Binding of the C-terminal Sterile α Motif (SAM) Domain of Human p73 to Lipid Membranes
2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês
10.1074/jbc.m307846200
ISSN1083-351X
AutoresFrancisco N. Barrera, José A. Poveda, José M. González‐Ros, José L. Neira,
Tópico(s)Cell death mechanisms and regulation
ResumoThe α splice variant of p73 (p73α), a homologue of the tumor suppressor p53, has close to its C terminus a sterile α motif (SAM), SAMp73, that is thought to be involved in protein-protein interactions. Here, we report the lipid binding properties of this domain. Binding was assayed against zwitterionic (phosphatidylcholine) and anionic (phosphatidic acid) lipids and was studied by different biophysical techniques, namely, circular dichroism and fluorescence spectroscopies and differential scanning calorimetry. These techniques unambiguously indicate that SAMp73 binds to lipids. The binding involves protein surface attachment and partial membrane penetration, accompanied by changes in SAMp73 structure. The α splice variant of p73 (p73α), a homologue of the tumor suppressor p53, has close to its C terminus a sterile α motif (SAM), SAMp73, that is thought to be involved in protein-protein interactions. Here, we report the lipid binding properties of this domain. Binding was assayed against zwitterionic (phosphatidylcholine) and anionic (phosphatidic acid) lipids and was studied by different biophysical techniques, namely, circular dichroism and fluorescence spectroscopies and differential scanning calorimetry. These techniques unambiguously indicate that SAMp73 binds to lipids. The binding involves protein surface attachment and partial membrane penetration, accompanied by changes in SAMp73 structure. p73 and p63 are members of the p53 gene family (1Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1533) Google Scholar, 2Jost C.A. Marin M.C. Kaelin Jr., W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (897) Google Scholar). As the tumor suppressor p53, p73 and p63 are also transcription factors that contain an N-terminal transactivation domain, a sequence-specific DNA-binding domain, and an oligomerization domain with a high sequence homology to the corresponding domains of p53. For instance, p73 shares 63% identity with the DNA-binding region of p53 (including the conservation of all DNA-binding residues), 38% identity with the tetramerization domain, and 29% with the transactivation domain. Furthermore, p73 and p63 share a relative functional homology with p53, because they can both activate transcription from p53-responsive genes, stop the cell cycle, and induce apoptosis when overexpressed. Moreover, p73 is positively regulated in p53-deficient tumors in response to oncogene overexpression, and its expression is increased in several tumor types (1Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1533) Google Scholar, 3Zhu J. Jiang J. Zhou W. Chen X. Cancer Res. 1998; 58: 5061-5065PubMed Google Scholar, 4Di Como C.J. Gaiddon C. Prives C. Mol. Cell Biol. 1999; 19: 1438-1449Crossref PubMed Scopus (379) Google Scholar, 5Zaika A. Irwin M. Sansome C. Moll U.M. J. Biol. Chem. 2001; 276: 11310-11316Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 6Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1830) Google Scholar, 7Strano S. Munarriz E. Rossi M. Cristofanelli B. Shaul Y. Castagnoli L. Levine A.J. Sacchi A. Cesareni G. Oren M. Blandino G. J. Biol. Chem. 2000; 275: 29503-29512Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). It seems that in the absence of p53, p73 can take its place and induce apoptosis in tumoral cells, although the ultimate role of p73 in tumor suppression is still unclear (5Zaika A. Irwin M. Sansome C. Moll U.M. J. Biol. Chem. 2001; 276: 11310-11316Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Unlike p53, p73 and p63 are only rarely mutated in the large number of tumors examined to date and thus they are unlikely to be classical tumor suppressor genes. Also in contrast to p53, in mice lacking p73 there have been described severe developmental abnormalities, such as hippocampal dysgenesis, hydrocephalus, chronic infections, and inflammation, as well as abnormalities in pheromone sensory pathways; however, no increase in the tumor formation rate is detected (as it happened in p53 knockout mice (8Yang A. Walker N. Bronson R. Kaghad M. Oosterwegel M. Bonnin J. Vagner C. Bonnet H. Dikkes P. Sharpe A. McKeon F. Caput D. Nature. 