cis-trans isomerization of proteins phosphorylated by proline-directed kinases is proposed to control numerous signaling molecules and is implicated in the pathogenesis of Alzheimer’s and other diseases. However, there is no direct evidence for the existence of cis-trans protein isomers in vivo or for their conformation-specific function or regulation. Here we develop peptide chemistries that allow the generation of cis- and trans-specific antibodies and use them to raise antibodies specific for isomers of phosphorylated tau. cis, but not trans, p-tau appears early in the brains of humans with mild cognitive impairment, accumulates exclusively in degenerated neurons, and localizes to dystrophic neurites during Alzheimer’s progression. Unlike trans p-tau, the cis isomer cannot promote microtubule assembly, is more resistant to dephosphorylation and degradation, and is more prone to aggregation. Pin1 converts cis to trans p-tau to prevent Alzheimer’s tau pathology. Isomer-specific antibodies and vaccines may therefore have value for the early diagnosis and treatment of Alzheimer’s disease.
Nanobodies are single-domain antibodies found in camelids. These are the smallest naturally occurring binding domains and derive functionality via three hypervariable loops (H1, H2 and H3) which form the binding surface. They are excellent candidates for antibody engineering due to their favorable characteristics like small-size, high solubility, and stability. To rationally engineer antibodies with affinity for a specific target, one can tailor the hypervariable loops to obtain the desired binding surface. As a first step toward such a goal, we consider the design of loops with a desired conformation. In this study, we focus on the H1 loop of the anti-hCG llama nanobody which exhibits a non-canonical conformation. We aim to “tilt” the balance of the H1 loop conformation from a non-canonical conformation to a (humanized) type-1 canonical conformation by correlating the effect of selected mutations to the amino-acid sequence of the H1, H2 and proximal residues on the H1 loop conformation. We use all-atomistic, explicit-solvent, biased molecular dynamic simulations to simulate the wildtype and mutant loops in a pre-folded framework. We thus find mutants with increasing propensity to form a stable type-1 canonical conformation of the H1 loop. Free-energy landscapes reveal the existence of conformational isomers of the canonical conformation which may play a role in binding different antigenic surfaces. We also elucidate the approximate mechanism and kinetics of transitions between such conformational isomers by using a Markovian model. We find that a particular three-point mutant has the largest thermodynamic propensity to form the H1 type-1 canonical structure but also to exhibit transitions between conformational isomers, while a different, more rigid three-point mutant has the largest propensity to be kinetically trapped in such a canonical structure.
Novel enantiopure pseudopeptide models containing a central -(beta-lactam)-(Aa)- scaffold characterized by the combined presence of an alpha-alkyl-alpha-amino-beta-lactam (i+1) residue and a alpha-substituted (i+2) amino acid have been readily synthesized from alpha-alkyl serines. The conformational analysis of such beta-lactam pseudopeptides conducted in CDCl3 and DMSOd6 solutions using 1D and 2D-NMR techniques revealed an equilibrium between beta-II turn and gamma-turn conformers, which was ultimately modulated by the relative configuration of the -(beta-lactam)-(Aa)- residues. Long range chiral effects on the alpha-lactam pseudopeptide conformers were also found when two (i) and (i+3) chiral residues were attached to the termini of a central -(beta-lactam)-(Aib)- segment. In such mimetics, heterochiral (i) and (i+3) residues reinforced a beta-II turn conformer, whereas homochiral corner residues stabilized an overlapped beta-II/ beta-I double turn motif. No beta-hairpin nucleation was observed in any instance. In good agreement with the conformers found in solution, beta-turned and open structures were also characterized by X-ray crystallography. Relative stabilities of the different conformers were estimated computationally at a B3LYP/6-31++G** calculation level and, finally, a conformation equilibrium model based on steric inter-residual interactions around the -(beta-lactam)-(i+2)- segment was proposed to account for the observed chiral effects.
The haloalkane dehalogenase LinB from Sphingobium indicum B90A converts β-hexachlorocyclohexane (β-HCH), the most persistent HCH stereoisomer, to mono- and dihydroxylated metabolites. Recently, we reported that LinB also transforms α-, β- and γ-hexabromocyclododecanes (HBCDs), which are structurally related to HCHs. Here, we show that LinB catalyzes the hydroxylation of δ-HBCD to two pentabromocyclododecanols (PBCDOHs) and two tetrabromocyclododecadiols (TBCDDOHs). The stereochemistry of this enzymatic transformation was deduced from XRD crystal structure data of the substrate δ-HBCD and α(2)-PBCDOH, one of the biotransformation products. Five stereocenters of δ-HBCD are unchanged but the one at C6 is converted to an alcohol with inversion from S- to R-configuration in a nucleophilic, S(N)2-like substitution reaction. Only α(2)-PBCDOH with the 1R,2R,5S,6R,9R,10S-configuration is obtained but not its enantiomer. With only two of the 64 PBCDOHs formed, these transformations indeed are regio- and stereoselective. A conformational analysis revealed that the triple-turn motive, which is predominant in δ-HBCD and in several other HBCD stereoisomers, is also found in the product. This shows that LinB preferentially converted reactive bromine atoms but not those in the conserved triple-turn motive. The widespread contamination with HCHs triggered the bacterial evolution of dehalogenases which acquired the ability to convert these pollutants and their metabolites. We here demonstrate that LinB of S. indicum also transforms HBCDs regio- and stereoselectively following a similar mechanism.
A series of novel naphthalene attached bis-oxazines were synthesized and characterized. The bis-oxazines were studied by VT-NMR analysis to assess the possibility of conformational twist. The bis-oxazine prepared from (l)-methylvalinate show a helical conformational twist in the single crystal X-ray analysis. Three isomers of bis-oxazines were prepared from chiral α-methylbenzyl amines, the meso isomer showed small optical rotation probably indicating the helical conformational twist in the molecule.
