Half-Gaussian-shaped pulses yield an almost ideal excitation profile, if the relatively broad dis... more Half-Gaussian-shaped pulses yield an almost ideal excitation profile, if the relatively broad dispersion component is eliminated by a simple nonselective 90" purge pulse. In contrast to Gaussian-shaped pulses, the half-Gaussian excitation scheme does not create significant amounts of antiphase components, but leads almost quantitatively to in-phase magnetization of the excited signal. This feature is especially useful in experiments where a semiselective excitation is followed by a mixing of in-phase components (e.g., in NOESY, ROESY, or TOCSY type experiments). In such a case even signals with large or numerous couplings give rise to a very efficient transfer. In addition, the magnetization vectors in the transverse plane show an almost perfect alignment after semiselective excitation with a purged half-Gaussian pulse. Thus it can be employed advantageously in all experiments where a semiselective excitation is directly followed by the I, evolution period. In such experiments the use of Gaussian pulses leads to very large first-order phase gradients, the correction of which causes severe distortions of the baseline. The alternative use of purged half-Gaussian-shaped pulses results only in very small phase gradients, comparable to those in experiments with nonselective excitation. C 1489 Academy Press. Inc.
The sequential assignment of backbone resonances is the first step in the structure determination... more The sequential assignment of backbone resonances is the first step in the structure determination of proteins by heteronuclear NMR. For larger proteins, an assignment strategy based on proton side-chain information is no longer suitable for the use in an automated procedure. Our program PASTA (Protein ASsignment by Threshold Accepting) is therefore designed to partially or fully automate the sequential assignment of proteins, based on the analysis of NMR backbone resonances plus C beta information. In order to overcome the problems caused by peak overlap and missing signals in an automated assignment process, PASTA uses threshold accepting, a combinatorial optimization strategy, which is superior to simulated annealing due to generally faster convergence and better solutions. The reliability of this algorithm is shown by reproducing the complete sequential backbone assignment of several proteins from published NMR data. The robustness of the algorithm against misassigned signals, no...
The introduction of deuterated and partially deuterated protein samples has greatly facilitated t... more The introduction of deuterated and partially deuterated protein samples has greatly facilitated the 13C assignment of larger proteins. Here we present a new version of the HC(CO)NH-TOCSY experiment, the ed-H(CCO)NH-TOCSY experiment for partially deuterated samples, introducing a multi-quantum proton evolution period. This approach removes the main relaxation source (the dipolar coupling to the directly bound 13C spin) and leads to a significant reduction of the proton and carbon relaxation rates. Thus, the indirect proton dimension can be acquired with high resolution, combined with a phase labeling of the proton resonances according to the C-C spin system topology. This editing scheme, independent of the CHn multiplicity, allows to distinguish between proton side-chain positions occurring within a narrow chemical shift range. Therefore this new experiment facilitates the assignment of the proton chemical shifts of partially deuterated samples even of high molecular weights, as demo...
International Journal of Peptide and Protein Research, 2009
NMR spectroscopy has been employed for the conformational analysis of the cyclic hexapeptide cycl... more NMR spectroscopy has been employed for the conformational analysis of the cyclic hexapeptide cyclo(-D-Pro1-Ala2-Ser3(Bzl)-Trp4-Orn5(Z)-Tyr 6-) with and without protecting groups on Ser3 and Orn5. This peptide sequence was derived from the active loop sequence of the alpha-amylase inhibitor Tendamistat (HOE 467). The aim was to investigate the role of serine in position i of a standard beta-turn on the conformation and stabilization of this turn. Based on distance and torsion constraints from 2D NMR spectroscopic measurements in DMSO-d6 solution, structure refinement was accomplished by restrained molecular dynamics (MD) simulations in vacuo and in DMSO. The analysis of both structures in solution reveals a considerable effect of the unprotected serine sidechain on the adjacent beta-turn conformation. While in the protected peptide with Ser3(Bzl) a beta II-turn is observed between Trp4 and Orn5, the deprotected compound reveals a beta I-turn in this region. The beta I-turn is stabilized by a backbone-sidechain hydrogen bond from Orn5N alpha H to Ser3O gamma. Comparisons with other NMR-derived solution structures of cyclic model peptides and in some protein structures from literature reveal a general structural motif in the stabilization of beta I-turns by serine in the i position through backbone-sidechain interactions.
