A Novel S100 Target Conformation Is Revealed by the Solution Structure of the Ca2+-S100B-TRTK-12 Complex

The Alzheimer-linked neural protein S100B is a signaling molecule shown to control the assembly of intermediate filament proteins in a calcium-sensitive manner. Upon binding calcium, a conformational change occurs in S100B exposing a hydrophobic surface for target protein interactions. The synthetic peptide TRTK-12 (TRTKIDWNKILS), derived from random bacteriophage library screening, bears sequence similarity to several intermediate filament proteins and has the highest calcium-dependent affinity of any target molecule for S100B to date (K d <1 μm). In this work, the three-dimensional structure of the Ca2+-S100B-TRTK-12 complex has been determined by NMR spectroscopy. The structure reveals an extended, contiguous hydrophobic surface is formed on Ca2+-S100B for target interaction. The TRTK-12 peptide adopts a coiled structure that fits into a portion of this surface, anchored at Trp7, and interacts with multiple hydrophobic contacts in helices III and IV of Ca2+-S100B. This interaction is strikingly different from the α-helical structures found for other S100 target peptides. By using the TRTK-12 interaction as a guide, in combination with other available S100 target structures, a recognition site on helix I is identified that may act in concert with the TRTK-12-binding site from helices III and IV. This would provide a larger, more complex site to interact with full-length target proteins and would account for the promiscuity observed for S100B target protein interactions. The Alzheimer-linked neural protein S100B is a signaling molecule shown to control the assembly of intermediate filament proteins in a calcium-sensitive manner. Upon binding calcium, a conformational change occurs in S100B exposing a hydrophobic surface for target protein interactions. The synthetic peptide TRTK-12 (TRTKIDWNKILS), derived from random bacteriophage library screening, bears sequence similarity to several intermediate filament proteins and has the highest calcium-dependent affinity of any target molecule for S100B to date (K d <1 μm). In this work, the three-dimensional structure of the Ca2+-S100B-TRTK-12 complex has been determined by NMR spectroscopy. The structure reveals an extended, contiguous hydrophobic surface is formed on Ca2+-S100B for target interaction. The TRTK-12 peptide adopts a coiled structure that fits into a portion of this surface, anchored at Trp7, and interacts with multiple hydrophobic contacts in helices III and IV of Ca2+-S100B. This interaction is strikingly different from the α-helical structures found for other S100 target peptides. By using the TRTK-12 interaction as a guide, in combination with other available S100 target structures, a recognition site on helix I is identified that may act in concert with the TRTK-12-binding site from helices III and IV. This would provide a larger, more complex site to interact with full-length target proteins and would account for the promiscuity observed for S100B target protein interactions. The S100 proteins are low molecular weight (10–12 kDa) members of the EF-hand family of calcium-binding proteins. Many of these proteins, including several of the S100s, the muscle contractile protein troponin C, and the ubiquitous protein calmodulin act as signaling molecules by converting an influx of cellular calcium into a biological response. Calcium binding to these EF-hand proteins triggers a conformational change and allows the protein to interact with an appropriate target molecule. The S100 proteins are unique among this family because, unlike troponin C or calmodulin, they exist in solution as homo- or heterodimers. Each S100 monomer contains two helix-loop-helix calcium-binding motifs as follows: a basic N-terminal pseudo EF-hand comprising 14 residues (site I), and a canonical and acidic C-terminal EF-hand of 12 residues (site II). A central linker region joining the two EF-hands along with the extreme N and C termini of these proteins exhibit the most sequence divergence among family members and are therefore believed to provide specificity for target protein interactions. This feature, along with the dimeric state of the S100 proteins, likely indicates these calcium-signaling proteins have the distinctive ability to interact with more than one target molecule at a time. S100B, 1The abbreviations used are: S100B, dimeric S100β; GFAP, glial fibrillary acidic protein; TRTK-12, acetyl-TRTKIDWNKILS-NH2; NOE, nuclear Overhauser effect; MOPS, 4-morpholinepropanesulfonic acid a homodimer of 91-residue S100β monomers, is found primarily in glial cells and has been implicated in neurological diseases including Alzheimer's disease and Down's syndrome (1Marshak D.R. Pesce S.A. Stanley L.C. Griffin W.S.T. Neurobiol. Aging. 