NP-MRD: the Natural Products Magnetic Resonance Database.

The Natural Products Magnetic Resonance Database (NP-MRD) is a comprehensive, freely available electronic resource for the deposition, distribution, searching and retrieval of nuclear magnetic resonance (NMR) data on natural products, metabolites and other biologically derived chemicals. NMR spectroscopy has long been viewed as the 'gold standard' for the structure determination of novel natural products and novel metabolites. NMR is also widely used in natural product dereplication and the characterization of biofluid mixtures (metabolomics). All of these NMR applications require large collections of high quality, well-annotated, referential NMR spectra of pure compounds. Unfortunately, referential NMR spectral collections for natural products are quite limited. It is because of the critical need for dedicated, open access natural product NMR resources that the NP-MRD was funded by the National Institute of Health (NIH). Since its launch in 2020, the NP-MRD has grown quickly to become the world's largest repository for NMR data on natural products and other biological substances. It currently contains both structural and NMR data for nearly 41,000 natural product compounds from >7400 different living species. All structural, spectroscopic and descriptive data in the NP-MRD is interactively viewable, searchable and fully downloadable in multiple formats. Extensive hyperlinks to other databases of relevance are also provided. The NP-MRD also supports community deposition of NMR assignments and NMR spectra (1D and 2D) of natural products and related meta-data. The deposition system performs extensive data enrichment, automated data format conversion and spectral/assignment evaluation. Details of these database features, how they are implemented and plans for future upgrades are also provided. The NP-MRD is available at https://np-mrd.org.

Practical Aspects of NMR-Based Metabolomics.

While NMR-based metabolomics is only about 20 years old, NMR has been a key part of metabolic and metabolism studies for >40 years. Historically, metabolic researchers used NMR because of its high level of reproducibility, superb instrument stability, facile sample preparation protocols, inherently quantitative character, non-destructive nature, and amenability to automation. In this chapter, we provide a short history of NMR-based metabolomics. We then provide a detailed description of some of the practical aspects of performing NMR-based metabolomics studies including sample preparation, pulse sequence selection, and spectral acquisition and processing. The two different approaches to metabolomics data analysis, targeted vs. untargeted, are briefly outlined. We also describe several software packages to help users process NMR spectra obtained via these two different approaches. We then give several examples of useful or interesting applications of NMR-based metabolomics, ranging from applications to drug toxicology, to identifying inborn errors of metabolism to analyzing the contents of biofluids from dairy cattle. Throughout this chapter, we will highlight the strengths and limitations of NMR-based metabolomics. Additionally, we will conclude with descriptions of recent advances in NMR hardware, methodology, and software and speculate about where NMR-based metabolomics is going in the next 5-10 years.

Automated identification and quantification of metabolites in human fecal extracts by nuclear magnetic resonance spectroscopy.

We report the development of a software program, called MagMet-F, that automates the processing and quantification of 1D 1 H NMR of human fecal extracts. To optimize the program, we identified 82 potential fecal metabolites using 1D 1 H NMR of six human fecal extracts using manual profiling and a literature review of known fecal metabolites. We acquired pure versions of those metabolites and then acquired their 1D 1 H NMR spectra at 700 MHz to generate a fecal metabolite spectral library for MagMet-F. The fitting of these metabolites by MagMet-F was iteratively optimized to replicate manual profiling. We validated MagMet-F's automated profiling using a test set of six fecal extracts. It correctly identified 80% of the compounds and quantified those within <20% of the values determined by manual profiling using Chenomx. We also compared MagMet-F's profiling performance to two other open-access NMR profiling tools, Bayesil and Batman. MagMet-F outperformed both. Bayesil repeatedly overestimated metabolite concentrations by 10% to 40% while Batman was unable to properly quantify any compounds and took 10-20× longer. We have implemented MagMet-F as a freely accessible web server to enable automated, fast and convenient 1D 1 H NMR spectral profiling of fecal samples. MagMet-F is available at https://www.magmet.ca.

MagMet: A fully automated web server for targeted nuclear magnetic resonance metabolomics of plasma and serum.