The product of the nusB gene of Escherichia coli modulates the efficiency of transcription termin... more The product of the nusB gene of Escherichia coli modulates the efficiency of transcription termination at I Z M~ (N utilization) sites of various bacterial and bacteriophage i genes. Similar control mechanisms operate in eukaryotic viruses (e.g. human immunodeficiency virus). A recombinant strain of E. coli producing relatively large amounts of NusB protein (about 10% of cell protein) was constructed. The protein could be purified with high yield by anion-exchange Chromatography followed by gel-permeation chromatography. The protein is a monomer of 15.6 kDa as shown by analytical ultracentrifugation. Structural studies were performed using protein samples labelled with "N, "C and *H in various combinations.
The NusB protein of Escherichia coli is involved in the regulation of rRNA biosynthesis by transc... more The NusB protein of Escherichia coli is involved in the regulation of rRNA biosynthesis by transcriptional antitermination. In cooperation with several other proteins, it binds to a dodecamer motif designated rrn boxA on the nascent rRNA. The antitermination proteins of E.coli are recruited in the replication cycle of bacteriophage λ, where they play an important role in switching from the lysogenic to the lytic cycle. Multidimensional heteronuclear NMR experiments were performed with recombinant NusB protein labelled with 13 C, 15 N and 2 H. The three-dimensional structure of the protein was solved from 1926 NMRderived distances and 80 torsion angle restraints. The protein folds into an α/α-helical topology consisting of six helices; the arginine-rich N-terminus appears to be disordered. Complexation of the protein with an RNA dodecamer equivalent to the rrn boxA site results in chemical shift changes of numerous amide signals. The overall packing of the protein appears to be conserved, but the flexible N-terminus adopts a more rigid structure upon RNA binding, indicating that the N-terminus functions as an arginine-rich RNA-binding motif (ARM).
E. coli Par10 is a peptidyl-prolyl cis/trans isomerase (PPIase) from Escherichia coli catalyzing ... more E. coli Par10 is a peptidyl-prolyl cis/trans isomerase (PPIase) from Escherichia coli catalyzing the isomerization of Xaa-Pro bonds in oligopeptides with a broad substrate specificity. The structure of E. coli Par10 has been determined by multidimensional solution-state NMR spectroscopy based on 1207 conformational constraints (1067 NOE-derived distances, 42 vicinal coupling-constant restraints, 30 hydrogen-bond restraints, and 68 / restraints derived from the Chemical Shift Index). Simulated-annealing calculations with the program ARIA and subsequent refinement with XPLOR yielded a set of 18 convergent structures with an average backbone RMSD from mean atomic coordinates of 0.50 Å within the well-defined secondary structure elements. E. coli Par10 is the smallest known PPIase so far, with a high catalytic efficiency comparable to that of FKBPs and cyclophilins. The secondary structure of E. coli Par10 consists of four helical regions and a four-stranded antiparallel -sheet. The N terminus forms a -strand, followed by a large stretch comprising three ␣-helices. A loop region containing a short -strand separates these helices from a fourth ␣-helix. The C terminus consists of two more -strands completing the four-stranded antiparallel -sheet with strand order 2143. Interestingly, the third -strand includes a Gly-Pro cis peptide bond. The curved -strand forms a hydrophobic binding pocket together with ␣-helix 4, which also contains a number of highly conserved residues. The three-dimensional structure of Par10 closely resembles that of the human proteins hPin1 and hPar14 and the plant protein Pin1At, belonging to the same family of highly homologous proteins.
