Human mitochondrial glutathione transferases: Kinetic parameters and accommodation of a mitochondria-targeting group in substrates.

Glutathione-S-transferases are key to the cellular detoxification of xenobiotics and products of oxidative damage. GSTs catalyse the reaction of glutathione (GSH) with electrophiles to form stable thioether adducts. GSTK1-1 is the main GST isoform in the mitochondrial matrix, but the GSTA1-1 and GSTA4-4 isoforms are also thought to be in the mitochondria with their distribution altering in transformed cells, thus potentially providing a cancer specific target. A mitochondria-targeted version of the GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), MitoCDNB, has been used to manipulate the mitochondrial GSH pool. To finesse this approach to target particular GST isoforms in the context of cancer, here we have determined the kcat/Km for the human isoforms of GSTK1-1, GSTA1-1 and GSTA4-4 with respect to GSH and CDNB. We show how the rate of the GST-catalysed reaction between GSH and CDNB analogues can be modified by both the electron withdrawing substituents, and by the position of the mitochondria-targeting triphenylphosphonium on the chlorobenzene ring to tune the activity of mitochondria-targeted substrates. These findings can now be exploited to selectively disrupt the mitochondrial GSH pools of cancer cells expressing particular GST isoforms.

Multiplexed Biosensing of Proteins and Virions with Disposable Plasmonic Assays.

Our growing ability to tailor healthcare to the needs of individuals has the potential to transform clinical treatment. However, the measurement of multiple biomarkers to inform clinical decisions requires rapid, effective, and affordable diagnostics. Chronic diseases and rapidly evolving pathogens in a larger population have also escalated the need for improved diagnostic capabilities. Current chemical diagnostics are often performed in centralized facilities and are still dependent on multiple steps, molecular labeling, and detailed analysis, causing the result turnaround time to be over hours and days. Rapid diagnostic kits based on lateral flow devices can return results quickly but are only capable of detecting a handful of pathogens or markers. Herein, we present the use of disposable plasmonics with chiroptical nanostructures as a platform for low-cost, label-free optical biosensing with multiplexing and without the need for flow systems often required in current optical biosensors. We showcase the detection of SARS-CoV-2 in complex media as well as an assay for the Norovirus and Zika virus as an early developmental milestone toward high-throughput, single-step diagnostic kits for differential diagnosis of multiple respiratory viruses and any other emerging diagnostic needs. Diagnostics based on this platform, which we term "disposable plasmonics assays," would be suitable for low-cost screening of multiple pathogens or biomarkers in a near-point-of-care setting.

Superchiral near fields detect virus structure.

Optical spectroscopy can be used to quickly characterise the structural properties of individual molecules. However, it cannot be applied to biological assemblies because light is generally blind to the spatial distribution of the component molecules. This insensitivity arises from the mismatch in length scales between the assemblies (a few tens of nm) and the wavelength of light required to excite chromophores (≥150 nm). Consequently, with conventional spectroscopy, ordered assemblies, such as the icosahedral capsids of viruses, appear to be indistinguishable isotropic spherical objects. This limits potential routes to rapid high-throughput portable detection appropriate for point-of-care diagnostics. Here, we demonstrate that chiral electromagnetic (EM) near fields, which have both enhanced chiral asymmetry (referred to as superchirality) and subwavelength spatial localisation (∼10 nm), can detect the icosahedral structure of virus capsids. Thus, they can detect both the presence and relative orientation of a bound virus capsid. To illustrate the potential uses of the exquisite structural sensitivity of subwavelength superchiral fields, we have used them to successfully detect virus particles in the complex milieu of blood serum.

Probing Specificity of Protein-Protein Interactions with Chiral Plasmonic Nanostructures.

Protein-protein interactions (PPIs) play a pivotal role in many biological processes. Discriminating functionally important well-defined protein-protein complexes formed by specific interactions from random aggregates produced by nonspecific interactions is therefore a critical capability. While there are many techniques which enable rapid screening of binding affinities in PPIs, there is no generic spectroscopic phenomenon which provides rapid characterization of the structure of protein-protein complexes. In this study we show that chiral plasmonic fields probe the structural order and hence the level of PPI specificity in a model antibody-antigen system. Using surface-immobilized Fab' fragments of polyclonal rabbit IgG antibodies with high specificity for bovine serum albumin (BSA), we show that chiral plasmonic fields can discriminate between a structurally anisotropic ensemble of BSA-Fab' complexes and random ovalbumin (OVA)-Fab' aggregates, demonstrating their potential as the basis of a useful proteomic technology for the initial rapid high-throughput screening of PPIs.

