NMR detection of side chain–side chain hydrogen bonding interactions in 13C/15N-labeled proteins

We describe the direct observation of side chain–side chain hydrogen bonding interactions in proteins with sensitivity-enhanced NMR spectroscopy. Specifically, the remote correlation between the guanidinium nitrogen ¹⁵N of arginine 71, which serves as the hydrogen donor, and the acceptor carboxylate carbon ¹³CO₂ of aspartate 100 in a 12 kDa protein, human FKBP12, is detected via the trans-hydrogen bond ^3h J _{N CO2} coupling by employing a novel HNCO-type experiment, soft CPD-HNCO. The ^3h J _{N CO2} coupling constant appears to be even smaller than the average value of backbone ^3h J _NC couplings, consistent with more extensive local dynamics in protein side chains. The identification of trans-hydrogen bond J-couplings between protein side chains should provide useful markers for monitoring hydrogen bonding interactions that contribute to the stability of protein folds, to alignments within enzyme active sites and to recognition events at macromolecular interfaces.     

Systematic SAR study of the side chain of nonsecosteroidal vitamin D₃ analogs

A series of nonsecosteroidal vitamin D(3) analogs with carboxylic acid were explored. Through our systematic SAR studies on the side chain moiety, compound 6b was identified as the optimal compound showing excellent vitamin D receptor (VDR) agonistic activity. Compound 6b had the diethyl group in the terminal which was bound by (E)-olefin linker to the bisphenyl core. Calculating the volume of the side chain showed that the diethyl group in 6b filled the hydrophobic region of VDR with the ideal packing coefficient based on the 55% rule, and that this resulted in the most potent in vitro activity.     

Structural origins of nitroxide side chain dynamics on membrane protein α-helical sites

Understanding the structure and dynamics of membrane proteins in their native, hydrophobic environment is important to understanding how these proteins function. EPR spectroscopy in combination with site-directed spin labeling (SDSL) can measure dynamics and structure of membrane proteins in their native lipid environment; however, until now the dynamics measured have been qualitative due to limited knowledge of the nitroxide spin label's intramolecular motion in the hydrophobic environment. Although several studies have elucidated the structural origins of EPR line shapes of water-soluble proteins, EPR spectra of nitroxide spin-labeled proteins in detergents or lipids have characteristic differences from their water-soluble counterparts, suggesting significant differences in the underlying molecular motion of the spin label between the two environments. To elucidate these differences, membrane-exposed α-helical sites of the leucine transporter, LeuT, from Aquifex aeolicus, were investigated using X-ray crystallography, mutational analysis, nitroxide side chain derivatives, and spectral simulations in order to obtain a motional model of the nitroxide. For each crystal structure, the nitroxide ring of a disulfide-linked spin label side chain (R1) is resolved and makes contacts with hydrophobic residues on the protein surface. The spin label at site I204 on LeuT makes a nontraditional hydrogen bond with the ortho-hydrogen on its nearest neighbor F208, whereas the spin label at site F177 makes multiple van der Waals contacts with a hydrophobic pocket formed with an adjacent helix. These results coupled with the spectral effect of mutating the i ± 3, 4 residues suggest that the spin label has a greater affinity for its local protein environment in the low dielectric than on a water-soluble protein surface. The simulations of the EPR spectra presented here suggest the spin label oscillates about the terminal bond nearest the ring while maintaining weak contact with the protein surface. Combined, the results provide a starting point for determining a motional model for R1 on membrane proteins, allowing quantification of nitroxide dynamics in the aliphatic environment of detergent and lipids. In addition, initial contributions to a rotamer library of R1 on membrane proteins are provided, which will assist in reliably modeling the R1 conformational space for pulsed dipolar EPR and NMR paramagnetic relaxation enhancement distance determination.     