Lithium Chloride Perturbation of Cis-Trans Peptide Bond ... Equilibria: Effect on Conformational ... more Lithium Chloride Perturbation of Cis-Trans Peptide Bond ... Equilibria: Effect on Conformational Equilibria in Cyclosporin ... A and on Time-Dependent Inhibition of Cyclophilin ... James L. Kofron,? Petr KuzmiE,t Vimal &shore,? Gerd Gemmecker,t Stephen W. Fesik,t and Daniel ...
The solution structure and the dimerization behavior of the lipophilic, highly C R -methylated mo... more The solution structure and the dimerization behavior of the lipophilic, highly C R -methylated model peptide, mBrBz-Iva 1 -Val 2 -Iva 3 -(RMe)Val 4 -(RMe)Phe 5 -(RMe)Val 6 -Iva 7 -NHMe, was studied by NMR spectroscopy and molecular dynamics simulations. The conformational analysis resulted in a right-handed 3 10 /Rhelical equilibrium fast on the NMR time scale with a slight preference for the R-helical conformation. The NOESY spectrum showed intermolecular NOEs due to an aggregation of the heptapeptide. In addition, temperature-dependent diffusion measurements were performed to calculate the hydrodynamic radius. All these findings are consistent with an antiparallel side-by-side dimerization. The structure of the dimeric peptide was calculated with a simulated annealing strategy. The lipophilic dimer is held together by favorable van der Waals interactions in the sense of a bulge fitting into a groove. The flexibility of the helical conformations concerning an R/3 10 -helical equilibrium is shown in a 3 ns molecular dynamics simulation of the resulting dimeric structure. Both overall helical structures of each monomer and the antiparallel mode of dimerization are stable. However, transitions were seen of several residues from a 3 10 -helical into an R-helical conformation and vice versa. Hence, this peptide represents a good model in which two often-discussed aspects of hierarchical transmembrane protein folding are present: i r i + 3 and i r i + 4 local H-bonding interactions cause a specific molecular shape which is then recognized as attractive by other surrounding structures.
In the past decade, multidimensional NMR has developed into the premier method for the determinat... more In the past decade, multidimensional NMR has developed into the premier method for the determination of structures of biomacromolecules in solution. In addition to the commonly used structural parameters derived from dipolar and scalar coupling, the measurement of hydrogen exchange rates has recently gained increasing importance for providing additional information about the structural and dynamical features of molecules, such as solvent accessibility, complexation sites, and hydrogen bonding.1" We propose here a new NMR technique for the convenient determination of fast amide proton exchange rates in uniformly 13Cand ISN-labeled compounds.
Conformational analysis by NMR spectroscopy and restrained molecular dynamics (MD) of the 0-glyco... more Conformational analysis by NMR spectroscopy and restrained molecular dynamics (MD) of the 0-glycosylated cyclic hexapeptide cyclo(~-Prol-Phe~-Ala'-Ser~[ 0-2-deoxy-~-lactopyranosyl-a-( 1-3)]-Phe5-Phe6) (I) and the cyclic hexapeptide precursor cyclo(~-Pro'-Phe~-Ala~-Ser~-Phe~-Phe~) (11) is described. For II, an X-ray structure was obtained and compared with the structure in solution. For both compounds, the distance constraints derived from 2D NMR measurements could not be completely satisfied by a single conformation, but distance violations occurred only in the PheS region of the peptide. The specific pattern of NOE-derived distances in this part of the molecule suggested an equilibrium between two conformers containing 01and BII-type turns, respectively, with PheS at i + 2. MD simulations with time-dependent distance constraints support the assumption of a bI/BII flip in I and 11. The conformations were refined using restrained MD simulations in vacuo, in water, and in DMSO. To study the exoanomeric effect of b(1-4)-and a-glycosidic linkages on conformation, new force field parameters derived from literature data were incorporated, leading to greater flexibility and significantly populated alternate conformers around the b( 1-4)-glycosidic bond, in agreement with literature data. The a-glycosidic linkage connecting the disaccharide moiety to the peptide prefers only one conformation. Both I and I1 have similar backbone conformations and hydrogen-bonding patterns. Therefore, the 0-glycosylation does not affect the conformation or the overall shape of the peptide backbone or side chains. ~ ~~ ~~~ (1) (a) Feizi, T. Nature 1985, 314, 53-57. (b) Feizi, T.; Childs, R. A.
