NMR spectroscopy is a powerful tool for probing molecular motions in solids. In this work we used solid-state 17O NMR to probe the sulfonate rotational dynamics in the crystal of L-cysteic acid monohydrate. Variable temperature 17O solid-state NMR spectra were recorded for L-[17O3]cysteic acid monohydrate at 11.7 and 16.4T. Spectral changes in the central-transition 17O NMR signal suggest that the SO3- undergoes a 3-fold jump. The rotational barrier found from the Arrhenius plot is 34.6 ± 1.9kJ/mol. The value is considerably smaller than those previously reported for other related sulfonic acids, suggesting that the hydrogen bonds around the SO3- group in L-cysteic acid monohydrate are relatively weak.
Wrapping (or aciniform) silk is the toughest of the six fibrillar spider silks (i.e., it absorbs more energy before failure), with a toughness per unit mass that is among the highest of known materials. In Argiope trifasciata, wrapping silk has a repetitive domain containing 14 or more identical 200 amino acid units (“W” units). Using solution-state NMR spectroscopy, we have shown that each W unit has globular domain of 5 α-helices connected to the neighbouring W units by intrinsically disordered linkers. During fibrillogenesis, a partial α-helix to β-sheet conversion occurs, although the atomic-level nature of this conversion remains elusive. As we gain clues, this is in turn allowing us to modulate and optimize protein structuring and properties. Fibre property modification and optimization is also being explored. As with a number of other spider silks, increasing the size of the starting material (i.e., adding W units to the protein) correlates with an improved strength and, correspondingly, toughness. Addition of non-repetitive silk domains from wrapping silk or spider dragline silk also changes the resulting fibre mechanical properties. The observed changes are not predicable on the basis of prior studies of other types of silks, indicative of distinctive self-assembly behaviour by wrapping silk. We have also determined that spinning conditions and fibrillar stretching following spinning modulate wrapping silk fibre mechanical properties, providing us a further means of customization of material properties from the same starting material. As a whole, we are targeting development of comprehensive understanding of wrapping silk self-assembly and fibre formation as a means to develop new biomaterials.
“Patchy particles” where the surface is anisotropically patterned through variation in the surface composition, can form different colloidal crystal structures and have applications as interface stabilizers, in catalysis and targeted drug delivery. Spontaneous phase separation has been detected in mixed ligand shells of nanoparticles for both, low molecular weight and polymeric ligands.
Although there is growing interest in such “patchy nanoparticles”, the majority of the studies have been theoretical rather than experimental due to difficulties in preparing significant quantities of nanoparticles with controlled ligand compositions.  Experimental validation has also lagged to due to the lack of appropriate tools to detect nanoscale phase separation. Here we apply NMR methods developed for characterizing heterogeneities in complex materials on different length scales to nanoparticles with polymer and low molecular weight mixed ligand shells. The mixed ligand shells consisted of different ratios of aromatic (polystyrene or phenyl) and aliphatic (polyethylene oxide or dodecyl) phosphonic acids on 4 nm ZrO2 nanoparticles.
The results for 1H NOESY, 1H double quantum MAS and 1H spin diffusion experiments on the nanoparticles with polymer versus small molecule ligand shells were compared. In the case of the low molecular weight mixed ligand shells, model systems consisting of Janus particles or physical mixtures of nanoparticles functionalized with only one ligand were studied to calibrate the 1H spin diffusion experiments originally developed for polymeric materials.
1. Chen et al. J. Poly. Sci, B: Poly. Phys. 2014, 52, 1583.
Magnetic resonance spectroscopy with the infusion of 13C-enriched substrates allows the characterization of brain oxidative metabolism and neurotransmission by following the incorporation of the 13C label into the different carbon positions of amino acid neurotransmitters and metabolites. In 2004, Boumezbeur used dynamic 1H MRS to detect the metabolism of infused 13C glucose in brain with standard MR sequence and hardware available to conventional preclinical scanner. In this study we aimed to revisit the idea of dynamic 1H MRS for detection of 13C labelled glucose metabolism in the brain, and demonstrate for the first time its application in human subjects.