2000; 404: 99-103Crossref PubMed Scopus (878) Google Scholar)).Conversely to p53, p73 and p63 contain additional C-terminal extensions. In both proteins, these extensions show alternative splicing, which results in at least six C-terminal variants for p73 (α–ϕ) and three for p63 (α–γ) (1Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1533) Google Scholar, 9De Laurenzi V. Costanzo A. Barcaroli D. Terrinoni A. Falco M. Annicchiarico-Petruzzelli M. Levero M. Mellino G. Exp. Med. 1998; 188: 1763-1768Crossref PubMed Scopus (361) Google Scholar, 10Irwin M.S. Kaelin W G. Cell Growth Differ. 2001; 12: 337-349PubMed Google Scholar). These isoforms have different transcription and biological properties, and their expression patterns change among normal tissues (9De Laurenzi V. Costanzo A. Barcaroli D. Terrinoni A. Falco M. Annicchiarico-Petruzzelli M. Levero M. Mellino G. Exp. Med. 1998; 188: 1763-1768Crossref PubMed Scopus (361) Google Scholar). For example, p73β transactivates many p53-responsive promoters, and p73α does so to a lesser extent (2Jost C.A. Marin M.C. Kaelin Jr., W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (897) Google Scholar, 3Zhu J. Jiang J. Zhou W. Chen X. Cancer Res. 1998; 58: 5061-5065PubMed Google Scholar, 4Di Como C.J. Gaiddon C. Prives C. Mol. Cell Biol. 1999; 19: 1438-1449Crossref PubMed Scopus (379) Google Scholar). Nonetheless, the role of the several isoforms in cellular function is far from being fully understood, and it has been shown that their differential regulatory roles are highly cell context-dependent (11Freebern W.J. Smith J.L. Chaudhry S.S. Haggerty C.M. Gardner K. J. Biol. Chem. 2003; 278: 2249-2255Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar).The α variants of p73 and p63 have close to their C terminus a SAM 1The abbreviations used are: SAMsterile α motifDMPA1,2-dimyristoyl-sn-glycero-3-phosphateDMPC1,2-dimyristoyl-sn-glycero-3-phosphocolineDSCdifferential scanning calorimetryPAphosphatidic acidPCphosphatidylcholineRETresonance energy transferTMA-DPH1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5hexatriene.1The abbreviations used are: SAMsterile α motifDMPA1,2-dimyristoyl-sn-glycero-3-phosphateDMPC1,2-dimyristoyl-sn-glycero-3-phosphocolineDSCdifferential scanning calorimetryPAphosphatidic acidPCphosphatidylcholineRETresonance energy transferTMA-DPH1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5hexatriene. domain, which is thought to be responsible for regulating p53-like functions (12Bork P. Koonin E.V. Nat. Genet. 1998; 18: 313-318Crossref PubMed Scopus (260) Google Scholar). SAM domains are protein modules of ∼65–70 amino acids found in diverse proteins whose functions range from signal transduction to transcriptional repression (12Bork P. Koonin E.V. Nat. Genet. 1998; 18: 313-318Crossref PubMed Scopus (260) Google Scholar). Interestingly enough, it has been reported that the α isoform of p73 (and also that of p63) has its p53-like function dramatically reduced in comparison with other non-SAM-containing isoforms, suggesting that SAM domain could be responsible for those functional differences (12Bork P. Koonin E.V. Nat. Genet. 1998; 18: 313-318Crossref PubMed Scopus (260) Google Scholar, 13Thanos C.D. Bowie J.U. Protein Sci. 1999; 8: 1708-1710Crossref PubMed Scopus (126) Google Scholar). The structure of the SAM domain of p73, SAMp73 (the C-terminal region of the p73α protein comprising residues 487–554 of the intact protein), has been resolved by NMR (14Chi S.-W. Ayed A. Arrowsmith C.H. EMBO J. 1999; 18: 4438-4445Crossref PubMed Scopus (149) Google Scholar) and x-ray crystallography (15Wang W.K. Proctor M.R. Buckle A.M. Bycroft M. Chen Y.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 769-771Crossref PubMed Scopus (15) Google Scholar, 16Wang W.K. Bycroft M. Foster N.W. Buckle A.M. Fersht A.R. Chen Y.