Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epito... more Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias.
Heptakis(2,3-di-O-acetyl-6-bromo-6-deoxy)cyclomaltoheptaose has been characterized in aqueous sol... more Heptakis(2,3-di-O-acetyl-6-bromo-6-deoxy)cyclomaltoheptaose has been characterized in aqueous solution by 1D and 2D NMR spectroscopy and in the solid state by X-ray crystallography. In methanol solution, the acetyl groups were found to interact with both inward and outward-pointing protons. This and the strong deshielding of the bridging carbons, relative to the nonacetylated precursor, indicate macrocyclic flexibility. In the crystalline state the macrocycle exists as a methanol complex. It exhibits elliptical distortion, all glucose residues been tilted with their primary side toward the cavity. The existing strain due to the congestion of 14 acetyl groups at the secondary site is relieved by two glucose rings acquiring the rarely observed skew-boat conformation, (0)S(2), by the increased tilting of two glucose residues, as well as by minor variations of the torsion angles of the acetyl groups. The seven bromine atoms are quite accessible to nucleophiles.
Octakis(6-bromo-6-deoxy)cyclomaltooctaose, perbrominated gamma-cyclodextrin at the primary side, ... more Octakis(6-bromo-6-deoxy)cyclomaltooctaose, perbrominated gamma-cyclodextrin at the primary side, crystallises from methanol in a very unique manner. The macrocycles are quite distorted in contrast to their beta-cyclodextrin analogue, heptakis(6-bromo-6-deoxy)cyclomaltoheptaose. The two monomers, arranged head-to-head, form a completely new kind of dimer by mutually entering into each other, both at the primary and the secondary sides. At the primary, hydrophobic side, they interact by Br...Br interactions and at the secondary, hydrophilic side, by direct H-bonds between hydroxylic groups. The short contacts of the Br atoms contribute to the macrocycle's distortion, which is considerable compared to the few available structures of gamma-CDs persubstituted at the primary side with bulkier and in some occasions charged substituents. Water and methanol molecules are entrapped in the cyclodextrin cavity, mostly in the area of the secondary hydroxylic groups connecting the macrocycles by indirect H-bonds. Thus the solvent molecules strengthen the association of the two monomers and contribute to the stabilisation of the cavity. The monomers stack along the a-axis and form columns that align in parallel lines along the same axis resulting in the formation of alternating hydrophobic and hydrophilic layers perpendicular to the a-axis resembling in this respect, the structure of the analogous perbrominated beta-cyclodextrin.
... PETROS GIASTAS, NIKOLAOS MOURTZIS, KONSTANTINA YANNAKOPOULOU ∗ and IRENE M. MAVRIDIS ∗ Instit... more ... PETROS GIASTAS, NIKOLAOS MOURTZIS, KONSTANTINA YANNAKOPOULOU ∗ and IRENE M. MAVRIDIS ∗ Institute of Physical Chemistry, National Center for Scientific Research Demokritos, Aghia Paraskevi 15310, Athens, Greece ...
Protein science : a publication of the Protein Society, 2003
A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the i... more A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the inhibitor, and the new allosteric sites are currently under investigation as targets for inhibition of hepatic glycogenolysis under high glucose concentrations; in some cases specific inhibitors are under evaluation in human clinical trials for therapeutic intervention in type 2 diabetes. In an attempt to investigate whether the storage site can be exploited as target for modulating hepatic glucose production, alpha-, beta-, and gamma-cyclodextrins were identified as moderate mixed-type competitive inhibitors of GPb (with respect to glycogen) with K(i) values of 47.1, 14.1, and 7.4 mM, respectively. To elucidate the structural basis of inhibition, we determined the structure of GPb complexed with beta- and gamma-cyclodextrins at 1.94 A and 2.3 A resolution, respectively. The structures of the two complexes reveal that the inhibitors can be accommodated in the glycogen storage site of T-st...
