Structure Determination of Protein−Protein Complexes Using NMR Chemical Shifts: Case of an Endonuclease Colicin−Immunity Protein Complex
Rinaldo W. Montalvao†‡, Andrea Cavalli†, Xavier Salvatella†, Tom L. Blundell‡ and Michele Vendruscolo*†
Abstract
Nuclear magnetic resonance (NMR) spectroscopy provides a range of powerful techniques for determining the structures and the dynamics of proteins. The high-resolution determination of the structures of protein−protein complexes, however, is still a challenging problem for this approach, since it can normally provide only a limited amount of structural information at protein−protein interfaces. We present here the determination using NMR chemical shifts of the structure (PDB code 2K5X) of the cytotoxic endonuclease domain from bacterial toxin colicin (E9) in complex with its cognate immunity protein (Im9). In order to achieve this result, we introduce the CamDock method, which combines a flexible docking procedure with a refinement that exploits the structural information provided by chemical shifts. The results that we report thus indicate that chemical shifts can be used as structural restraints for the determination of the conformations of protein complexes that are difficult to obtain by more standard NMR approaches.
Friday, November 28, 2008
J. Am. Chem. Soc., 2008, 130 (47), pp 15927–15937
16-Fold Degeneracy of Peptide Plane Orientations from Residual Dipolar Couplings: Analytical Treatment and Implications for Protein Structure Determination
Jean-Christophe Hus#†‡, Loïc Salmon‡, Guillaume Bouvignies‡, Johannes Lotze‡, Martin Blackledge*‡ and Rafael Brüschweiler*§
Abstract
Residual dipolar couplings (RDCs) measured for internally rigid molecular fragments provide important information about the relative orientations of these fragments. Dependent on the symmetry of the alignment tensor and the symmetry of the molecular fragment, however, there generally exist more than one solution for the fragment orientation consistent with the measured RDCs. Analytical solutions are presented that describe the complete set of orientations of internally rigid fragments that are consistent with multiple dipolar couplings measured in a single alignment medium that is rhombic. For the first time, it is shown that, for a planar fragment such as the peptide plane, there generally exist 16 different solutions with their analytical expressions presented explicitly. The presence of these solutions is shown to be highly relevant for standard structure determination protocols using RDCs to refine molecular structures. In particular, when using standard protein structure refinement with RDCs that were measured in a single alignment medium as constraints, it is found that often more than one of the peptide plane solutions is physically viable; i.e., despite being consistent with measured RDCs, the local backbone structure can be incorrect. On the basis of experimental and simulated examples, it is rationalized why protein structures that are refined against RDCs measured in a single medium can have lower resolution (precision) than one would expect on the basis of the experimental accuracy of the RDCs. Conditions are discussed under which the correct solution can be identified.
Jean-Christophe Hus#†‡, Loïc Salmon‡, Guillaume Bouvignies‡, Johannes Lotze‡, Martin Blackledge*‡ and Rafael Brüschweiler*§
Abstract
Residual dipolar couplings (RDCs) measured for internally rigid molecular fragments provide important information about the relative orientations of these fragments. Dependent on the symmetry of the alignment tensor and the symmetry of the molecular fragment, however, there generally exist more than one solution for the fragment orientation consistent with the measured RDCs. Analytical solutions are presented that describe the complete set of orientations of internally rigid fragments that are consistent with multiple dipolar couplings measured in a single alignment medium that is rhombic. For the first time, it is shown that, for a planar fragment such as the peptide plane, there generally exist 16 different solutions with their analytical expressions presented explicitly. The presence of these solutions is shown to be highly relevant for standard structure determination protocols using RDCs to refine molecular structures. In particular, when using standard protein structure refinement with RDCs that were measured in a single alignment medium as constraints, it is found that often more than one of the peptide plane solutions is physically viable; i.e., despite being consistent with measured RDCs, the local backbone structure can be incorrect. On the basis of experimental and simulated examples, it is rationalized why protein structures that are refined against RDCs measured in a single medium can have lower resolution (precision) than one would expect on the basis of the experimental accuracy of the RDCs. Conditions are discussed under which the correct solution can be identified.
Thursday, November 20, 2008
J. Am. Chem. Soc., 2008, 130 (46), pp 15726–15731
17O and 15N Solid State NMR Studies on Ligand-Assisted Templating and Oxygen Coordination in the Walls of Mesoporous Nb, Ta and Ti Oxides
Yuxiang Rao†, Tom F. Kemp‡, Michel Trudeau§, Mark E. Smith*‡ and Dave M. Antonelli*†
Abstract
A multinuclear solid state NMR approach is applied to four templated mesoporous oxides (silica, titania, niobia and tantala) to include 15N and 17O magic angle spinning (MAS) NMR and double resonance 15N−93Nb, 17O Rotational-Echo Adiabatic Passage Double Resonance (REAPDOR). The templated samples were ramped in steps of 20 °C for 2 days up to typically 110 °C where the samples were left for 2−4 days. 15N MAS NMR shows that amines are the only species present in the TiO2, Nb2O5, and Ta2O5. In SiO2, amines are only present as a minor coordination (10 ± 2%), but there are several strong ammonium 15N resonances. The REAPDOR experiments show that the nitrogen interacts with niobium, confirming a ligand interaction between the Nb and N, as previously believed. In the case of silica, the amine is quaternized and there is apparently no interaction with the Si, suggesting a RNH3+ −O−Si- hydrogen-bonding interaction with the walls. 17O MAS NMR provides the clearest indication of the local wall structure. In the aged, templated samples in all cases only OM2 coordinations are present which is very different from the pure bulk oxides (apart from SiO2) and must be due to the effects of amine coordination at the metal centers. On removal of the template, these oxides behave differently, with Ta2O5 showing a mixture of OTa2 (85 ± 5%) and OTa3 (15 ± 5%) which is similar to the types of coordination found in the bulk oxide. The previously reported 17O MAS NMR data from heat-treated mesoporous niobia shows only ONb2, which is very highly ordered. In contrast for titania, the OTi2 coordination is immediately lost on removal of the template to be replaced by a mixture of OTi3 (60 ± 5%) and OTi4 (40 ± 5%), with the OTi4 becoming dominant above 250 °C, very different behavior from the corresponding bulk oxide. In summary, this NMR study shows that the local oxygen coordination in amine-templated mesoporous transition metal oxides is present as OM2 which is relatively rare in bulk oxides. The data indicates that the template interaction is largely controlled by the N−M dative bond to the wall, suppressing higher oxygen coordination numbers. Qualitatively it appears that the strength of this interaction varies greatly in the different mesoporous oxides.