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 545-551Crossref PubMed Scopus (33) Google Scholar). The domain (residues 487–554 of the full p73α protein) contains a single tryptophan residue, which could be used as a spectroscopic probe to monitor the protein conformational changes. The structure of the domain reveals a small five-helix bundle composed of four α-helices (residues 491–499 (helix 1), 506–511 (helix 2), 525–531 (helix 4), and 538–550 (helix 5)) and a small 310-helix (residues 517–520 (helix 3)). The SAMp73 has structural similarity with two ephrin receptors tyrosine kinases (14Chi S.-W. Ayed A. Arrowsmith C.H. EMBO J. 1999; 18: 4438-4445Crossref PubMed Scopus (149) Google Scholar), and the spatial arrangement of the bundle is similar to that of SAM domains found in other proteins (13Thanos C.D. Bowie J.U. Protein Sci. 1999; 8: 1708-1710Crossref PubMed Scopus (126) Google Scholar). SAM domains are putatively considered to be responsible for regulating protein functions via self-association or by association with other domains (17Schultz J. Pointing C.P. Hoffmann K. Bork P. Protein Sci. 1997; 6: 249-253Crossref PubMed Scopus (264) Google Scholar), but the exact function of SAMp73 is not known. The crystal structure of SAMp73 reveals a dimeric organization (15Wang W.K. Proctor M.R. Buckle A.M. Bycroft M. Chen Y.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 769-771Crossref PubMed Scopus (15) Google Scholar), but the NMR structure is monomeric (14Chi S.-W. Ayed A. Arrowsmith C.H. EMBO J. 1999; 18: 4438-4445Crossref PubMed Scopus (149) Google Scholar), suggesting that dimer formation in the crystal is an effect of crystal packing rather than a real physiological state; furthermore, equilibrium sedimentation experiments have shown that SAMp73 is monomeric under a wide range of experimental conditions (14Chi S.-W. Ayed A. Arrowsmith C.H. EMBO J. 1999; 18: 4438-4445Crossref PubMed Scopus (149) Google Scholar, 18Barrera F.N. Garzon M.T. Gomez F.J. Neira J.L. Biochemistry. 2002; 41: 743-753Google Scholar).Because of its small size (67 residues long), we are using SAMp73 as a model for folding, stability, and macromolecular binding studies. We have embarked in the study of the interactions of SAMp73 with other macromolecules, namely proteins, nucleic acids, and lipids. Here, we explore its lipid binding properties toward PA (an anionic lipid) and PC (a zwitterionic lipid). The results indicate that SAMp73 interacts with both lipids. To the best of our knowledge, this study represents the first report of the lipid binding properties of a SAM domain, and it raises new questions about the role of this domain in p73 function.EXPERIMENTAL PROCEDURESMaterialsImidazole, Trizma base, and NaCl were from Sigma. The Ni2+-nitrilotriacetic acid resin was from Invitrogen. Egg yolk PC and PA, DMPA, and DMPC were obtained from Avanti Polar lipids (Birminghman, AL). TMA-DPH was from Molecular Probes. Spectroscopy grade N,N-dimethylformamide was from Merck. Dialysis tubing was from Spectrapore, with a molecular mass cut-off of 3500 Da. Standard suppliers were used for all other chemicals. Water was deionized and purified on a Millipore system.Protein Expression and PurificationThe SAMp73 clone, comprising residues 487–554 of the intact p73α and a His6 tag at the N terminus, was kindly donated by C. H. Arrowsmith. We have carried out all the studies with this construct because its structure is well known by NMR (14Chi S.-W. Ayed A. Arrowsmith C.H. EMBO J. 1999; 18: 4438-4445Crossref PubMed Scopus (149) Google Scholar), and no differences were observed with that obtained by x-ray, where the His6 tag had been removed (14Chi S.-W. Ayed A. Arrowsmith C.H. EMBO J. 1999; 18: 4438-4445Crossref PubMed Scopus (149) Google Scholar, 15Wang W.K. Proctor M.R. Buckle A.M. Bycroft M. Chen Y.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 769-771Crossref PubMed Scopus (15) Google Scholar, 16Wang W.K. Bycroft M. Foster N.W. Buckle A.M. Fersht A.R. Chen Y.