The crystal of the 1:1 complex of 4-tert-butyltoluene with cyclomaltoheptaose (beta-cyclodextrin,... more The crystal of the 1:1 complex of 4-tert-butyltoluene with cyclomaltoheptaose (beta-cyclodextrin, beta CD) is triclinic P1 with a = 15.562(2), b = 15.564(4), c = 15.835(3) A, alpha = 102.11(2), beta = 102.15(1), gamma = 103.64(2) degrees, V = 3505(1) A3, and Z = 2. The two independent beta CD molecules in the asymmetric unit form a dimer by hydrogen bonding involving HO-3, which accommodates two molecules of the guest. The hydrophobic guests are enclosed completely in the beta CD cavities with the tert-butyl groups in the hydrophobic region beneath the primary hydroxyl groups. The aromatic rings have two orientations and their toluene methyl moieties could not be located but were calculated to be at the interface of the two monomers. The dimers form channels along the c axis. The inter-dimer space is filled with 17 molecules of water distributed over 25 sites. A dense network of hydrogen bonds is formed, involving the beta CD hydroxyl groups and water molecules.
The crystal of the 1:1 complex of 4-tert-butyltoluene with cyclomaltoheptaose (beta-cyclodextrin,... more The crystal of the 1:1 complex of 4-tert-butyltoluene with cyclomaltoheptaose (beta-cyclodextrin, beta CD) is triclinic P1 with a = 15.562(2), b = 15.564(4), c = 15.835(3) A, alpha = 102.11(2), beta = 102.15(1), gamma = 103.64(2) degrees, V = 3505(1) A3, and Z = 2. The two independent beta CD molecules in the asymmetric unit form a dimer by hydrogen bonding involving HO-3, which accommodates two molecules of the guest. The hydrophobic guests are enclosed completely in the beta CD cavities with the tert-butyl groups in the hydrophobic region beneath the primary hydroxyl groups. The aromatic rings have two orientations and their toluene methyl moieties could not be located but were calculated to be at the interface of the two monomers. The dimers form channels along the c axis. The inter-dimer space is filled with 17 molecules of water distributed over 25 sites. A dense network of hydrogen bonds is formed, involving the beta CD hydroxyl groups and water molecules.
The crystal structure of the 1 : 1 inclusion complex of hexakis(2,3,6-tri-O-methyl)- a-cyclodextr... more The crystal structure of the 1 : 1 inclusion complex of hexakis(2,3,6-tri-O-methyl)- a-cyclodextrin (TM aCD) with 1,7-dioxaspiro[5.5]undecane (spiroacetal) is orthorhombic, space group C2221, with a = 24.002(2), b = 14.812(1), c = 21.792(2) Å V = 7747.3(11) Å3 and Z = 8. The molecular six-fold axis of TM aCD coincides with the a two-fold crystallographic axis and the guest is
β-Cyclodextrin (β-CD) dimers have been prepared using the bioorthogonal Staudinger ligation for t... more β-Cyclodextrin (β-CD) dimers have been prepared using the bioorthogonal Staudinger ligation for the first time. In addition to a known linker, methyl 2-(diphenylphosphanyl)terephthalate, a doubly active linker was specifically developed that enabled connection of two β-CD units in a single step and in aqueous/organic media, under mild conditions and with good yields. A three-carbon spacer between the β-CD torus and the azido group was required for facile dimer formation. The products, as studied by NMR spectroscopy, were found to adopt closed conformations by intramolecular self-inclusion. On the other hand, association via intermolecular binding was also observed in aqueous solution, confirmed by DOSY NMR experiments. Despite self-inclusion, the β-CD cavities were capable of guest encapsulation, as shown by titration experiments: the binding constant with 1-adamantylamine was similar to that of natural β-CD. Theoretical calculations for isolated molecules (PM3 level of theory) and in the presence of solvent [water, PM3(COSMO)] as well as DFT calculations suggested that the compounds prefer to adopt conformations which bring the phenyl groups either inside the β-CD cavity (inclusion) or over its narrow side (vicinal). Thus, Staudinger ligation could be the method of choice for linking CDs exhibiting (i) ease of preparation in aqueous media, in short steps, under mild conditions and in good yields, (ii) satisfactory aqueous solubility and independent binding capacity of the cavities.