Yuxiang Rao†, Tom F. Kemp‡, Michel Trudeau§, Mark E. Smith*‡ and Dave M. Antonelli*†
Abstract
A multinuclear solid state NMR approach is applied to four templated mesoporous oxides (silica, titania, niobia and tantala) to include 15N and 17O magic angle spinning (MAS) NMR and double resonance 15N−93Nb, 17O Rotational-Echo Adiabatic Passage Double Resonance (REAPDOR). The templated samples were ramped in steps of 20 °C for 2 days up to typically 110 °C where the samples were left for 2−4 days. 15N MAS NMR shows that amines are the only species present in the TiO2, Nb2O5, and Ta2O5. In SiO2, amines are only present as a minor coordination (10 ± 2%), but there are several strong ammonium 15N resonances. The REAPDOR experiments show that the nitrogen interacts with niobium, confirming a ligand interaction between the Nb and N, as previously believed. In the case of silica, the amine is quaternized and there is apparently no interaction with the Si, suggesting a RNH3+ −O−Si- hydrogen-bonding interaction with the walls. 17O MAS NMR provides the clearest indication of the local wall structure. In the aged, templated samples in all cases only OM2 coordinations are present which is very different from the pure bulk oxides (apart from SiO2) and must be due to the effects of amine coordination at the metal centers. On removal of the template, these oxides behave differently, with Ta2O5 showing a mixture of OTa2 (85 ± 5%) and OTa3 (15 ± 5%) which is similar to the types of coordination found in the bulk oxide. The previously reported 17O MAS NMR data from heat-treated mesoporous niobia shows only ONb2, which is very highly ordered. In contrast for titania, the OTi2 coordination is immediately lost on removal of the template to be replaced by a mixture of OTi3 (60 ± 5%) and OTi4 (40 ± 5%), with the OTi4 becoming dominant above 250 °C, very different behavior from the corresponding bulk oxide. In summary, this NMR study shows that the local oxygen coordination in amine-templated mesoporous transition metal oxides is present as OM2 which is relatively rare in bulk oxides. The data indicates that the template interaction is largely controlled by the N−M dative bond to the wall, suppressing higher oxygen coordination numbers. Qualitatively it appears that the strength of this interaction varies greatly in the different mesoporous oxides.
J. Phys. Chem. B, 2008, 112 (45), pp 14312–14318
Anesthetic Modulation of Protein Dynamics: Insight from an NMR Study
Christian G. Canlas†, Tanxing Cui†, Ling Li†, Yan Xu†‡ and Pei Tang
Abstract
Mistic (membrane integrating sequence for translation of integral membrane protein constructs) comprises the four-α-helix bundle scaffold found in the transmembrane domains of the Cys-loop receptors that are plausible targets for general anesthetics. Nuclear magnetic resonance (NMR) studies of anesthetic halothane interaction with Mistic in dodecyl phosphocholine (DPC) micelles provide an experimental basis for understanding molecular mechanisms of general anesthesia. Halothane was found to interact directly with Mistic, mostly in the interfacial loop regions. Although the presence of halothane had little effect on Mistic structure, 15N NMR relaxation dispersion measurements revealed that halothane affected Mistic’s motion on the microsecond−millisecond time scale. Halothane shifted the equilibrium of chemical exchange in some residues and made the exchange faster or slower in comparison to the original state in the absence of halothane. The motion on the microsecond−millisecond time scale in several residues disappeared in response to the addition of halothane. Most of the residues experiencing halothane-induced dynamics changes also exhibited profound halothane-induced changes in chemical shift, suggesting that dynamics modification of these residues might result from their direct interaction with halothane molecules. Allosteric modulation by halothane also contributed to dynamics changes, as reflected in residues I52 and Y82 where halothane introduction brought about dynamics changes but not chemical shift changes. The study suggests that inhaled general anesthetics could act on proteins via altering protein motion on the microsecond−millisecond time scale, especially motion in the flexible loops that link different alpha helices. The validation of anesthetic effect on protein dynamics that are potentially correlated with protein functions is a critical step in unraveling the mechanisms of anesthetic action on proteins.
Christian G. Canlas†, Tanxing Cui†, Ling Li†, Yan Xu†‡ and Pei Tang
Abstract
Mistic (membrane integrating sequence for translation of integral membrane protein constructs) comprises the four-α-helix bundle scaffold found in the transmembrane domains of the Cys-loop receptors that are plausible targets for general anesthetics. Nuclear magnetic resonance (NMR) studies of anesthetic halothane interaction with Mistic in dodecyl phosphocholine (DPC) micelles provide an experimental basis for understanding molecular mechanisms of general anesthesia. Halothane was found to interact directly with Mistic, mostly in the interfacial loop regions. Although the presence of halothane had little effect on Mistic structure, 15N NMR relaxation dispersion measurements revealed that halothane affected Mistic’s motion on the microsecond−millisecond time scale. Halothane shifted the equilibrium of chemical exchange in some residues and made the exchange faster or slower in comparison to the original state in the absence of halothane. The motion on the microsecond−millisecond time scale in several residues disappeared in response to the addition of halothane. Most of the residues experiencing halothane-induced dynamics changes also exhibited profound halothane-induced changes in chemical shift, suggesting that dynamics modification of these residues might result from their direct interaction with halothane molecules. Allosteric modulation by halothane also contributed to dynamics changes, as reflected in residues I52 and Y82 where halothane introduction brought about dynamics changes but not chemical shift changes. The study suggests that inhaled general anesthetics could act on proteins via altering protein motion on the microsecond−millisecond time scale, especially motion in the flexible loops that link different alpha helices. The validation of anesthetic effect on protein dynamics that are potentially correlated with protein functions is a critical step in unraveling the mechanisms of anesthetic action on proteins.
J. Am. Chem. Soc., 2008, 130 (45), pp 14990–15001
Structural Insights into the Polymorphism of Amyloid-Like Fibrils Formed by Region 20−29 of Amylin Revealed by Solid-State NMR and X-ray Fiber Diffraction
Jillian Madine†, Edward Jack‡, Peter G. Stockley§, Sheena E. Radford§, Louise C. Serpell∥ and David A. Middleton*†
Abstract
Many unrelated proteins and peptides can assemble into amyloid or amyloid-like nanostructures, all of which share the cross-β motif of repeat arrays of β-strands hydrogen-bonded along the fibril axis. Yet, paradoxically, structurally polymorphic fibrils may derive from the same initial polypeptide sequence. Here, solid-state nuclear magnetic resonance (SSNMR) analysis of amyloid-like fibrils of the peptide hIAPP20−29, corresponding to the region S20NNFGAILSS29 of the human islet amyloid polypeptide amylin, reveals that the peptide assembles into two amyloid-like forms, (1) and (2), which have distinct structures at the molecular level. Rotational resonance SSNMR measurements of 13C dipolar couplings between backbone F23 and I26 of hIAPP20−29 fibrils are consistent with form (1) having parallel β-strands and form (2) having antiparallel strands within the β-sheet layers of the protofilament units. Seeding hIAPP20−29 with structurally homogeneous fibrils from a 30-residue amylin fragment (hIAPP8−37) produces morphologically homogeneous fibrils with similar NMR properties to form (1). A model for the architecture of the seeded fibrils is presented, based on the analysis of X-ray fiber diffraction data, combined with an extensive range of SSNMR constraints including chemical shifts, torsional angles, and interatomic distances. The model features a cross-β spine comprising two β-sheets with an interface defined by residues F23, A25, and L27, which form a hydrophobic zipper. We suggest that the energies of formation for fibril form containing antiparallel and parallel β-strands are similar when both configurations can be stabilized by a core of hydrophobic contacts, which has implications for the relationship between amino acid sequence and amyloid polymorphism in general.