One healthy human participant was studied after giving informed written consent. A single bolus infusion of 0.23 g/kg body weight of 99% [1-13C] glucose was administrated intravenously into the vein at a constant rate over 15 min. All MR data were acquired with a 1H body coil transmitter and a 32-channel 1H receive head array implemented on a 3-T Siemens TIM Trio scanner. To guide placement of the voxels of interest (VOIs) for MRS acquisition, a three-dimensional T1-weighted anatomical image was acquired using a magnetization prepared rapid gradient echo acquisition. Two VOIs, each measuring 30 ml, were positioned for 1H MRS acquisition; one in anterior cingulate cortex (ACC) and one in precuneus/posterior cingulate cortex (PCC). First and second order shimming within each region of interest was achieved using the FASTESTMAP sequence, and 1H MRS acquisitions were acquired using the short-TE SPECIAL sequence. Outer volume suppression was applied to minimize unwanted signal from outside the VOI, and the water signal was suppressed using the VAPOR scheme. Prior to the 13C glucose infusion, one baseline water-suppressed scan and one water unsuppressed scan was performed in each VOI. During and following the infusion, repeated water-suppressed MRS scans were acquired, alternating between ACC and PCC voxels. Spectral pre-processing was performed in MATLAB using the FID-A toolkit. To allow visualization of the dynamic post-infusion changes in 1H MRS signal, the processed pre-infusion scan was then subtracted from the each timepoint’s processed post-infusion scan, resulting in four difference spectra in ACC and three difference spectra in PCC. LC Model was used for quantification of the fully processed difference spectra using in-house simulated basis sets. The post-infusion difference spectra were quantified in LCModel using the generated post-infusion basis sets that include the components to accounting for enrichments of glutamate C4 and C3.
Following a low-dose infusion of 1-13C glucose, the presence of the 13C label was clearly detectable, owing to the pronounced effect of heteronuclear (13C-1H) scalar coupling on the observed 1H spectral patterns. The 13C labelling of glutamate was characterized by a decrease in signal from 12C-bonded protons and an increase in signal from 13C-coupled protons.
The fractional enrichments of Glu-C4 and Glu-C3 rose rapidly in both in both PCC and ACC regions, reaching a plateau enrichment of 13 % and 4- 5%, respectively, during approximately 40 min following glucose infusion. The similar fractional enrichment of Glu-C4 and Glu-C3 in PCC and ACC regions demonstrate the repeatability of this approach at different in vivo conditions such as localization and shimming.
These preliminary results confirm the feasibility of the use of dynamic conventional 1H MRS to monitor the conversion of 13C labelled glucose into downstream metabolic products in the human brain. This approach avoids the need for expensive 13C hardware and complicated acquisition sequences, and is straightforward to implement on most clinical grade scanners.
Spider silk is a strong, extensible and mechanically resistant fiber. It is therefore particularly interesting in various fields of application as in medicine or in the textile industry. The structure of silk is dictated by the primary structure of the spidroins and by the spinning process, notably by the spinning speed. Thus, we study the effect of spinning rate on the molecular structure and the dynamics of various amino acids located in the crystalline and amorphous phases of silk.
The response of silk to water, more precisely its contraction amplitude when the fiber is exposed to high humidity (a phenomenon called "supercontraction"), is also modified by spinning speed. It seems that the spinning speed influences the structure of the silk, especially in terms of molecular orientation of the polypeptide chains. Moreover, the outstanding properties of silk can be attributed to the protein conformation and packing structures. When supercontraction occurs, a decrease in strength and an increase in elasticity can be observed. The question is what controls the amplitude of supercontraction, how amino acids regulate this phenomenon and how are they affected by water or humidity? To date, however, only a few studies have examined those questions in detail, systematically and quantitatively
We want to answer these uncertainties and to this aim, we plan to analyze the molecular structure of dragline silk of two spider species, Nephila clavipes and Araneus diadematus, which differ in the proteins that make up the fiber. The fibers are obtained by forced spinning at speeds varying between 0,3 and 20 cm/s and are subjected to a relative humidity greater than 90%. We characterize the fibers by Raman spectromicroscopy and by solid state nuclear magnetic resonance, more precisely with the sequence ROCSA (recoupling of chemical shift anisotropy). To do so, different amino acids labeled solution (1-13C-Gly and 1-13C-Ala) were administered to two groups of spiders. Raman spectroscopy allows quantifying the orientation of the protein chains and determining the proportions of secondary structures. Solid state NMR can provide information about the secondary structure and dynamics of amino acids. The two techniques are therefore complementary and should provide a global vision and a better understanding of the impact of spinning speed on the structure of spider silk, its response to water and its properties.