An α-glucoside of 1,4-dideoxy-1,4-imino-d-lyxitol with an eleven carbon side chain

The distribution of pyrrolidine-type iminosugars with a long-side chain appears to be restricted to the relatively unrelated plant families Moraceae, Campanulaceae, and Hyacinthaceae. In a search for glycosidase inhibitors in these plant families, we isolated the 1,4-dideoxy-1,4-imino-d-lyxitol (DIL) glucoside bearing the 1,2,11-trihydroxyundec-4-ene side chain at the C-1α position from the roots of Adenophora triphylla. This iminosugar was a powerful and selective inhibitor of coffee bean α-galactosidase, with an IC₅₀ value of 8 μM.     

Proline-glutamate chimera’s side chain conformation directs the type of β-hairpin structure

Our aim was to study the impact of two proline chimeras, containing a glutamic acid side chain in cis- or trans-configuration, on secondary structure formation. We further investigated to what extent the configuration of the side chain contributes to the overall peptide conformation. We used a 10 residue peptide (IYSNPDGTWT) that forms a β-hairpin in water. The turn-forming proline was substituted with either a cis- or trans-proline-glutamic acid chimera, resulting in the peptides IYSNP ^{cis -E} DGTWT (P1_P ^cis-E ) and IYSNP ^{trans -E} DGTWT (P1_P ^trans-E ). We studied the conformation of the modified peptides by circular dichroism (CD) and NMR-spectroscopy, and SEC/static light scattering (SLS) analysis. NMR analysis reveals that the modified peptides maintain the β-hairpin conformation in aqueous solution. At 5 °C and pH 4.3, the peptide (P1_P ^cis-E ) was found to adopt two coexisting β-hairpin conformations (2:2 β-hairpin, and 3:5 β-hairpin). In contrast to that, the peptide (P1_P ^trans-E ) adopts a 2:2 β-hairpin that exists in equilibrium with a 4:4 β-hairpin conformation. The adoption of ordered β-hairpin structures for both modified peptides could be confirmed by CD spectroscopy, while SEC/SLS analysis showed a monomeric oligomerization state for all three investigated peptides. With the combination of several NMR methods, we were able to elucidate that even small alterations in the side chain conformation of the proline-glutamate chimera (cis or trans) can significantly influence the conformation of the adopted β-hairpin.     

Modulating amyloid-β aggregation: The effects of peptoid side chain placement and chirality

Alzheimer’s disease (AD) is characterized by the buildup of insoluble aggregated amyloid-β protein (Aβ) into plaques that accumulate between the neural cells in the brain. AD is the sixth leading cause of death in the United States and is the only cause of death among the top ten that cannot currently be treated or cured (Alzheimer’s Association, 2011; Selkoe, 1996). Researchers have focused on developing small molecules and peptides to prevent Aβ aggregation; however, while some compounds appear promising in vitro, the research has not resulted in a viable therapeutic treatment. We previously reported a peptoid-based mimic (JPT1) of the peptide KLVFF (residues 16–20 of Aβ) that modulates Aβ40 aggregation, specifically reducing the total number of fibrillar, β-sheet structured aggregates formed. In this study, we investigate two new variants of JPT1 that probe the importance of aromatic side chain placement (JPT1s) and side chain chirality (JPT1a). Both JPT1s and JPT1a modulate Aβ40 aggregation by reducing total β-sheet aggregates. However, JPT1a also has a pronounced effect on the morphology of fibrillar Aβ40 aggregates. These results suggest that Aβ40 aggregation may follow a different pathway in the presence of peptoids with different secondary structures. A better understanding of the interactions between peptoids and Aβ will allow for improved design of AD treatments.     

Decoupled side chain and backbone dynamics for proton translocation – M2 of influenza A

The 97 amino acid bitopic membrane protein M2 of influenza A forms a tetrameric bundle in which two of the monomers are covalently linked via a cysteine bridge. In its tetrameric assembly the protein conducts protons across the viral envelope and within intracellular compartments during the infectivity cycle of the virus. A key residue in the translocation of the protons is His-37 which forms a planar tetrad in the configuration of the bundle accepting and translocating the incoming protons from the N terminal side, exterior of the virus, to the C terminal side, inside the virus. With experimentally available data from NMR spectroscopy of the transmembrane domains of the tetrameric M2 bundle classical MD simulations are conducted with the protein bundle in different protonation stages in respect to His-37. A full correlation analysis (FCA) of the data sets with the His-37 tetrad either in a fully four times unprotonated or protonated state, assumed to mimic high and low pH in vivo, respectively, in both cases reveal asymmetric backbone dynamics. His-37 side chain rotation dynamics is increased at full protonation of the tetrad compared to the dynamics in the fully unprotonated state. The data suggest that proton translocation can be achieved by decoupled side chain or backbone dynamics.