... Acknowledgment. Discussions with Prof. B. D. Nageswara Rao and Prof. P. Balaram, who also pro... more ... Acknowledgment. Discussions with Prof. B. D. Nageswara Rao and Prof. P. Balaram, who also provided the peptide sample, are gratefully acknowledged. We thank Profs. C. L. Khetrapal and J. Tropp for perusal of the manuscript. ...
The somatostatin analogue DOTATOC, DOTA-[Tyr(3)]octreotide, is used for in vivo diagnosis and tar... more The somatostatin analogue DOTATOC, DOTA-[Tyr(3)]octreotide, is used for in vivo diagnosis and targeted therapy of somatostatin-receptor-positive tumors. DOTATOC consists of a disulfide-bridged octapeptide, d-Phe(1)-Cys(2)-Tyr(3)-d-Trp(4)-Lys(5)-Thr(6)-Cys(7)-Thr(8)-ol, connected to the metal chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Two metal complexes, Ga(III)- and Y(III)-DOTATOC, were reported to differ significantly in somatostatin receptor affinity and tumor uptake. Our (1)H and (13)C solution NMR data and modeling studies of both compounds are in agreement with a fast conformational equilibrium of the peptide part, as previously reported for octreotide itself. However, the different coordination geometry of Ga(3+) and Y(3+) (6-fold and 8-fold, respectively, as known from model compounds) causes pronounced differences for the d-Phe(1) residue. For Y(III)-DOTATOC this leads to two conformers exchanging slowly on the NMR time scale. From various NMR measurements, they could be identified as cis-trans isomers at the amide bond between DOTA chelator and first residue (d-Phe(1)H(N)) of the peptide.
Half-Gaussian-shaped pulses yield an almost ideal excitation profile, if the relatively broad dis... more Half-Gaussian-shaped pulses yield an almost ideal excitation profile, if the relatively broad dispersion component is eliminated by a simple nonselective 90" purge pulse. In contrast to Gaussian-shaped pulses, the half-Gaussian excitation scheme does not create significant amounts of antiphase components, but leads almost quantitatively to in-phase magnetization of the excited signal. This feature is especially useful in experiments where a semiselective excitation is followed by a mixing of in-phase components (e.g., in NOESY, ROESY, or TOCSY type experiments). In such a case even signals with large or numerous couplings give rise to a very efficient transfer. In addition, the magnetization vectors in the transverse plane show an almost perfect alignment after semiselective excitation with a purged half-Gaussian pulse. Thus it can be employed advantageously in all experiments where a semiselective excitation is directly followed by the I, evolution period. In such experiments the use of Gaussian pulses leads to very large first-order phase gradients, the correction of which causes severe distortions of the baseline. The alternative use of purged half-Gaussian-shaped pulses results only in very small phase gradients, comparable to those in experiments with nonselective excitation. C 1489 Academy Press. Inc.
The sequential assignment of backbone resonances is the first step in the structure determination... more The sequential assignment of backbone resonances is the first step in the structure determination of proteins by heteronuclear NMR. For larger proteins, an assignment strategy based on proton side-chain information is no longer suitable for the use in an automated procedure. Our program PASTA (Protein ASsignment by Threshold Accepting) is therefore designed to partially or fully automate the sequential assignment of proteins, based on the analysis of NMR backbone resonances plus C beta information. In order to overcome the problems caused by peak overlap and missing signals in an automated assignment process, PASTA uses threshold accepting, a combinatorial optimization strategy, which is superior to simulated annealing due to generally faster convergence and better solutions. The reliability of this algorithm is shown by reproducing the complete sequential backbone assignment of several proteins from published NMR data. The robustness of the algorithm against misassigned signals, no...
