The Crystal Structure of a Novel Unmethylated Form of C-phycocyanin, a Possible Connector Between Cores and Rods in Phycobilisomes

Artigo Acesso aberto Revisado por pares

The Crystal Structure of a Novel Unmethylated Form of C-phycocyanin, a Possible Connector Between Cores and Rods in Phycobilisomes

2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês

10.1074/jbc.m302838200

ISSN

1083-351X

Autores

Noam Adir, Natalia Lerner,

Tópico(s)

Algal biology and biofuel production

Resumo

A novel fraction of c-phycocyanin from the thermophilic cyanobacterium Thermosynechcoccus vulcanus, with an absorption maxima blue-shifted to 612 nm (PC612), has been purified from allophycocyanin and crystallized. The crystals belong to the P63 space group with cell dimensions of 153 Å × 153 Å × 59 Å with a single (αβ) monomer in the asymmetric unit, resulting in a solvent content of 65%, and diffract to 2.7 Å. The PC612 crystal structure has been determined by molecular replacement and refined to a crystallographic R-factor of 20.9% (R free = 27.8%). The crystal packing in this form shows that the PC612 form of phycocyanin does not associate into hexamers and that its association with adjacent trimers in the unit cell is very different from that found in a previously determined structure of the normal form of T. vulcanus phycocyanin, which absorbs at 620 nm. Analysis of the PC612 structure shows that the α subunits, which typically form the interface between two trimers within a hexamer, have a high degree of flexibility, as indicated by elevated B-factors in portions of helices B, E, and G. Examination of calculated electron density omit maps shows that unlike all other structures of phycobiliproteins determined so far, the Asnβ72 residue is not methylated, explaining the blue-shift in its absorption spectra. On the basis of the results presented here, we suggest that this new form of trimeric phycocyanin may constitute a special minor component of the phycobilisome and may form the contact between the phycocyanin rods and the allophycocyanin core. A novel fraction of c-phycocyanin from the thermophilic cyanobacterium Thermosynechcoccus vulcanus, with an absorption maxima blue-shifted to 612 nm (PC612), has been purified from allophycocyanin and crystallized. The crystals belong to the P63 space group with cell dimensions of 153 Å × 153 Å × 59 Å with a single (αβ) monomer in the asymmetric unit, resulting in a solvent content of 65%, and diffract to 2.7 Å. The PC612 crystal structure has been determined by molecular replacement and refined to a crystallographic R-factor of 20.9% (R free = 27.8%). The crystal packing in this form shows that the PC612 form of phycocyanin does not associate into hexamers and that its association with adjacent trimers in the unit cell is very different from that found in a previously determined structure of the normal form of T. vulcanus phycocyanin, which absorbs at 620 nm. Analysis of the PC612 structure shows that the α subunits, which typically form the interface between two trimers within a hexamer, have a high degree of flexibility, as indicated by elevated B-factors in portions of helices B, E, and G. Examination of calculated electron density omit maps shows that unlike all other structures of phycobiliproteins determined so far, the Asnβ72 residue is not methylated, explaining the blue-shift in its absorption spectra. On the basis of the results presented here, we suggest that this new form of trimeric phycocyanin may constitute a special minor component of the phycobilisome and may form the contact between the phycocyanin rods and the allophycocyanin core. Cyanobacteria and red algae efficiently harvest light used for photo-induced electron transfer by a variety of pigment-protein complexes called phycocbilisomes (PB) 1The abbreviations used are: PB, phycobilisomes; APC, allophycocyanin; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PC, C-phycocyanin; PC612, phycocyanin absorbing at 612 nm; PC620, phycocyanin absorbing at 620 nm; Tv-PC, phycocyanin from T. vulcanus; HPLC, high pressure liquid chromatography; CNS, crystallography NMR software. (reviewed in Refs. 1Glazer A.N. Annu. Rev. Biophys. Biophys. Chem. 1985; 14: 47-77Crossref PubMed Scopus (461) Google Scholar, 2Glazer A.N. J. Biol. Chem. 1989; 264: 1-4Abstract Full Text PDF PubMed Google Scholar, 3Huber R. EMBO J. 1989; 8: 2125-2147Crossref PubMed Scopus (49) Google Scholar, 4MacColl R. J. Struct. Biol. 1998; 124: 311-334Crossref PubMed Scopus (529) Google Scholar, 5Anderson L.K. Toole C.M. Mol. Microbiol. 