Nuclear magnetic resonance (NMR) spectral analysis of biofluids can be a time-consuming process, requiring the expertise of a trained operator. With NMR becoming increasingly popular in the field of metabolomics, there is a growing need to change this paradigm and to automate the process. Here we introduce MagMet, an online web server, that automates the processing and quantification of 1D 1 H NMR spectra from biofluids-specifically, human serum/plasma metabolites, including those associated with inborn errors of metabolism (IEM). MagMet uses a highly efficient data processing procedure that performs automatic Fourier Transformation, phase correction, baseline optimization, chemical shift referencing, water signal removal, and peak picking/peak alignment. MagMet then uses the peak positions, linewidth information, and J-couplings from its own specially prepared standard metabolite reference spectral NMR library of 85 serum/plasma compounds to identify and quantify compounds from experimentally acquired NMR spectra of serum/plasma. MagMet employs linewidth adjustment for more consistent quantification of metabolites from higher field instruments and incorporates a highly efficient data processing procedure for more rapid and accurate detection and quantification of metabolites. This optimized algorithm allows the MagMet webserver to quickly detect and quantify 58 serum/plasma metabolites in 2.6 min per spectrum (when processing a dataset of 50-100 spectra). MagMet's performance was also assessed using spectra collected from defined mixtures (simulating other biofluids), with >100 previously measured plasma spectra, and from spiked serum/plasma samples simulating known IEMs. In all cases, MagMet performed with precision and accuracy matching the performance of human spectral profiling experts. MagMet is available at http://magmet.ca.

Molecular basis for impaired DNA damage response function associated with the RAP80 ΔE81 defect.

Signal transduction within the DNA damage response is driven by the flux of protein-protein interaction cascades that ultimately recruit repair complexes to sites of damage. The protein RAP80 plays a central role in the damage response by targeting BRCA1/BRCA2 tumor suppressors to DNA damage foci through multivalent binding of Lys-63-linked polyubiquitin chains. Mutations within the high penetrance BRCA1/BRCA2 genes account for ∼20% of familial breast cancers. The genetic basis for the remaining cancers remains unknown, but may involve defects in binding partners for BRCA1 and BRCA2 that lead to impaired targeting to foci and a concomitant role in the pathogenesis of cancer. Recently, an in-frame deletion mutation (ΔE81) in a conserved region from the first ubiquitin interaction motif of RAP80 has been linked to an increase in chromosomal abnormalities. Using NMR spectroscopy, we demonstrate that the N-cap motif within the α-helix of the first ubiquitin interaction motif from ΔE81 undergoes a structural frameshift that leads to abolishment of multivalent binding of polyubiquitin chains. Loss of this single glutamate residue disrupts favorable electrostatic interactions between RAP80 and ubiquitin, establishing a plausible molecular basis for a potential predisposition to cancer unrelated to mutations within BRCA1/BRCA2 genes.

Stochastic gate dynamics regulate the catalytic activity of ubiquitination enzymes.

Initiation of the DNA damage and innate immune responses is dependent upon the flow of chemical information through coupled protein-protein interaction networks and driven by the synthesis and recognition of Lys 63 linked polyubiquitin (polyUb) chains on adaptor proteins. The central chemical step in Lys 63-linked protein ubiquitination involves the reaction of a specific lysine on a target protein with Ub that is covalently attached as a thioester conjugate to the Ub conjugating enzyme (E2) Ubc13. The active site cysteine of Ubc13, and E2 enzymes in general, is buttressed by a flexible loop. The role of loop dynamics in catalysis was investigated by mutating the central and hinge residues to glycine. The loop dynamics were experimentally characterized through measurement of enzyme kinetics, main chain NMR relaxation, X-ray crystallographic studies, and in vivo studies in yeast. The experimental data were complemented by analysis of MD simulations of the dynamics and kinetics for the loop motion. The results show that fast pico- to nanosecond time scale active site loop fluctuations play a crucial role in regulating the catalytic activity of Ubc13 by functioning as a stochastic active site gate, which is characterized by precisely balanced rates of opening and closing. In vivo functional complementation assays in yeast demonstrate that defects within this regulatory mechanism can have profound biological consequences, given that Ubc13 is the only E2 dedicated to synthesizing Lys 63-linked polyUb chains.

Effects of thermal denaturation on the solid-state structure and molecular mobility of glycinin.