Reaction of NO with amorphous Mn(TPP) layers gives two Mn(TPP)(NO) isomers with linear and bent Mn-N-O geometries that reversibly interconvert with changes in temperature. DFT computations predict that the linear complex is the singlet ground state while the bent structure is a triplet state.
Solvothermal reaction of Zn(NO(3) )(2) ⋅4 H(2) O, 1,4-bis[2-(4-pyridyl)ethenyl]benzene (bpeb) and 4,4'-oxybisbenzoic acid (H(2) obc) in the presence of dimethylacetamide (DMA) as one of the solvents yielded a threefold interpenetrated pillared-layer porous coordination polymer with pcu topology, [Zn(2) (bpeb)(obc)(2) ]⋅5 H(2) O (1), which comprised an unusual isomer of the well-known paddle-wheel building block and the trans-trans-trans isomer of the bpeb pillar ligand. When dimethylformamide (DMF) was used instead of DMA, a supramolecular isomer [Zn(2) (bpeb)(obc)(2) ]⋅2 DMF⋅H(2) O (2), with the trans-cis-trans isomer of the bpeb ligand with a slightly different variation of the paddle-wheel repeating unit, was isolated. In MeOH, single crystals of 2 were transformed by solvent exchange in a single-crystal-to-single-crystal (SCSC) manner to yield [Zn(2) (bpeb)(obc)(2) ]⋅2 H(2) O (3), which is a polymorph of 1. SCSC conversion of 3 to 2 was achieved by soaking 3 in DMF. Compounds 1 and 2 as well as 2 and 3 are supramolecular isomers.
Perfluorooctane sulfonate (PFOS) found extensive use for over 60years up until its restriction in the early 2000s, culminating in its listing under the Stockholm Convention on Persistent Organic Pollutants (POPs) in 2009. Efforts to minimise human body burdens are hindered by uncertainty over their precise origins. While diet appears the principal source for the majority of western populations (with other pathways like dust ingestion, drinking water and inhalation also important contributors); the role played by exposure to PFOS-precursor compounds followed by in vivo metabolism to PFOS as the ultimate highly stable end-product is unclear. Such PFOS-precursor compounds include perfluorooctane sulfonamide derivates, e.g., perfluorooctane sulfonamides (FOSAs) and sulfonamidoethanols (FOSEs). Understanding the indirect contribution of such precursors to human body burdens of PFOS is important as a significant contribution from this pathway would render the margin of safety between the current exposure limits and estimates of external exposure to PFOS alone, narrower than hitherto appreciated. Estimates derived from mathematical modelling studies, put the contribution of so-called “precursor exposure” at between 10% and 40% of total PFOS body burdens. However, there are substantial uncertainties associated with such approaches. This paper reviews current understanding of human exposure to PFOS, with particular reference to recent research highlighting the potential of environmental forensics approaches based on the relative abundance and chiral signatures of branched chain PFOS isomers to provide definitive insights into the role played by “precursor exposure”.
Perfluoroalkyl substances are globally distributed in both urban and remote settings, and routinely are detected in wildlife, humans, and the environment. One of the most prominent and routinely detected perfluoroalkyl substances is perfluorooctanoic acid (PFOA), which has been shown to be toxic to both humans and animals. PFOA exists as both linear and branched isomers; some of the branched isomers are chiral. A novel GC-NCI-MS method was developed to allow for isomer/enantiomer separation, which was achieved using two columns working in tandem; a 30-m DB-5MS column and a 30-m BGB-172 Analytik column. Samples were derivatized with diazomethane to form methyl esters of the PFOA isomers. In standards, at least eight PFOA isomers were detected, of which at least four were enantiomers of chiral isomers; one chiral isomer (P3) was sufficiently separated to allow for enantiomer-fraction calculations. Soil, sediment and plant samples from contaminated locations in Alabama and Georgia were analyzed. P3 was observed in most of these environmental samples, and was non-racemic in at least one sediment, suggesting the possibility of chirally selective generation from precursors or enantioselective sorption. In addition, the ratio of P3/linear PFOA was inversely related to distance from source, which we suggest might reflect a higher sorption affinity for the P3 over the linear isomer. This method focuses on PFOA, but preliminary results suggest that it should be broadly applicable to other chiral and achiral perfluorocarboxylic acids (PFCAs); e.g., we detected several other homologous PFCA isomers in our PFCA standards and some environmental samples.
The ability to decrypt volatile plant signals is essential if herbivorous insects are to optimize their choice of host plants for their offspring. Green leaf volatiles (GLVs) constitute a widespread group of defensive plant volatiles that convey a herbivory-specific message via their isomeric composition: feeding of the tobacco hornworm Manduca sexta converts (Z)-3- to (E)-2-GLVs thereby attracting predatory insects. Here we show that this isomer-coded message is monitored by ovipositing M. sexta females. We detected the isomeric shift in the host plant Datura wrightii and performed functional imaging in the primary olfactory center of M. sexta females with GLV structural isomers. We identified two isomer-specific regions responding to either (Z)-3- or (E)-2-hexenyl acetate. Field experiments demonstrated that ovipositing Manduca moths preferred (Z)-3-perfumed D. wrightii over (E)-2-perfumed plants. These results show that (E)-2-GLVs and/or specific (Z)-3/(E)-2-ratios provide information regarding host plant attack by conspecifics that ovipositing hawkmoths use for host plant selection. DOI:http://dx.doi.org/10.7554/eLife.00421.001.