Twelve Schiff bases of methoxy-substituted salicylaldehyde have been examined by crystallographic... more Twelve Schiff bases of methoxy-substituted salicylaldehyde have been examined by crystallographic and spectroscopic methods, as well as by DFT theoretical calculations in order to investigate the effect of the substituent's position on the keto-enol equilibrium in the crystalline state. Four out of the 10 structurally characterized compounds with methoxy substitution on the para and/or ortho positions with respect to the aldimine bridge and deriving from aliphatic amines or alkylarylamines are found as cis-keto tautomers and form dimers. In contrast, the five pure enol tautomers derive either from aliphatic or alkylarylamines and are meta substituted or from aniline or benzylamine and are para and/or ortho methoxy substituted. The DFT calculations support the crystallographic results and, moreover, they have shown that keto and enol tautomers are affected differently by the relative arrangement of the monomers. Overall, the DFT calculations point to a plausible hypothesis for the stabilization of the keto form in the crystalline state: In cases with a sufficiently low enol-keto energy difference of the isolated monomers, as when the methoxy group is at ortho and/or para positions with respect to the aldimino group, extra stabilization of the keto form is derived from molecular association, thus leading to its crystallization.
Active muscles generate substantial mechanical forces by the contraction/relaxation cycle, and, t... more Active muscles generate substantial mechanical forces by the contraction/relaxation cycle, and, to maintain an ordered state, they require molecular structures of extraordinary stability. These forces are sensed and buffered by unusually long and elastic filament proteins with highly repetitive domain arrays. Members of the myomesin protein family function as molecular bridges that connect major filament systems in the central M-band of muscle sarcomeres, which is a central locus of passive stress sensing. To unravel the mechanism of molecular elasticity in such filament-connecting proteins, we have determined the overall architecture of the complete C-terminal immunoglobulin domain array of myomesin by X-ray crystallography, electron microscopy, solution X-ray scattering, and atomic force microscopy. Our data reveal a dimeric tail-to-tail filament structure of about 360 Å in length, which is folded into an irregular superhelical coil arrangement of almost identical α-helix/domain modules. The myomesin filament can be stretched to about 2.5-fold its original length by reversible unfolding of these linkers, a mechanism that to our knowledge has not been observed previously. Our data explain how myomesin could act as a highly elastic ribbon to maintain the overall structural organization of the sarcomeric M-band. In general terms, our data demonstrate how repetitive domain modules such as those found in myomesin could generate highly elastic protein structures in highly organized cell systems such as muscle sarcomeres.
Novel -type cyclodextrin (CD) derivatives, , and , bearing 6, 7 and 8 bis(carboxymethyl)amino (im... more Novel -type cyclodextrin (CD) derivatives, , and , bearing 6, 7 and 8 bis(carboxymethyl)amino (iminodiacetic acid) groups, respectively, were prepared, and their complexation with Eu(iii), Tb(iii) and Gd(iii) ions was studied. Luminescence titrations and mass spectrometry showed formation of multimetal complexes ( 2 to 3, mainly 3 and exactly 4 metal ions), whereas luminescence lifetime measurements revealed the presence of exchangeable water molecules. Semiempirical quantum mechanical calculations, performed by the PM3 method and assessed by DFT calculations on model ligands, indicated efficient multi-metal complexation, in agreement with the experiment. The structures showed coordination of the metal ions in the outer primary side of the CDs via 4 carboxylate O atoms, 2 N atoms and a glucopyranose O atom per metal ion. Coordination of water molecules was also predicted, in accordance with experimental results. Calculated bond lengths and angles were in agreement with literature experimental values of lanthanide complexes. Calculated energies showed that complex stability decreases in the order > > . (1)H NMR molecular relaxivity measurements for the Gd(iii) complexes of , or in water afforded values 4 to 10 times higher than the relaxivity of a commercial contrast agent at 12 MHz, and 6 to 20 times higher at 100 MHz. Solutions of and Gd(iii) complexes in human blood plasma displayed relaxivity values at 100 MHz 7 and 12 times, respectively, higher than the commercial agent. MTT tests of the Gd(iii) complexes using human skin fibroblasts did not show toxicity. Attempts to supramolecularly sensitize the luminescence of the lanthanide complexes using various aromatic CD guests were ineffective, evidently due to large guest-metal distances and inefficient inclusion. The described lanthanide complexes, could be useful as contrast agents in MRI.
Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epito... more Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias.
Heptakis(2,3-di-O-acetyl-6-bromo-6-deoxy)cyclomaltoheptaose has been characterized in aqueous sol... more Heptakis(2,3-di-O-acetyl-6-bromo-6-deoxy)cyclomaltoheptaose has been characterized in aqueous solution by 1D and 2D NMR spectroscopy and in the solid state by X-ray crystallography. In methanol solution, the acetyl groups were found to interact with both inward and outward-pointing protons. This and the strong deshielding of the bridging carbons, relative to the nonacetylated precursor, indicate macrocyclic flexibility. In the crystalline state the macrocycle exists as a methanol complex. It exhibits elliptical distortion, all glucose residues been tilted with their primary side toward the cavity. The existing strain due to the congestion of 14 acetyl groups at the secondary site is relieved by two glucose rings acquiring the rarely observed skew-boat conformation, (0)S(2), by the increased tilting of two glucose residues, as well as by minor variations of the torsion angles of the acetyl groups. The seven bromine atoms are quite accessible to nucleophiles.
Octakis(6-bromo-6-deoxy)cyclomaltooctaose, perbrominated gamma-cyclodextrin at the primary side, ... more Octakis(6-bromo-6-deoxy)cyclomaltooctaose, perbrominated gamma-cyclodextrin at the primary side, crystallises from methanol in a very unique manner. The macrocycles are quite distorted in contrast to their beta-cyclodextrin analogue, heptakis(6-bromo-6-deoxy)cyclomaltoheptaose. The two monomers, arranged head-to-head, form a completely new kind of dimer by mutually entering into each other, both at the primary and the secondary sides. At the primary, hydrophobic side, they interact by Br...Br interactions and at the secondary, hydrophilic side, by direct H-bonds between hydroxylic groups. The short contacts of the Br atoms contribute to the macrocycle's distortion, which is considerable compared to the few available structures of gamma-CDs persubstituted at the primary side with bulkier and in some occasions charged substituents. Water and methanol molecules are entrapped in the cyclodextrin cavity, mostly in the area of the secondary hydroxylic groups connecting the macrocycles by indirect H-bonds. Thus the solvent molecules strengthen the association of the two monomers and contribute to the stabilisation of the cavity. The monomers stack along the a-axis and form columns that align in parallel lines along the same axis resulting in the formation of alternating hydrophobic and hydrophilic layers perpendicular to the a-axis resembling in this respect, the structure of the analogous perbrominated beta-cyclodextrin.
... PETROS GIASTAS, NIKOLAOS MOURTZIS, KONSTANTINA YANNAKOPOULOU ∗ and IRENE M. MAVRIDIS ∗ Instit... more ... PETROS GIASTAS, NIKOLAOS MOURTZIS, KONSTANTINA YANNAKOPOULOU ∗ and IRENE M. MAVRIDIS ∗ Institute of Physical Chemistry, National Center for Scientific Research Demokritos, Aghia Paraskevi 15310, Athens, Greece ...
Protein science : a publication of the Protein Society, 2003
A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the i... more A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the inhibitor, and the new allosteric sites are currently under investigation as targets for inhibition of hepatic glycogenolysis under high glucose concentrations; in some cases specific inhibitors are under evaluation in human clinical trials for therapeutic intervention in type 2 diabetes. In an attempt to investigate whether the storage site can be exploited as target for modulating hepatic glucose production, alpha-, beta-, and gamma-cyclodextrins were identified as moderate mixed-type competitive inhibitors of GPb (with respect to glycogen) with K(i) values of 47.1, 14.1, and 7.4 mM, respectively. To elucidate the structural basis of inhibition, we determined the structure of GPb complexed with beta- and gamma-cyclodextrins at 1.94 A and 2.3 A resolution, respectively. The structures of the two complexes reveal that the inhibitors can be accommodated in the glycogen storage site of T-st...