1991; 13: 1-7Google Scholar, 2Van Eldik L.J. Griffin W.S.T. Biochim. Biophys. Acta. 1994; 1223: 398-403Google Scholar, 3Griffin W.S.T. Stanley L.C. Ling C. White L. MacLeod V. Perrot L.J. White C.L. Araoz C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7611-7615Google Scholar). More than 20 calcium-sensitive in vitro binding partners have been identified for S100B (4Donato R. Biochim. Biophys. Acta. 1999; 1450: 191-231Google Scholar) including several cellular architecture proteins such as tubulin (5Donato R. J. Biol. Chem. 1988; 263: 106-110Google Scholar) and GFAP (6Bianchi R. Garbuglia M. Verzini M. Giambanco I. Ivanenkov V.V. Dimlich R.V.W. Jamieson Jr., G.A. Donato R. Biochim. Biophys. Acta. 1996; 1313: 258-267Google Scholar), where S100B can inhibit polymerization of these oligomeric molecules. Furthermore, S100B inhibits the phosphorylation of multiple kinase substrates including the Alzheimer protein tau (7Baudier J. Mochly-Rosen D. Newton A. Lee S.-H. Koshland D.E. Cole R.D. Biochemistry. 1987; 26: 2886-2893Google Scholar,8Baudier J. Cole R.D. J. Biol. Chem. 1988; 263: 5876-5883Google Scholar) and neuromodulin (GAP-43) (9Lin L.-H. Van Eldik L.J. Osheroff N. Norden J.J. Mol. Brain Res. 1994; 25: 297-304Google Scholar) through a calcium-sensitive interaction with the protein substrates. Consistent with the calcium-induced conformational change mechanism, a comparison of three-dimensional structures of apo- and calcium-bound S100B reveals that binding of calcium leads to the exposure of a hydrophobic surface(s) for protein-protein interactions (10Smith S.P. Shaw G.S. Structure. 1998; 6: 211-222Google Scholar). The 12-residue peptide TRTK-12, derived from random bacteriophage library experiments, has been used in previous studies as a model for S100B-target protein interactions. Experiments utilizing this peptide have indicated that peptides containing the consensus sequence (R/K)(L/I)(XWXXIL) bind specifically to S100B in a calcium-sensitive manner (11Ivanenkov V.V. Jamieson Jr., G.A. Gruenstein E. Dimlich R.V.W. J. Biol. Chem. 1995; 270: 14651-14658Google Scholar). Furthermore, this consensus sequence is conservatively found in the cytoskeletal proteins tubulin, desmin, vimentin, and GFAP, shown previously to interact with Ca2+-S100B (12McClintock K.A. Shaw G.S. Protein Sci. 2000; 9: 2043-2046Google Scholar). The TRTK-12 peptide competes with S100B target proteins including CapZ-α and GFAP for Ca2+-S100B binding (6Bianchi R. Garbuglia M. Verzini M. Giambanco I. Ivanenkov V.V. Dimlich R.V.W. Jamieson Jr., G.A. Donato R. Biochim. Biophys. Acta. 1996; 1313: 258-267Google Scholar, 11Ivanenkov V.V. Jamieson Jr., G.A. Gruenstein E. Dimlich R.V.W. J. Biol. Chem. 1995; 270: 14651-14658Google Scholar) and has the highest affinity (K d∼260 nm) (13McClintock K.A. Van Eldik L.J. Shaw G.S. Biochemistry. 2002; 41: 5421-5428Google Scholar) of any known S100B target. Fluorescence studies (13McClintock K.A. Van Eldik L.J. Shaw G.S. Biochemistry. 2002; 41: 5421-5428Google Scholar) have shown that Trp7 of TRTK-12 is a key residue for the calcium-sensitive interaction with S100B becoming buried at the protein-peptide interface. In addition, deletion of residues 85–91 from S100B leads to a >2000-fold decrease in affinity for TRTK-12 indicating the C-terminal helix is an important site of interaction. To date, only three structures are available for S100-target peptide complexes. In each case the target peptide adopts a 2.5 turn α-helical structure that interacts via two distinct modes with the S100 protein. The structures of human S100A10 and S100A11 in complex with peptides from the binding regions of annexin II and I, respectively, are nearly identical (14Rety S. Sopkova J. Renouard M. Osterloh D. Gerke V. Tabaries S. Russo- Marie F. Lewit-Bentley A. Nat. Struct. Biol. 1999; 6: 89-95Google Scholar, 15Rety S. Osterloh D. Arie J.