The addition of boronic acid groups to silicone polymers results in the formation of highly bio-compatible elastomeric films.1 Film formation is attributed to crosslinking at the boronic acid site. However, this interaction and the structure of the resultant boron coordination environments remain poorly understood. Boron coordination environments can be diverse as three- and four-coordinate sites are possible in these materials. It is also unknown whether these linkages are B-O-B or B-O-Si bonds or some combination of the two. Quadrupolar NMR lineshape fitting has been extensively used to characterize boron coordination environments in amorphous glasses2 and in mineral samples3 as three- and four-coordinate environments possess widely different quadrupole parameters. This technique is not often used to determine boron coordination environment in polymeric materials but is used here to characterize boron environments in terms of their symmetry and coordination number. Chain extension in these materials is attributed to hydrogen bonding between boronic acids and is indicated by the presence of 3-coordinate boron sites. Elastomers are formed via crosslinking between boronic acid sites through 4-coordinate, dative bonded boron centers. The proportion of boronic acid in the material and boronic acid density both influence crosslinking in these materials.
1. Dodge, L., Chen, Y. & Brook, M. A. Silicone boronates reversibly crosslink using Lewis acid-Lewis base amine complexes. Chem. - A Eur. J. 20, 9349–9356 (2014).
2. Kroeker, S. & Stebbins, J. F. Three-coordinated boron-11 chemical shifts in borates. Inorg. Chem. 40, 6239–6246 (2001).
3. MacKenzie, K.J.D., Smith, M. . Multinuclear Solid-State NMR of Inorganic Materials. (Elsevier Science Ltd, 2002).
The protozoan parasite Giardia intestinalis does not synthesize heme and lacks many heme proteins common to eukaryotes, yet it expresses four isotypes of the cytochrome b5 family of small redox proteins. Giardia cytochromes b5 (CYTB5s) have low reduction potentials and distinct subcellular locations, which is consistent with structural features and biological roles that differ markedly from their thoroughly studied mammalian counterparts. Our goal is to determine the structure of gCYTB5-III, which unusually for proteins of this type is found solely in the nucleus. Heme proteins are especially interesting targets for NMR studies as 1H-1D NMR can provide useful information on the local heme environment owing to the significant effects on the chemical shifts of nearby protons caused by the strong porphyrin aromatic ring current and the paramagnetism of the ferric state iron. To obtain recombinant, heme-replete gCYTB5-III for NMR experiments, we optimized expression with a lactose-driven auto-induction system, which mitigates user intervention and can lead to higher protein yields. To establish the general utility of our expression system and for comparison purposes, we also studied bovine microsomal cytochrome b5 (CYTB5A). Both cytochromes were expressed in E. coli BL21; optimal expression in unlabeled rich media yielded 160 mg / L culture, while the yield from 15N-labelled minimal media was fourth-fold lower. The 1H-1D and 1H-15N HSQC NMR spectra of CYTB5A compared well to those reported previously, while those of gCYTB5-III indicated significant differences in its heme-binding pocket, despite a similar coordination environment consisting of a pair of axial histidine ligands. These results and other NMR experiments in progress extends auto-induction and isotope-labelling methods to heme proteins and helps reveal structural features of the enigmatic gCYTB5s.
Stable isotopic labeling is an important part of structural biology by multidimensional NMR spectroscopy. Improvements and novel stable isotopic labeling methods have continuously been developed to navigate the possibilities to enhance protein NMR spectroscopy. Generating 3D structures from 15N- and 13C-labeled proteins is already well established. In this project, we explore the efficiency of 17O-labeling of proteins for 17O NMR studies. We used yeast ubiquitin (8.6 kDa) as a model system for studying the incorporation of 17O-labeled amino acid residues. The glycine, alanine and phenylalanine residues in yeast ubiquitin are selectively labeled using 17O isotopes. These residue-specifically 17O-labeled ubiquitins will be used for further 17O NMR studies.