The introduction of deuterated and partially deuterated protein samples has greatly facilitated t... more The introduction of deuterated and partially deuterated protein samples has greatly facilitated the 13C assignment of larger proteins. Here we present a new version of the HC(CO)NH-TOCSY experiment, the ed-H(CCO)NH-TOCSY experiment for partially deuterated samples, introducing a multi-quantum proton evolution period. This approach removes the main relaxation source (the dipolar coupling to the directly bound 13C spin) and leads to a significant reduction of the proton and carbon relaxation rates. Thus, the indirect proton dimension can be acquired with high resolution, combined with a phase labeling of the proton resonances according to the C-C spin system topology. This editing scheme, independent of the CHn multiplicity, allows to distinguish between proton side-chain positions occurring within a narrow chemical shift range. Therefore this new experiment facilitates the assignment of the proton chemical shifts of partially deuterated samples even of high molecular weights, as demo...
International Journal of Peptide and Protein Research, 2009
NMR spectroscopy has been employed for the conformational analysis of the cyclic hexapeptide cycl... more NMR spectroscopy has been employed for the conformational analysis of the cyclic hexapeptide cyclo(-D-Pro1-Ala2-Ser3(Bzl)-Trp4-Orn5(Z)-Tyr 6-) with and without protecting groups on Ser3 and Orn5. This peptide sequence was derived from the active loop sequence of the alpha-amylase inhibitor Tendamistat (HOE 467). The aim was to investigate the role of serine in position i of a standard beta-turn on the conformation and stabilization of this turn. Based on distance and torsion constraints from 2D NMR spectroscopic measurements in DMSO-d6 solution, structure refinement was accomplished by restrained molecular dynamics (MD) simulations in vacuo and in DMSO. The analysis of both structures in solution reveals a considerable effect of the unprotected serine sidechain on the adjacent beta-turn conformation. While in the protected peptide with Ser3(Bzl) a beta II-turn is observed between Trp4 and Orn5, the deprotected compound reveals a beta I-turn in this region. The beta I-turn is stabilized by a backbone-sidechain hydrogen bond from Orn5N alpha H to Ser3O gamma. Comparisons with other NMR-derived solution structures of cyclic model peptides and in some protein structures from literature reveal a general structural motif in the stabilization of beta I-turns by serine in the i position through backbone-sidechain interactions.
The product of the nusB gene of Escherichia coli modulates the efficiency of transcription termin... more The product of the nusB gene of Escherichia coli modulates the efficiency of transcription termination at I Z M~ (N utilization) sites of various bacterial and bacteriophage i genes. Similar control mechanisms operate in eukaryotic viruses (e.g. human immunodeficiency virus). A recombinant strain of E. coli producing relatively large amounts of NusB protein (about 10% of cell protein) was constructed. The protein could be purified with high yield by anion-exchange Chromatography followed by gel-permeation chromatography. The protein is a monomer of 15.6 kDa as shown by analytical ultracentrifugation. Structural studies were performed using protein samples labelled with "N, "C and *H in various combinations.
The NusB protein of Escherichia coli is involved in the regulation of rRNA biosynthesis by transc... more The NusB protein of Escherichia coli is involved in the regulation of rRNA biosynthesis by transcriptional antitermination. In cooperation with several other proteins, it binds to a dodecamer motif designated rrn boxA on the nascent rRNA. The antitermination proteins of E.coli are recruited in the replication cycle of bacteriophage λ, where they play an important role in switching from the lysogenic to the lytic cycle. Multidimensional heteronuclear NMR experiments were performed with recombinant NusB protein labelled with 13 C, 15 N and 2 H. The three-dimensional structure of the protein was solved from 1926 NMRderived distances and 80 torsion angle restraints. The protein folds into an α/α-helical topology consisting of six helices; the arginine-rich N-terminus appears to be disordered. Complexation of the protein with an RNA dodecamer equivalent to the rrn boxA site results in chemical shift changes of numerous amide signals. The overall packing of the protein appears to be conserved, but the flexible N-terminus adopts a more rigid structure upon RNA binding, indicating that the N-terminus functions as an arginine-rich RNA-binding motif (ARM).