Chiral Plasmonic Fields Probe Structural Order of Biointerfaces.

The structural order of biopolymers, such as proteins, at interfaces defines the physical and chemical interactions of biological systems with their surroundings and is hence a critical parameter in a range of biological problems. Known spectroscopic methods for routine rapid monitoring of structural order in biolayers are generally only applied to model single-component systems that possess a spectral fingerprint which is highly sensitive to orientation. This spectroscopic behavior is not a generic property and may require the addition of a label. Importantly, such techniques cannot readily be applied to real multicomponent biolayers, have ill-defined or unknown compositions, and have complex spectroscopic signatures with many overlapping bands. Here, we demonstrate the sensitivity of plasmonic fields with enhanced chirality, a property referred to as superchirality, to global orientational order within both simple model and "real" complex protein layers. The sensitivity to structural order is derived from the capability of superchiral fields to detect the anisotropic nature of electric dipole-magnetic dipole response of the layer; this is validated by numerical simulations. As a model study, the evolution of orientational order with increasing surface density in layers of the antibody immunoglobulin G was monitored. As an exemplar of greater complexity, superchiral fields are demonstrated, without knowledge of exact composition, to be able to monitor how qualitative changes in composition alter the structural order of protein layers formed from blood serum, thereby establishing the efficacy of the phenomenon as a tool for studying complex biological interfaces.

Superchiral Plasmonic Phase Sensitivity for Fingerprinting of Protein Interface Structure.

The structure adopted by biomaterials, such as proteins, at interfaces is a crucial parameter in a range of important biological problems. It is a critical property in defining the functionality of cell/bacterial membranes and biofilms (i.e., in antibiotic-resistant infections) and the exploitation of immobilized enzymes in biocatalysis. The intrinsically small quantities of materials at interfaces precludes the application of conventional spectroscopic phenomena routinely used for (bio)structural analysis due to a lack of sensitivity. We show that the interaction of proteins with superchiral fields induces asymmetric changes in retardation phase effects of excited bright and dark modes of a chiral plasmonic nanostructure. Phase retardations are obtained by a simple procedure, which involves fitting the line shape of resonances in the reflectance spectra. These interference effects provide fingerprints that are an incisive probe of the structure of interfacial biomolecules. Using these fingerprints, layers composed of structurally related proteins with differing geometries can be discriminated. Thus, we demonstrate a powerful tool for the bioanalytical toolbox.

Spectrum of Slow and Super-Slow (Picosecond to Nanosecond) Water Dynamics around Organic and Biological Solutes.

Water dynamics in the solvation shell of solutes plays a very important role in the interaction of biomolecules and in chemical reaction dynamics. However, a selective spectroscopic study of the solvation shell is difficult because of the interference of the solute dynamics. Here we report on the observation of heavily slowed down water dynamics in the solvation shell of different solutes by measuring the low-frequency spectrum of solvation water, free from the contribution of the solute. A slowdown factor of ∼50 is observed even for relatively low concentrations of the solute. We go on to show that the effect can be generalized to different solutes including proteins.

The molecular relationship between antigenic domains and epitopes on hCG.