The crystal of the 1:1 complex of 4-tert-butyltoluene with cyclomaltoheptaose (beta-cyclodextrin,... more The crystal of the 1:1 complex of 4-tert-butyltoluene with cyclomaltoheptaose (beta-cyclodextrin, beta CD) is triclinic P1 with a = 15.562(2), b = 15.564(4), c = 15.835(3) A, alpha = 102.11(2), beta = 102.15(1), gamma = 103.64(2) degrees, V = 3505(1) A3, and Z = 2. The two independent beta CD molecules in the asymmetric unit form a dimer by hydrogen bonding involving HO-3, which accommodates two molecules of the guest. The hydrophobic guests are enclosed completely in the beta CD cavities with the tert-butyl groups in the hydrophobic region beneath the primary hydroxyl groups. The aromatic rings have two orientations and their toluene methyl moieties could not be located but were calculated to be at the interface of the two monomers. The dimers form channels along the c axis. The inter-dimer space is filled with 17 molecules of water distributed over 25 sites. A dense network of hydrogen bonds is formed, involving the beta CD hydroxyl groups and water molecules.
The crystal of the 1:1 complex of 4-tert-butyltoluene with cyclomaltoheptaose (beta-cyclodextrin,... more The crystal of the 1:1 complex of 4-tert-butyltoluene with cyclomaltoheptaose (beta-cyclodextrin, beta CD) is triclinic P1 with a = 15.562(2), b = 15.564(4), c = 15.835(3) A, alpha = 102.11(2), beta = 102.15(1), gamma = 103.64(2) degrees, V = 3505(1) A3, and Z = 2. The two independent beta CD molecules in the asymmetric unit form a dimer by hydrogen bonding involving HO-3, which accommodates two molecules of the guest. The hydrophobic guests are enclosed completely in the beta CD cavities with the tert-butyl groups in the hydrophobic region beneath the primary hydroxyl groups. The aromatic rings have two orientations and their toluene methyl moieties could not be located but were calculated to be at the interface of the two monomers. The dimers form channels along the c axis. The inter-dimer space is filled with 17 molecules of water distributed over 25 sites. A dense network of hydrogen bonds is formed, involving the beta CD hydroxyl groups and water molecules.
The crystal structure of the 1 : 1 inclusion complex of hexakis(2,3,6-tri-O-methyl)- a-cyclodextr... more The crystal structure of the 1 : 1 inclusion complex of hexakis(2,3,6-tri-O-methyl)- a-cyclodextrin (TM aCD) with 1,7-dioxaspiro[5.5]undecane (spiroacetal) is orthorhombic, space group C2221, with a = 24.002(2), b = 14.812(1), c = 21.792(2) Å V = 7747.3(11) Å3 and Z = 8. The molecular six-fold axis of TM aCD coincides with the a two-fold crystallographic axis and the guest is
β-Cyclodextrin (β-CD) dimers have been prepared using the bioorthogonal Staudinger ligation for t... more β-Cyclodextrin (β-CD) dimers have been prepared using the bioorthogonal Staudinger ligation for the first time. In addition to a known linker, methyl 2-(diphenylphosphanyl)terephthalate, a doubly active linker was specifically developed that enabled connection of two β-CD units in a single step and in aqueous/organic media, under mild conditions and with good yields. A three-carbon spacer between the β-CD torus and the azido group was required for facile dimer formation. The products, as studied by NMR spectroscopy, were found to adopt closed conformations by intramolecular self-inclusion. On the other hand, association via intermolecular binding was also observed in aqueous solution, confirmed by DOSY NMR experiments. Despite self-inclusion, the β-CD cavities were capable of guest encapsulation, as shown by titration experiments: the binding constant with 1-adamantylamine was similar to that of natural β-CD. Theoretical calculations for isolated molecules (PM3 level of theory) and in the presence of solvent [water, PM3(COSMO)] as well as DFT calculations suggested that the compounds prefer to adopt conformations which bring the phenyl groups either inside the β-CD cavity (inclusion) or over its narrow side (vicinal). Thus, Staudinger ligation could be the method of choice for linking CDs exhibiting (i) ease of preparation in aqueous media, in short steps, under mild conditions and in good yields, (ii) satisfactory aqueous solubility and independent binding capacity of the cavities.