Jillian Madine†, Edward Jack‡, Peter G. Stockley§, Sheena E. Radford§, Louise C. Serpell∥ and David A. Middleton*†
Abstract
Many unrelated proteins and peptides can assemble into amyloid or amyloid-like nanostructures, all of which share the cross-β motif of repeat arrays of β-strands hydrogen-bonded along the fibril axis. Yet, paradoxically, structurally polymorphic fibrils may derive from the same initial polypeptide sequence. Here, solid-state nuclear magnetic resonance (SSNMR) analysis of amyloid-like fibrils of the peptide hIAPP20−29, corresponding to the region S20NNFGAILSS29 of the human islet amyloid polypeptide amylin, reveals that the peptide assembles into two amyloid-like forms, (1) and (2), which have distinct structures at the molecular level. Rotational resonance SSNMR measurements of 13C dipolar couplings between backbone F23 and I26 of hIAPP20−29 fibrils are consistent with form (1) having parallel β-strands and form (2) having antiparallel strands within the β-sheet layers of the protofilament units. Seeding hIAPP20−29 with structurally homogeneous fibrils from a 30-residue amylin fragment (hIAPP8−37) produces morphologically homogeneous fibrils with similar NMR properties to form (1). A model for the architecture of the seeded fibrils is presented, based on the analysis of X-ray fiber diffraction data, combined with an extensive range of SSNMR constraints including chemical shifts, torsional angles, and interatomic distances. The model features a cross-β spine comprising two β-sheets with an interface defined by residues F23, A25, and L27, which form a hydrophobic zipper. We suggest that the energies of formation for fibril form containing antiparallel and parallel β-strands are similar when both configurations can be stabilized by a core of hydrophobic contacts, which has implications for the relationship between amino acid sequence and amyloid polymorphism in general.
Cryst. Growth Des., 2008, 8 (11), pp 3941–3950
1H, 13C, and 15N Solid-State NMR Studies of Imidazole- and Morpholine-Based Model Compounds Possessing Halogen and Hydrogen Bonding Capabilities
Karim Bouchmella†, Sylvain G. Dutremez*†, Bruno Alonso‡, Francesco Mauri§ and Christel Gervais*#
Abstract
The halogen and hydrogen bonding interactions present in solid 1-(2,3,3-triiodoallyl)imidazole (1), morpholinium iodide (2), the 1:1 cocrystal 1-(2,3,3-triiodoallyl)imidazole·morpholinium iodide (3), morpholine (4), imidazole (5), and 1-(3-iodopropargyl)imidazole (6) have been investigated by solid-state 1H, 13C, and 15N NMR spectroscopies. Comparison of the 15N CP MAS NMR spectrum of 3 with that of 2 indicates that protonated morpholine is present in solid 3, but this conclusion must be taken with caution as GIPAW calculations predict a 15N chemical shift for morpholine similar to that of the morpholinium cation. Conclusive evidence for the presence of a morpholinium cation in crystalline 3 was obtained by recording the static 15N NMR spectrum of this host−guest complex and comparing the morpholinium/morpholine part of the spectrum with the static spectra of 3 and 4 as obtained from ab initio calculations of NMR parameters based on the X-ray structures of these compounds. Concerning the imidazolyl group, 15N NMR spectroscopy has proven quite valuable to identify changes in the bonding situation of the C−NC nitrogen on passing from 1 to 3. In addition, slight differences are observed between the 15N chemical shifts of 1 and 6 that are ascribed to differences in halogen bond strengths between the two compounds. Attempts have also been made to study halogen bonding by 13C NMR spectroscopy, but this method did not provide exploitable results as signals corresponding to the sp and sp2 carbon atoms bonded to iodine could not be observed experimentally. 1H NMR spectroscopy is a powerful tool to study hydrogen bonding interactions of moderate energies such as +NH2···X (X = N, O, I). Indeed, we have found that the chemical shifts of the NH hydrogens were quite sensitive to the nature of X and to the N−H···X distance. This is demonstrated by the fact that the chemical shifts of the +NH2 protons of the morpholinium cation in 2 and 3 are noticeably different.
Karim Bouchmella†, Sylvain G. Dutremez*†, Bruno Alonso‡, Francesco Mauri§ and Christel Gervais*#
Abstract
The halogen and hydrogen bonding interactions present in solid 1-(2,3,3-triiodoallyl)imidazole (1), morpholinium iodide (2), the 1:1 cocrystal 1-(2,3,3-triiodoallyl)imidazole·morpholinium iodide (3), morpholine (4), imidazole (5), and 1-(3-iodopropargyl)imidazole (6) have been investigated by solid-state 1H, 13C, and 15N NMR spectroscopies. Comparison of the 15N CP MAS NMR spectrum of 3 with that of 2 indicates that protonated morpholine is present in solid 3, but this conclusion must be taken with caution as GIPAW calculations predict a 15N chemical shift for morpholine similar to that of the morpholinium cation. Conclusive evidence for the presence of a morpholinium cation in crystalline 3 was obtained by recording the static 15N NMR spectrum of this host−guest complex and comparing the morpholinium/morpholine part of the spectrum with the static spectra of 3 and 4 as obtained from ab initio calculations of NMR parameters based on the X-ray structures of these compounds. Concerning the imidazolyl group, 15N NMR spectroscopy has proven quite valuable to identify changes in the bonding situation of the C−NC nitrogen on passing from 1 to 3. In addition, slight differences are observed between the 15N chemical shifts of 1 and 6 that are ascribed to differences in halogen bond strengths between the two compounds. Attempts have also been made to study halogen bonding by 13C NMR spectroscopy, but this method did not provide exploitable results as signals corresponding to the sp and sp2 carbon atoms bonded to iodine could not be observed experimentally. 1H NMR spectroscopy is a powerful tool to study hydrogen bonding interactions of moderate energies such as +NH2···X (X = N, O, I). Indeed, we have found that the chemical shifts of the NH hydrogens were quite sensitive to the nature of X and to the N−H···X distance. This is demonstrated by the fact that the chemical shifts of the +NH2 protons of the morpholinium cation in 2 and 3 are noticeably different.
J. Med. Chem., 2008, 51 (22), pp 7205–7215
Discovery of Ligands for a Novel Target, the Human Telomerase RNA, Based on Flexible-Target Virtual Screening and NMR
Irene Gómez Pinto†‡, Christophe Guilbert†, Nikolai B. Ulyanov†, Jay Stearns§ and Thomas L. James*†
Abstract
The human ribonucleoprotein telomerase is a validated anticancer drug target, and hTR-P2b is a part of the human telomerase RNA (hTR) essential for its activity. Interesting ligands that bind hTR-P2b were identified by iteratively using a tandem structure-based approach: docking of potential ligands from small databases to hTR-P2b via the program MORDOR, which permits flexibility in both ligand and target, with subsequent NMR screening of high-ranking compounds. A high percentage of the compounds tested experimentally were found via NMR to bind to the U-rich region of hTR-P2b; most have MW < 500 Da and are from different compound classes, and several possess a charge of 0 or +1. Of the 48 ligands identified, 24 exhibit a decided preference to bind hTR-P2b RNA rather than A-site rRNA and 10 do not bind A-site rRNA at all. Binding affinity was measured by monitoring RNA imino proton resonances for some of the compounds that showed hTR binding preference.
