Xanthophyll Binding Sites of the CP29 (Lhcb4) Subunit of Higher Plant Photosystem II Investigated by Domain Swapping and Mutation Analysis

Abstract

The binding sites for xanthophylls in the CP29 antenna protein of higher plant Photosystem II have been investigated using recombinant proteins refolded in vitro. Despite the presence of three xanthophyll species CP29 binds two carotenoids per polypeptide. The localization of neoxanthin was studied producing a chimeric protein constructed by swapping the C-helix domain from CP29 to LHCII. The resulting holoprotein did not bind neoxanthin, confirming that the N1 site is not present in CP29. Neoxanthin in CP29 was, instead, bound to the L2 site, which is thus shown to have a wider specificity with respect to the homologous site L2 in LHCII. Lutein was found in the L1 site of CP29. For each site the selectivity for individual xanthophyll species was studied as well as its role in protein stabilization, energy transfer, and photoprotection. Putative xanthophyll binding sequences, identified by primary structure analysis as a stretch of hydrophobic residues including an acidic term, were analyzed by site-directed mutagenesis or, in one case, by deleting the entire sequence. The mutant proteins were unaffected in their xanthophyll composition, thus suggesting that the target motifs had little influence in determining xanthophyll binding, whereas hydrophobic sequences in the membrane-spanning helices are important. The binding sites for xanthophylls in the CP29 antenna protein of higher plant Photosystem II have been investigated using recombinant proteins refolded in vitro. Despite the presence of three xanthophyll species CP29 binds two carotenoids per polypeptide. The localization of neoxanthin was studied producing a chimeric protein constructed by swapping the C-helix domain from CP29 to LHCII. The resulting holoprotein did not bind neoxanthin, confirming that the N1 site is not present in CP29. Neoxanthin in CP29 was, instead, bound to the L2 site, which is thus shown to have a wider specificity with respect to the homologous site L2 in LHCII. Lutein was found in the L1 site of CP29. For each site the selectivity for individual xanthophyll species was studied as well as its role in protein stabilization, energy transfer, and photoprotection. Putative xanthophyll binding sequences, identified by primary structure analysis as a stretch of hydrophobic residues including an acidic term, were analyzed by site-directed mutagenesis or, in one case, by deleting the entire sequence. The mutant proteins were unaffected in their xanthophyll composition, thus suggesting that the target motifs had little influence in determining xanthophyll binding, whereas hydrophobic sequences in the membrane-spanning helices are important. Carotenoids are involved in many aspects of higher plants photosynthesis. Reaction center complexes bind β-carotene for light harvesting, chlorophyll a (Chl a) 1The abbreviations used are: Chl, chlorophyll; Car, carotenoid; Lhc, light-harvesting complex; L, lutein; LD, linear dichroism; N, neoxanthin; V, violaxanthin; WT, wild type; PSII, Photosystem II; HPLC, high performance liquid chromatography; CTL, control. triplet quenching, and electron transport between cytochrome b559 and P680+ (1Tracewell C.A. Cua A. Stewart D.H. Bocian D.F. Brudvig G.W. Biochemistry. 2001; 40: 193-203Crossref PubMed Scopus (107) Google Scholar, 2Faller P. Pascal A. Rutherford A.W. Biochemistry. 2001; 40: 6431-6440Crossref PubMed Scopus (105) Google Scholar). In the peripheral antenna, Lhc proteins bind a number of xanthophyll species, namely lutein, neoxanthin, and violaxanthin, acting in the harvesting of light and transfer of excitation energy to Chls (3Gradinaru C.C. van Stokkum I.H.M. Pascal A.A. van Grondelle R. Van Amerongen H. J. Phys. Chem. B. 2000; 104: 9330-9342Crossref Scopus (181) Google Scholar, 4Croce R. Muller M.G. Bassi R. Holzwarth A.R. Biophys. J. 2001; 80: 901-915Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 5Das S.K. Frank H.A. Biochemisty. 2002; 41: 13087-13095Crossref PubMed Scopus (25) Google Scholar, 6Croce R. Müller M.G. Caffarri S. Bassi R. Holzwarth A.R. Biophys. J. 2003; 84: 2517-2532Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The photoprotection function of xanthophylls is accomplished through multiple mechanisms including the quenching of Chl a triplet states, scavenging of singlet oxygen produced by the reaction of Chl triplets with O2, and participation to non-photochemical quenching, a mechanism in which violaxanthin is de-epoxidated to zeaxanthin and excess energy is dissipated into heat (7Demmig B. Winter K. Kruger A. Czygan F.