The effects of moisture and thermal denaturation on the solid-state structure and molecular mobility of soy glycinin powder were investigated using multiple techniques that probe over a range of length and time scales. In native glycinin, increased moisture resulted in a decrease in both the glass transition temperature and the denaturation temperature. The sensitivity of the glass transition temperature to moisture is shown to follow the Gordon-Taylor equation, while the sensitivity of the denaturation temperature to moisture is modeled using Flory's melting point depression theory. While denaturation resulted in a loss of long-range order, the principal conformational structures as detected by infrared are maintained. The temperature range over which the glass to rubber transition occurred was extended on the high temperature side, leading to an increase in the midpoint glass transition temperature and suggesting that the amorphous regions of the newly disordered protein are less mobile. (13)C NMR results supported this hypothesis.

Cortisol modulates calcium release-activated calcium channel gating in fish hepatocytes.

Glucocorticoids (GCs) are rapidly released in response to stress and play an important role in the physiological adjustments to re-establish homeostasis. The mode of action of GCs for stress coping is mediated largely by the steroid binding to the glucocorticoid receptor (GR), a ligand-bound transcription factor, and modulating the expression of target genes. However, GCs also exert rapid actions that are independent of transcriptional regulation by modulating second messenger signaling. However, a membrane-specific protein that transduces rapid GCs signal is yet to be characterized. Here, using freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) and fura2 fluorescence microscopy, we report that stressed levels of cortisol rapidly stimulate the rise in cytosolic free calcium ([Ca2+]i). Pharmacological manipulations using specific extra- and intra-cellular calcium chelators, plasma membrane and endoplasmic reticulum channel blockers and receptors, indicated extracellular Ca2+ entry is required for the cortisol-mediated rise in ([Ca2+]i). Particularly, the calcium release-activated calcium (CRAC) channel gating appears to be a key target for the rapid action of cortisol in the ([Ca2+]i) rise in trout hepatocytes. To test this further, we carried out in silico molecular docking studies using the Drosophila CRAC channel modulator 1 (ORAI1) protein, the pore forming subunit of CRAC channel that is highly conserved. The result predicts a putative binding site on CRAC for cortisol to modulate channel gating, suggesting a direct, as well as an indirect regulation (by other membrane receptors) of CRAC channel gating by cortisol. Altogether, CRAC channel may be a novel cortisol-gated Ca2+ channel transducing rapid nongenomic signalling in hepatocytes during acute stress.

Candidate serum metabolite biomarkers of residual feed intake and carcass merit in sheep.

Mutton and lamb sales continue to grow globally at a rate of 5% per year. However, sheep farming struggles with low profit margins due to high feed costs and modest carcass yields. Selecting those sheep expected to convert feed efficiently and have high carcass merit, as early as possible in their life cycle, could significantly improve the profitability of sheep farming. Unfortunately, direct measurement of feed conversion efficiency (via residual feed intake [RFI]) and carcass merit is a labor-intensive and expensive procedure. Thus, indirect, marker-assisted evaluation of these traits has been explored as a means of reducing the cost of its direct measurement. One promising and potentially inexpensive route to discover biomarkers of RFI and/or carcass merit is metabolomics. Using quantitative metabolomics, we profiled the blood serum metabolome (i.e., the sum of all measurable metabolites) associated with sheep RFI and carcass merit and identified candidate biomarkers of these traits. The study included 165 crossbred ram-lambs that underwent direct measurement of feed consumption to determine their RFI classification (i.e., low vs. high) using the GrowSafe System over a period 40 d. Carcass merit was evaluated after slaughter using standardized methods. Prior to being sent to slaughter, one blood sample was drawn from each animal, and serum prepared and frozen at -80 °C to limit metabolite degradation. A subset of the serum samples was selected based on divergent RFI and carcass quality for further metabolomic analyses. The analyses were conducted using three analytical methods (nuclear magnetic resonance spectroscopy, liquid chromatography mass spectrometry, and inductively coupled mass spectrometry), which permitted the identification and quantification of 161 unique metabolites. Biomarker analyses identified three significant (P < 0.05) candidate biomarkers of sheep RFI (AUC = 0.80), seven candidate biomarkers of carcass yield grade (AUC = 0.77), and one candidate biomarker of carcass muscle-to-bone ratio (AUC = 0.74). The identified biomarkers appear to have roles in regulating energy metabolism and protein synthesis. These results suggest that serum metabolites could be used to categorize and predict sheep for their RFI and carcass merit. Further validation using a larger (3×) and more diverse cohort of sheep is required to confirm these findings.

Fluctuating partially native-like topologies in the acid denatured ensemble of autolysis resistant HIV-1 protease.