Irene Gómez Pinto†‡, Christophe Guilbert†, Nikolai B. Ulyanov†, Jay Stearns§ and Thomas L. James*†
Abstract
The human ribonucleoprotein telomerase is a validated anticancer drug target, and hTR-P2b is a part of the human telomerase RNA (hTR) essential for its activity. Interesting ligands that bind hTR-P2b were identified by iteratively using a tandem structure-based approach: docking of potential ligands from small databases to hTR-P2b via the program MORDOR, which permits flexibility in both ligand and target, with subsequent NMR screening of high-ranking compounds. A high percentage of the compounds tested experimentally were found via NMR to bind to the U-rich region of hTR-P2b; most have MW < 500 Da and are from different compound classes, and several possess a charge of 0 or +1. Of the 48 ligands identified, 24 exhibit a decided preference to bind hTR-P2b RNA rather than A-site rRNA and 10 do not bind A-site rRNA at all. Binding affinity was measured by monitoring RNA imino proton resonances for some of the compounds that showed hTR binding preference.
J. Am. Chem. Soc., Article ASAP
Combined NMR and DFT Studies for the Absolute Configuration Elucidation of the Spore Photoproduct, a UV-Induced DNA Lesion
Claire Mantel§, Alexia Chandor†‡, Didier Gasparutto†, Thierry Douki†, Mohamed Atta‡, Marc Fontecave‡, Pierre-Alain Bayle§, Jean-Marie Mouesca*§ and Michel Bardet*§
Abstract
By irradiation of bacterial spores under UV radiation, a photoproduct (SP) bearing a covalent methylene link between two adjacent thymines is formed in DNA. Because of the presence of an asymmetric carbon on the aglycone and of two possible orientations for the formation of the cross-link, four isomers could in principle be obtained. Currently, no conclusive structural information of this photoproduct is available. The structure of the isolated SPTpT dinucleotide was revisited in order to determine the type of cross-link and the absolute configuration of the C5a carbon. For this purpose, a study combining NMR spectroscopy and DFT calculations was pursued on the spore photoproduct of the dinucleoside TpT since its structure was previously shown to be identical to the one produced in DNA. A full characterization of SPTpT by NMR analyses was performed in D2O and DMSO. 2D NMR measurements (1H−13C, 1H−31P, COSY, NOESY, and ROESY) and DFT calculations (geometries optimization of R and S isomers and theoretical chemical shifts) lead us to conclude without ambiguity that the absolute configuration of the C5a carbon is R and that the methylene bridge of the photoproduct corresponds to the methyl group of the thymine located on the 3′-end of the dinucleoside monophosphate.
Claire Mantel§, Alexia Chandor†‡, Didier Gasparutto†, Thierry Douki†, Mohamed Atta‡, Marc Fontecave‡, Pierre-Alain Bayle§, Jean-Marie Mouesca*§ and Michel Bardet*§
Abstract
By irradiation of bacterial spores under UV radiation, a photoproduct (SP) bearing a covalent methylene link between two adjacent thymines is formed in DNA. Because of the presence of an asymmetric carbon on the aglycone and of two possible orientations for the formation of the cross-link, four isomers could in principle be obtained. Currently, no conclusive structural information of this photoproduct is available. The structure of the isolated SPTpT dinucleotide was revisited in order to determine the type of cross-link and the absolute configuration of the C5a carbon. For this purpose, a study combining NMR spectroscopy and DFT calculations was pursued on the spore photoproduct of the dinucleoside TpT since its structure was previously shown to be identical to the one produced in DNA. A full characterization of SPTpT by NMR analyses was performed in D2O and DMSO. 2D NMR measurements (1H−13C, 1H−31P, COSY, NOESY, and ROESY) and DFT calculations (geometries optimization of R and S isomers and theoretical chemical shifts) lead us to conclude without ambiguity that the absolute configuration of the C5a carbon is R and that the methylene bridge of the photoproduct corresponds to the methyl group of the thymine located on the 3′-end of the dinucleoside monophosphate.
Tuesday, November 18, 2008
Al's Update
Consolidated silica glass from nanoparticles
Journal of Solid State Chemistry 181 (2008) 2442– 2447
Thomas G. Mayerhöfer, Zhijian Shen, Ekaterina Leonova, Mattias Edén, Antje Kriltz, Jürgen Popp
Abstract:
A dense silica glass was prepared by consolidating a highly dispersed silicic acid powder (particle size less than 10 nm) with the Spark Plasma Sintering (SPS) technique. The glass was characterized by ellipsometry, transmission electron microscopy (TEM), infrared reflectance and transmittance spectroscopy, as well as by Raman, UV–Vis–NIR and solid-state nuclear magnetic resonance (NMR) spectroscopy. The prototypic sample showed a transmittance of about 63% compared to silica glass in the UV–Vis spectral range. Based on the results of infrared transmittance spectroscopy this lower transparency is due to the comparably high water content, which is about 40 times higher than that in silica glass. 1H magic-angle spinning (MAS) NMR confirmed an increase in hydroxyl groups in the sample prepared by SPS relative to that of the conventional SiO2 reference glass. Aside from the comparably high water content, we conclude from the similarity of the IR-reflectance and the 29Si MAS NMR spectra of the SPS sample and the corresponding spectra of the conventionally prepared silica glass, that the short- and medium-range order is virtually the same in both materials. Raman spectroscopy, however, suggests that the number of three- and four-membered rings is significantly smaller in the SPS sample compared to the conventionally prepared sample. Based on these results we conclude that it is possible to prepare glasses by compacting amorphous powders by the SPS process. The SPS process may therefore enable the preparation of glasses with compositions inaccessible by conventional methods.
The mechanism of paramagnetic NMR relaxation produced by Mn(II): Role of orthorhombic and fourth-order zero field splitting terms
J. Chem. Phys. 129, 144307 (2008)
Robert Sharp
Abstract:
Mn(II) is a spin-5/2 paramagnetic ion that mediates a characteristically large NMR paramagnetic relaxation enhancement (NMR-PRE) of nuclear spins in solution. In the range of high magnetic field strengths (above about 0.3 T), where the electronic Zeeman interaction provides the largest term of the electron spin Hamiltonian, NMR relaxation mechanism is well understood. In the lower field range, the physical picture is more complex because of the presence in the spin Hamiltonian of zero field splitting (ZFS) terms that are comparable to or greater than the Zeeman term. This work describes a systematic study of the relaxation mechanism in the low field range, particularly aspects involving the dependence of NMR-PRE on the orthorhombic (E) and fourth-order (a q(4)">, q=0,2,4) ZFS tensor components. It is shown that the fourfold (a4(4)"> ) and twofold (a2(4)"> ) fourth-order components exert large orientation-dependent influences on the NMR-PRE. Thus, fourth-order terms with magnitudes equal to only a few percent of the quadratic ZFS terms (D,E) produce large changes in the shape of the magnetic field profile of the PRE. Effects arising from the orthorhombic quadratic ZFS term (E) are much smaller than those of the fourth-order terms and can in most cases be neglected. However, effects due to a4(4)"> and a2(4)"> need to be included in simulations of low field data.