Antigenic domains are defined to contain a limited number of neighboring epitopes recognized by antibodies (Abs) but their molecular relationship remains rather elusive. We thoroughly analyzed the antigenic surface of the important pregnancy and tumor marker human chorionic gonadotropin (hCG), a cystine knot (ck) growth factor, and set antigenic domains and epitopes in molecular relationships to each other. Antigenic domains on hCG, its free hCGα and hCGß subunits are dependent on appropriate inherent molecular features such as molecular accessibility and protrusion indices that determine bulging structures accessible to Abs. The banana-shaped intact hCG comprises ∼7500Å(2) of antigenic surface with minimally five antigenic domains that encompass a continuum of overlapping non-linear composite epitopes, not taking into account the C-terminal peptide extension of hCGß (hCGßCTP). Epitopes within an antigenic domain are defined by specific Abs, that bury nearly 1000Å(2) of surface accessible area on the antigen and recognize a few up to 15 amino acid (aa) residues, whereby between 2 and 5 of these provide the essential binding energy. Variability in Ab binding modes to the contact aa residues are responsible for the variation in affinity and intra- and inter-species specificity, e.g. cross-reactions with luteinizing hormone (LH). Each genetically distinct fragment antigen binding (Fab) defines its own epitope. Consequently, recognition of the same epitope by different Abs is only possible in cases of genetically identical sequences of its binding sites. Due to combinatorial V(D)J gene segment variability of heavy and light chains, Abs defining numerous epitopes within an antigenic domain can be generated by different individuals and species. Far more than hundred Abs against the immuno-dominant antigenic domains of either subunit at both ends of the hCG-molecule, the tips of peptide loops one and three (L1+3) protruding from the central ck, encompassing hCGßL1+3 (aa 20-25+64+68-81) and hCGαL1 (aa 13-22; Pro16, Phe17, Phe18) plus hCGαL3 (Met71, Phe74), respectively, have been identified in the two "ISOBM Tissue Differentiation-7 Workshops on hCG and Related Molecules" and in other studies. These Abs recognize distinct but overlapping epitopes with slightly different specificity profiles and affinities. Heterodimeric-specific epitopes involve neighboring αL1 plus ßL2 (hCGß44/45 and 47/48). Diagnostically important Abs recognize the middle of the molecule, the ck (aa Arg10, Arg60 and possibly Gln89) and the linear hCGßCTP "tail" (aa 135-145; Asp139, Pro144, Gln145), respectively. Identification of antigenic domains and of specific epitopes is essential for harmonization of Abs in methods that are used for reliable and robust hCG measurements for the management of pregnancy, pregnancy-related disease and tumors.

Observation of coherent delocalized phonon-like modes in DNA under physiological conditions.

Underdamped terahertz-frequency delocalized phonon-like modes have long been suggested to play a role in the biological function of DNA. Such phonon modes involve the collective motion of many atoms and are prerequisite to understanding the molecular nature of macroscopic conformational changes and related biochemical phenomena. Initial predictions were based on simple theoretical models of DNA. However, such models do not take into account strong interactions with the surrounding water, which is likely to cause phonon modes to be heavily damped and localized. Here we apply state-of-the-art femtosecond optical Kerr effect spectroscopy, which is currently the only technique capable of taking low-frequency (GHz to THz) vibrational spectra in solution. We are able to demonstrate that phonon modes involving the hydrogen bond network between the strands exist in DNA at physiologically relevant conditions. In addition, the dynamics of the solvating water molecules is slowed down by about a factor of 20 compared with the bulk.

Standardization of Epitopes for Human Chorionic Gonadotropin (hCG) Immunoassays.

hCG and its variants are markers for pregnancy tests, pregnancyrelated complications, trophoblastic diseases, pre-natal screening of Down's syndrome and doping controls. Strong demands are imposed on diagnostic methods by the dynamic changes in the absolute and relative levels of hCG protein backbone variants and glycosylation isoforms in serum and urine during development of pregnancy or the progression/remission of tumors. Observed differences in the results between commercial diagnostic immunoassays reflect the unequal molar recognition of the different metabolic hCG variants, in particular the hCG beta core fragment (hCGßcf), by the diagnostic antibodies (Abs), as their epitopes are not standardized, and the fact that suboptimal hCG standards are used. To rapidly characterize Abs by their epitope recognition and specificity to evaluate their suitability for diagnostic immunoassays a procedure of comparative epitope mapping has been developed using epitope-defined reference Abs. Comparative epitope mapping of diagnostic Abs will provide the basis for the standardization of diagnostic antigenic domains/epitopes and consequently for improved reliability of hCG measurements. Diagnostic first line assays likely consist of pairs of Abs that recognize specific epitopes at the top of the neighboring peptide loops 1 and 3 (L1+3) and the cystine knot (ck) of hCGß, respectively. In future, significant improvements of reliability, robustness and comparability of the results of immunoassays for complex glycoproteins such as hCG will be achieved by the use (i) of standardized diagnostic Abs against welldefined epitopes and (ii) of the new International Standards for hCG and for five hCG variants established by WHO, that are calibrated in molar (SI) units.

