CuP,12 i.e. in the presence of 0.15 M NaCl. TheFig. 2 (a) Changes of CD signal vs. pH for PLG solutions, with orwithout added t-CuP (black and red, respectively). (b) Changes inextinction, CD and RLS signals vs. pH for PLG/t-CuP. ([Glu] = 100 mM,FW B 17 kD; [t-CuP] = 0–5 mM; [buﬀer] = 2 mM.)390 | Chem. Commun., 2010, 46, 389–391 This journal is c The Royal Society of Chemistry 2010Published on 19 November 2009. Downloaded by Swarthmore College on 28/04/2015 14:29:23. View Article Onlineaddition of salt to the porphyrin/protein assemblies formed atpH B 4.5 (5 mM t-CuP, 100 mM protein) does not lead toappreciable changes in the optical features (Fig. 4, S4z, solidlines). In the UV/vis spectrum the B-band is still found at 410 nm(Fig. 4a), the corresponding ICD signal is centered at the samewavelength and exhibits a positive Cotton eﬀect (Fig. 4b), andthe RLS signal is as weak as under low IS conditions(Fig. S4z). Two main comments can be made about theseresults: (i) the hypothesis that the interaction of t-CuP dimerswith a-helical PLG consists not only of an electrostaticcomponent, but also of a hydrophobic one, is substantiatedby the fact that the porphyrins do not detach from thebiopolymers upon salt addition, and (ii) the stability oft-CuP/protein adducts is such that the typical aggregationphenomena shown by this porphyrin upon addition of0.15 M NaCl are suppressed in the presence of the helicalbiopolymer. Also at pH B 7 (Fig. 4, S4z, dashed lines) theoptical properties of t-CuP/PLG adducts are not aﬀectedsigniﬁcantly by the presence of 0.15 M NaCl: the UV/visextinction spectrum (Fig. 4a) is almost unchanged and,together with a strong RLS signal (Fig. S4z), point to largelyaggregated dyes, as in the absence of added salt. However,diﬀerent from the batch titrations carried out under low ISthat displayed a smooth transition between the two bindingmodes of t-CuP onto PLG, in the presence of 0.15 M NaClvery unusual optical features appear over a narrow pH rangenear 5.5 (Fig. 4, S4z, dotted lines). The very broad UV/visextinction and RLS signals (Fig. 4a, S4z, respectively), coveringalmost completely the screened spectral range, point to theformation of extremely large J-aggregates. More remarkable isthe presence of the most intense, unusual ICD signals observedso far for this system (Fig. 4b), with proﬁles that seem largelyaﬀected by a diﬀerential scattering component. These resultsare interpreted as due to t-CuP aggregating at a completelydiﬀerent binding site, e.g. interface regions where both helicaland random coil conformations are present on the same strandof PLG. This hypothesis is also supported by pH dependencestudies on polymers of diﬀerent lengths in which higherintensity ICD signals are obtained for shorter peptides withrespect to longer ones at pH 4.5 (data not shown). Onincreasing the polymer length, the interface regions areexpected to decrease in importance due to cooperativity eﬀects.In summary, t-CuP is able to bind to PLG as a supra-molecular aggregate, revealing it to be a sensitive reporter forthe major conformational structures of this polymeric scaﬀold.Its sensitivity opens the way to a variety of potential applicationsas a non-covalent chiroptical sensor.Notes and references1 M. Balaz, A. E. Holmes, M. Benedetti, P. C. Rodriguez,N. Berova, K. Nakanishi and G. Proni, J. Am. Chem. Soc.,2005, 127, 4172; R. F. Pasternack, Chirality, 2003, 15, 