1998; 30: 467-474Crossref PubMed Scopus (65) Google Scholar). The PB is the largest type of all photosynthetic antenna pigment-protein complexes attached to photosynthetic reaction centers. The sizes of such complexes vary between species and growth conditions and can easily reach molecular masses in excess of 2 MDa. The self-association of the protein subunits has been studied and documented for a variety of species and shows different complex forms. All pigment-binding protein species show a canonical first level quaternary structure of the association of two pigment-binding subunits termed α and β into the basic (αβ) monomer. The monomers further assemble into higher organizational levels of (αβ)3 trimers and (αβ)6 hexamers. The trimers (and hexamers) are round disks with dimensions of about 110 × 30 Å (or 60 Å for the hexamers). The hexamer encloses a large internal cavity with triangular shaped openings on both sides. The most prevalent PB forms, found in many cyanobacteria, are made up of a core assembly of three hexamers of allophycocyanin (APC) that are arranged with their ring planes perpendicular to the membrane surface directly above Photosystem II complexes (4MacColl R. J. Struct. Biol. 1998; 124: 311-334Crossref PubMed Scopus (529) Google Scholar). On this core are arranged six rod-like structures, made up of phycocyanin (PC) hexamers. In some species, and under certain environmental conditions, additional phycoerythrin (or other phycobiliprotein variants) hexamers attach at the terminal ends of the PC rods (12Duerring M. Schmidt G.B. Huber R. J. Mol. Biol. 1991; 217: 577-592Crossref PubMed Scopus (191) Google Scholar). The planes of the rings that make up the hexamers in the rods are perpendicular to both the APC core and to the membrane, so that in essence the APC hexamer disks bind the PC rods by their outer circumference. In addition to the pigment-binding pycobiliproteins, a number of linker proteins have been found associated with PB components (6Tandeau de Marsac N. Cohen-Bazire G. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 1635-1639Crossref PubMed Scopus (154) Google Scholar, 7Glauser M. Bryant D.A. Frank G. Wehrli E. Rusconi S.S. Sidler W. Zuber H. Eur. J. Biochem. 1992; 205: 907-915Crossref PubMed Scopus (68) Google Scholar). It has been suggested that these linker proteins occupy positions running through the internal cavities of the disks and may play roles in complex stabilization, rod-core assembly, and in inducing the directionality of energy transfer toward Photosystem II. Light energy trapped by the most prevalent pigments (phycoerythrobilin, λmax = 560 nm; phycocyanobilin, λmax = 620 nm) traverses down through the rods to the APC pigments (λmax = 652 nm) and from these pigments to the chlorophyll pigments of the reaction center (λmax = 674–680 nm) (3Huber R. EMBO J. 1989; 8: 2125-2147Crossref PubMed Scopus (49) Google Scholar, 4MacColl R. J. Struct. Biol. 1998; 124: 311-334Crossref PubMed Scopus (529) Google Scholar). A significant number of PB protein structures have been determined over the past few years (8Schirmer T. Bode W. Huber R. Sidler W. Zuber H. J. Mol. Biol. 1985; 184: 257-277Crossref PubMed Scopus (296) Google Scholar, 9Schirmer T. Huber R. Schneider M. Bode W. Miller M. Hackert M.L. J. Mol. Biol. 1986; 188: 651-676Crossref PubMed Scopus (212) Google Scholar, 10Schirmer T. Bode W. Huber R. J. Mol. Biol. 1987; 196: 677-695Crossref PubMed Scopus (284) Google Scholar, 11Duerring M. Huber R. Bode W. Ruembeli R. Zuber H. J. Mol. Biol. 1990; 211: 633-644Crossref PubMed Scopus (143) Google Scholar, 12Duerring M. Schmidt G.B. Huber R. J. Mol. Biol. 1991; 217: 577-592Crossref PubMed Scopus (191) Google Scholar, 13Ficner R. Lobeck K. Schmidt G. Huber R. J. Mol. Biol. 1992; 228: 935-950Crossref PubMed Scopus (129) Google Scholar, 14Brejc K. Ficner R. Huber R. Steinbacher S. J. Mol. Biol. 1995; 249: 424-440Crossref PubMed Scopus (196) Google Scholar, 15Chang W.R. Jiang T. Wan Z.L. Zhang J.P. Yang Z.X. Liang D.C. J. Mol. Biol. 1996; 262: 721-731Crossref PubMed Scopus (114) Google Scholar, 16Jiang T. Zhang J. Liang D. Proteins. 