Ab initio molecular dynamics-based assignment of the protonation state of pepstatin A/HIV-1 protease cleavage site

A recent 13C NMR experiment (Smith et al. Nature Struct. Biol. 1996, 3, 946-950) on the Asp 25-Asp25' dyad in pepstatin A/HIV-1 protease measured two separate resonance lines, which were interpreted as being a singly protonated dyad. We address this issue by performing ab initio molecular dynamics calculations on models for this site accompanied by calculations of 13C NMR chemical shifts and isotopic shifts. We find that already on the picosecond time-scale the model proposed by Smith et al. is not stable and evolves toward a different monoprotonated form whose NMR pattern differs from the experimental one. We suggest, instead, a different protonation state in which both aspartic groups are protonated. Despite the symmetric protonation state, the calculated 13C NMR properties are in good agreement with the experiment. We rationalize this result using a simple valence bond model, which explains the chemical inequality of the two C sites. The model calculations, together with our calculations on the complex, allow also the rationalization of 13C NMR properties on other HIV-1 PR/inhibitor complexes. Both putative binding of the substrate to the free enzyme, which has the dyad singly protonated (Piana, S.; Carloni, P. Proteins: Struct., Funct., Genet. 2000, 39, 26-36), and pepstatin A binding to the diprotonated form are consistent with the inverse solvent isotope effect on the onset of inhibition of pepsin by pepstatin and the kinetic iso-mechanism proposed for aspartic proteases (Cho, T.-K.; Rebholz, K.; Northrop, D.B. Biochemistry 1994, 33, 9637-9642).     

Comparisons of NMR spectral quality and success in crystallization demonstrate that NMR and X-ray crystallography are complementary methods for small protein structure determination

X-ray crystallography and NMR spectroscopy provide the only sources of experimental data from which protein structures can be analyzed at high or even atomic resolution. The degree to which these methods complement each other as sources of structural knowledge is a matter of debate; it is often proposed that small proteins yielding high quality, readily analyzed NMR spectra are a subset of those that readily yield strongly diffracting crystals. We have examined the correlation between NMR spectral quality and success in structure determination by X-ray crystallography for 159 prokaryotic and eukaryotic proteins, prescreened to avoid proteins providing polydisperse and/or aggregated samples. This study demonstrates that, across this protein sample set, the quality of a protein's [15N-1H]-heteronuclear correlation (HSQC) spectrum recorded under conditions generally suitable for 3D structure determination by NMR, a key predictor of the ability to determine a structure by NMR, is not correlated with successful crystallization and structure determination by X-ray crystallography. These results, together with similar results of an independent study presented in the accompanying paper (Yee, et al., J. Am. Chem. Soc., accompanying paper), demonstrate that X-ray crystallography and NMR often provide complementary sources of structural data and that both methods are required in order to optimize success for as many targets as possible in large-scale structural proteomics efforts.     

Synthesis and structure determination of kahalalide F (1,2).

Kahalalide F, the only member of the family of peptides called kahalalides, isolated from the sacoglossan mollusc Elysia rufescens and the green alga Bryopsis sp., with important bioactivity, is in clinical trials for treatment of prostate cancer. An efficient solid-phase synthetic approach is reported. Kahalalide F presents several synthetic difficulties: (i) an ester bond between two beta-branched and sterically hindered amino acids; (ii) a didehydroamino acid; and (iii) a rather hydrophobic sequence with two fragments containing several beta-branched amino acids in a row, one of them terminated with a saturated aliphatic acid. The cornerstones of our strategy were (i) a quasiorthogonal protecting system with allyl, tert-butyl, fluorenyl, and trityl-based groups, (ii) azabenzotriazole coupling reagents, (iii) formation of the didehydroamino acid residue on the solid phase, and (iv) cyclization and final purification in solution. HPLC, high-field NMR, and biological activity studies showed that the correct stereochemistry of the natural product is that proposed by Rinehart et al. whereas the stereochemistry proposed by Scheuer et al. is that of a biologically less active diastereoisomer.     