Half of Canadians above the age of 85 suffer from Alzheimer’s Disease (AD), a neurodegenerative disorder characterized by memory loss and progressive cognitive impairment. Despite being the focus of enormous research efforts, there remains a lack of accurate pre-mortem diagnosis and effective treatments against AD. Carbon-13 (13C) magnetic resonance spectroscopy (MRS) is a powerful non-invasive technique that enables quantitative assessment of metabolic fluxes such as glucose tricarboxylic acid (TCA) cycling and glutamate/glutamine neurotransmitter cycling rates in vivo. This is achieved by infusing a 13C labelled substrate such as glucose, and subsequently detecting the uptake of 13C label into downstream metabolic products. Since the 13C-labelled substrates used in MRS are metabolized normally by the brain, 13C-MRS enables a direct measure of the rate of conversion of glucose into downstream metabolic products such as glutamate and glutamine.
The aim of this study was to apply 1H and 13C MRS to detect neurochemical and metabolic alterations in the brain in a rat model of Alzheimer’s disease and to develop quantitative modelling methods for reproducible assessment of neurochemical and metabolic rates in the rat brain. Specifically, we studied the TgF344-AD rodent model, which exhibits both amyloid and tau pathology as well as neurodegeneration mirroring human AD pathology. We hypothesized that TgF344-AD rats will exhibit reduced TCA cycle and glutamate/glutamine neurotransmitter cycling rates relative to wild type rats.
Scans were performed on a 7T Bruker Biospec 70/30 horizontal bore preclinical scanner. High resolution structural image was taken using the RARE sequence, TR/TE = 2713/10.8 ms, with a 2-minute acquisition time for VOI selection (4 x 5 x 5 mm3 voxel), selecting for the hippocampus and corpus collosum. We acquired localized water-suppressed 1H spectra using the POCE PRESS sequence, TR/TE = 4000/8.13 ms, 6000 Hz Spectral width, 128 averages, with 8 min 48s acquisition time. Spectra were processed using the FID-A toolkit and fitted using LCModel. Plasma samples were taken every thirty minutes through the femoral artery for metabolic modelling and 13C labelled glucose was infused through the femoral vein using a bolus infusion protocol.
Preliminary results show that this method can be used to follow and quantitatively measure the rate of 13C label transfer from glucose into glutamate and glutamine in the brain. Fractional enrichment was calculated and plotted for 13C labeling on the 3rd and 4th carbon of both glutamate and glutamine. Initial results comparing fractional enrichment between wildtype and transgenic rats agrees that TgF344-AD rats exhibit slower 13C labeling, suggesting a lowered glucose metabolic rate in AD pathology. The 13C MRS methods developed here will provide valuable complimentary information to FDG-PET by revealing, in vivo, which specific glucose metabolism pathways are altered in AD and will allow non-invasive detection of pre-symptomatic changes in the brain. We expect both our experimental methods and findings to be translatable to humans and that translational 1H and 13C MRS methods will allow for early detection of decreased glucose metabolism in pre-symptomatic stages of AD.
α-Synuclein (AS) is an amyloid protein involved in Parkinson’s disease. In pathological cases, aggregates of this protein form in the dopaminergic neuronal network, leading to its progressive degeneration accompanied with a dramatic decrease in dopamine levels. Under physiological conditions, AS is disordered in solution or weakly bound to neuronal membranes, via the formation of α-helices. The triggers and steps underlying the formation of insoluble β-sheet rich fibrils are still unclear. In our work, we focus on a central 12 amino acids segment of AS in the amyloidogenic part of the protein that is believed to be responsible for the fibrillization of the whole protein: AS71-82. Interactions between AS and neuronal membranes are thought to be the starting point of the fibrillization process, triggering the pathogenic amyloid cascade. In order to investigate and probe the mechanisms responsible for this fibrillization, model membranes composed of different ratios of zwitterionic (PC) and anionic (PG) phospholipids were used in our work. Infrared spectroscopy allowed the identification of irreversible changes in the β-sheet structure of AS71-82 upon the gel→fluid phase transition of the lipids, underlining the critical role of peptide/membrane interactions. Furthermore, the 31P solid-state NMR study of phospholipid polar headgroups is arguably a powerful method to probe the interactions between peptides and model membranes. Recently, a 2D pulse sequence named PROCSA (phosphorus recoupling of chemical shift anisotropy) was developed in order to study model membranes composed of a mixture of phospholipids1. This MAS (magic-angle spinning) pulse sequence gives rise to spectra with isotropic chemical shifts in the direct dimension and the powder spectra of each phospholipid in the indirect dimension. This feature is the main advantage of PROCSA when compared to standard static 31P spectra where the powder spectra of all phospholipids are superimposed and hard to separate. Eventually, PROCSA provides insights into the peptide/membrane interactions with structural and dynamical information on the preferentially interacting phospholipid in the mixture composing the membrane.