Spatial control of chemical processes on nanostructures through nano-localized water heating.

Optimal performance of nanophotonic devices, including sensors and solar cells, requires maximizing the interaction between light and matter. This efficiency is optimized when active moieties are localized in areas where electromagnetic (EM) fields are confined. Confinement of matter in these 'hotspots' has previously been accomplished through inefficient 'top-down' methods. Here we report a rapid 'bottom-up' approach to functionalize selective regions of plasmonic nanostructures that uses nano-localized heating of the surrounding water induced by pulsed laser irradiation. This localized heating is exploited in a chemical protection/deprotection strategy to allow selective regions of a nanostructure to be chemically modified. As an exemplar, we use the strategy to enhance the biosensing capabilities of a chiral plasmonic substrate. This novel spatially selective functionalization strategy provides new opportunities for efficient high-throughput control of chemistry on the nanoscale over macroscopic areas for device fabrication.

Terahertz underdamped vibrational motion governs protein-ligand binding in solution.

Low-frequency collective vibrational modes in proteins have been proposed as being responsible for efficiently directing biochemical reactions and biological energy transport. However, evidence of the existence of delocalized vibrational modes is scarce and proof of their involvement in biological function absent. Here we apply extremely sensitive femtosecond optical Kerr-effect spectroscopy to study the depolarized Raman spectra of lysozyme and its complex with the inhibitor triacetylchitotriose in solution. Underdamped delocalized vibrational modes in the terahertz frequency domain are identified and shown to blue-shift and strengthen upon inhibitor binding. This demonstrates that the ligand-binding coordinate in proteins is underdamped and not simply solvent-controlled as previously assumed. The presence of such underdamped delocalized modes in proteins may have significant implications for the understanding of the efficiency of ligand binding and protein-molecule interactions, and has wider implications for biochemical reactivity and biological function.

The diversity of microbial aldo/keto reductases from Escherichia coli K12.

The genome of Escherichia coli K12 contains 9 open reading frames encoding aldo/keto reductases (AKRs) that are differentially regulated and sequence diverse. A significant amount of data is available for the E. coli AKRs through the availability of gene knockouts and gene expression studies, which adds to the biochemical and kinetic data. This together with the availability of crystal structures for nearly half of the E. coli AKRs and homologues of several others provides an opportunity to look at the diversity of these representative bacterial AKRs. Based around the common AKR fold of (ß/α)8 barrel with two additional α-helices, the E. coli AKRs have a loop structure that is more diverse than their mammalian counterparts, creating a variety of active site architectures. Nearly half of the AKRs are expected to be monomeric, but there are examples of dimeric, trimeric and octameric enzymes, as well as diversity in specificity for NAD as well as NADP as a cofactor. However in functional assignments and characterisation of enzyme activities there is a paucity of data when compared to the mammalian AKR enzymes.

Nanomolar competitive inhibitors of Mycobacterium tuberculosis and Streptomyces coelicolor type II dehydroquinase.

Isomeric nitrophenyl and heterocyclic analogues of the known inhibitor (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid have been synthesized and tested as inhibitors of M. tuberculosis and S. coelicolor type II dehydroquinase, the third enzyme of the shikimic acid pathway. The target compounds were synthesized by a combination of Suzuki and Sonogashira cross-coupling and copper(I)-catalyzed 2,3-dipolar cycloaddition reactions from a common vinyl triflate intermediate. These studies showed that a para-nitrophenyl derivative is almost 20-fold more potent as a competitive inhibitor against the S. coelicolor enzyme than that of M. tuberculosis. The opposite results were obtained with the meta isomer. Five of the bicyclic analogues reported herein proved to be potent competitive inhibitors of S. coelicolor dehydroquinase, with inhibition constants in the low nanomolar range (4-30 nM). These derivatives are also competitive inhibitors of the M. tuberculosis enzyme, but with lower affinities. The most potent inhibitor against the S. coelicolor enzyme, a 6-benzothiophenyl derivative, has a K(i) value of 4 nM-over 2000-fold more potent than the best previously known inhibitor, (1R,4R,5R)-1,5-dihydroxy-4-(2-nitrophenyl)cyclohex-2-en-1-carboxylic acid (8 microM), making it the most potent known inhibitor against any dehydroquinase. The binding modes of the analogues in the active site of the S. coelicolor enzyme (GOLD 3.0.1), suggest a key pi-stacking interaction between the aromatic rings and Tyr 28, a residue that has been identified as essential for enzyme activity.