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 545-551Crossref PubMed Scopus (33) Google Scholar). Recombinant protein was expressed in Escherichia coli C43 strain (19Miroux B. Walker J.E. J. Mol. Biol. 1996; 260: 289-298Crossref PubMed Scopus (1559) Google Scholar) and purified using Ni2+-nitrilotriacetic acid chromatography. To eliminate any protein or DNA bound to the resin, co-eluting with the protein, an additional gel filtration chromatography step, was carried out by using a Superdex 75 16/60 gel filtration column (Amersham Biosciences) running on an AKTA FPLC system (Amersham Biosciences). Protein purity was larger than 95% as concluded from visual inspection in the SDS protein-denaturing gels and from matrix-assisted laser desorption ionization time-of-flight experiments (data not shown). The yield was 30–35 mg of protein/liter of culture. The samples were dialyzed extensively against 0.2 m NaCl, lyophilized, and stored at –80 °C. The protein concentration was calculated from the absorbance of a stock solution measured at 280 nm, using the extinction coefficients of model compounds (20Pace C.N. Scholtz J.M. Creighton T.E. Protein Structure. 2nd Ed. Oxford University Press, Oxford1997: 253-259Google Scholar).Lipid Vesicles and Sample PreparationLipid vesicles were prepared by dissolving the required amount of lipid (PA, PC, DMPA, and DMPC) in chloroform/methanol (1:1 v/v) and drying, first under a gaseous nitrogen stream and then under vacuum for 3 h to remove all traces of organic solvents. The thin layer formed was resuspended in water (resulting in a 27 mm lipid concentration) while being vortexed and warmed gently (usually 10 °C over the Tc for DMPA and DMPC and at 55 °C for PA and PC). After lipid hydration, the PA and PC resulting multilamellar liposome suspensions were sonicated, using a Branson Sonifier model 250 fitted with a microtip (Branson, Shelton, CT) until a clear suspension of small unilamellar vesicles was obtained (typically 1 min for PA and 5 min, in bursts of 1 min, for PC while being cooled in ice). These preparations have been shown to yield vesicles with diameters ranging from 300 to 600 Å (21Sanghera N. Pinheiro T.J.T. J. Mol. Biol. 2002; 315: 1241-1256Crossref PubMed Scopus (166) Google Scholar). The sonicated samples were then centrifuged in a microcentrifuge at 10,000 rpm for 2 min to remove any titanium particle shed from the microtip during sonication. No significant lipid pellet was observed after the centrifugation.The lipid-protein samples were prepared by mixing the corresponding volumes of protein and lipid solutions, NaCl and Tris, pH 7; salt and buffer concentrations were carefully set to 200 and 10 mm, respectively, from 10-fold concentrated stocks, and the final volume was adjusted with water. All of the samples were mixed thoroughly and equilibrated usually for 20 min before the corresponding experiment was carried out. The pH of the samples was measured with an ultrathin Aldrich electrode in a Radiometer (Copenhagen) pH meter to discard differences between calculated and measured pH values.Far-UV CD MeasurementsCircular dichroism spectra were collected on a Jasco J810 spectropolarimeter fitted with a thermostated cell holder and interfaced with a Neslab RTE-111 water bath. The instrument was periodically calibrated with (+)10-camphorsulphonic acid. Isothermal wavelength spectra were acquired at a scan speed of 50 nm/min with a response time of 2 s and averaged over seven scans at 25 °C. Far-UV measurements were performed using 30 μm of protein in 10 mm of the above described buffer, using 0.1-cm-pathlength cells (Hellma). The corresponding backgrounds were subtracted from the final spectra, and they usually accounted less than the 5% of the sample ellipticities. Only at lipid concentrations over 5 mm were more intense backgrounds observed, but they never accounted for more than the 15% of the total protein ellipticity signal (data not shown). Subtracted spectra were smoothed avoiding alteration of spectral intensities. To further check that the measurements were not influenced by the scattering derived from the presence of the lipid, data were also analyzed without making subtractions, and similar results were obtained (data not shown). Ellipticities are expressed as mean residue ellipticities, [Θ], in units of deg cm2 dmol–1, according to Equation 1,[Θ]=Θ10lcN(Eq. 1) where Θ is the observed ellipticity, c is the molar concentration of the protein, l is the cell pathlength (in cm), and N is