329.2 R. F. Pasternack and E. J. Gibbs in Metal Ions inBiological Systems, vol. 33, ed. A. Sigel and H. Sigel, MarcelDekker, 1996, p. 367; A. D’Urso, A. Mammana, M. Balaz,A. E. Holmes, N. Berova, R. Lauceri and R. Purrello, J. Am.Chem. Soc., 2009, 131, 2046.3 R. F. Pasternack, A. Giannetto, P. Pagano and E. J. Gibbs, J. Am.Chem. Soc., 1991, 113, 7799.4 R. F. Pasternack, S. Ewen, A. Rao, A. S. Meyer, M. A. Freedman,P. J. Collings, S. L. Frey, M. C. Ranen and J. C. de Paula, Inorg.Chim. Acta, 2001, 317, 59.5 A metal free species would show acid/base behavior on varying thesolution pH. A Zn derivative is labile at low pH, and there is achange from an aquo to a hydroxo axial ligand (with a concomitantspectral change) at high pH. Mn, Fe and Co derivatives are allaﬀected by pH with the possibility of m-oxo aggregation for the Feand possibly the Mn species. The Ni derivative coexists as diamagneticfour-coordinate and paramagnetic six-coordinate species, eachwith its own Soret maximum (see: A. Pasternack, E. G. Spiroand M. Teach, J. Inorg. Nucl. Chem., 1974, 36, 599.6 R. Purrello, A. Raudino, L. M. Scolaro, A. Loisi, E. Bellacchioand R. Lauceri, J. Phys. Chem. B, 2000, 104, 10900.7 R. F. Pasternack and P. J. Collings, Science, 1995, 269, 935.8 F. Mallamace, N. Micali, S. Trusso, L. M. Scolaro, A. Romeo,A. Terracina and R. F. Pasternack, Phys. Rev. Lett., 1996, 76,4741.9 M. Nagasawa and A. Holtzer, J. Am. Chem. Soc., 1964, 86, 538.10 E. Bellacchio, R. Lauceri, S. Gurrieri, L. M. Scolaro, A. Romeoand R. Purrello, J. Am. Chem. Soc., 1998, 120, 12353.11 J. Parkash, J. H. Robblee, J. Agnew, E. Gibbs, P. Collings,R. F. Pasternack and J. C. de Paula, Biophys. J., 1998, 74, 2089.12 R. F. Pasternack, C. Bustamante, P. J. Collings, A. Giannetto andE. J. Gibbs, J. Am. Chem. Soc., 1993, 115, 5393.13 L. Stryer and E. R. Blout, J. Am. Chem. Soc., 1961, 83, 1411.14 R. Purrello, L. M. Scolaro, E. Bellacchio, S. Gurrieri andA. Romeo, Inorg. Chem., 1998, 37, 3647; L. Rosaria, A. D’Urso,A. Mammana and R. Purrello, Chirality, 2008, 20, 411.15 L. M. Scolaro, A. Romeo and A. Terracina, Chem. Commun.,1997, 1451.16 A. V. Mikhonin, N. S. Myshakina, S. V. Bykov and S. A. Asher,J. Am. Chem. Soc., 2005, 127, 7712.17 C. L. Phillips, W. Mickols, M. F. Maestre and I. Tinoco, Jr,Biochemistry, 1986, 25, 7803.18 C. R. Cantor and P. R. Schimmel, Biophysical Chemistry ofMacromolecules, Pt. 1–3, W. H. Freeman, San Francisco, 1980.19 E. J. Spek, Y. Gong and N. R. Kallenbach, J. Am. Chem. Soc.,1995, 117, 10773.Fig. 3 CD spectrum of t-CuP in the presence of short PLG at pH= 4.7and [Glu] = 20 mM, 2 mM (solid and dashed curve, respectively).(PLG FW B 1.5–3 kD; [t-CuP] = 5 mM; [buﬀer] = 2 mM.)Fig. 4 Extinction (a) and CD (b) spectra of 5 mM t-CuP in thepresence of PLG (pH = 4.58, solid line; pH = 5.5, dotted line; pH =6.86, dashed line), at high IS. ([Glu] = 500 mM, FW B 17 kD;[buﬀer] = 2 mM; [NaCl] = 0.15 M.)This journal is c The Royal Society of Chemistry 2010 Chem. Commun., 2010, 46, 389–391 | 391Published on 19 November 2009. Downloaded by Swarthmore College on 28/04/2015 14:29:23. View Article Online

Conformations Of A Model Protein Revealed By An Aggregating Cuᴵᴵ Porphyrin: Sensing The Difference

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Conformations Of A Model Protein Revealed By An Aggregating Cuᴵᴵ Porphyrin: Sensing The Difference

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