A magic-angle-spinning NMR method for 1H–1H distance measurement using coherent polarization transfer in 13C-labeled organic solids
J. Chem. Phys. 129, 154504 (2008)
Hiroki Takahashi, Hideo Akutsu, and Toshimichi Fujiwara
Abstract:
We have developed a theory for 1H–1H distance measurements from the direct polarization transfer in 13C-labeled solids under magic-angle spinning. The polarization transfer caused by the 1H–1H dipolar interactions was analyzed with zeroth-order average Hamiltonian for a 1H–13C–13C–1H spin system in the frame modulated by 13C–1H dipolar interactions and chemical shifts. Strong 13C–1H dipolar couplings primarily determine the recovery of the 1H–1H coupling as a function of sample spinning frequency. The effect of additional 1H spins on the polarization transfer was also taken into account. We have applied this method to the distance measurements for uniformly 13C-, 15N-labeled L-valine and adenosine. Experimental 1H polarization transfer was monitored through high-resolution 13C-NMR. The theoretical analysis provided the distances up to about 3 Å with an accuracy of about 0.2 Å and those of about 4 Å with 1 Å even from the transfer amplitudes at a few mixing times. The longer distances are partly affected by the relayed polarization transfer which makes apparent 1H–1H distances shorter. Our theory based on the coherent polarization transfer in the initial build-up regime was compared to the description by the rate equations with spin diffusion time constants.
Symmetry-based recoupling in double-rotation NMR spectroscopy
J. Chem. Phys. 129, 174507 (2008)
Andreas Brinkmann, Arno P. M. Kentgens, Tiit Anupõld, and Ago Samoson
Abstract:
In this contribution, we extend the theory of symmetry-based pulse sequences of types CN νn and RN νn in magic-angle-spinning nuclear resonance spectroscopy [M. H. Levitt, in Encyclopedia of Nuclear Magnetic Resonance, edited by D. M. Grant and R. K. Harris (Wiley, Chichester, 2002), Vol. 9]. to the case of rotating the sample simultaneously around two different angles with respect to the external magnetic field (double-rotation). We consider the case of spin-1/2 nuclei in general and the case of half-integer quadrupolar nuclei that are subjected to weak radio frequency pulses operating selectively on the central-transition polarizations. The transformation properties of the homonuclear dipolar interactions and J-couplings under central-transition-selective spin rotations are presented. We show that the pulse sequence R221R22-1 originally developed for homonuclear dipolar recoupling of half-integer quadrupolar nuclei under magic-angle-spinning conditions [M. Edén, D. Zhou, and J. Yu, Chem. Phys. Lett. 431, 397 (2006)] may be used for the same purpose in the case of double rotation, if the radio frequency pulses are synchronized with the outer rotation of the sample. We apply this sequence, sandwiched by central-transition selective 90° pulses, to excite double-quantum coherences in homonuclear spin systems consisting of 23Na and 27Al nuclei.
Three Polymorphic Forms of the Co-Crystal 4,4-Bipyridine/Pimelic Acid and their Structural, Thermal, and Spectroscopic Characterization
Chem. Eur. J., Volume 14 Issue 32, Pages 10149 - 10159
Dario Braga, Prof., Giuseppe Palladino, Dr., Marco Polito, Dr., Katia Rubini, Dr., Fabrizia Grepioni, Prof., Michele R. Chierotti, Dr., Roberto Gobetto, Prof.
Abstract:
Three crystal forms of the co-crystal 4,4-bipy/pimelic acid (bipy: bipyridine), [NH4C5-C5H4N][HOOC(CH2)5COOH], have been prepared and their relationship investigated by single-crystal X-ray diffraction, variable-temperature X-ray powder diffraction, differential scanning calorimetry and solid-state NMR spectroscopy. Both X-ray and NMR spectroscopic results indicate that no proton transfer takes place, that is, the three crystal forms are true co-crystals of neutral molecules. Forms I and II both convert into Form III at high temperature, Forms II and III being the thermodynamically stable forms at room and high temperature, respectively.
Journal of Solid State Chemistry 181 (2008) 2442– 2447
Thomas G. Mayerhöfer, Zhijian Shen, Ekaterina Leonova, Mattias Edén, Antje Kriltz, Jürgen Popp
Abstract:
A dense silica glass was prepared by consolidating a highly dispersed silicic acid powder (particle size less than 10 nm) with the Spark Plasma Sintering (SPS) technique. The glass was characterized by ellipsometry, transmission electron microscopy (TEM), infrared reflectance and transmittance spectroscopy, as well as by Raman, UV–Vis–NIR and solid-state nuclear magnetic resonance (NMR) spectroscopy. The prototypic sample showed a transmittance of about 63% compared to silica glass in the UV–Vis spectral range. Based on the results of infrared transmittance spectroscopy this lower transparency is due to the comparably high water content, which is about 40 times higher than that in silica glass. 1H magic-angle spinning (MAS) NMR confirmed an increase in hydroxyl groups in the sample prepared by SPS relative to that of the conventional SiO2 reference glass. Aside from the comparably high water content, we conclude from the similarity of the IR-reflectance and the 29Si MAS NMR spectra of the SPS sample and the corresponding spectra of the conventionally prepared silica glass, that the short- and medium-range order is virtually the same in both materials. Raman spectroscopy, however, suggests that the number of three- and four-membered rings is significantly smaller in the SPS sample compared to the conventionally prepared sample. Based on these results we conclude that it is possible to prepare glasses by compacting amorphous powders by the SPS process. The SPS process may therefore enable the preparation of glasses with compositions inaccessible by conventional methods.
The mechanism of paramagnetic NMR relaxation produced by Mn(II): Role of orthorhombic and fourth-order zero field splitting terms
J. Chem. Phys. 129, 144307 (2008)
Robert Sharp
Abstract:
Mn(II) is a spin-5/2 paramagnetic ion that mediates a characteristically large NMR paramagnetic relaxation enhancement (NMR-PRE) of nuclear spins in solution. In the range of high magnetic field strengths (above about 0.3 T), where the electronic Zeeman interaction provides the largest term of the electron spin Hamiltonian, NMR relaxation mechanism is well understood. In the lower field range, the physical picture is more complex because of the presence in the spin Hamiltonian of zero field splitting (ZFS) terms that are comparable to or greater than the Zeeman term. This work describes a systematic study of the relaxation mechanism in the low field range, particularly aspects involving the dependence of NMR-PRE on the orthorhombic (E) and fourth-order (a q(4)">, q=0,2,4) ZFS tensor components. It is shown that the fourfold (a4(4)"> ) and twofold (a2(4)"> ) fourth-order components exert large orientation-dependent influences on the NMR-PRE. Thus, fourth-order terms with magnitudes equal to only a few percent of the quadratic ZFS terms (D,E) produce large changes in the shape of the magnetic field profile of the PRE. Effects arising from the orthorhombic quadratic ZFS term (E) are much smaller than those of the fourth-order terms and can in most cases be neglected. However, effects due to a4(4)"> and a2(4)"> need to be included in simulations of low field data.