the number of amino acid residues in the sequence.Fluorescence MeasurementsFluorescence spectra for SAMp73 were collected either on a SLM 8000 spectrofluorometer (Spectronics Instruments, Urbana, IL) interfaced with a Haake water bath or in a Cary Eclipse spectrofluorometer (Varian) interfaced with a Peltier cell. A 0.5-cm-pathlength quartz cell (Hellma) was used for measurements in the SLM spectrofluorometer, and a 1-cm-pathlength quartz cell (Hellma) was used in the Varian spectrofluorometer. All of the experiments were carried out at 25 °C.Steady State Fluorescence Measurements—The protein samples were excited at 280 and 295 nm to characterize possible different behaviors of tryptophan and/or tyrosine residues (20Pace C.N. Scholtz J.M. Creighton T.E. Protein Structure. 2nd Ed. Oxford University Press, Oxford1997: 253-259Google Scholar). It was observed that both spectra were similar. The rest of the experiments were acquired by excitation at 280 nm. The slit width was 4 nm for the excitation light and 8 nm for the emission light; the integration time was 1 s, and the increment of wavelength was set to 1 nm between 310 and 400 nm. Blank corrections were made in all spectra. The protein concentration was 3 μm.Resonance Energy Transfer (RET) Measurements—The efficiency of energy transfer (E) can be defined according to Equation 2 (22Ferrer-Montiel A. González-Ros J.M. Ferragut J.A. Biochim. Biophys. Acta. 1988; 937: 379-386Crossref PubMed Scopus (26) Google Scholar),E=1-(IF/IFo)(Eq. 2) where IF and IFo are the fluorescence emission intensity of the donor in the presence and in the absence of the energy acceptor, respectively. Aliquots of a concentrated TMA-DPH stock (in N,N-dimethylformamide) were added to a cuvette containing the lipid-protein mixtures, and after 90 min of incubation, the changes of tryptophan emission fluorescence were monitored upon excitation at 280 nm. The effect of the acceptor (TMA-DPH) absorption at donor (tryptophan) emission maximum (340 nm) was corrected as described by Coutinho and Prieto (23Coutinho A. Prieto M. J. Chem. Ed. 1993; 70: 425Crossref Scopus (109) Google Scholar). Monitoring of the emission wavelength at 330 nm (where TMA-DPH absorption is clearly reduced) yielded similar results (data not shown). No correction for the acceptor absorption at donor excitation wavelength was made because TMA-DPH absorbance at 280 nm was negligible. The extinction molar coefficient, ϵ, for TMA-DPH was that provided by Molecular Probes (ϵ = 75000 m–1 cm–1). The slit width was 2 nm for the excitation light and 4 nm for the emission light.The approach developed by Wolber and Hudson (24Wolber P.K. Hudson B.S. Biophys. J. 1979; 28: 197-210Abstract Full Text PDF PubMed Scopus (350) Google Scholar) was used to obtain the theoretical expected value for the efficiency of energy transfer in a bilayer two-dimensional system. This theoretical model represents an analytical solution of the Förster energy transfer problem when: (i) both the donors and acceptors are randomly distributed in a plane and (ii) donors are excluded from a region surrounding each acceptor. According to this model, the relative quantum yield, qr, is defined as follows.qr=1-E(Eq. 3) The relative quantum yield was theoretically calculated for different Re/Ro ratios at increasing C values, where Re is the distance between donor and acceptor at their closest approach, Ro is the critical radius of transfer (also defined as the distance at which the transfer efficiency is 50%), and C is the concentration of acceptors per Ro2 (where the area of one PA molecule is 70 Å2 and that of PC molecule is 80 Å2). Ro was calculated according to the equations developed by Förster and others (25Förster T. A. Naurforsch. A. Astrophys. Phys. Chem. 1959; 4: 321-327Google Scholar, 26Rapaport D. Shai Y. J. Biol. Chem. 1992; 267: 6502-6509Abstract Full Text PDF PubMed Google Scholar),Ro=9876(Jκ2n-4ΦD)1/6(in̊)(Eq. 4) where J is the overlap integral, which measures the degree of overlap between the donor emission spectrum and the acceptor absorption spectrum; κ2 is the orientation factor, which was taken to be 2/3 (27Stryer L. Annu. Rev. Biochem. 