-P. Tabaries S. Seeman J. Russo-Marie F. Gerke V. Lewit-Bentley A. Structure. 2000; 8: 175-184Google Scholar) with the annexin peptides bridging the two S100 monomers through contacts in the linker region and C terminus of one monomer and the N terminus of the other monomer. Surprisingly, this similarity of interaction occurs despite little sequence similarity between the annexin peptides. In contrast, the interaction of rat S100B with a 23-residue peptide from the tumor suppressor protein p53 shows each S100β monomer binds to a single peptide through interactions with helix III and a portion of helix IV (16Rustandi R.R. Baldisseri D.M. Weber D.J. Nat. Struct. Biol. 2000; 7: 570-574Google Scholar). This orients the α-helical p53 peptide about 90° from that found for the annexin peptides with respect to their S100 partners. These structural variations of the S100A10, S100A11, and S100B complexes indicate that recognition differences in the protein and the target must exist for S100 proteins. In an effort to clarify these interactions, we present the three-dimensional solution structure of Ca2+-S100B in complex with the TRTK-12 peptide. The amino acid sequence of TRTK-12 does not correspond to a natural sequence for any known S100B target. However, its unusually high affinity for S100B may indicate that it contains structural determinants that remain to be uncovered for recognition of an S100 binding partner. The binding interaction of TRTK-12 is unexpected with the peptide adopting an extended and reversed orientation compared with the p53 interaction. Furthermore, there is no interaction with helix I of S100B as found in the S100A11/S100A10 annexin structures thus providing a novel third mode of recognition for an S100-target protein complex. Human S100B protein was expressed inEscherichia coli (strain N99) and purified as described previously (17Smith S.P. Barber K.R. Dunn S.D. Shaw G.S. Biochemistry. 1996; 35: 8805-8814Google Scholar). Uniformly 15N-labeled or15N/13C-labeled S100B was prepared using M9 minimal media containing 1 g/liter 99% 15NH4Cl or 1 g/liter 99% 15NH4Cl and 2 g/liter [13C]glucose. Uniformly2H/15N-labeled S100B was prepared in M9 minimal media containing 1 g/liter 99% 15NH4Cl and 99% D2O. Unlabeled TRTK-12 peptide (Ac-TRTKIDWNKILS-NH2) and [13C]isoleucine/[13C]acetyl-labeled peptide (*Ac-TRTK*IDWNK*ILS-NH2) were synthesized by the Queen's Peptide Synthesis Lab (Queen's University, Kingston, Canada). Purity was confirmed using reversed phase-high pressure liquid chromatography and mass spectrometry. Typically, NMR samples contained 1 mm S100β monomer, 1.2 mm TRTK-12 peptide, 35 mm KCl, 4 mm CaCl2, and 5 mm dithiothreitol in 90% H2O, 10% D2O (v/v), pH 7.05. For a single sample, a mixed13C/12C S100B dimer was prepared by incubating 20 mg each of 13C-labeled S100B and unlabeled S100B (total S100B concentration) in D2O at 37 °C for 150 h. Evolution of the mixed dimer over time was monitored by mass spectrometry. The final concentration of13C/12C S100B dimer in the NMR sample was ∼1.5 mm, and the sample was prepared in 100% D2O. Experimental conditions were otherwise identical to those reported above. NMR experiments were performed at 35 °C on Varian 500-, 600-, and 800-MHz spectrometers with pulsed field gradient probes. Backbone resonances for Ca2+-S100B in the complex were sequentially assigned using HNCACB (18Wittekind M. Mueller L. J. Magn. Reson. Ser. B. 1993; 101: 171-180Google Scholar), CBCA(CO)NH (19Grzesiek S. Bax A. J. Am. Chem. Soc. 1992; 114: 6291-6293Scopus (919) Google Scholar), and HNCO (20Kay L.E. Xu G.Y. Yamazaki T. J. Magn. Reson. Ser. A. 1994; 109: 129-133Google Scholar) experiments. Side chain assignments were made using C(CO)NH (21Grzesiek S. J. Bax A. J. Reson. 1993; 101: Scholar), (21Grzesiek S. J. Bax A. J. Reson. 