Evaluation of the precision of drop-size determination in oil/water emulsions by low-resolution NMR spectroscopy

The accuracy of the recently reported low-resolution NMR method (Goudappel, G. J. W.; et al. J. Colloid Interface Sci. 2001, 239, 535) for the determination of drop-size distribution in oil-in-water emulsions is evaluated by comparing the NMR results with precise data from video-enhanced optical microscopy. A series of 27 soybean-oil-in-water emulsions, differing in their mean drop size, polydispersity, oil volume fraction, and emulsifier, is studied. Soybean oil is selected as a typical component of food emulsions. The experimental error of our optical procedure for drop-size determination is estimated to be around 0.3 microm, which allows us to use the microscopy data as a reference for the mean drop-size and distribution width of the studied emulsions, with known experimental error. The main acquisition parameters in the NMR experiment are varied to find their optimal values and to check how the experimental conditions affect the NMR results. Comparison of the results obtained by the two methods shows that the low-resolution NMR method underestimates the mean drop size, d33, by approximately 20%. For most of the samples, NMR measures relatively precisely the distribution width (+/-0.1 to 0.2 dimensionless units), but for approximately 20% of the samples, larger systematic deviation was registered (underestimate by 0.3-0.4 units). No correlation is found between the emulsion properties and the relative difference between the microscopy and NMR data. Possible reasons for the observed discrepancy between NMR and optical microscopy are discussed, and some advantages and limitations of the low-resolution NMR method are considered.     

Studying posttranslational modifications by in-cell NMR

Functional in vivo investigation of posttranslational modifications is a problem that a number of analytical techniques are trying to tackle. Below, we briefly discuss the breakthroughs and challenges in placing NMR spectroscopy on the map, as illustrated by a recent report by Selenko et al. (2008).     

Density functional theory calculations of hydrogen-bond-mediated NMR J coupling in the solid state

A recently developed method for calculating NMR J coupling in solid-state systems is applied to calculate hydrogen-bond-mediated (2h) J NN couplings across intra- or intermolecular N-H...N hydrogen bonds in two 6-aminofulvene-1-aldimine derivatives and the ribbon structure formed by a deoxyguanosine derivative. Excellent quantitative agreement is observed between the calculated solid-state J couplings and those previously determined experimentally in two recent spin-echo magic-angle-spinning NMR studies ( Brown, S. P. ; et al. Chem. Commun. 2002, 1852-1853 and Pham, T. N. ; et al. Phys. Chem. Chem. Phys. 2007, 9, 3416-3423 ). For the 6-aminofulvene-1-aldimines, the differences in (2h) J NN couplings in pyrrole and triazole derivatives are reproduced, while for the guanosine ribbons, an increase in (2h) J NN is correlated with a decrease in the N-H...N hydrogen-bond distance. J couplings are additionally calculated for isolated molecules of the 6-aminofulevene-1-aldimines extracted from the crystal with and without further geometry optimization. Importantly, it is shown that experimentally observed differences between J couplings determined by solution- and solid-state NMR are not solely due to differences in geometry; long-range electrostatic effects of the crystal lattice are shown to be significant also. J couplings that are yet to be experimentally measured are calculated. Notably, (2h) J NO couplings across N-H...O hydrogen bonds are found to be of a similar magnitude to (2h) J NN couplings, suggesting that their utilization and quantitative determination should be experimentally feasible.     

NMR and X-ray crystallography, complementary tools in structural proteomics of small proteins

NMR spectroscopy and X-ray crystallography, the two primary experimental methods for protein structure determination at high resolution, have different advantages and disadvantages in terms of sample preparation and data collection and analysis. It is therefore of interest to assess their complementarity when applied to small proteins. Structural genomics/proteomics projects provide an ideal opportunity to make such comparisons as they generate data in a systematic manner for large enough numbers of proteins to allow firm conclusions to be drawn. Here we report a comparison for 263 unique proteins screened by both NMR spectroscopy and X-ray crystallography in our structural proteomics pipeline. Only 21 targets (8%) were deemed amenable to both methods based on an initial 2D 15N-HSQC NMR spectrum and optimized crystallization trials. However, the use of both methods in the pipeline increased the total number of targets amenable to structure determination to 107, with 43 amenable to NMR only and 43 amenable to X-ray crystallographic methods only. We did not observe a correlation between 15N-HSQC spectral quality and the success of the same protein in crystallization screens. Similar results were found for an independent set of 159 proteins as reported in the accompanying paper by Snyder et al. Thus, we conclude that both methods are highly complementary, and in order to increase the number of proteins suited for structure determination, we suggest that both methods be used in parallel in screening of all small proteins for structure determination.     