1999; 34: 224-231Crossref PubMed Scopus (54) Google Scholar, 17Liu J.Y. Jiang T. Zhang J.P. Liang D.C. J. Biol. Chem. 1999; 274: 16945-16952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 18Ritter S. Hiller R.G. Wrench P.M. Welte W. Diederichs K. J. Struct. Biol. 1999; 126: 86-97Crossref PubMed Scopus (75) Google Scholar, 19Reuter W. Wiegand G. Huber R. Than M.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1363-1368Crossref PubMed Scopus (126) Google Scholar, 20Stec B. Troxler R.F. Teeter M.M. Biophys. J. 1999; 76: 2912-2921Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 21Jiang T. Zhang J.P. Chang W.R. Liang D.C. Biophys. J. 2001; 81: 1171-1179Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 22Wang X.Q. Li L.N. Chang W.R. Zhang J.P. Gui L.L. Guo B.J. Liang D.C. Acta Crystallogr. D Biol. Crystallogr. 2001; 57: 784-792Crossref PubMed Scopus (73) Google Scholar, 23Adir N. Dobrovetsky Y. Lerner N. J. Mol. Biol. 2001; 313: 71-81Crossref PubMed Scopus (61) Google Scholar, 24Padyana A.K. Bhat V.B. Madyastha K.M. Rajashankar K.R. Ramakumar S. Biochem. Biophys. Res. Commun. 2001; 282: 893-898Crossref PubMed Scopus (92) Google Scholar, 25Adir N. Vainer R. Lerner N. Biochim. Biophys. Acta. 2002; 1556: 168-174Crossref PubMed Scopus (47) Google Scholar). All of these structures show a very high degree of similarity in the overall structures, the details of the pigment surroundings, the solvent interactions, and the protein residue positions in each structure, and thus have given us an excellent molecular view of the constituents of the PBs. However, a true structural description of how these different components interact is still lacking, due to the fact that the structures of each of the components has been determined separately. Because there appears to be a certain degree of correlation between the formation of hexamers and rods during the crystallization process and the actual PB rods, it has been suggested that the overall packing of the crystal unit cell can help identify energy transfer pathways between pigments at all levels, including inter-rod energy transfer (9Schirmer T. Huber R. Schneider M. Bode W. Miller M. Hackert M.L. J. Mol. Biol. 1986; 188: 651-676Crossref PubMed Scopus (212) Google Scholar, 20Stec B. Troxler R.F. Teeter M.M. Biophys. J. 1999; 76: 2912-2921Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 21Jiang T. Zhang J.P. Chang W.R. Liang D.C. Biophys. J. 2001; 81: 1171-1179Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 24Padyana A.K. Bhat V.B. Madyastha K.M. Rajashankar K.R. Ramakumar S. Biochem. Biophys. Res. Commun. 2001; 282: 893-898Crossref PubMed Scopus (92) Google Scholar). Biochemical, biophysical, and electron microscopic studies of the PB have been interpreted and promoted a number of models of the entire PB (5Anderson L.K. Toole C.M. Mol. Microbiol. 1998; 30: 467-474Crossref PubMed Scopus (65) Google Scholar, 26Yu M.H. Glazer A.N. Williams R.C. J. Biol. Chem. 1981; 256: 13130-13136Abstract Full Text PDF PubMed Google Scholar, 27Yamanaka G. Lundell D.J. Glazer A.N. J. Biol. Chem. 1982; 257: 4077-4086Abstract Full Text PDF PubMed Google Scholar, 28Anderson L.K. Eiserling F.A. J. Mol. Biol. 1986; 191: 441-451Crossref PubMed Scopus (43) Google Scholar, 29Maxson P. Sauer K. Zhou J.H. Bryant D.A. Glazer A.N. Biochim. Biophys. Acta. 1989; 977: 40-51Crossref PubMed Scopus (42) Google Scholar). These studies were performed on a variety of PB forms, from different species, with different numbers of APC core disks. There are, however, geometric problems with