A magic-angle-spinning NMR method for 1H–1H distance measurement using coherent polarization transfer in 13C-labeled organic solids
J. Chem. Phys. 129, 154504 (2008)
Hiroki Takahashi, Hideo Akutsu, and Toshimichi Fujiwara
Abstract:
We have developed a theory for 1H–1H distance measurements from the direct polarization transfer in 13C-labeled solids under magic-angle spinning. The polarization transfer caused by the 1H–1H dipolar interactions was analyzed with zeroth-order average Hamiltonian for a 1H–13C–13C–1H spin system in the frame modulated by 13C–1H dipolar interactions and chemical shifts. Strong 13C–1H dipolar couplings primarily determine the recovery of the 1H–1H coupling as a function of sample spinning frequency. The effect of additional 1H spins on the polarization transfer was also taken into account. We have applied this method to the distance measurements for uniformly 13C-, 15N-labeled L-valine and adenosine. Experimental 1H polarization transfer was monitored through high-resolution 13C-NMR. The theoretical analysis provided the distances up to about 3 Å with an accuracy of about 0.2 Å and those of about 4 Å with 1 Å even from the transfer amplitudes at a few mixing times. The longer distances are partly affected by the relayed polarization transfer which makes apparent 1H–1H distances shorter. Our theory based on the coherent polarization transfer in the initial build-up regime was compared to the description by the rate equations with spin diffusion time constants.
Symmetry-based recoupling in double-rotation NMR spectroscopy
J. Chem. Phys. 129, 174507 (2008)
Andreas Brinkmann, Arno P. M. Kentgens, Tiit Anupõld, and Ago Samoson
Abstract:
In this contribution, we extend the theory of symmetry-based pulse sequences of types CN νn and RN νn in magic-angle-spinning nuclear resonance spectroscopy [M. H. Levitt, in Encyclopedia of Nuclear Magnetic Resonance, edited by D. M. Grant and R. K. Harris (Wiley, Chichester, 2002), Vol. 9]. to the case of rotating the sample simultaneously around two different angles with respect to the external magnetic field (double-rotation). We consider the case of spin-1/2 nuclei in general and the case of half-integer quadrupolar nuclei that are subjected to weak radio frequency pulses operating selectively on the central-transition polarizations. The transformation properties of the homonuclear dipolar interactions and J-couplings under central-transition-selective spin rotations are presented. We show that the pulse sequence R221R22-1 originally developed for homonuclear dipolar recoupling of half-integer quadrupolar nuclei under magic-angle-spinning conditions [M. Edén, D. Zhou, and J. Yu, Chem. Phys. Lett. 431, 397 (2006)] may be used for the same purpose in the case of double rotation, if the radio frequency pulses are synchronized with the outer rotation of the sample. We apply this sequence, sandwiched by central-transition selective 90° pulses, to excite double-quantum coherences in homonuclear spin systems consisting of 23Na and 27Al nuclei.
Three Polymorphic Forms of the Co-Crystal 4,4-Bipyridine/Pimelic Acid and their Structural, Thermal, and Spectroscopic Characterization
Chem. Eur. J., Volume 14 Issue 32, Pages 10149 - 10159
Dario Braga, Prof., Giuseppe Palladino, Dr., Marco Polito, Dr., Katia Rubini, Dr., Fabrizia Grepioni, Prof., Michele R. Chierotti, Dr., Roberto Gobetto, Prof.
Abstract:
Three crystal forms of the co-crystal 4,4-bipy/pimelic acid (bipy: bipyridine), [NH4C5-C5H4N][HOOC(CH2)5COOH], have been prepared and their relationship investigated by single-crystal X-ray diffraction, variable-temperature X-ray powder diffraction, differential scanning calorimetry and solid-state NMR spectroscopy. Both X-ray and NMR spectroscopic results indicate that no proton transfer takes place, that is, the three crystal forms are true co-crystals of neutral molecules. Forms I and II both convert into Form III at high temperature, Forms II and III being the thermodynamically stable forms at room and high temperature, respectively.
Monday, November 10, 2008
Journal of Magnetic Resonance - Vol 195 Issue 2
Band selective small flip angle COSY: A simple experiment for the analyses of 1H NMR spectra of small chiral molecules
Uday Ramesh Prabhu and N. Suryaprakash
The NMR spectroscopic discrimination of enantiomers in the chiral liquid crystalline solvent is more often carried out using 2H detection in its natural abundance. The employment of 1H detection for such a purpose is severely hampered due to significant loss of resolution in addition to indistinguishable overlap of the spectra from the two enantiomers. This study demonstrates that the band selected small flip angle homonuclear correlation experiment is a simple and robust technique that provides unambiguous discrimination, very high spectral resolution, reduced multiplicity of transitions, relative signs of the couplings and enormous saving of instrument time.
Biomolecular solid state NMR with magic-angle spinning at 25 K
Kent R. Thurber and Robert Tycko
A magic-angle spinning (MAS) probe has been constructed which allows the sample to be cooled with helium, while the MAS bearing and drive gases are nitrogen. The sample can be cooled to 25 K using roughly 3 L/h of liquid helium, while the 4-mm diameter rotor spins at 6.7 kHz with good stability (±5 Hz) for many hours. Proton decoupling fields up to at least 130 kHz can be applied. This helium-cooled MAS probe enables a variety of one-dimensional and two-dimensional NMR experiments on biomolecular solids and other materials at low temperatures, with signal-to-noise proportional to 1/T. We show examples of low-temperature 13C NMR data for two biomolecular samples, namely the peptide Aβ14–23 in the form of amyloid fibrils and the protein HP35 in frozen glycerol/water solution. Issues related to temperature calibration, spin–lattice relaxation at low temperatures, paramagnetic doping of frozen solutions, and 13C MAS NMR linewidths are discussed.
Single-scan 2D DOSY NMR spectroscopy
Yoav Shrot, and Lucio Frydman
Spatial encoding is a particular kind of spin manipulation that enables the acquisition of multidimensional NMR spectra within a single scan. This encoding has been shown to possess a general applicability and to enable the completion of arbitrary nD NMR acquisitions within a single transient. The present study explores its potential towards the acquisition of 2D DOSY spectra, where the indirect dimension is meant to encode molecular displacements rather than a coherent spin evolution. We find that in its simplest form this extension shows similarities with methods that have been recently discussed for the single-scan acquisition of this kind of traces; still, a number of advantageous features are also evidenced by the “ultrafast” modality hereby introduced. The principles underlying the operation of this new single-scan 2D DOSY approach are discussed, its use is illustrated with a variety of sequences and of samples, the limitations of this new experiment are noted, and potential extensions of the methodology are mentioned.