1978; 47: 819-846Crossref PubMed Scopus (1952) Google Scholar); n is the refractive index of the medium, which was taken as 1.44 (that is, the value of the bilayer interior) (28Davenport J. Dale R.E. Bisby R.H. Cundal R.B. Biochemistry. 1985; 24: 4097-4108Crossref PubMed Scopus (165) Google Scholar); and ΦD is the quantum yield of the donor in the absence of acceptor. The quantum yield of the sole tryptophan was determined using 5-metoxiindole as a quantum yield standard, as described by Lakowicz (29Lakowicz J.R. Principles of Fluorescence Spectroscopy. 2nd Ed. Kluwer Academic/Plenum Press, New York1999Crossref Google Scholar). In the presence of PA, the calculated ΦD was 0.228, and in the presence of PC, ΦD was 0.179. The overlap integral, J, was calculated by Equation 5,J=∫fD(λ)εA(λ)λ4dλ∫fD(λ)dλ(inM-1cm3)(Eq. 5) where fD(λ) is the donor fluorescence intensity at each wavelength, λ, and ϵA (λ) is the acceptor molar extinction coefficient at each wavelength. The absorption spectra were taken in a Beckman DU 640 spectrophotometer.Partition Coefficient DeterminationA parameter that can be used to quantify the extent of lipid-protein interactions is the partition coefficient, Kp, which is described by the following equation,Kp=n1/V1nw/Vw(Eq. 6) where nl is the number of moles of the protein in lipid, nw is the number of moles of the protein in aqueous solution, Vl is the volume of the lipid phase, and Vw is the volume of aqueous phase. The variation in a spectroscopic parameter, if it is proportional to the concentration of protein bound to the membrane, can be used to determine the lipid partition coefficient according to Refs. 30Eftink M.R. Methods Enzymol. 1997; 278: 221-257Crossref PubMed Scopus (234) Google Scholar and 31Mateo C.R. Prieto M. Micol V. Shapiro S. Villalaín J. Biochim. Biophys. Acta. 2000; 1509: 167-175Crossref PubMed Scopus (27) Google Scholar,X=XmaxKpγ[lipid]1+Kpγ[lipid](Eq. 7) where X is the spectroscopic parameter that changes upon addition of increasing amounts of lipid (in our studies, the [Θ], or the fluorescence emission maximum and the fluorescence intensity); Xmax is the maximum value of X, and γ is the molar volume of the lipid (which has values of 0.7 m–1 for PA and 0.8 m–1 for PC (32Marsh D. CRC Handbook of Lipid Bilayers. CRC Press, Boca Raton, FL1990Google Scholar)). Fitting by nonlinear least squares analysis was carried out by using the general curve fit option of Kaleidagraph (Abelbeck software) on a personal computer.Differential Scanning CalorimetryDSC experiments were performed with a MicroCal MC-2 (Microcal Inc., Northampton, MA) differential scanning calorimeter interfaced to a computer equipped with a Data Translation DT-2801 A/D converter board for instrument control and automatic data collection. The samples were prepared as described (see before), but DMPA-containing samples were prepared with 0.1 mm EDTA to avoid perturbations in the phase transition created by the presence of Ca2+. NaCl concentration was carefully kept constant to avoid artifacts caused by the positive ions during the DMPA phase transition. The samples were degassed under vacuum for 10 min with gentle stirring prior to being loaded into the calorimetric cell. Differences in the heat capacity between the sample and the reference cell, filled with buffer solution, were obtained by raising the temperature at a constant rate of 60 °C/h over a temperature range of 10–60 °C. At these temperatures thermal unfolding of the protein is not expected (18Barrera F.N. Garzon M.T. Gomez F.J. Neira J.L. Biochemistry. 2002; 41: 743-753Google Scholar). The excess heat capacity functions were obtained after base-line subtraction and correction for the instrument time response. A series of three consecutive scans were at least acquired to ensure scan-to-scan reproducibility. Although the second and third scans were identical, only the third scan was used for calculation of the transition temperature and the enthalpy. The Microcal Origin software was used for data acquisition and analysis.RESULTSFar-UV CD ExperimentsWe used far-UV CD in the analysis of the protein-lipid binding as a spectroscopic probe that is sensitive to protein secondary structure (33Woody R.W. Methods Enzymol. 