the schematic models of the PB structure. To assemble six PC rods around the three APC disks, interpenetration of the rods must occur, because the circumference if the core is significantly smaller than the sum of the rod diameters. Interpenetration of the circumference of one PC hexamer into the molecular circumference of an adjacent hexamer would necessitate the presence of large cavities in the circumference. Analysis of the crystal structures of many PC hexamers has not shown such cavities to exist, indicating that such interpenetration is not possible. We have recently determined the structure of the PC component of phycobilisomes from the thermophilic cyanobacteria Thermosynchococcus vulcanus (Tv-PC, formally Synechococcus vulcanus) at high resolution (1.6 Å, Protein Data Bank code 1KTP; see Ref. 25Adir N. Vainer R. Lerner N. Biochim. Biophys. Acta. 2002; 1556: 168-174Crossref PubMed Scopus (47) Google Scholar). This structure, along with a room-temperature structure at lower resolution (2.5 Å, Protein Data Bank code 1I7Y; see Ref. 23Adir N. Dobrovetsky Y. Lerner N. J. Mol. Biol. 2001; 313: 71-81Crossref PubMed Scopus (61) Google Scholar) allowed the detailed analysis of a number of important structural details pertaining to the structure, function, and stability of this protein. In the present study we have identified and determined the structure of a novel form of Tv-PC, which we believe may prove to be a functional and structural link between the rods and cores of the phycobilisomes, helping to avoid the interpenetration problem in PB assembly. Protein Isolation, Characterization, and Crystallization—Phycocyanin with an absorption maxima of 612 nm from the thermophilic cyanobacterium T. vulcanus (PC612) was isolated by the following procedure. Cyanobacterial cells were grown at 55 °C for 3–4 days with 5% CO2 in air added continuously to the growth medium. Cells were harvested by centrifugation, washed in Buffer A (20 mm HEPES, pH 8.0) and then treated with 1 mg/ml lysozyme (Sigma) at 55 °C for 60 min. The cells were then ruptured using a Yeda Pressure cell using 25 atmospheres of N2. Broken cell debris was separated from the photosynthetic membranes by centrifugation, and the membranes were then pelleted. The supernatant contained large amounts of PC absorbing at 620 nm (PC620). The membranes were then treated with Buffer A with 2 m KCl, which removed the remaining phycobiliproteins. Following removal of the membranes by centrifugation, the soluble fraction was treated with polyethylene glycol 4000 to precipitate contaminating PC620, and fractions that absorbed at 651 nm (APC) were pooled. The APC-rich fraction was further fractionated by anion-exchange chromatography (DEAE, Toyohaas), using Buffer A as the mobile phase. A salt gradient of 0–300 mm NaCl in Buffer A was used to separate between different protein fractions, with a fraction containing PC612 eluting at 130 mm NaCl and APC eluting at 200 mm NaCl. Protein fractions were analyzed for purity by SDS-PAGE, and the aggregation state was determined by size-exclusion HPLC (PL-GFC 1000 Å, Polymer Laboratories Ltd.). The final PC612 fractions were dialyzed against Buffer A and concentrated to a protein concentration of 20 mg/ml. PC612 crystals were obtained by mixing 6.7% polyethylene glycol 4000 with 6.3 mg/ml purified PC612 in the presence of 70 mm bis-Tris (pH 7.0). 6 μl hanging drops were equilibrated against a 1-ml reservoir containing 10% polyethylene glycol 4000 in 100 mm bis-Tris (pH 7.0). Data Collection and Structure Determination—PC612 crystallized in the P63 space group with cell dimensions of a = b = 153 Å = 39 Å and γ= 120o and diffracted maximally to 2.7 Å. A data set was collected using a single crystal on beamline X11 of the EMBL-Hamburg outstation at DESY using an MAR CCD detector (Table I). The data was scaled and merged using the DENZO/SCALEPACK suite (30Otwinowski Z. Sawyer L. Isaacs L. Baily S. Data Collection and Processing. SERC Daresbury Laboratory, Daresbury, UK1993: 56-62Google Scholar). The final data was 84.7% complete to 2.7 Å and was