Twelve Schiff bases of methoxy-substituted salicylaldehyde have been examined by crystallographic... more Twelve Schiff bases of methoxy-substituted salicylaldehyde have been examined by crystallographic and spectroscopic methods, as well as by DFT theoretical calculations in order to investigate the effect of the substituent's position on the keto-enol equilibrium in the crystalline state. Four out of the 10 structurally characterized compounds with methoxy substitution on the para and/or ortho positions with respect to the aldimine bridge and deriving from aliphatic amines or alkylarylamines are found as cis-keto tautomers and form dimers. In contrast, the five pure enol tautomers derive either from aliphatic or alkylarylamines and are meta substituted or from aniline or benzylamine and are para and/or ortho methoxy substituted. The DFT calculations support the crystallographic results and, moreover, they have shown that keto and enol tautomers are affected differently by the relative arrangement of the monomers. Overall, the DFT calculations point to a plausible hypothesis for the stabilization of the keto form in the crystalline state: In cases with a sufficiently low enol-keto energy difference of the isolated monomers, as when the methoxy group is at ortho and/or para positions with respect to the aldimino group, extra stabilization of the keto form is derived from molecular association, thus leading to its crystallization.
Active muscles generate substantial mechanical forces by the contraction/relaxation cycle, and, t... more Active muscles generate substantial mechanical forces by the contraction/relaxation cycle, and, to maintain an ordered state, they require molecular structures of extraordinary stability. These forces are sensed and buffered by unusually long and elastic filament proteins with highly repetitive domain arrays. Members of the myomesin protein family function as molecular bridges that connect major filament systems in the central M-band of muscle sarcomeres, which is a central locus of passive stress sensing. To unravel the mechanism of molecular elasticity in such filament-connecting proteins, we have determined the overall architecture of the complete C-terminal immunoglobulin domain array of myomesin by X-ray crystallography, electron microscopy, solution X-ray scattering, and atomic force microscopy. Our data reveal a dimeric tail-to-tail filament structure of about 360 Å in length, which is folded into an irregular superhelical coil arrangement of almost identical α-helix/domain modules. The myomesin filament can be stretched to about 2.5-fold its original length by reversible unfolding of these linkers, a mechanism that to our knowledge has not been observed previously. Our data explain how myomesin could act as a highly elastic ribbon to maintain the overall structural organization of the sarcomeric M-band. In general terms, our data demonstrate how repetitive domain modules such as those found in myomesin could generate highly elastic protein structures in highly organized cell systems such as muscle sarcomeres.
Novel -type cyclodextrin (CD) derivatives, , and , bearing 6, 7 and 8 bis(carboxymethyl)amino (im... more Novel -type cyclodextrin (CD) derivatives, , and , bearing 6, 7 and 8 bis(carboxymethyl)amino (iminodiacetic acid) groups, respectively, were prepared, and their complexation with Eu(iii), Tb(iii) and Gd(iii) ions was studied. Luminescence titrations and mass spectrometry showed formation of multimetal complexes ( 2 to 3, mainly 3 and exactly 4 metal ions), whereas luminescence lifetime measurements revealed the presence of exchangeable water molecules. Semiempirical quantum mechanical calculations, performed by the PM3 method and assessed by DFT calculations on model ligands, indicated efficient multi-metal complexation, in agreement with the experiment. The structures showed coordination of the metal ions in the outer primary side of the CDs via 4 carboxylate O atoms, 2 N atoms and a glucopyranose O atom per metal ion. Coordination of water molecules was also predicted, in accordance with experimental results. Calculated bond lengths and angles were in agreement with literature experimental values of lanthanide complexes. Calculated energies showed that complex stability decreases in the order > > . (1)H NMR molecular relaxivity measurements for the Gd(iii) complexes of , or in water afforded values 4 to 10 times higher than the relaxivity of a commercial contrast agent at 12 MHz, and 6 to 20 times higher at 100 MHz. Solutions of and Gd(iii) complexes in human blood plasma displayed relaxivity values at 100 MHz 7 and 12 times, respectively, higher than the commercial agent. MTT tests of the Gd(iii) complexes using human skin fibroblasts did not show toxicity. Attempts to supramolecularly sensitize the luminescence of the lanthanide complexes using various aromatic CD guests were ineffective, evidently due to large guest-metal distances and inefficient inclusion. The described lanthanide complexes, could be useful as contrast agents in MRI.
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