Studies on the Cotelomerization of Ethylene and Vinyl Chloride with Carbon Tetrachloride

The cotelomerization of ethylene and vinyl chloride with carbon tetrachloride was studied under various reaction conditions by using the amine-metallic salt initiating system. From the GC and NMR spectra, we found that no cotelomerization occured. This is contrary to the findings determined by Rosin et al. The modified insertion mechanism proposed by Asahada and Wu was used to explain the experimental results.     

Combined experimental-theoretical characterization of the hydrido-cobaloxime [HCo(dmgH)2(PnBu3)

A combined theoretical and experimental approach has been employed to characterize the hydrido-cobaloxime [HCo(dmgH)(2)(PnBu(3))] compound. This complex was originally investigated by Schrauzer et al. [Schrauzer et al., J. Am. Chem. Soc. 1971, 93,1505] and has since been referred to as a key, stable analogue of the hydride intermediate involved in hydrogen evolution catalyzed by cobaloxime compounds [Artero, V. et al. Angew. Chem., Int. Ed. 2011, 50, 7238-7266]. We employed quantum chemical calculations, using density functional theory and correlated RI-SCS-MP2 methods, to characterize the structural and electronic properties of the compound and observed important differences between the calculated (1)H NMR spectrum and that reported in the original study by Schrauzer and Holland. To calibrate the theoretical model, the stable hydrido tetraamine cobalt(III) complex [HCo(tmen)(2)(OH(2))](2+) (tmen = 2,3-dimethyl-butane-2,3-diamine) [Rahman, A. F. M. M. et al. Chem. Commun. 2003, 2748-2749] was subjected to a similar analysis, and, in this case, the calculated results agreed well with those obtained experimentally. As a follow-up to the computational work, the title hydrido-cobaloxime compound was synthesized and recharacterized experimentally, together with the Co(I) derivative, giving results that were in agreement with the theoretical predictions.     

Transannular Addition of Phenols to 1,1′‐Dialkynylferrocenes: Unanticipated Formation of Phenoxy[4]ferrocenophanedienes

The reaction of some 1,1′‐dialkynylferrocenes with a variety of phenols in the presence as well as in the absence of [Mo(CO)₆] yields good to high yields of phenoxy[4]ferrocenophanedienes. Similar reactivity was observed with a thiophenol and with acetic acid. Reaction under basic reaction conditions led to the formation of the [4]ferrocenophanone 17 . The phenoxy[4]ferrocenophanedienes obtained show dynamic behavior as a result of a torsional twist of the carbon bridge as indicated by the ¹H and ¹³C NMR spectra. The reaction mechanism is discussed in view of recent related results of Sato et al. as well as of Pudelski et al. A vinyl cation intermediate is postulated in this context, whose relative stability is evident from the mass spectra of the compounds prepared.     

Calculating the response of NMR shielding tensor σ(31P) and 2J(31P,13C) coupling constants in nucleic acid phosphate to coordination of the Mg2+ cation