Uday Ramesh Prabhu and N. Suryaprakash
The NMR spectroscopic discrimination of enantiomers in the chiral liquid crystalline solvent is more often carried out using 2H detection in its natural abundance. The employment of 1H detection for such a purpose is severely hampered due to significant loss of resolution in addition to indistinguishable overlap of the spectra from the two enantiomers. This study demonstrates that the band selected small flip angle homonuclear correlation experiment is a simple and robust technique that provides unambiguous discrimination, very high spectral resolution, reduced multiplicity of transitions, relative signs of the couplings and enormous saving of instrument time.
Biomolecular solid state NMR with magic-angle spinning at 25 K
Kent R. Thurber and Robert Tycko
A magic-angle spinning (MAS) probe has been constructed which allows the sample to be cooled with helium, while the MAS bearing and drive gases are nitrogen. The sample can be cooled to 25 K using roughly 3 L/h of liquid helium, while the 4-mm diameter rotor spins at 6.7 kHz with good stability (±5 Hz) for many hours. Proton decoupling fields up to at least 130 kHz can be applied. This helium-cooled MAS probe enables a variety of one-dimensional and two-dimensional NMR experiments on biomolecular solids and other materials at low temperatures, with signal-to-noise proportional to 1/T. We show examples of low-temperature 13C NMR data for two biomolecular samples, namely the peptide Aβ14–23 in the form of amyloid fibrils and the protein HP35 in frozen glycerol/water solution. Issues related to temperature calibration, spin–lattice relaxation at low temperatures, paramagnetic doping of frozen solutions, and 13C MAS NMR linewidths are discussed.
Single-scan 2D DOSY NMR spectroscopy
Yoav Shrot, and Lucio Frydman
Spatial encoding is a particular kind of spin manipulation that enables the acquisition of multidimensional NMR spectra within a single scan. This encoding has been shown to possess a general applicability and to enable the completion of arbitrary nD NMR acquisitions within a single transient. The present study explores its potential towards the acquisition of 2D DOSY spectra, where the indirect dimension is meant to encode molecular displacements rather than a coherent spin evolution. We find that in its simplest form this extension shows similarities with methods that have been recently discussed for the single-scan acquisition of this kind of traces; still, a number of advantageous features are also evidenced by the “ultrafast” modality hereby introduced. The principles underlying the operation of this new single-scan 2D DOSY approach are discussed, its use is illustrated with a variety of sequences and of samples, the limitations of this new experiment are noted, and potential extensions of the methodology are mentioned.
Friday, November 07, 2008
J. Am. Chem. Soc., 130 (44), 14521–14532, 2008.
Raftlike Mixtures of Sphingomyelin and Cholesterol Investigated by Solid-State 2H NMR Spectroscopy
Tim Bartels, Ravi S. Lankalapalli, Robert Bittman, Klaus Beyer, and Michael F. Brown
Abstract:
Sphingomyelin is a lipid that is abundant in the nervous systems of mammals, where it is associated with putative microdomains in cellular membranes and undergoes alterations due to aging or neurodegeneration. We investigated the effect of varying the concentration of cholesterol in binary and ternary mixtures with N-palmitoylsphingomyelin (PSM) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using deuterium nuclear magnetic resonance (2H NMR) spectroscopy in both macroscopically aligned and unoriented multilamellar dispersions. In our experiments, we used PSM and POPC perdeuterated on the N-acyl and sn-1 acyl chains, respectively. By measuring solid-state 2H NMR spectra of the two lipids separately in mixtures with the same compositions as a function of cholesterol mole fraction and temperature, we obtained clear evidence for the coexistence of two liquid-crystalline domains in distinct regions of the phase diagram. According to our analysis of the first moments M1 and the observed 2H NMR spectra, one of the domains appears to be a liquid-ordered phase. We applied a mean-torque potential model as an additional tool to calculate the average hydrocarbon thickness, the area per lipid, and structural parameters such as chain extension and thermal expansion coefficient in order to further define the two coexisting phases. Our data imply that phase separation takes place in raftlike ternary PSM/POPC/cholesterol mixtures over a broad temperature range but vanishes at cholesterol concentrations equal to or greater than a mole fraction of 0.33. Cholesterol interacts preferentially with sphingomyelin only at smaller mole fractions, above which a homogeneous liquid-ordered phase is present. The reasons for these phase separation phenomena seem to be differences in the effects of cholesterol on the configurational order of the palmitoyl chains in PSM-d31 and POPC-d31 and a difference in the affinity of cholesterol for sphingomyelin observed at low temperatures. Hydrophobic matching explains the occurrence of raftlike domains in cellular membranes at intermediate cholesterol concentrations but not saturating amounts of cholesterol.
Tim Bartels, Ravi S. Lankalapalli, Robert Bittman, Klaus Beyer, and Michael F. Brown
Abstract:
Sphingomyelin is a lipid that is abundant in the nervous systems of mammals, where it is associated with putative microdomains in cellular membranes and undergoes alterations due to aging or neurodegeneration. We investigated the effect of varying the concentration of cholesterol in binary and ternary mixtures with N-palmitoylsphingomyelin (PSM) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using deuterium nuclear magnetic resonance (2H NMR) spectroscopy in both macroscopically aligned and unoriented multilamellar dispersions. In our experiments, we used PSM and POPC perdeuterated on the N-acyl and sn-1 acyl chains, respectively. By measuring solid-state 2H NMR spectra of the two lipids separately in mixtures with the same compositions as a function of cholesterol mole fraction and temperature, we obtained clear evidence for the coexistence of two liquid-crystalline domains in distinct regions of the phase diagram. According to our analysis of the first moments M1 and the observed 2H NMR spectra, one of the domains appears to be a liquid-ordered phase. We applied a mean-torque potential model as an additional tool to calculate the average hydrocarbon thickness, the area per lipid, and structural parameters such as chain extension and thermal expansion coefficient in order to further define the two coexisting phases. Our data imply that phase separation takes place in raftlike ternary PSM/POPC/cholesterol mixtures over a broad temperature range but vanishes at cholesterol concentrations equal to or greater than a mole fraction of 0.33. Cholesterol interacts preferentially with sphingomyelin only at smaller mole fractions, above which a homogeneous liquid-ordered phase is present. The reasons for these phase separation phenomena seem to be differences in the effects of cholesterol on the configurational order of the palmitoyl chains in PSM-d31 and POPC-d31 and a difference in the affinity of cholesterol for sphingomyelin observed at low temperatures. Hydrophobic matching explains the occurrence of raftlike domains in cellular membranes at intermediate cholesterol concentrations but not saturating amounts of cholesterol.