used for structure determination by molecular replacement with CNS (31Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) using the 1.6 Å Tv-PC structure (Protein Data Bank code 1KTP; see Ref. 25Adir N. Vainer R. Lerner N. Biochim. Biophys. Acta. 2002; 1556: 168-174Crossref PubMed Scopus (47) Google Scholar) as the search model (see “Results” for further details).Table IData collectionData collectionSpace groupP63Cell dimensionsa = b = 153.29 Å c = 39.05 Å, γ = 120°Matthews coefficient3.55Resolution range (Å)30-2.7ObservationsaNumbers in parenthesis correspond to the highest resolution shell (2.8-2.7Å).12540 (1036) / aNumbers in parenthesis correspond to the highest resolution shell (2.8-2.7Å).7.4 (1.2)R symaNumbers in parenthesis correspond to the highest resolution shell (2.8-2.7Å).bR sym = ΣhklΣi |Ii(hkl) — 〈Ii(hkl)〉|/ΣhklΣi Ii(hkl). (%)0.065 (0.55)CompletenessaNumbers in parenthesis correspond to the highest resolution shell (2.8-2.7Å). (%)84.7 (70.2)RedundancyaNumbers in parenthesis correspond to the highest resolution shell (2.8-2.7Å).2.4 (1.8)a Numbers in parenthesis correspond to the highest resolution shell (2.8-2.7Å).b R sym = ΣhklΣi |Ii(hkl) — 〈Ii(hkl)〉|/ΣhklΣi Ii(hkl). Open table in a new tab Refinement—The structure was refined using CNS (31Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Following simulated annealing, B-factor refinement, and water molecule addition, the structure was inspected against electron density maps calculated in CNS and examined visually using Quanta (Accelrys). Extensive use of calculated omit maps were used to manually adjust and confirm the positions of all residues and co-factors. The final model had a crystallographic R-factor of 20.9% and a R free of 27.8%. The coordinates and structure factors were deposited in the Protein Data Bank under the code 1ON7. PC 612 Isolation and Characterization—We have previously determined the structure of Tv-PC in a rhombohedral crystal space group. Protein for crystallization was isolated in trimeric form from the thylakoid membranes following detergent treatment and anion-exchange chromatography (23Adir N. Dobrovetsky Y. Lerner N. J. Mol. Biol. 2001; 313: 71-81Crossref PubMed Scopus (61) Google Scholar). The isolated protein had an absorption maxima at 620 nm, as has been reported previously (4MacColl R. J. Struct. Biol. 1998; 124: 311-334Crossref PubMed Scopus (529) Google Scholar) for PC. This form of Tv-PC resulted in two crystal structures, the first from data collected at room temperature (Protein Data Bank code 1I7Y, 2.5 Å resolution; see Ref. 23Adir N. Dobrovetsky Y. Lerner N. J. Mol. Biol. 2001; 313: 71-81Crossref PubMed Scopus (61) Google Scholar) and the second from data collected at 100K from frozen crystals (Protein Data Bank code 1KTP, 1.6 Å resolution; see Ref. 25Adir N. Vainer R. Lerner N. Biochim. Biophys. Acta. 2002; 1556: 168-174Crossref PubMed Scopus (47) Google Scholar). Following our successful determination of the Tv-PC structure at high resolution, we decided to obtain a more complete picture of this species phycobilisomes by determination of the structure of the APC component of the phycobilisome core (5Anderson L.K. Toole C.M. Mol. Microbiol. 1998; 30: 467-474Crossref PubMed Scopus (65) Google Scholar). To obtain APC, we modified the isolation procedure to separate the APC fraction from the bulk PC that is in large excess (see “Experimental Procedures” for details). In the course of isolation of APC, we obtained three fractions: i) APC, with an absorption at λ = 651 nm; ii) a fraction of PC that had an absorption blue-shifted to λ = 612 nm (hence referred to as PC612); and iii), a fraction that appeared to be a mixture of the other two fractions (Fig. 1). All three fractions appeared to have less than 5% non-pigmented protein contaminants by SDS-PAGE analysis (data not shown). Both PC and APC fractions exclusively contained (αβ)3 trimers as identified by size-exclusion HPLC (data not shown). PC 612 Crystallization and Structure Determination—Crystallization trials of all three fractions were performed using various