Quantification of free ligand conformational preferences by NMR and their relationship to the bioactive conformation

Accurate unbound solution 3D-structures of ligands provide unique opportunities for medicinal chemistry and, in particular, a context to understand binding thermodynamics and kinetics. Previous methods of deriving these 3D-structures have had neither the accuracy nor resolution needed for drug design and have not yet realized their potential. Here, we describe and apply a NMR methodology to the aminoglycoside streptomycin that can accurately quantify accessible 3D-space and rank the occupancy of observed conformers to a resolution that enables medicinal chemistry understanding and design. Importantly, it is based upon conventional small molecule NMR techniques and can be performed in physiologically-relevant solvents. The methodology uses multiple datasets, an order of magnitude more experimental data than previous NMR approaches and a dynamic model during refinement, is independent of computational chemistry and avoids the problem of virtual conformations. The refined set of solution 3D-shapes for streptomycin can be grouped into two major families, of which the most populated is almost identical to the 30S ribosomal subunit bioactive shape. We therefore propose that accurate unbound ligand solution conformations may, in some cases, provide a subsidiary route to bioactive shape without crystallography. This experimental technique opens up new opportunities for drug design and more so when complemented with protein co-crystal structures, SAR data and pharmacophore modeling.     

The role of terminal amino group and histidine at the fourth position in the metal ion binding of oligopeptides revisited: Copper(II) and nickel(II) complexes of glycyl-glycyl-glycyl-histamine and its N-Boc protected derivative

Copper(II) and nickel(II) binding properties of two pseudo tetrapeptides, N-Boc-Gly-Gly-Gly-Histamine (BGGGHa) and Gly-Gly-Gly-Histamine (GGGHa) have been investigated by pH-potentiometric titrations, UV-visible-, EPR-, NMR- and ESI-HRMS (electrospray ionization high resolution MS) spectroscopies, in order to compare the role of N-terminal amino group and imidazole moiety at the fourth position in the complex formation processes. Substantially higher stabilities were determined for the ML complexes of GGGHa, compared to those of BGGGHa, supporting the coordination of the terminal amino group and the histamine imidazole of the non-protected ligand. A dimeric Cu₂H_− 2L₂ species, formed through the deprotonation of peptide groups of the ligands, was found in the GGGHa-copper(II) system. Deprotonation and coordination of further amide nitrogens led to CuH_− 2L and, above pH ~ 10, CuH_− 3L. Experimental data supports a {NH₂,2 × N_amide,N_im} macrochelate structure in CuH_− 2L whereas a {NH₂,3 × N_amide} coordination environment in CuH_− 3L. The first two amide deprotonation processes were found to be strongly cooperative with nickel(II) and spectroscopic studies proved the transformation of the octahedral parent complexes to square planar, yellow, diamagnetic species, NiH_− 2L and above pH ~ 9, NiH_− 3L. In the basic pH-range deprotonation and coordination of the amide groups also took place in the BGGGHa containing systems, leading to complexes with a {3 × N_amide,N_im} donor set, and in parallel the re-dissolving of precipitate. Above pH ~ 11, a further proton release from the pyrrolic NH group of the imidazole ring of BGGGHa occurred providing an additional proof for the different binding modes of the two ligands.     

Effect of protein structural integrity on cross-linking by tyrosinase evidenced by multidimensional heteronuclear magnetic resonance spectroscopy