-C. Plant Physiol. 1987; 84: 218-224Crossref PubMed Google Scholar). In addition, xanthophylls are essential for Lhc protein folding (8Plumley F.G. Schmidt G.W. Proc. Natl. Acad. Sci. S. A. 1987; 84: PubMed Google whereas their binding to a site the between and protein Bassi R. Biochemistry. 2001; 40: PubMed Scopus Google Scholar, Bassi R. J. 2001; PubMed Scopus Google Scholar). The Lhc proteins a number of xanthophyll species reaction center proteins β-carotene is not species have in each case, light harvesting, triplet quenching, and singlet oxygen scavenging R. Caffarri S. 2000; PubMed Scopus Google Scholar). the is one of the in higher suggesting a function for each The xanthophyll binding protein is the light harvesting of Photosystem II for which binding sites have been to bound into of the L1 and L2 the in the center of the Lhc structure PubMed Scopus Google Scholar). L1 is for lutein, whereas L2 bind violaxanthin R. S. Bassi R. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). N1 is for neoxanthin and is the domain of R. R. J. Bassi R. PubMed Scopus (105) Google Scholar). a binding site been its in light P. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. R. J. Bassi R. J. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). binding site was shown to a structure and Chl a triplet quenching are by in site L1 Bassi R. J. 2001; PubMed Scopus Google Scholar, R. R. Proc. Natl. Acad. Sci. S. A. PubMed Scopus Google Scholar). is not involved in singlet in triplet energy transfer and was to a of for the violaxanthin S. R. J. Bassi R. J. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google bound to the site L2 R. Bassi R. J. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). N1 is in light harvesting and singlet oxygen scavenging R. S. Bassi R. J. Chem. Full Text Full Text PDF PubMed Scopus Google and the of Bassi R. Biochemistry. 2002; 41: PubMed Scopus Google Scholar). Despite the high in the which a the number of xanthophyll binding their and their in the structure to in each Lhc the of individual sites and the of the Lhc protein is that the function of individual Lhc is by the of individual xanthophyll sites and by their selectivity and analysis of for xanthophyll binding sites been in the of Chl binding to the of Chls of to the of binding for xanthophylls R. S. Bassi R. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. R. J. Bassi R. PubMed Scopus (105) Google Scholar). sequences, in the and into hydrophobic and a involved in the binding by a hydrophobic xanthophylls whereas the was to with S. The Scholar). binding by between hydrophobic residues with the in the as in the A.A. Scopus Google Scholar). In the of a analysis of the chlorophyll protein CP29 with respect to the xanthophyll and function as well as of the involved in their with the and of CP29 and were as in Bassi R. R. Proc. Natl. Acad. Sci. S. A. PubMed Scopus Google Scholar). The chimeric was using two and which are present in the of of sites and from the for of The between sites was with the of CP29 using two and in which are the In the of the chimeric is with the sequences of and CP29. The were and as in Bassi R. R. Proc. Natl. Acad. Sci. S. A. PubMed Scopus Google Scholar). and of and and were as in R. Bassi R. J. PubMed Scopus Google using for CP29 a chlorophyll of The was in the using a chlorophyll of as CP29 and as and the present in the with were in the with a xanthophyll in the CP29 was from as in R. J. Bassi R. Biochemistry. PubMed Scopus Google Scholar). of the holoprotein was analyzed by Plant Physiol. PubMed Scopus Google and of the with the of the individual R. Bassi R. Biochemistry. 