1995; 246: 34-71Crossref PubMed Scopus (698) Google Scholar, 34Kelly S.M. Price N.C. Curr. Protein Peptide Sci. 2000; 1: 349-384Crossref PubMed Scopus (815) Google Scholar). SAMp73 in solution shows an intense far-UV CD spectrum with the features of an α-helical protein, with minima at 222 and 208 nm (Fig. 1), although interference from the aromatic residues cannot be ruled out (33Woody R.W. Methods Enzymol. 1995; 246: 34-71Crossref PubMed Scopus (698) Google Scholar, 34Kelly S.M. Price N.C. Curr. Protein Peptide Sci. 2000; 1: 349-384Crossref PubMed Scopus (815) Google Scholar). To study the lipid binding properties of SAMp73, we first tested whether its CD spectrum changed upon incubation with either anionic (PA) or zwitterionic (PC) lipid membranes. It was observed that the negatively charged lipid vesicles of PA induced a significant change in the SAMp73 CD spectrum, with a marked increase in the ellipticity (in absolute value) at 222 nm and a larger change in that at 208 nm (Fig. 1). The latter could indicate either an increase in the random coil population or changes in the environment of the aromatic residues (33Woody R.W. Methods Enzymol. 1995; 246: 34-71Crossref PubMed Scopus (698) Google Scholar, 34Kelly S.M. Price N.C. Curr. Protein Peptide Sci. 2000; 1: 349-384Crossref PubMed Scopus (815) Google Scholar). Binding of the protein to zwitterionic PC vesicles induced also an enhancement in the ellipticity of the SAMp73 spectrum, but its extent was smaller than that observed in PA. In this case, the ellipticity at 208 and 222 nm increased to nearly the same extent.The partition coefficients, Kp, obtained from the ellipticity changes at 208 nm upon increase in lipid concentration were 1360 ± 190 for PA and 3967 ± 970 for PC (Fig. 2) (Equation 6). Similar partition coefficients were also determined from the ellipticity changes at 222 nm. These partition coefficients allowed us to determine the population of SAMp73 distributed between the aqueous and lipid phases at a given lipid concentration. The molar fraction of a protein in the aqueous solution, Xw, can be calculated as follows (35Contreras L.M. de Almeida R.F. Villalaín J. Fedorov A. Prieto M. Biophys. J. 2001; 80: 2273-2283Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar).Xw=11+Kpγ[lipid](Eq. 8) Fig. 2Binding constant determination by CD. Mean residue ellipticity changes at 208 nm (Δ[Θ]208) were followed at increasing lipid concentration in PC (A) and PA (B). Kp values were obtained by fitting to Equation 6 (solid lines). The protein concentration was 30 μm. The conditions were 10 mm Tris, pH 7, 0.2 m NaCl, 25 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)For instance, in PC at 3 mm, the percentage of SAMp73 in the aqueous phase was 9.5%, whereas for PA at 3 mm, it was 26%.Fluorescence ExperimentsSteady State Measurements—The intrinsic protein fluorescence is a highly sensitive probe to monitor protein-lipid binding (36Surewicz W.K. Epand R.M. Biochemistry. 1984; 23: 6072-6077Crossref PubMed Scopus (96) Google Scholar, 37Rankin S.E. Watts A. Pinheiro T.J.T. Biochemistry. 1998; 37: 12588-12595Crossref PubMed Scopus (48) Google Scholar, 38Prendergast F.G. Haugland R.P. Callahan P.J. Bodeau D. Biochemistry. 1981; 20: 7333-7400Crossref PubMed Scopus (453) Google Scholar). SAMp73 has one tryptophan and five tyrosine residues. Trp542, in the numbering of intact p73α, is in the middle of helix 5 forming the hydrophobic core of the protein; Tyr487 is the N-terminal residue; Tyr508 is in the middle of helix 2; Tyr518 and Tyr537 are at the beginnings of helices 3 and 5, respectively; and Tyr554 is the C-terminal residue. The emission fluorescence spectrum of native SAMp73 is dominated by the emission of the sole tryptophan residue (18Barrera F.N. Garzon M.T. Gomez F.J. Neira J.L. Biochemistry. 2002; 41: 743-753Google Scholar), with a maximum at 336 nm at neutral pH, which indicates that the tryptophan is partially buried within the protein structure, as concluded from the x-ray (15Wang W.K. Proctor M.R. Buckle A.M. Bycroft M. Chen Y.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 769-771Crossref PubMed Scopus (15)
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