Folding, in-vivo, starts from a denatured state and thus the nature of the denatured state would play an important role in directing the folding of a protein. We report here NMR characterization of the acid-denatured state of a mutant of HIV-1 protease, designed to prevent autolysis (Q7K, L33I, L63I) and to prevent cysteine oxidation (C67A and C95A). Secondary chemical shifts, TALOS analysis of chemical shifts and (15)N relaxation data (R(1), R(2), NOE) coupled with AABUF and hydrophobicity calculations, suggest formation of hydrophobic clusters and possibility of some partially native-like topologies in the acid denatured state of the protease. The structural and dynamics characteristics of the acid denatured PR seem to be considerably different from those of the guanidine or urea denatured states of some variants of PR. These would have implications for the folding and auto-processing of the enzyme in-vivo.

Automated Tools for the Analysis of 1D-NMR and 2D-NMR Spectra.

Nuclear magnetic resonance (NMR) spectroscopy is becoming increasingly automated. Most modern NMR spectrometers are now equipped with auto-tune/auto-match probes along with automated locking and shimming systems. Likewise, more and more instruments, especially for NMR-based metabolomics applications, are equipped with automated sample changers. All this instrumental automation allows NMR data to be collected at a rate of >100 samples/day. However, a continuing bottleneck in NMR-based metabolomics has been the time required to manually analyze and annotate the collected NMR spectra. In many cases, manual spectral annotation and analysis can take one or more hours per spectrum. Fortunately, over the past few years, several software tools have been developed that largely automate the spectral deconvolution or spectral annotation process. Using these tools requires that the samples must be prepared and the NMR spectra must be acquired in a very specific manner. In this chapter, we will describe the step-by-step preparation of biofluid samples along with the required protocols for acquiring optimal spectra for automated NMR metabolomics analysis. We will also discuss the use of three common tools (Chenomx NMR Suite, Bayesil, and COLMARm) for (semi-) automated profiling, and annotation of 1D- and 2D-NMR spectra of biofluids.

Structure and molecular mobility of soy glycinin in the solid state.

We report a multitechnique study of structural organization and molecular mobility for soy glycinin at a low moisture content (<30% w/w) and relate these to its glass-to-rubber transition. Small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy are used to probe structure and mobility on different length and time scales. NMR (approximately 10(-6) to 10(-3) s) reveals transitions at a higher moisture content (>17%) than DSC or SAXS, which sample for much longer times (approximately 10 to 10(3) s) and where changes are detected at >13% water content at 20 degrees C. The mobility transitions are accompanied by small changes in unit-cell parameters and IR band intensities and are associated with the enhanced motion of the polypeptide backbone. This study shows how characteristic features of the ordered regions of the protein (probed by SAXS and FTIR) and mobile segments (probed by NMR and DSC) can be separately monitored and integrated within a mobility transformation framework.

Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K.

The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a three-component enzyme system. Ubiquitination, or E2 enzymes, catalyze the central step by facilitating reaction of a target protein lysine with the C-terminus of Ub that is attached to the active site cysteine of the E2 through a thioester bond. E2 reactivity is modulated by dynamics of an active site gate, whose central residue packs against the active site cysteine in a closed conformation. Interestingly, for the E2 Ubc13, which specifically catalyzes K63-linked ubiquitination, the central gate residue adopts an open conformation. We set out to determine if active site gate dynamics play a role in catalysis for E2-25K, which adopts the canonical, closed gate conformation, and which selectively synthesizes K48-linked ubiquitin chains. Gate dynamics were characterized using mutagenesis of key residues, combined with enzyme kinetics measurements, and main chain NMR relaxation. The experimental data were interpreted with all atom MD simulations. The data indicate that active site gate opening and closing rates for E2-25K are precisely balanced.

Production of particulates from transducer erosion: implications on food safety.