E. coli Par10 is a peptidyl-prolyl cis/trans isomerase (PPIase) from Escherichia coli catalyzing ... more E. coli Par10 is a peptidyl-prolyl cis/trans isomerase (PPIase) from Escherichia coli catalyzing the isomerization of Xaa-Pro bonds in oligopeptides with a broad substrate specificity. The structure of E. coli Par10 has been determined by multidimensional solution-state NMR spectroscopy based on 1207 conformational constraints (1067 NOE-derived distances, 42 vicinal coupling-constant restraints, 30 hydrogen-bond restraints, and 68 / restraints derived from the Chemical Shift Index). Simulated-annealing calculations with the program ARIA and subsequent refinement with XPLOR yielded a set of 18 convergent structures with an average backbone RMSD from mean atomic coordinates of 0.50 Å within the well-defined secondary structure elements. E. coli Par10 is the smallest known PPIase so far, with a high catalytic efficiency comparable to that of FKBPs and cyclophilins. The secondary structure of E. coli Par10 consists of four helical regions and a four-stranded antiparallel -sheet. The N terminus forms a -strand, followed by a large stretch comprising three ␣-helices. A loop region containing a short -strand separates these helices from a fourth ␣-helix. The C terminus consists of two more -strands completing the four-stranded antiparallel -sheet with strand order 2143. Interestingly, the third -strand includes a Gly-Pro cis peptide bond. The curved -strand forms a hydrophobic binding pocket together with ␣-helix 4, which also contains a number of highly conserved residues. The three-dimensional structure of Par10 closely resembles that of the human proteins hPin1 and hPar14 and the plant protein Pin1At, belonging to the same family of highly homologous proteins.
Lithium Chloride Perturbation of Cis-Trans Peptide Bond ... Equilibria: Effect on Conformational ... more Lithium Chloride Perturbation of Cis-Trans Peptide Bond ... Equilibria: Effect on Conformational Equilibria in Cyclosporin ... A and on Time-Dependent Inhibition of Cyclophilin ... James L. Kofron,? Petr KuzmiE,t Vimal &shore,? Gerd Gemmecker,t Stephen W. Fesik,t and Daniel ...
The solution structure and the dimerization behavior of the lipophilic, highly C R -methylated mo... more The solution structure and the dimerization behavior of the lipophilic, highly C R -methylated model peptide, mBrBz-Iva 1 -Val 2 -Iva 3 -(RMe)Val 4 -(RMe)Phe 5 -(RMe)Val 6 -Iva 7 -NHMe, was studied by NMR spectroscopy and molecular dynamics simulations. The conformational analysis resulted in a right-handed 3 10 /Rhelical equilibrium fast on the NMR time scale with a slight preference for the R-helical conformation. The NOESY spectrum showed intermolecular NOEs due to an aggregation of the heptapeptide. In addition, temperature-dependent diffusion measurements were performed to calculate the hydrodynamic radius. All these findings are consistent with an antiparallel side-by-side dimerization. The structure of the dimeric peptide was calculated with a simulated annealing strategy. The lipophilic dimer is held together by favorable van der Waals interactions in the sense of a bulge fitting into a groove. The flexibility of the helical conformations concerning an R/3 10 -helical equilibrium is shown in a 3 ns molecular dynamics simulation of the resulting dimeric structure. Both overall helical structures of each monomer and the antiparallel mode of dimerization are stable. However, transitions were seen of several residues from a 3 10 -helical into an R-helical conformation and vice versa. Hence, this peptide represents a good model in which two often-discussed aspects of hierarchical transmembrane protein folding are present: i r i + 3 and i r i + 4 local H-bonding interactions cause a specific molecular shape which is then recognized as attractive by other surrounding structures.