Enzymatic cross-linking of proteins can be catalyzed either by transferase-type enzymes, e.g., transglutaminases, or by oxidoreductases, e.g., tyrosinases or laccases. Three-dimensional structure of protein substrate plays a key role in these reactions, that is, the reactivity and end product are strongly modulated by the accessibility of target amino acid residues to the cross-linking enzyme. Typically structural integrity of protein can be distorted by heat, pH, or mechanical action, as well as by varying ionic concentration of the solution. In this study we used partially unfolded protein (wild-type DrkN SH3) and its structurally stabilized mutant (T22G) to investigate the impact of folded/unfolded conformations on cross-linking by Trichoderma reesei tyrosinase. Our results clearly showed formation of intermolecular cross-links solely between unfolded conformations, making them superior substrates to folded proteins when using tyrosinase as a cross-linking enzyme. Multidimensional heteronuclear magnetic resonance experiments in solution state were employed to investigate cross-linked end-products. The results presented in this study form basis for application development in food, medical, cosmetic, textile, packing and other sectors. In addition, the outcome of this study has a high value for the basic understanding of reaction mechanism of tyrosinases on proteins.     

The role of formic acid in solution stability and crystallization of silk protein polymer

In this paper, the regenerated silk fibroin (SF) solution dissolved in formic acid was used as a model protein to understand the role of formic acid in solution stability and crystallization of protein-based materials. The molecular decomposition of SF did not occur for the dissolution process in formic acid within 1–2 days of storage times. The β-sheet crystallization of SF molecules was occurred by the elimination of formic acid upon drying. The SF molecules in formic acid solution are stable and have low hydrodynamic radius values. This may be closely related to the fact that formic acid has two opposite functions of dissolution and crystallization simultaneously. The turbidity, dynamic light scattering and FTIR measurements elucidate that the solution stability and crystallization of SF are attributed to compact molecular shape of SF in formic acid, resulted from the molecular interactions between formic acid and polar groups in SF molecules.     

Contryphan-Vn: a modulator of Ca2+-dependent K+ channels

Contryphan-Vn is a D-tryptophan-containing disulfide-constrained nonapeptide isolated from the venom of Conus ventricosus, the single Mediterranean cone snail species. The structure of the synthetic Contryphan-Vn has been determined by NMR spectroscopy. Unique among Contryphans, Contryphan-Vn displays the peculiar presence of a Lys-Trp dyad, reminiscent of that observed in several voltage-gated K(+) channel blockers. Electrophysiological experiments carried out on dorsal unpaired median neurons isolated from the cockroach (Periplaneta americana) nerve cord on rat fetal chromaffin cells indicate that Contryphan-Vn affects both voltage-gated and Ca(2+)-dependent K(+) channel activities, with composite and diversified effects in invertebrate and vertebrate systems. Voltage-gated and Ca(2+)-dependent K(+) channels represent the first functional target identified for a conopeptide of the Contryphan family. Furthermore, Contryphan-Vn is the first conopeptide known to modulate the activity of Ca(2+)-dependent K(+) channels.     

Expression of mRNAs Encoding Subunits of the NMDA Receptor in Developing Rat Brain

Abstract: Developmental changes in the levels of N -methyl- d -aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low-affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5' insert and with or without both 3' inserts. In cerebellum, however, the major NR1 variants were those containing the 5' insert and lacking both 3' inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Syntheses, structures, thermal behavior and solution studies of two types of Hg(II) complexes with [1,3-di(2-methoxy)benzene]triazene

The reaction of [1,3-di(2-methoxy)benzene]triazene, [HL], with Hg(CH₃COO)₂ and Hg(SCN)₂ in methanol as solvent, resulted in the formation of [HgL₂] (1) and [HgL(SCN)] (2), respectively. These compounds were characterized by means of X-ray diffraction, FT-IR spectroscopy, CHN and TGA-DTA analysis. In the lattice of the compound 1, the mono-nuclear complexes were connected to dimer structure by intermolecular non-classical C-H···O hydrogen bonds. Also, weak Hg-η²-arene π-interactions link the dimers into 1D supramolecular chains. The compound 2 is a 2D coordination polymer induced by C-H···π stacking interactions between 1D chains produced by weak Hg-η³-η³-arene π-interactions. The results of studies of the stoichiometry and formation of complexes of 1 and 2 in methanol solution were found to be in support of their solid state stoichiometry.