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Graphical abstract Visualization of the tetrameric bundle of the transmembrane domains of M2 of influenza A after 200 ns of MD simulations (upper left). The four histidine residues 37 are either not protonated as in M2⁰ or fully protonated is in M2⁴⁺. The asymmetric dynamics of the backbones are shown after a full correlation analysis (FCA) in blue (lower left). The rotational dynamics of the χ2 dihedral angles of the histidines in M2⁰ (upper right) are less than those in M2⁴⁺ (lower right)

     

Cytotoxicity and DNA binding property of the dimers of triphenylethylene–coumarin hybrid with one amino side chain

Novel dimers of triphenylethylene–coumarin hybrid containing one amino side chain were designed and synthesized by the condensation of four dicarboxylic acids with the amino monomeric hybrids catalyzed by HATU and DIPEA at room temperature. The adding order of the reactants had a significant effect on the condensation reaction when the malonic acid was used. The dimeric compounds 7a and 7b linked by the malonic acid, showed a broad-spectrum and good anti-proliferative activity against four tumor cells and low cytotoxicity in osteoblast. UV–vis, fluorescence, and circular dichroism (CD) spectroscopies and thermal denaturation exhibited that compounds 7b, 8b, 9b, and 10b had significant interactions with Ct-DNA by the intercalative mode of binding. Both the DNA binding properties and the anti-proliferative activities would be enhanced by dimerization of the monomeric hybrid with one amino side chain, and were significantly affected by the length of the linker (dicarboxylic acids).     

How strong are side chain interactions in the folding intermediate?

Influence of 12 nonpolar amino acids residues from the hydrophobic core of apomyoglobin on stability of its native state and folding intermediate was studied. Six of the selected residues are from the A, G and H helices; these are conserved in structure of the globin family, although nonfunctional, that is, not involved in heme binding. The rest are nonconserved hydrophobic residues that belong to the B, C, D, and E helices. Each residue was substituted by alanine, and equilibrium pH‐induced transitions in apomyoglobin and its mutants were studied by circular dichroism and fluorescent spectroscopy. The obtained results allowed estimating changes in their free energy during formation of the intermediate state. It was first shown that the strength of side chain interactions in the apomyoglobin intermediate state amounts to 15–50% of that in its native state for conserved residues, and practically to 0% for nonconserved residues. These results allow a better understanding of interactions occurring in the intermediate state and shed light on involvement of certain residues in protein folding at different stages.     

Microbial metabolism of alkylbenzene sulphonates effect of α-methyl branching of the alkyl side chain on oxidation by a Bacillus species

A Bacillus species originally elected for the ability to utilise unbranched-alkyl-side-chain-alkylbenzene-sulphonate (ABS) isomers as the sole source of carbon and sulphur was found to be able to utilise various α-methyl-branched-alkyl-side-chain(ABS) isomers in a similar minimal nutrient role. The enzymic mechanism involved in α-methyl-branched-alkyl-side-chain biodegradation of various ABS isomers by the Bacillus was demonstrated to involve the classical β-oxidation sequence characteristic of unbranched-fatty-acid oxidation, by appropriate enzyme induction experiments. Results obtained from such enzyme induction studies plus an examination of the behaviour of these induced enzymes during separation by gel-filtration indicated a single set of enzymes to be responsible for the β-oxidation of long-chain fatty acid isomers, unbranched-alkyl-side-chain (ABS) isomers and α-methyl-branched-alkyl-side-chain (ABS) isomers in the Bacillus species. The substrate-specificity of partially purified enzymes after growth on appropriate substrates confirmed the operation in this microorganism of a single β-oxidation pathway capable of catalysing the oxidation of a wide range of different chemicals containing either unbranched or α-methyl-branched alkyl side chains.     