modifications of the conditions optimized previously for Tv-PC. Very thin plate-like crystals of purified APC were obtained that were not amenable to structure determination. However, the PC612 fraction crystallized readily, and large blue hexagonal crystals were obtained. X-ray diffraction analysis, performed on an R-Axis IIc diffractometer, showed that these crystals belonged to a hexagonal space group and were unlike the previously described crystal forms of Tv-PC. We thus undertook the task of high-resolution data collection at cryogenic temperature using synchrotron radiation (the EMBL PX beam-line (X11) at the DORIS storage ring, DESY, Hamburg, Germany). The crystals did not diffract as well as the rhombohedral form (25Adir N. Vainer R. Lerner N. Biochim. Biophys. Acta. 2002; 1556: 168-174Crossref PubMed Scopus (47) Google Scholar); however, a complete data set to 2.7 Å was obtained (Table I). Analysis of the diffraction pattern showed that the crystal unit cell dimensions were about 153 × 153 × 39 Å. Following data processing, using the DENZO/SCALEPACK suite, it became apparent that reflections of the type h = 0, k = 0 had not been collected, and so the exact space group could not be determined by identification of systematic absences. However, processing of the data to higher symmetry hexagonal space groups (P622, P6122, etc.) resulted in high R sym values (> 0.12), whereas those of lower symmetry had R merge values of about 0.06. Molecular replacement (CNS, Ref. 31Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) was thus performed using the 1KTP structure in all six hexagonal space groups of type P6, P61, etc. The only space group for which a translation function solution and correct proper packing could be obtained was P63. The crystallographic R-factor for the molecular replacement solution after rigid-body refinement was 0.39. Calculation of the Matthews coefficient (32Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7927) Google Scholar) indicated the possibility of either one or two (αβ) monomers in the asymmetric unit (V m = 3.55 or 1.78, respectively). Thus at this stage, CNS was used to search for a possible second monomer in the asymmetric unit. No such solution could be obtained, and calculated electron density maps using the unrefined monomer as the source of phases showed clear density for the monomer, without additional protein. Thus the hexagonal crystal form of PC612 has a single monomer in the asymmetric unit, with two (αβ)3 trimers in the unit cell (Fig. 2). Interestingly, the PC612 form of Tv-PC is similar to the first PC structure determined that from Mastigocladus laminosus (this structure has not been deposited in the Protein Data Bank; see Ref. 10Schirmer T. Bode W. Huber R. J. Mol. Biol. 1987; 196: 677-695Crossref PubMed Scopus (284) Google Scholar). Refinement—The PC612 structure was refined using maximum likelihood simulated annealing (31Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar), followed by multiple rounds of B-factor, coordinate minimization, and manual fitting. The locations of 36 water molecules were identified, and the final refined structure (Table II) had a crystallographic R-factor of 20.9% (R free = 27.8%). All geometric constraints were within or better than the mean values as determined by the Protein Data Bank ADIT validation server. Only the highly conserved Thrβ77 residue has non-typical peptide geometry (Φ = 76.7°, Ψ = 128.3°), as has been found for this residue in all previously determined PC structures (10Schirmer T. Bode W. Huber R. J. Mol. Biol. 1987; 196: 677-695Crossref PubMed Scopus (284) Google Scholar, 23Adir N. Dobrovetsky Y. Lerner N. J. Mol. Biol. 2001; 313: 71-81Crossref PubMed Scopus (61) Google Scholar).Table IIRefinement statisticsaStatistics for data with F > 1.3σ.Resolution range (Å)30-2.7 (2.8-2.70)Reflections in work set (test set)9088 (1000)R crystbNumbers in parenthesis correspond to the highest resolution shell (2.8-2.7Å).,cR cryst,free = Σhkl∥Fobs — |Fcalc∥/Σhkl |Fobs| where R cryst and R free are calculated using the working