In the past decade, multidimensional NMR has developed into the premier method for the determinat... more In the past decade, multidimensional NMR has developed into the premier method for the determination of structures of biomacromolecules in solution. In addition to the commonly used structural parameters derived from dipolar and scalar coupling, the measurement of hydrogen exchange rates has recently gained increasing importance for providing additional information about the structural and dynamical features of molecules, such as solvent accessibility, complexation sites, and hydrogen bonding.1" We propose here a new NMR technique for the convenient determination of fast amide proton exchange rates in uniformly 13Cand ISN-labeled compounds.
Conformational analysis by NMR spectroscopy and restrained molecular dynamics (MD) of the 0-glyco... more Conformational analysis by NMR spectroscopy and restrained molecular dynamics (MD) of the 0-glycosylated cyclic hexapeptide cyclo(~-Prol-Phe~-Ala'-Ser~[ 0-2-deoxy-~-lactopyranosyl-a-( 1-3)]-Phe5-Phe6) (I) and the cyclic hexapeptide precursor cyclo(~-Pro'-Phe~-Ala~-Ser~-Phe~-Phe~) (11) is described. For II, an X-ray structure was obtained and compared with the structure in solution. For both compounds, the distance constraints derived from 2D NMR measurements could not be completely satisfied by a single conformation, but distance violations occurred only in the PheS region of the peptide. The specific pattern of NOE-derived distances in this part of the molecule suggested an equilibrium between two conformers containing 01and BII-type turns, respectively, with PheS at i + 2. MD simulations with time-dependent distance constraints support the assumption of a bI/BII flip in I and 11. The conformations were refined using restrained MD simulations in vacuo, in water, and in DMSO. To study the exoanomeric effect of b(1-4)-and a-glycosidic linkages on conformation, new force field parameters derived from literature data were incorporated, leading to greater flexibility and significantly populated alternate conformers around the b( 1-4)-glycosidic bond, in agreement with literature data. The a-glycosidic linkage connecting the disaccharide moiety to the peptide prefers only one conformation. Both I and I1 have similar backbone conformations and hydrogen-bonding patterns. Therefore, the 0-glycosylation does not affect the conformation or the overall shape of the peptide backbone or side chains. ~ ~~ ~~~ (1) (a) Feizi, T. Nature 1985, 314, 53-57. (b) Feizi, T.; Childs, R. A.
... Acknowledgment. Discussions with Prof. B. D. Nageswara Rao and Prof. P. Balaram, who also pro... more ... Acknowledgment. Discussions with Prof. B. D. Nageswara Rao and Prof. P. Balaram, who also provided the peptide sample, are gratefully acknowledged. We thank Profs. C. L. Khetrapal and J. Tropp for perusal of the manuscript. ...
The somatostatin analogue DOTATOC, DOTA-[Tyr(3)]octreotide, is used for in vivo diagnosis and tar... more The somatostatin analogue DOTATOC, DOTA-[Tyr(3)]octreotide, is used for in vivo diagnosis and targeted therapy of somatostatin-receptor-positive tumors. DOTATOC consists of a disulfide-bridged octapeptide, d-Phe(1)-Cys(2)-Tyr(3)-d-Trp(4)-Lys(5)-Thr(6)-Cys(7)-Thr(8)-ol, connected to the metal chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Two metal complexes, Ga(III)- and Y(III)-DOTATOC, were reported to differ significantly in somatostatin receptor affinity and tumor uptake. Our (1)H and (13)C solution NMR data and modeling studies of both compounds are in agreement with a fast conformational equilibrium of the peptide part, as previously reported for octreotide itself. However, the different coordination geometry of Ga(3+) and Y(3+) (6-fold and 8-fold, respectively, as known from model compounds) causes pronounced differences for the d-Phe(1) residue. For Y(III)-DOTATOC this leads to two conformers exchanging slowly on the NMR time scale. From various NMR measurements, they could be identified as cis-trans isomers at the amide bond between DOTA chelator and first residue (d-Phe(1)H(N)) of the peptide.
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Papers by Gerd Gemmecker