1993; 101: Scholar), and L.E. Xu D.R. J. Magn. Reson. Ser. B. 1993; 101: Scholar) experiments. were assigned using and experiments T. L.E. J. Am. Chem. Soc. 1993; Scholar). TRTK-12 1The abbreviations used are: S100B, dimeric S100β; GFAP, glial fibrillary acidic protein; TRTK-12, acetyl-TRTKIDWNKILS-NH2; NOE, nuclear Overhauser effect; MOPS, 4-morpholinepropanesulfonic assignments were determined from and experiments with and respectively, using S100B and unlabeled Side chain resonances were from a natural and 1The abbreviations used are: S100B, dimeric S100β; GFAP, glial fibrillary acidic protein; TRTK-12, acetyl-TRTKIDWNKILS-NH2; NOE, nuclear Overhauser effect; MOPS, 4-morpholinepropanesulfonic assignments for the two residues and were confirmed using specifically were and on a using F. S. J. Bax A. J. 1995; 263: Scholar), and and R. J. Magn. Reson. 1991; Scholar) in S100B were determined using L.E. J. 1994; 150 and three-dimensional D.R. Yamazaki T. L.E. J. Reson. Ser. B. 1994; experiments. derived from were on and three-dimensional was to or on In where was not the of was and for Ca2+-S100B and the TRTK-12 peptide were where than of the region of the on the F. Bax A. J. 1999; 13: Scholar). of and were used for and were confirmed derived from an Bax A. J. Am. Chem. Soc. 1993; Scholar). of were on the of regions in between the two S100β monomers were assigned using a mixed13C/12C S100B dimer and a M. C. L.E. 1994; Scholar) only between a and a These were in the as from three-dimensional structure of the TRTK-12 peptide was determined using a and S100B protein. for the peptide were using the as for Ca2+-S100B. between S100B and TRTK-12 were identified using the M. C. L.E. 1994; Scholar) described S100B protein and unlabeled TRTK-12 as as a three-dimensional using 15N-labeled S100B and specifically structures were using the and in the and NMR The two of S100B were to be identical using the and a of The was for the two molecules of Experiments were performed at °C on a For the TRTK-12, and Ca2+-S100B-TRTK-12 from three nm) in a were and the was Protein samples were prepared in mm KCl, mm MOPS, pH 1 mm 1 mm to final protein of for S100β and for TRTK-12 and S100β; TRTK-12 for the complex. of the TRTK-12 peptide has been monitored previously by NMR and and found to have d (13McClintock K.A. Van Eldik L.J. Shaw G.S. Biochemistry. 2002; 41: 5421-5428Google Scholar). In this work, of the complex was using a 15N-labeled S100B sample and the change in resonances as a of TRTK-12 peptide. was at a of indicating that two TRTK-12 peptides bind to each S100B dimer protein. The 1The abbreviations used are: S100B, dimeric S100β; GFAP, glial fibrillary acidic protein; TRTK-12, acetyl-TRTKIDWNKILS-NH2; NOE, nuclear Overhauser effect; MOPS, 4-morpholinepropanesulfonic of this complex a single of resonances in the NMR for most residues indicating the of the Ca2+-S100B-TRTK-12 complex dimer is were for residues 1The abbreviations used are: S100B, dimeric S100β; GFAP, glial fibrillary acidic protein; TRTK-12, acetyl-TRTKIDWNKILS-NH2; NOE, nuclear Overhauser effect; MOPS, 4-morpholinepropanesulfonic and have from the of and N-terminal S100B as observed previously for apo- and Ca2+-S100B S.P. Barber K.R. Shaw G.S. Protein Sci. 6: Scholar). residues in the N-terminal calcium-binding have and or and in the 1The abbreviations used are: S100B, dimeric S100β; GFAP, glial fibrillary acidic protein; TRTK-12, acetyl-TRTKIDWNKILS-NH2; NOE, nuclear Overhauser effect; MOPS, 4-morpholinepropanesulfonic likely to with the a 1The abbreviations used are: S100B, dimeric S100β; GFAP, glial fibrillary acidic protein; TRTK-12, acetyl-TRTKIDWNKILS-NH2; NOE, nuclear Overhauser effect; MOPS, 4-morpholinepropanesulfonic of calcium binding to the C-terminal calcium A comparison of this with that for Ca2+-S100B indicated that several residues including and TRTK-12 To the interactions between TRTK-12 and human the solution structure of the complex kDa) was determined using in combination with and This a family of structures on the of the helices in the dimer The TRTK-12 peptide was between residues 4 and 12 a structure that on observed in the rat structure (16Rustandi R.R. Baldisseri D.M. Weber D.J. Nat. Struct. Biol. 2000; 7: 570-574Google Scholar), each S100B monomer binds one TRTK-12 and structure of is for the complex for the dimer and the from are the S.D. for the low from are the S.D. for the low from in was used to the of residues were in most in other and in was using the and was not in any of structure to the structure and in helices