Structure–activity relationship investigation of benzamide and picolinamide derivatives containing dimethylamine side chain as acetylcholinesterase inhibitors

A series of benzamide and picolinamide derivatives containing dimethylamine side chain (4a–4c and 7a–7i) were synthesised and evaluated for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity in vitro. Structure–activity relationship investigation revealed that the substituted position of dimethylamine side chain markedly influenced the inhibitory activity and selectivity against AChE and BChE. In addition, it seemed that the bioactivity of picolinamide amide derivatives was stronger than that of benzamide derivatives. Among them, compound 7a revealed the most potent AChE inhibitory activity (IC₅₀: 2.49?±?0.19?μM) and the highest selectivity against AChE over BChE (Ratio: 99.40). Enzyme kinetic study indicated that compound 7a show a mixed-type inhibition against AChE. The molecular docking study revealed that this compound can bind with both the catalytic site and the peripheral site of AChE.     

Detection of very weak side chain–main chain hydrogen bonding interactions in medium-size 13C/15N-labeled proteins by sensitivity-enhanced NMR spectroscopy

We describe the direct observation of very weak side chain–main chain hydrogen bonding interactions in medium-size ¹³C/¹⁵N-labeled proteins with sensitivity-enhanced NMR spectroscopy. Specifically, the remote correlation between the hydrogen acceptor side chain carboxylate carbon ¹³CO₂ of glutamate 54 and the hydrogen donor backbone amide ¹⁵N of methionine 49 in a 12 kDa protein, human FKBP12, is detected via the trans-hydrogen bond ^3h J _NCO2 coupling by employing a novel sensitivity-enhanced HNCO-type experiment, CPD-HNCO. The ^3h J _NCO2 coupling constant appears to be even smaller than the average value of backbone ^3h J _NC couplings, consistent with more extensive local dynamics in protein side chains.     

FT-ICR-MS characterization of intermediates in the biosynthesis of the α-methylbutyrate side chain of lovastatin by the 277 kDa polyketide synthase LovF

There are very few fungal polyketide synthases that have been characterized by mass spectrometry. In this paper we describe the in vitro reconstitution and FT-ICR-MS verification of the full activity of an intact 277 kDa fungal polyketide synthase LovF of the lovastatin biosynthetic pathway. We report here both the verification of the reconstitution of fully functional holo-LovF by using (13)C-labeled malonyl-CoA to form α-methylbutyrate functionality and also detection of five predicted intermediates covalently bound to the 4'-phosphopantetheine at the acyl carrier protein (ACP) active site utilizing the phosphopantetheine ejection assay and high-resolution mass spectrometry. Under in vitro conditions, the diketide acetoacetyl intermediate did not accumulate on the ACP active site of holo-LovF following incubation with malonyl-CoA substrate. We found that incubation of holo-LovF with acetoacetyl-CoA served as an effective means of loading the diketide intermediate onto the ACP active site of LovF. Our results demonstrate that subsequent α-methylation of the acetoacetyl intermediate stabilizes the intermediate onto the ACP active site and facilitates the formation and mass spectrometric detection of additional intermediates en route to the formation of α-methylbutyrate.     