and test reflections, respectively. The test reflections were held aside and not used during the entire refinement process. (%)20.9 (34.6)R freebNumbers in parenthesis correspond to the highest resolution shell (2.8-2.7Å).,cR cryst,free = Σhkl∥Fobs — |Fcalc∥/Σhkl |Fobs| where R cryst and R free are calculated using the working and test reflections, respectively. The test reflections were held aside and not used during the entire refinement process. (%)27.8 (32.0)RMS deviationsbond length deviation (Å)0.0085angle deviation (degrees)1.26Average B-factor (Å2)50.7α subunit59.8β subunit38.8co-factors58.5Final modelTotal number of atoms3035Protein2499Non-protein atomsCo-factor129Water36Number of amino acids332Cofactor molecules3a Statistics for data with F > 1.3σ.b Numbers in parenthesis correspond to the highest resolution shell (2.8-2.7Å).c R cryst,free = Σhkl∥Fobs — |Fcalc∥/Σhkl |Fobs| where R cryst and R free are calculated using the working and test reflections, respectively. The test reflections were held aside and not used during the entire refinement process. Open table in a new tab The PC612 structure shows the typical globin-like fold identified in the past (8Schirmer T. Bode W. Huber R. Sidler W. Zuber H. J. Mol. Biol. 1985; 184: 257-277Crossref PubMed Scopus (296) Google Scholar). The overall structure is similar to the previously determined high-resolution structure 1KTP with a root mean square deviation coordinate difference of 0.82 and 1.02 Å over all α carbon and all atoms respectively. However, a number of characteristics make the PC612 structure unique. The (αβ) monomers are organized into (αβ)3 trimers, which were also the basic unit in solution prior to crystallization. The packing of the unit cell (Fig. 2) shows that the trimers are not associated further into (αβ)6 hexamers, as was previously shown in the 1KTP structure, as well as for many other PC structures (8Schirmer T. Bode W. Huber R. Sidler W. Zuber H. J. Mol. Biol. 1985; 184: 257-277Crossref PubMed Scopus (296) Google Scholar, 9Schirmer T. Huber R. Schneider M. Bode W. Miller M. Hackert M.L. J. Mol. Biol. 1986; 188: 651-676Crossref PubMed Scopus (212) Google Scholar, 11Duerring M. Huber R. Bode W. Ruembeli R. Zuber H. J. Mol. Biol. 1990; 211: 633-644Crossref PubMed Scopus (143) Google Scholar, 12Duerring M. Schmidt G.B. Huber R. J. Mol. Biol. 1991; 217: 577-592Crossref PubMed Scopus (191) Google Scholar, 13Ficner R. Lobeck K. Schmidt G. Huber R. J. Mol. Biol. 1992; 228: 935-950Crossref PubMed Scopus (129) Google Scholar, 14Brejc K. Ficner R. Huber R. Steinbacher S. J. Mol. Biol. 1995; 249: 424-440Crossref PubMed Scopus (196) Google Scholar, 15Chang W.R. Jiang T. Wan Z.L. Zhang J.P. Yang Z.X. Liang D.C. J. Mol. Biol. 1996; 262: 721-731Crossref PubMed Scopus (114) Google Scholar, 16Jiang T. Zhang J. Liang D. Proteins. 1999; 34: 224-231Crossref PubMed Scopus (54) Google Scholar, 17Liu J.Y. Jiang T. Zhang J.P. Liang D.C. J. Biol. Chem. 1999; 274: 16945-16952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 18Ritter S. Hiller R.G. Wrench P.M. Welte W. Diederichs K. J. Struct. Biol. 1999; 126: 86-97Crossref PubMed Scopus (75) Google Scholar, 19Reuter W. Wiegand G. Huber R. Than M.E. Proc. Natl. 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The immediate result of this form of crystal packing is a high solvent content (65%) as opposed to only 42% for the 1KTP structure. One consequence of the high solvent content is the relatively high B-factors, especially those of certain stretches of the α subunit. In crystal structures made up of (αβ)6 hexamers (i.e. 1KTP), the α subunit forms most of the trimer-trimer contacts, whereas the β subunits are involved in the formation of both the (αβ) monomer-monomer and in (αβ)6 hexamer-(αβ)6 hexamer interactions. In the PC612 structure, the α subunits have few intermolecular contacts (Fig. 3), whereas the β subunits link one ano

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The Crystal Structure of a Novel Unmethylated Form of C-phycocyanin, a Possible Connector Between Cores and Rods in Phycobilisomes