in S100B and residues of of is for the complex for the dimer and the are the S.D. for the low J. 1993; 26: Scholar) was used to the of residues were in most in other and in was using the and was not in any of structure and in helices in S100B and residues of in a the structure of human Ca2+-S100B in the TRTK-12 complex structural of the calcium-bound S.P. Shaw G.S. J. Scholar). The dimer is by interactions between helices I and and helices IV and an helix Each S100β monomer of an N-terminal pseudo EF-hand (site comprising helix I calcium binding I and helix II and a C-terminal canonical EF-hand (site comprising helix III calcium binding II and helix IV Calcium binding I and II are through a and The of helices I and II in site I is to that of Ca2+-S100B and indicating that TRTK-12 binding to Ca2+-S100B in little conformational change to this In contrast, are identified in site The helix is by more than 90° than found in Furthermore, the of helices shows distinct differences from human Ca2+-S100B more to the found in the I complex and more compared with the or II complexes. These that the of helices III and IV may be a for target previous studies K.R. K.A. Jamieson Jr., G.A. Dimlich Shaw G.S. J. Biol. Chem. 1999; Scholar) have shown that the TRTK-12 peptide is in the of Ca2+-S100B. Consistent with the of TRTK-12 an at of a random coiled Upon TRTK-12 a coiled that The structure of TRTK-12 was by using a peptide at and and the N-terminal thus providing the peptide The coiled structure of TRTK-12 was by between the of residues and peptide were by an of α-helical and for and not with α-helical These are by of Ca2+-S100B that at and of an α-helical protein. Upon of 1 of TRTK-12 S100β monomer little in this occurs indicating the TRTK-12 peptide does not an α-helical Furthermore, the for the complex nearly identical to that of indicating little change in interactions The between Ca2+-S100B and TRTK-12 is by several residues in helix III and and helix IV and In 13C-labeled and interactions to residues in helix IV and and in helix Furthermore, residues and in the C terminus of TRTK-12 have multiple contacts in helix and 1The abbreviations used are: S100B, dimeric S100β; GFAP, glial fibrillary acidic protein; TRTK-12, acetyl-TRTKIDWNKILS-NH2; NOE, nuclear Overhauser effect; MOPS, 4-morpholinepropanesulfonic and are in to in helix IV. These interactions the N terminus of the TRTK-12 peptide helix IV and the C terminus helix III in a peptide orientation to that observed for the complex. of the N-terminal in TRTK-12 was no from this were observed indicating the extreme N terminus of TRTK-12 is to and does not interact with residues in helix I as the annexin peptides in the S100A10 and S100A11 A hydrophobic on Ca2+-S100B where TRTK-12 is This is with previous that calcium binding to human S100B in exposure of a hydrophobic surface (10Smith S.P. Shaw G.S. Structure. 1998; 6: 211-222Google Scholar). Trp7 of the peptide is the residue in a hydrophobic including residues and of This in the observed and an in for Trp7 protein binding K.R. K.A. Jamieson Jr., G.A. Dimlich Shaw G.S. J. Biol. Chem. 1999; Scholar). of the surface between Ca2+-S100B and in complex with TRTK-12 shows the of and decrease their surface exposure by and respectively, TRTK-12 residues Trp7 and of TRTK-12 have more than and respectively, of their surface buried in the complex. studies of the interaction have indicated that deletion of the C-terminal residues in S100B in a decrease in TRTK-12 binding affinity (13McClintock K.A. Van Eldik L.J. Shaw G.S. Biochemistry. 2002; 41: 5421-5428Google Scholar). This was to a in α-helical structure in the S100B protein. this would the of residues and in a surface the TRTK-12-binding site on Ca2+-S100B. is indicated in and is shown for Ca2+-S100B. of the two TRTK-12 molecules on of Ca2+-S100B. The TRTK-12 peptide fits into the hydrophobic by helices III and IV of each monomer. that on of the TRTK-12-binding site a region of the from TRTK-12 interactions with S100B residues in helix III and and IV and of the structure of Ca2+-S100B-TRTK-12 and comparison to the structures of I, and allows a to be made between target recognition by these S100 proteins. Furthermore, the TRTK-12 sequence was identified from random bacteriophage peptide and therefore may unique binding in comparison to the natural for the annexin and p53 peptides. In the the TRTK-12 peptide in a hydrophobic formed between helices III and IV the interaction of the hydrophobic residues Trp7, and with residues in helices of the interaction is hydrophobic in that the interaction is at (13McClintock K.A. Van Eldik L.J. Shaw G.S. Biochemistry. 