The formation of metallic particulates from erosion was investigated by running a series of transducers at various frequencies in water. Two low frequency transducer sonotrodes were run for 7.5h at 18kHz and 20kHz. Three high frequency plates operating at megasonic frequencies of 0.4MHz, 1MHz, and 2MHz were run over a 7days period. Electrical conductivity and pH of the solution were measured before and after each run. A portion of the non-sonicated and treated water was partially evaporated to achieve an 80-fold concentration of particles and then sieved through nano-filters of 0.1µm, 0.05µm, and 0.01µm. An aliquot of the evaporated liquid was also completely dried on strips of carbon tape to determine the presence of finer particles post sieving. An aliquot was analyzed for detection of 11 trace elements by Inductively Coupled Plasma Mass Spectroscopy (ICPMS). The filters and carbon tapes were analyzed by FE-SEM imaging to track the presence of metals by EDS (Energy Dispersive Spectroscopy) and measure the particle size and approximate composition of individual particles detected. Light microscopy visualization was used to calculate the area occupied by the particles present in each filter and high resolution photography was used for visualization of sonotrode surfaces. The roughness of all transducers before and after sonication was tested through profilometry. No evidence of formation of nano-particles was found at any tested frequency. High amounts of metallic micron-sized particles at 18kHz and 20kHz formed within a day, while after 7day runs only a few metallic micro particles were detected above 0.4MHz. Erosion was corroborated by an increase in roughness in the 20kHz tip after ultrasound. The elemental analysis showed that metal leach occurred but values remained below accepted drinking water limits, even after excessively long exposure to ultrasound. With the proviso that the particles measured here were only characterized in two dimensions and could be nanoparticulate in terms of the third dimension, this research suggests that there are no serious health implications resulting from the formation of nanoparticles under the evaluation conditions. Therefore, high frequency transducer plates can be safely operated in direct contact with foods. However, due to significant production of metallic micro-particulates, redesign of lower frequency sonotrodes and reaction chambers is advised to enable operation in various food processing direct-contact applications.

Structural basis of prion inhibition by phenothiazine compounds.

Conformational transitions of the cellular form of the prion protein, PrP(C), into an infectious isoform, PrP(Sc), are considered to be central events in the progression of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies. Tricyclic phenothiazine compounds exhibit antiprion activity; however, the underlying molecular mechanism of PrP(Sc) inhibition remains elusive. We report the molecular structures of two phenothiazine compounds, promazine and chlorpromazine bound to a binding pocket formed at the intersection of the structured and the unstructured domains of the mouse prion protein. Promazine binding induces structural rearrangement of the unstructured region proximal to ß1, through the formation of a "hydrophobic anchor." We demonstrate that these molecules, promazine in particular, allosterically stabilize the misfolding initiator-motifs such as the C terminus of α2, the α2-α3 loop, as well as the polymorphic ß2-α2 loop. Hence, the stabilization effects of the phenothiazine derivatives on initiator-motifs induce a PrP(C) isoform that potentially resists oligomerization.

Single point mutation induced alterations in the equilibrium structural transitions on the folding landscape of HIV-1 protease.

Equilibrium folding-unfolding transitions are hard to study in HIV-1 protease (PR) because of its autolytic properties. Further, the protease exhibits many tolerant point mutations some of which also impart drug resistance to the protein. It is conceivable that the mutations affect protein's function by altering its folding characteristics; these would clearly depend on the nature of the mutations themselves. In this background, we report here NMR studies on the effects of D25 N mutation, which removes one negative charge from the protein at the active site, on the equilibrium folding behaviour of PR starting from its acetic acid denatured state. It is observed that in PRD25N two slowly exchanging conformations are present at the N-terminal. One of them is similar to that of PR. Though the conformational and dynamics preferences of PR and PRD25N are fairly similar in 9 M acetic acid, they seem to undergo different folding transitions when acetic acid concentration is reduced. The differences are seen in the active site, in the flap, and in the hinge of the flap regions. The present study suggests that such differences, though different in detail, would occur for other mutations as well, and also for different initial denatured states. These would have significant regulatory implications for the efficacy of protease function.

Creaming enhancement in a liter scale ultrasonic reactor at selected transducer configurations and frequencies.

Recent research has shown that high frequency ultrasound (0.4-3 MHz), can enhance milkfat separation in small scale systems able to treat only a few milliliters of sample. In this work, the effect of ultrasonic standing waves on milkfat creaming was studied in a 6L reactor and the influence of different frequencies and transducer configurations in direct contact with the fluid was investigated. A recombined coarse milk emulsion with fat globules stained with oil-red-O dye was selected for the separation trials. Runs were performed with one or two transducers placed in vertical (parallel or perpendicular) and horizontal positions (at the reactor base) at 0.4, 1 and/or 2 MHz (specific energy 8.5 ± 0.6 kJ/kg per transducer). Creaming behavior was assessed by measuring the thickness of the separated cream layer. Other methods supporting this assessment included the measurement of fat content, backscattering, particle size distribution, and microscopy of samples taken at the bottom and top of the reactor. Most efficient creaming was found after treatment at 0.4 MHz in single and double vertical transducer configurations. Among these configurations, a higher separation rate was obtained when sonicating at 0.4 MHz in a vertical perpendicular double transducer setup. The horizontal transducer configuration promoted creaming at 2 MHz only. Fat globule size increase was observed when creaming occurred. This research highlights the potential for enhanced separation of milkfat in larger scale systems from selected transducer configurations in contact with a dairy emulsion, or emulsion splitting in general.