Thursday, November 06, 2008
J. Am. Chem. Soc., 130 (43), 14280–14286, 2008.
NMR-Based Mapping of Disulfide Bridges in Cysteine-Rich Peptides: Application to the μ-Conotoxin SxIIIA
Aleksandra Walewska,†‡ Jack J. Skalicky,§ Darrell R. Davis,∥ Min-Min Zhang,† Estuardo Lopez-Vera,†⊥ Maren Watkins,# Tiffany S. Han,† Doju Yoshikami,† Baldomero M. Olivera,† and Grzegorz Bulaj*∥
Abstract:
Disulfide-rich peptides represent a megadiverse group of natural products with very promising therapeutic potential. To accelerate their functional characterization, high-throughput chemical synthesis and folding methods are required, including efficient mapping of multiple disulfide bridges. Here, we describe a novel approach for such mapping and apply it to a three-disulfide-bridged conotoxin, μ-SxIIIA (from the venom of Conus striolatus), whose discovery is also reported here for the first time. μ-SxIIIA was chemically synthesized with three cysteine residues labeled 100% with 15N/13C, while the remaining three cysteine residues were incorporated using a mixture of 70%/30% unlabeled/labeled Fmoc-protected residues. After oxidative folding, the major product was analyzed by NMR spectroscopy. Sequence-specific resonance assignments for the isotope-enriched Cys residues were determined with 2D versions of standard triple-resonance (1H, 13C, 15N) NMR experiments and 2D [13C, 1H] HSQC. Disulfide patterns were directly determined with cross-disulfide NOEs confirming that the oxidation product had the disulfide connectivities characteristic of μ-conotoxins. μ-SxIIIA was found to be a potent blocker of the sodium channel subtype NaV1.4 (IC50 = 7 nM). These results suggest that differential incorporation of isotope-labeled cysteine residues is an efficient strategy to map disulfides and should facilitate the discovery and structure−function studies of many bioactive peptides.
Aleksandra Walewska,†‡ Jack J. Skalicky,§ Darrell R. Davis,∥ Min-Min Zhang,† Estuardo Lopez-Vera,†⊥ Maren Watkins,# Tiffany S. Han,† Doju Yoshikami,† Baldomero M. Olivera,† and Grzegorz Bulaj*∥
Abstract:
Disulfide-rich peptides represent a megadiverse group of natural products with very promising therapeutic potential. To accelerate their functional characterization, high-throughput chemical synthesis and folding methods are required, including efficient mapping of multiple disulfide bridges. Here, we describe a novel approach for such mapping and apply it to a three-disulfide-bridged conotoxin, μ-SxIIIA (from the venom of Conus striolatus), whose discovery is also reported here for the first time. μ-SxIIIA was chemically synthesized with three cysteine residues labeled 100% with 15N/13C, while the remaining three cysteine residues were incorporated using a mixture of 70%/30% unlabeled/labeled Fmoc-protected residues. After oxidative folding, the major product was analyzed by NMR spectroscopy. Sequence-specific resonance assignments for the isotope-enriched Cys residues were determined with 2D versions of standard triple-resonance (1H, 13C, 15N) NMR experiments and 2D [13C, 1H] HSQC. Disulfide patterns were directly determined with cross-disulfide NOEs confirming that the oxidation product had the disulfide connectivities characteristic of μ-conotoxins. μ-SxIIIA was found to be a potent blocker of the sodium channel subtype NaV1.4 (IC50 = 7 nM). These results suggest that differential incorporation of isotope-labeled cysteine residues is an efficient strategy to map disulfides and should facilitate the discovery and structure−function studies of many bioactive peptides.
J. Am. Chem. Soc., 130 (43), 14179–14188, 2008.
Microsolvation and 13C−Li NMR Coupling
Rudolf Knorr,* Thomas Menke, Kathrin Ferchland, Johann Mehlstäubl, and David S. Stephenson
Abstract:
The empirical expression 1JCLi = L[n(a + d)]−1 is proposed; it claims a reciprocal dependence of the NMR coupling constant 1J(13C, Li) in a C−Li compound on two factors: (i) the number n of lithium nuclei in bonding contact with the observed carbanion center and (ii) the sum (a + d) of the numbers a of anions and d of donor ligands coordinated at the Li nucleus that generates the observed 1JCLi value. The expression was derived from integrations of separate NMR resonances of coordinated and free monodentate donor ligands (t-BuOMe, Et2O, or THF) in toluene solutions of dimeric and monomeric 2-(α-aryl-α-lithiomethylidene)-1,1,3,3-tetramethylindan at moderately low temperatures. This unusually slow ligand interchange is ascribed to steric congestion in these compounds, which is further characterized by measurements of nuclear Overhauser correlations and by solid-state structures of the dimers bearing only one donor per lithium atom (d = 1). Increasing microsolvation numbers d are also accompanied by typical changes of the NMR chemical shifts δ (positive for the carbanionic 13Cα, negative for Cpara and p-H). The aforementioned empirical expression for 1JCLi appears to be applicable to other cases of solvated monomeric, dimeric, or tetrameric C−Li compounds (alkyl, alkenyl, alkynyl, and aryl) and even to unsolvated (d ≈ 0) trimeric, tetrameric, or hexameric organolithium aggregates, indicating that 1JCLi might serve as a tool for assessing unknown microsolvation numbers. The importance of obtaining evidence about the 13C NMR C−Li multiplet splitting of both the nonfluxional and fluxional aggregates is emphasized.
Rudolf Knorr,* Thomas Menke, Kathrin Ferchland, Johann Mehlstäubl, and David S. Stephenson
Abstract:
The empirical expression 1JCLi = L[n(a + d)]−1 is proposed; it claims a reciprocal dependence of the NMR coupling constant 1J(13C, Li) in a C−Li compound on two factors: (i) the number n of lithium nuclei in bonding contact with the observed carbanion center and (ii) the sum (a + d) of the numbers a of anions and d of donor ligands coordinated at the Li nucleus that generates the observed 1JCLi value. The expression was derived from integrations of separate NMR resonances of coordinated and free monodentate donor ligands (t-BuOMe, Et2O, or THF) in toluene solutions of dimeric and monomeric 2-(α-aryl-α-lithiomethylidene)-1,1,3,3-tetramethylindan at moderately low temperatures. This unusually slow ligand interchange is ascribed to steric congestion in these compounds, which is further characterized by measurements of nuclear Overhauser correlations and by solid-state structures of the dimers bearing only one donor per lithium atom (d = 1). Increasing microsolvation numbers d are also accompanied by typical changes of the NMR chemical shifts δ (positive for the carbanionic 13Cα, negative for Cpara and p-H). The aforementioned empirical expression for 1JCLi appears to be applicable to other cases of solvated monomeric, dimeric, or tetrameric C−Li compounds (alkyl, alkenyl, alkynyl, and aryl) and even to unsolvated (d ≈ 0) trimeric, tetrameric, or hexameric organolithium aggregates, indicating that 1JCLi might serve as a tool for assessing unknown microsolvation numbers. The importance of obtaining evidence about the 13C NMR C−Li multiplet splitting of both the nonfluxional and fluxional aggregates is emphasized.
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