2002; Scopus Google Scholar). were by an excitation and were using a were with a were in was for and and for are the in R. R. J. Bassi R. Biochemistry. PubMed Scopus Google Scholar). was as in Bassi R. J. 2001; PubMed Scopus Google Scholar). of the complexes was the of the by the between and was The was by The of the protein was by the of the of in the was as R. Holzwarth A.R. Bassi R. 2000; PubMed Scopus Google using a transfer from to Chls was from the of in excitation with respect to of individual In the the energy transfer from Chl a was to The of into the of the number of carotenoids bound to each CP29 polypeptide. the was by from as P. Bassi R. PubMed Google Scholar). The were with and the is shown in The of individual were used for the of the from two CP29 complexes binding Chls Chl a Chl R. R. Proc. Natl. Acad. Sci. S. A. PubMed Scopus Google Scholar, P. Bassi R. J. Chem. Full Text PDF PubMed Google and xanthophylls in the shown by lutein, neoxanthin, and violaxanthin R. Bassi R. Biochemistry. PubMed Scopus Google Scholar). The are shown in with the was two per whereas the of three was of CP29 with the to in CP29 is three xanthophyll species were found in the of one of the binding the of the binding sites for the recombinant CP29 in was in the presence of individual carotenoids a of complexes were with xanthophylls neoxanthin, to was in and R. S. Bassi R. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. Bassi R. Biochemistry. 2002; Scopus Google Scholar, H.A. S.K. S. R. Bassi R. Biochemistry. 2001; 40: PubMed Scopus Google Scholar). The of the was analyzed by and of the The are in a two xanthophylls per polypeptide. The with zeaxanthin was the a with two zeaxanthin per was in in a was found Neoxanthin in the one per suggesting that one site the of site not the folding of the and of complexes in the presence of were the species used in the in the presence of in the are the of The in the are For the the in the is in the of the of carotenoids have been which are and of carotenoids have been which are and in a was shown that in the of not into a (8Plumley F.G. Schmidt G.W. Proc. Natl. Acad. Sci. S. A. 1987; 84: PubMed Google that carotenoids a primary role in Lhc the influence of each of the two binding sites and of individual xanthophyll species in the of the were The are in The CP29 lutein, violaxanthin, and neoxanthin had a of the complexes in the presence of two xanthophyll species, and were whereas was thus that neoxanthin a with respect to one species were the their between and of complexes were The were in the that the binding of xanthophyll species not influence the Chl not were in the the by their In the antenna complexes of higher plants carotenoids have a light-harvesting the energy transfer of individual in the two the of the complexes were in of of individual using two Chl a two Chl and two the was for the the of was used to the excitation The of the in the and excitation were used to the of the energy transfer in the The is for Chl a transfer The are in transfer for individual in of the of the xanthophylls is the were with two The transfer for each of is The in the is transfer in the is to the with were for the for the for transfer are The in the is to the with For were for the for the for transfer are in a The to Chl a energy transfer the xanthophyll complexes with a xanthophyll species, was the with transfer whereas had the The two with zeaxanthin had energy transfer a an of whereas for two to thus that the zeaxanthin is to transfer energy to Chl a to a two xanthophyll species were the was in the of the with In the CP29 three xanthophyll species were with the with and violaxanthin and neoxanthin with R. Müller M.G. Caffarri S. Bassi R. Holzwarth A.R. Biophys. J. 2003; 84: 2517-2532Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). in Lhc complexes in quenching and scavenging of thus photoprotection. from the in Chl light in the presence of oxygen R. S. Bassi R. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of for the recombinant CP29 complexes are shown in and the complexes with two xanthophyll species high to whereas complexes with one xanthophyll species are thus a to the is to that violaxanthin is in photoprotection lutein; that a per whereas the of the CP29 bound xanthophyll binding sites have been in sites L1 and L2 PubMed Scopus Google in the and which is for neoxanthin R. S. Bassi R. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. R. J. Bassi R. PubMed Scopus (105) Google Scholar). The that two of sites are in CP29. that site L1 is in CP29 R. R. Proc. Natl. Acad. Sci. S. A. PubMed Scopus Google Scholar). is not site N1 L2 is sites present between the a chimeric was including the domain of and the domain of CP29. The and of the C-helix were in the domain the Lhc protein folding B. H. Biochemistry. PubMed Scopus Google The was in and the was in with was which had the in a of CP29 and complexes and a of between that of CP29 and The of the with its is in The in the is whereas a Chl a was In the the and in the were to Car, Chl and Chl analysis that the chimeric a Chl of in the of (Chl in the and in the of (Chl in the The Chl to was and the xanthophyll species bound were and of The chimeric binds neoxanthin of the xanthophyll in the and of chimeric in two for and CP29 in the are The number the the Chl in the The are from the of two in the Chl in a of in CP29 are bound to the L1 and L2 sites of their an and with respect to the to the PubMed Scopus Google Scholar). have used to the of xanthophyll a from the of the Frank H.A. J. Phys. Chem. B. 2001; Scopus Google in CP29. between the of xanthophylls in sites L1 and L2 to the have used the CP29 and shown to have of site L2 R. R. Proc. Natl. Acad. Sci. S. A. PubMed Scopus Google Scholar). The of xanthophylls to the as a is in the mutant and in the that the xanthophyll bound to the site L1 its an with the to the The of the in that the is to in the L2 In case, the between the and the to the is an is to the for In CP29 the of the Chls been from the analyzed the in R. R. J. Bassi R. Biochemistry. PubMed Scopus Google and the of the The of the xanthophyll in the L1 site was from the of the mutant in which the xanthophyll present is in site The analysis of L2 was the The were with the of the to the as in R. R. J. Bassi R. PubMed Scopus (105) Google Scholar). using the of from the analysis of the Chls R. R. J. Bassi R. Biochemistry. PubMed Scopus Google the for L1 and L2 were and L1 and L2 Putative primary structure analysis the Lhc sequences have been identified in the to and two the and two the which involved in xanthophyll binding S. The sequences are by an hydrophobic stretch of residues with a thus the of a hydrophobic for of whereas the of the with the have a S. The Scholar). the of sequences in xanthophyll binding, analysis was In three the in the center of the hydrophobic were by a one by a with an in one the binding was with the In the of the for the in L1 site, the the for Chl R. R. Proc. Natl. Acad. Sci. S. A. PubMed Scopus Google been with xanthophyll of the structure PubMed Scopus Google that the of the xanthophyll in the L2 site to with respect to the of with xanthophyll was to In a the structure of CP29 with of the binding sequences and of the is The mutant sequences were in and the were into complexes using a in which xanthophylls were with Chl a and Chl The mutant Lhc complexes were and by and complexes a high of to that mutant proteins have the of the The was the which a of In the analysis of the complexes is The in the binding in the with respect to the are and Chl and to the in the whereas a in neoxanthin and violaxanthin was by an in was to the and complexes a Chl in the of the not in the composition, suggesting that were to the of a Chl binding that protein motifs including the not a role in xanthophyll of the binding sites to the were three and mutant proteins were in using from the In the the in in each mutant is to the in the neoxanthin, and violaxanthin were the number of Chls in in a CP29 is the protein of the Lhc binds Chls per P. Bassi R. J. Chem. Full Text PDF PubMed Google each by a R. R. Proc. Natl. Acad. Sci. S. A. PubMed Scopus Google Scholar). The binding of xanthophylls to is a of have been the binding of three xanthophyll species, violaxanthin, lutein, and neoxanthin R. B. P. J. J. PubMed Scopus Google have been for the of each species to a binding is the binding of to site violaxanthin and neoxanthin have been to bind to as in P. J. Chem. Full Text Full Text PDF PubMed Scopus Google to for site L2 thus that site N1 is in CP29 R. R. Proc. Natl. Acad. Sci. S. A. PubMed Scopus Google Scholar). In an analysis of xanthophyll binding in CP29. In many carotenoids are bound to in which sites are which is the role of individual sites and individual and the binding sequences influence the the by using recombinant complexes in which the was the was the binding Carotenoids to that and recombinant CP29 bind two carotenoids per polypeptide. of and of neoxanthin and violaxanthin to one per were found in thus suggesting selectivity of one of the binding is by the analysis of CP29 from plant in the between the xanthophylls S.K. Frank H.A. Biochemisty. 