Application of a new vector system for efficient protein purification in the crystallization of PA5104/ORF from Pseudomonas aeruginosa.

We have developed a new T7-based vector system for rapid purification and high-throughput capability applicable for structural studies. The system allows purification of target proteins to homogeneity in two steps with a single Ni-affinity column. The first step relies on affinity purification of the N-terminal His-tagged protein in the conventional way, eluting the protein with imidazole. Addition of a His-tagged 3C protease to cleave the His-tag permits a second pass through the nickel column, this time all impurities bind to the column while the pure protein does not. This has the major advantage of quickly removing the residual contaminating proteins that are associated with nickel affinity purification as well as the protease and His-tag. Here, we describe the application of this system to over-express and purify ORF PA5104 from Pseudomonas aeruginosa. The protein was successfully crystallized and crystals were shown to diffract to atomic resolution. Additionally preliminary X-ray diffraction analysis of two crystals forms is presented, one diffracting to 1.9 A and the other to 0.96 A resolution.

Crystal structure of mouse succinic semialdehyde reductase AKR7A5: structural basis for substrate specificity.

The aldo-keto reductases make up a superfamily of enzymes which can reduce a variety of aldehydes and ketones to their corresponding alcohols. Within each family are distinct preferences for certain substrates, presumably reflecting their role within the cell. The original member of the AKR7A subfamily was purified from liver as an aflatoxin dialdehyde reductase AKR7A1. However, recent additions to the family have revealed that even closely related enzymes have clear substrate preferences with AKR7A2, AKR7A4, and AKR7A5 showing much higher affinities for succinic semialdehyde (SSA) than does AKR7A1. To investigate the structural basis of this specificity, the crystal structure of mouse AKR7A5 has been determined to better than 2.5 A resolution. The structure is of the ternary complex of the enzyme with NADP+ and tartrate as an inhibitor. This structure has the same overall fold as the previously determined structure of AKR7A1; however, there are a number of differences in loops around the active site that contribute to observed differences in the substrate specificity between the AKR7A enzymes. Several differences are the result of bulky hydrophobic residues found in AKR7A5, namely, Met44, Trp77, and Trp224, which significantly restrict the size and modify the architecture of the substrate-binding pocket, producing a tighter or less flexible binding site for SSA than in AKR7A1. Site-directed mutagenesis was used to introduce Met44, Trp77, and Trp224 individually into AKR7A1, to test if they improved the affinity of the enzyme for SSA. Each mutation showed improved affinity for SSA, with Trp77Met having the largest effect. This confirms the role of these amino acids as substrate determinants for SSA.

Crystal structures of Helicobacter pylori type II dehydroquinase inhibitor complexes: new directions for inhibitor design.

The crystal structures of the type II dehydroquinase (DHQase) from Helicobacter pylori in complex with three competitive inhibitors have been determined. The inhibitors are the substrate analogue 2,3-anhydroquinate (FA1), citrate, and an oxoxanthene sulfonamide derivative (AH9095). Despite the very different chemical nature of the inhibitors, in each case the primary point of interaction with the enzyme is via the residues that bind the C1 functionalities of the substrate, 3-dehydroquinate, i.e., N76, H102, I103, and H104. The DHQase/AH9095 complex crystal structure shows that sulfonamides can form a scaffold for nonsubstrate-like inhibitors and identifies a large conserved hydrophobic patch at the entrance to the active site as a locus that can be exploited in the development of new ligands.