Dependence of NMR (31)P shielding tensor and (2)J(P,C) coupling constants on solvation of nucleic acid phosphate by Mg(2+) and water was studied using methods of bioinformatic structural analyses of crystallographic data and DFT B3LYP calculations of NMR parameters. The effect of solvent dynamics on NMR parameters was calculated using molecular dynamic. The NMR calculations for representative solvation patterns determined in crystals of B-DNA and A-RNA molecules pointed out the crucial importance of local Mg(2+) coordination geometry, including hydration by explicit water molecules and necessity of dynamical averaging over the solvent reorientation. The dynamically averaged (31)P chemical shift decreased by 2-9.5 ppm upon Mg(2+) coordination, the chemical shielding anisotropy increased by 0-20 ppm, and the (2)J(P,C5') coupling magnitude decreased by 0.2-1.8 Hz upon Mg(2+) coordination. The calculated decrease of the (31)P chemical shift is in excellent agreement with the 1.5-10 ppm decrease of the phosphorothioate (31)P chemical shift upon Cd(2+) coordination probed experimentally in hammerhead ribozyme (Suzumura; et al. J. Am. Chem. Soc. 2002, 124, 8230-8236; Osborne; et al., Biochemistry 2009, 48, 10654-10664). None of the dynamically averaged NMR parameters unequivocally distinguishes the site-specific Mg(2+) coordination to one of the two nonesterified phosphate oxygen atoms of the phosphate determined by bioinformatic analyses. By comparing the limit cases of static and dynamically averaged solvation, we propose that mobility of the solvent has a dramatic impact on NMR parameters of nucleic acid phosphate and must be taken into account for their accurate modeling.     

Effects of the relative humidity and water droplet on adhesion of a bio-inspired nano-film

Inspired by geckos' adhesion, the effect of water membrane forming due to the environmental humidity, on the adhesion between a bio-inspired nano-film and a substrate is investigated first. The disjoining pressure is considered, which results in an enhancing adhesion between the nano-film and substrate. When the thickness of water membrane increases, water droplets will form and a repulsive capillary force between the nano-film and substrate is produced. The total adhesion force decreases with an increasing volume of water droplets. The two opposite results in the two different models are consistent well with two seemingly inconsistent experimental observations by Huber et al. (2005) [4] and Sun et al. (2005) [5], respectively, and may be significant for the development of artificial biomimetic attachment systems.     

Evaluation Of Some Methods For 17- Ketosteroids In Urine

Abstract

Three procedure for 17-ketosteroids are compared: The method of Callow et al., the method of Drekter et al. and the method of Corker et al. Good correlation has been found between results obtained by the methods of Callow and Corker. Analyses of single analytical steps showed that Drekter's method gives in some cases too high results because non-specific chromogens are included when readings are taken only at 520 nm. Of these three procedures that of Corker et al. is proposed as the method of choice.     

Synthetic Approaches to Triarylboranes from 1885 to 2020

In recent years, research in the fields of optoelectronics, anion sensors and bioimaging agents have been greatly influenced by novel compounds containing triarylborane motifs. Such compounds possess an empty p-orbital at boron which results in useful optical and electronic properties. Such a diversity of applications was not expected when the first triarylborane was reported in 1885. Synthetic approaches to triarylboranes underwent various changes over the following century, some of which are still used in the present day, such as the generally applicable routes developed by Krause et al. in 1922, or by Grisdale et al. in 1972 at Eastman Kodak. Some other developments were not pursued further after their initial reports, such as the synthesis of two triarylboranes bearing three different aromatic groups by Mikhailov et al. in 1958. This review summarizes the development of synthetic approaches to triarylboranes from their first report nearly 135 years ago to the present.     

Molecular architecture of nanocapsules, bilayer-enclosed solid particles of Cisplatin

Cisplatin nanocapsules represent a lipid formulation of the anticancer drug cis-diamminedichloroplatinum(II) (cisplatin) characterized by an unprecedented cisplatin-to-lipid ratio and exhibiting strongly improved cytotoxicity against tumor cells in vitro as compared to the free drug (Burger, K. N. J., et al. Nat. Med. 2002, 8, 81-84). Cisplatin nanocapsules are prepared by the repeated freezing and thawing of an equimolar dispersion of phosphatidylserine (PS) and phosphatidylcholine (PC) in a concentrated aqueous solution of cisplatin. Here, the molecular architecture of these novel nanostructures was elucidated by solid-state NMR techniques. 15N NMR and 2H NMR spectra of nanocapsules containing 15N- and 2H-labeled cisplatin, respectively, demonstrated that the core of the nanocapsules consists of solid cisplatin devoid of free water. Magic-angle spinning 15N NMR showed that approximately 90% of the cisplatin in the core is present as the dichloro species. The remaining 10% was accounted for by a newly discovered dinuclear Pt compound that was identified as the positively charged chloride-bridged dimer of cisplatin. NMR techniques sensitive to lipid organization, 31P NMR and 2H NMR, revealed that the cisplatin core is coated by phospholipids in a bilayer configuration and that the interaction between solid core and bilayer coat exerts a strong ordering effect on the phospholipid molecules. Compared to phospholipids in liposomal membranes, the motion of the phospholipid headgroups is restricted and the ordering of the acyl chains is increased, particularly in PS. The implications of these findings for the structural organization, the mechanism of formation, and the mode of action of cisplatin nanocapsules are discussed.     