2002; 41: 13087-13095Crossref PubMed Scopus (25) Google Scholar, R. A. Bassi R. Biophys. PubMed Scopus Google Scholar, A. P. van Grondelle R. B. Van Amerongen H. J. PubMed Scopus Google Scholar). in the presence of one xanthophyll species a of two xanthophyll species that lutein, violaxanthin, and zeaxanthin binding with and binding The neoxanthin, instead, in one of the two and its to the folding that the of site is not to the in xanthophyll binding sites have been in that three and and a one In CP29 two of the three sites are that the L1 site is present in R. R. Proc. Natl. Acad. Sci. S. A. PubMed Scopus Google Scholar). The binding site present in CP29 high for neoxanthin, not to the protein and the L2 N1 between two constructed a chimeric protein in which the two helices and from whereas the shown to the N1 binding site, from CP29 In a of the is The chimeric was and had and binding between CP29 and LHCII. neoxanthin was found in the high of used the that the N1 site is not present in CP29 and that the binding site in is homologous to the L2 site of LHCII. The analysis that the of L1 and L2 an of and with the to the is not from to the of the that the xanthophylls are with a was of CP29 structure have been the Lhc structure PubMed Scopus Google that Lhc proteins are of for the of Lhc protein structure and that neoxanthin the L2 site, whereas in that a one of neoxanthin and one of the to site the role by individual xanthophylls in the two to the to the influence of xanthophylls in the L1 and L2 the of and and a of with not into the of Chls to the the Chl of the was The a of for the of in site L1 to the of the whereas the xanthophyll for in site For violaxanthin the were and whereas neoxanthin in site L2 for an the of and that with the of the The the of in L1 site the in the from which xanthophyll is present in site The that site L1 is the in Lhc protein stabilization, as in Bassi R. J. 2001; PubMed Scopus Google and in site to the to the Lutein in the L2 site the whereas the of site by violaxanthin or, by neoxanthin the of excitation energy transfer from individual binding sites been by the and excitation in of the of individual The of which for the composition, to from is that were with the of and which a from Chl the was the used for the analysis been that binding to one the of the two sites into a of the energy of the xanthophylls R. Muller M.G. Bassi R. Holzwarth A.R. Biophys. J. 2001; 80: 901-915Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). that the xanthophyll in the two sites is In two for the were found for and violaxanthin, to the in the L1 and L2 In a neoxanthin was for the of and excitation with its binding to the L2 that in the neoxanthin, violaxanthin and in the L1 site, is to the two site L1 L2 the of the in the L1 site whereas in the site violaxanthin The found for the was to the analysis of excitation thus between the transfer from in L1 and is to the of II with the that and to xanthophyll binding sites L1 and that with high to in site its in Chl a is to is in the L2 from the L1 site, and the is for violaxanthin and neoxanthin from the L2 violaxanthin and high from the L1 site, the in the transfer from the L2 site is its by a of whereas its by a of that the of the two xanthophylls in the L2 site is The of zeaxanthin is its energy transfer from the L1 site is whereas from the L2 site is that the energy transfer from a site is from the site In the L2 site to in the energy transfer as with the L1 the of excitation energy transfer to Chl is by the xanthophyll species bound in the The transfer from the L2 site, to xanthophyll species, that in site the between is not for excitation energy been that of the transfer from to Chl the in the a between and is for energy transfer (3Gradinaru C.C. van Stokkum I.H.M. Pascal A.A. van Grondelle R. Van Amerongen H. J. Phys. Chem. B. 2000; 104: 9330-9342Crossref Scopus (181) Google Scholar, 4Croce R. Muller M.G. Bassi R. Holzwarth A.R. Biophys. J. 2001; 80: 901-915Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 6Croce R. Müller M.G. Caffarri S. Bassi R. Holzwarth A.R. Biophys. J. 2003; 84: 2517-2532Abstract Full Text Full Text PDF PubMed Scopus (47) Google and in influence the the that the xanthophylls bound to the L2 site that protein domain its folding in the presence of the that the L2 site Bassi R. J. 2001; PubMed Scopus Google Scholar). is thus to that for the site, which is by neoxanthin and violaxanthin, a the the transfer The in CP29 is thus as with the of L1 and L2 sites high of excitation energy transfer to Chl a R. Muller M.G. Bassi R. Holzwarth A.R. Biophys. J. 2001; 80: 901-915Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). the L2 site a function in the two In the of a and binding site in the between and was to as a for in the Rutherford A.W. Bassi R. J. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, P. Bassi R. PubMed Scopus Google Scholar). binding sites have been found in P. J. PubMed Scopus Google Scholar). the of the with an the violaxanthin to and zeaxanthin to the site R. Bassi R. J. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The high which is for thus the energy transfer from In the of zeaxanthin the and the structure for the function of excitation energy that with two xanthophyll species are to the one xanthophyll species, and are the to been that in site L2 is with the xanthophylls and in not transfer energy one xanthophyll in the L1 The thus that the L2 site in CP29 an role in photoprotection that role is by the site In CP29 from for which been shown that L1 is the site in Bassi R. J. 2001; PubMed Scopus Google Scholar). Putative in the of the binding sequences for the carotenoids in the L1 and L2 sites have been studied by between the primary of Lhc shown in the sequences by one hydrophobic residues in between that Lhc proteins bind was that sequences are involved in the xanthophyll binding by an between the in the stretch and the S. The Scholar). of the binding sequences not did not did not the of binding sites for the xanthophyll The in Chl for the mutant are to in the domain and of the Chl in site, as by the of and R. R. Proc. Natl. Acad. Sci. S. A. PubMed Scopus Google by not and the heat The two which the binding sequences for xanthophylls in the L2 site and the that by xanthophyll binding is not by of violaxanthin and neoxanthin by an of was, that the the a role in determining the selectivity of the L2 The binding of chimeric the that sequences have little site the presence of the CP29 in the between and in the L2 site, as is the in R. S. Bassi R. J. Chem. Full Text Full Text PDF PubMed Scopus Google violaxanthin and neoxanthin, as in CP29. the of a for chimeric in the of the for the of the binding sites for individual In in xanthophyll was by deleting the domain to by of the and the that the for the selectivity of the L2 site to in between two namely a from to in the been that the L2 as in CP29 R. Bassi R. Biochemistry. 2002; Scopus Google that the to in CP29 and and not in LHCII. The three sequences are in The is the in LHCII. in CP29 and In the structure of is from the in The presence in of a the between the binding of violaxanthin and neoxanthin with respect to to the in the between and violaxanthin and neoxanthin; is in its energy the is to the whereas the of violaxanthin and neoxanthin are a role in the selectivity of the binding an in site higher and the high as is to the L2 site the is in the of violaxanthin and with is the that the is the and in site L2 is in the as the binding sequences the of the protein involved in the selectivity of the sites for the is that the of the binding to have been for the light-harvesting the of the which the binding for L1 site, not influence the of the The for the binding is to found in hydrophobic in the of the the in a to that in A.A. J. PubMed Scopus Google Scholar). residues present in and helices involved in The of residues is not in of the Lhc in the from analysis the Chl binding residues in CP29 and have shown that the between Chls and carotenoids a role in the xanthophylls In the binding of neoxanthin in the N1 site of is by with Chls R. R. J. Bassi R. PubMed Scopus (105) Google Scholar). is with CP29 the N1 the number of Chls in the domain in between helices and is in The in between and of CP29 is with respect to the domain in to Chl and binding in CP29 and the chimeric CP29 is a of the Lhc which is in higher plants and and is involved in light harvesting and from of the and of the proteins to the of the of individual in that light the xanthophyll of site L2 is into zeaxanthin R. Bassi R. J. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google thus a to quenching, whereas the binding of neoxanthin in site N1 the Bassi R. Biochemistry. 2001; 40: PubMed Scopus Google Scholar, Bassi R. Biochemistry. 2002; 41: PubMed Scopus Google Scholar). The of site N1 in CP29 antenna for to Caffarri of for