2002; 41: 5421-5428Google Scholar). The interaction is distinct from that of the rat peptide (16Rustandi R.R. Baldisseri D.M. Weber D.J. Nat. Struct. Biol. 2000; 7: 570-574Google Scholar), likely to the sequence differences of the peptides. The target sequence for p53 bears little similarity to TRTK-12 containing a C terminus that orients the acidic N terminus of helix III In contrast, the residues in TRTK-12 are more its sequence and most are through interactions. between the target peptide molecules The residues for the p53 peptide are and only one residue as are and Trp7 in The of these two residues in each peptide is their with respect to In the in an region to in the chain of is that of in to a different The of these two interactions with the in the peptides likely in the of orientation of TRTK-12 to p53 binding to The regions of interaction of TRTK-12 and p53 from that of annexin I and II with S100A11 and S100A10, of the annexin peptides have interactions with several residues in the N-terminal helix I of these S100 proteins of the and interactions reveals three key residues in the annexin peptides and that have identical interactions with the N-terminal residues and in S100A10 and in This in the target peptide, where two are by three including a central acidic is not found in the TRTK-12 or p53 peptides. Furthermore, the chain of in S100A10 in has to the and in annexin II and in In S100B the of is by a its chain this is among the the S100 protein This sequence divergence in the S100B and proteins and in the peptide is likely for the different target peptide recognition between the proteins. Consistent with previous experiments have shown that TRTK-12 shows no interaction with S100A11 K.R. K.A. Jamieson Jr., G.A. Dimlich Shaw G.S. J. Biol. Chem. 1999; Scholar) monitored by The interaction of Trp7 in TRTK-12 likely to the binding for any S100B target to is that of in the p53 peptide with a residue its affinity for rat S100B by about R.R. Baldisseri D.M. Weber D.J. Biochemistry. 1998; Scholar). In addition, important exist between the of Trp7 and the orientation of the two target peptides is different with respect to the protein helices in each complex. A comparison of the Trp7 of TRTK-12 reveals chain interactions with and in a of the NMR The interactions, and exist for S100A11 this the differences of the peptide for TRTK-12 and annexin I in a hydrophobic surface for each protein that in the of the peptide 5 A and This may the target peptides used are or that the interaction can be through a region of interaction. a it has been J. C. R. Structure. 2002; Scholar) that S100 proteins may bind full-length using more than one The similarity of the for Trp7 and would provide a unique bridging residue to a larger, contiguous binding region that an α-helical annexin utilizing the residues found in annexin I and and an extended TRTK-12 interaction on hydrophobic interactions anchored by Trp7 is that the structure of the kinase kinase peptide complex these where the N-terminal portion of the peptide an α-helical structure and the C-terminal region is more extended M. N. M. M. J. Mol. Biol. Scholar), on the helix in to important protein in calmodulin, this interaction site would provide a for the promiscuity of S100B target interactions, including cellular architecture proteins such as tubulin, vimentin, desmin, GFAP, and the TRTK-12 consensus (12McClintock K.A. Shaw G.S. Protein Sci. 2000; 9: 2043-2046Google Scholar). to be the natural for the intermediate filament proteins interact in a manner as TRTK-12 or via the α-helical mode found for annexin or p53 peptides. assignments for S100B and TRTK-12 in the complex have been in the Barber for and of for and of for and The for experiments. for and NMR spectrometers was made by from the for the of and the of the of and from R. and of

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