The origin of protein sidechain order parameter distributions

Previous work by Wand et al. (Nature 2001, 411, 501-504) showed that the NMR order parameters characterizing the amplitude of motion of protein side chains seemed to form a multimodal distribution. At the time, no detailed explanation of this at the molecular level was offered, yet three "classes" of motion were inferred. We have analyzed a larger published data set and found that, although the distribution is multimodal, the evidence for three classes is weak. More significantly, we have been able to provide a simple physical explanation for the distributions based on the results of molecular dynamics simulations. This result will aid in the interpretation of data from NMR dynamics experiments.     

Simvastatin: structure solution of two new low-temperature phases from synchrotron powder diffraction and ss-NMR

Simvastatin is a substance used for the treatment of hypercholesterolemia. In addition to the already known room temperature structure of simvastatin (Čejka et al. in Acta Cryst C59:o428, 2003) two new low-temperature polymorphs were found by X-ray powder diffraction with the phase transition at 261 and 223 K (later confirmed by DSC to be 272 and 232 K). The main differences among three polymorphs consist in the side-chains conformation only and the phase changes are fully reversible. The structures of the polymorphs were studied by the powder diffraction based on synchrotron radiation as well as by solid-state NMR spectroscopy.     

Ω型分子筛中两种结晶学不等价四面体位上的铝分布

本文用Monte Carlo模拟实现Ω型分子筛中硅铝分布的物理模型,把由此计算获得的{Si_k(nAl)}结构单元相对数量与~(29)Si MAS NMR实验及Klinowski等的统计计算结果相比较,无论是B位{Si_B(nAl)},还是A位{Si_A(nAl)}分布,从总体上全面的逼近NMR实验分析值.对铝浓度不很高时~(29)SiMAS NMR实验分析呈现出的Isi_(?)(2Al)>Isi_(?)(1Al)的令人费解的情况,也得到了初步阐明.这说明,本文提出的T_B位硅有同时桥联两个骨架铝原子的倾向假设是合理和自洽的,也是影响Ω型分子筛中硅、铝分布的一个因素.     

基于3,5-二氨基苯甲酸(4-烷氧基)苯酯可溶性聚酰亚胺的合成与表征

本文合成了含长烷基侧链的二胺单体3,5-二氨基苯甲酸(4-烷氧基)苯酯,然后使它与2,2-双[4.(3,4-二羧酸基苯氧基)苯基]丙烷二酐在N甲基-2-吡咯烷酮溶液中聚合,再通过化学酰亚胺化得到一系列含有长烷基侧链的聚酰亚胺。聚酰亚胺的结构经红外光谱和核磁共振氢谱确认,并对它的溶解性能、特征粘数、光学性能和热性能等进行了表征。结果表明,所合成的聚酰亚胺具有好的溶解性能、较高的分子量、优异的透光率和较好的热稳定性。     

Synthesis and Insecticidal Activity of New 4-Hydroxy-2H-1-benzopyran-2-one Derivatives

Several stable and storable anticoagulant rodenticides involving both merits of acute and chronic rodenticides have been synthesized (Holbrook et al. in Arch Intern Med 165:1095–1106, 2005; Baglin et al. in Br J Haematol 132:277–285, 2006). The structures of synthesized compounds were confirmed by IR, ¹H NMR. The compounds were also evaluated for their anticoagulant and acute biologic activity (Lipton et al., JAMA 252:3004–3005, 3).