Denaturation of HIV-1 protease (PR) monomer by acetic acid: mechanistic and trajectory insights from molecular dynamics simulations and NMR.

Inside a living cell there can be a variety of interactions for any given protein, which serve to regulate denaturation and renaturation processes. Insights into some of them can be obtained by in vitro studies using various denaturing agents. In this study, all-atom MD simulations in explicit solvent and NMR relaxation studies were performed on HIV-1 Protease (PR) in 9 M acetic acid (AcOH) (the commonly used denaturant during PR preparation). Following previous reports that denaturation proceeds via dissociation of the dimer into monomers, unfolding of the monomer by acetic acid has been explicitly investigated here. Direct visualization of the denaturation process and evidence for the mechanism of denaturation have been presented. Our simulations reveal that the denaturation of the PR monomer is caused due to direct interaction between acetic acid molecules and PR. Autocorrelation of N-H vectors calculated from the simulations have revealed that the α-helix and the surrounding ß-strands represent the sensitive regions of the PR that respond maximally to the change in the solvent environment around the PR and are prone to disruption by acetic acid. This disruption is caused due to increased penetration of the acetic acid molecules into the PR structure by formation of preferred tertiary contacts and hydrogen bonds between the PR and acetic acid molecules. Following the loss of these critical interactions, the PR follows a random and non-equilibrating path on the conformation landscape and cycles between different denatured extended and compact states.

Reduced dimensionality 3D HNCAN for unambiguous HN, CA and N assignment in proteins.

We present here an improvisation of HNN (Panchal, Bhavesh et al., 2001) called RD 3D HNCAN for backbone (HN, CA and (15)N) assignment in both folded and unfolded proteins. This is a reduced dimensionality experiment which employs CA chemical shifts to improve dispersion. Distinct positive and negative peak patterns of various triplet segments along the polypeptide chain observed in HNN are retained and these provide start and check points for the sequential walk. Because of co-incrementing of CA and (15)N, peaks along one of the dimensions appear at sums and differences of the CA and (15)N chemical shifts. This changes the backbone assignment protocol slightly and we present this in explicit detail. The performance of the experiment has been demonstrated using Ubiquitin and Plasmodium falciparum P2 proteins. The experiment is particularly valuable when two neighboring amino acid residues have nearly identical backbone (15)N chemical shifts.

Modulation of in vitro activity of zymogenic and mature recombinant human ß-secretase by dietary plants.

The in vitro activity of human recombinant ß-secretase (BACE1) was studied using a fluorogenic substrate based on the cleavage site for the enzyme in the Swedish mutation of amyloid precursor protein. The enzyme was inhibited by a control peptide inhibitor with good repeatability. The enzyme preparation comprised a mixture of pro-enzyme or zymogen and mature enzyme whereby the pro-enzyme sequence forms a 'flap' that can obstruct the binding site. 'Open flap' forms of the zymogen and mature enzyme are active, but the 'closed flap' form of the zymogen is inactive. This mixture of enzyme populations permitted apparent stimulation of enzyme activity under particular conditions, presumably due to facilitating flap-opening of the zymogen. As reported for heparin, enzyme activation was stimulated in the presence of low concentrations of Tween 20 and dimethylsulfoxide before becoming inhibited at higher concentrations. Dietary plant extracts either consistently inhibited (e.g. clove, tea, cinammon) or consistently stimulated (e.g. mushroom, parsley, asparagus) BACE1. Common structural features identified by Fourier transform infrared spectroscopy revealed that BACE1 activity could be explained by differential interactions of either small molecule or polymeric species with mature versus zymogen forms of the enzyme, respectively. Further, enzyme activity could be reversed by mixtures of high and low mass species. These results may have implications for the regulation of ß-secretase activity in vivo by either endogenous or possibly dietary factors and for a potential role of BACE1 in stimulation of the production of amyloid beta peptide in sporadic Alzheimer's disease.