Accessing ns–μs side chain dynamics in ubiquitin with methyl RDCs

This study presents the first application of the model-free analysis (MFA) (Meiler in J Am Chem Soc 123:6098–6107, 2001; Lakomek in J Biomol NMR 34:101–115, 2006) to methyl group RDCs measured in 13 different alignment media in order to describe their supra-τ _c dynamics in ubiquitin. Our results indicate that methyl groups vary from rigid to very mobile with good correlation to residue type, distance to backbone and solvent exposure, and that considerable additional dynamics are effective at rates slower than the correlation time τ _c. In fact, the average amplitude of motion expressed in terms of order parameters S ² associated with the supra-τ _c window brings evidence to the existence of fluctuations contributing as much additional mobility as those already present in the faster ps-ns time scale measured from relaxation data. Comparison to previous results on ubiquitin demonstrates that the RDC-derived order parameters are dominated both by rotameric interconversions and faster libration-type motions around equilibrium positions. They match best with those derived from a combined J-coupling and residual dipolar coupling approach (Chou in J Am Chem Soc 125:8959–8966, 2003) taking backbone motion into account. In order to appreciate the dynamic scale of side chains over the entire protein, the methyl group order parameters are compared to existing dynamic ensembles of ubiquitin. Of those recently published, the broadest one, namely the EROS ensemble (Lange in Science 320:1471–1475, 2008), fits the collection of methyl group order parameters presented here best. Last, we used the MFA-derived averaged spherical harmonics to perform highly-parameterized rotameric searches of the side chains conformation and find expanded rotamer distributions with excellent fit to our data. These rotamer distributions suggest the presence of concerted motions along the side chains.     

Synthesis of α-trifluoromethyl α-amino acids with aromatic, heteroaromatic and ferrocenyl subunits in the side chain

Summary. 5-Benzyloxy-4-trifluoromethyl-1,3-oxazoles, obtained from 5-fluoro-4-trifluoromethyloxazoles and benzyl alcohols, are capable for rearrangements. A 1,3 shift of a benzyl group is the key step of a new general route toward α-trifluoromethyl substituted aromatic and heteroaromatic amino acids, demonstrating that 5-fluoro-4-trifluoromethyl-1,3-oxazole is a synthetic Tfm-Gly equivalent. On reaction with benzpinacol partially fluorinated oxazoles are transformed into bis(trifluoromethyl) substituted 2,5-diamino adipic acid and N-benzoyl-2-benzhydryl-3,3,3-trifluoroalanine.     

Role of the side chain stereochemistry in the α-glucosidase inhibitory activity of kotalanol, a potent natural α-glucosidase inhibitor

Synthesis and evaluation of four diastereomers (9a, 9b, 9c and 9d) of kotalanol, a potent α-glucosidase inhibitor isolated from an Ayurvedic medicinal plant Salacia species, are described. Stereo-inversion at C-3' and C-4' of kotalanol (2) caused significant decrease of the inhibitory activities against maltase and sucrase, whereas inhibitory activity against isomaltase sustained, thus resulted in exerting selectivity against isomaltase.     

NMR assignment of backbone and side chain resonances for a dockerin-containing C-terminal fragment of the putative μ-toxin from Clostridium perfringens

The μ-toxin of Clostridium perfringens, termed CpGH84A, is modular hydrolytic enzyme that contributes to the pathogenicity of this organism. Backbone and side chain ¹H, ¹³C, and ¹⁵N resonance assignments have been determined for the C-terminal 15.5 kDa FIVAR-Doc modular pair of CpGH84A.     

Structure–activity studies on the side chain of a simplified analog of aplysiatoxin (aplog-1) with anti-proliferative activity

We have recently developed a simplified analog of aplysiatoxin (aplog-1) as an activator of protein kinase C (PKC) with anti-proliferative activity like bryostain 1. To identify sites in aplog-1 that could be readily modified to optimize therapeutic performance and to develop a molecular probe for examining the analog’s mode of action, substituent effects on the phenol ring were systematically examined. Whereas hydrophilic acetamido derivatives were less active than aplog-1 in inhibiting cancer cell growth and binding to PKCδ, introduction of hydrophobic bromine and iodine atoms enhanced both biological activities. The anti-proliferative activity was found to correlate closely with molecular hydrophobicity, and maximal activity was observed at a log P value of 4.0–4.5. On the other hand, an induction test with Epstein–Barr virus early antigen demonstrated that these derivatives have less tumor-promoting activity in vitro than aplog-1 regardless of the hydrophobicity of their substituents. These results would facilitate rapid preparation of molecular probes to examine the mechanism of the unique biological activities of aplog-1.     

1H, 13C and 15N backbone and side chain resonance assignments of human interleukin 1α

As part of our NMR structure determination of the human Interleukin-1α, we report nearly complete NMR chemical shift assignments for the ¹H, ¹³C and ¹⁵N nuclei.