1 Warschawski, Dror E., Arnold, Alexandre A. and Marcotte, Isabelle (2018) Biophys. J. , 114 , 6, 1368-1376
Conventional methods for determination of meat composition are generally laborious, time-consuming, and destructive to the samples, which therefore are not suited for on-line applications. This work investigated the possibility of determining meat composition in fresh, thawed, and thawed–ground pork loins by using time-domain nuclear magnetic resonance (TD‑NMR).
By performing leave-one-out cross-validation and partial least squares regression, calibration models were established using NMR signals from 100 samples in two series. These signals yielded superior predictive performance for quantifying water and protein content than lipid content. For thawed samples, reducing the sample size impaired the lipid content determination. The modeling results were better for thawed–ground samples than for fresh samples. The best calibration result for thawed–ground samples was achieved with calibration correlation coefficient RC of 0.9992, 0.9500, and 0.9781 and root-mean-squared error of calibration (RMSEC) of 1.68, 1.77, and 2.77 g for water, lipid, and protein content, respectively.
Another calibration was performed on a smaller scale (30 samples) using weighed amounts of chicken breast and pork back fat as lean and fat components, respectively. RC of 0.9995 and 0.9966 and RMSEC of 1.29 and 0.80 g were obtained at 4°C for the lean and fat components, respectively, and comparable numbers were obtained for analyzed water, lipid, and protein content. New samples (8) were analyzed to validate the performance of the calibration. The explained variance of prediction RP2 was slightly reduced for the lean component compared to the explained variance of cross-validation RCV2 (RP2 = 0.9790 vs. RCV2 = 0.9978), but was lower for the fat component (RP2 = 0.6751 vs. RCV2 = 0.9677), likely due to the different composition of the fat used for sample preparation of the calibration and validation batches.
The calibration performed (6‑cm probe; samples between 60 and 230 g) could be validated further with turkey breast and lard (11‑cm probe; samples up to ca. 900 g).
Malaria is a life-threatening disease responsible for about a million fatalities per year worldwide and about half of the world population is at risk. Most of the malaria deaths are caused by Plasmodium falciparum. An essential regulator for the development of P. falciparum is the cyclic GMP (cGMP) dependent protein kinase (PfPKG). PfPKG is composed of a regulatory domain and a catalytic domain. In the absence of cGMP, the regulatory domain inhibits the kinase domain. Upon cGMP binding to the regulatory domain, the inhibition is released and PfPKG is activated. Targeting directly the active site of PfPKG poses a major selectivity challenge, since the kinase catalytic domains are highly conserved among eukaryotes. One approach to circumvent this problem is to selectively target less conserved allosteric sites of PfPKG, such as the cGMP-binding domains (CBDs), which can be achieved through cGMP-analogs. Here, we report the mechanism of action for a cGMP-antagonist and a cGMP-partial agonist of PfPKG to gain structural and dynamical insight on the otherwise elusive bound-inhibited state of PfPKG. This will provide another avenue to rationally design PfPKG-selective inhibitors for the treatment of malaria. NMR Chemical Shift Projection Analysis (CHESPA) of PfPKG CBD-D with cGMP-analogs shows that the cGMP-antagonist inhibits PfPKG not through a simple reversal of a two-state active-inactive equilibrium, but through multi-state equilibria sampling distinct holo-inactive intermediates that combine elements of both active and inactive states in different regions of CBD-D. NMR spin relaxation measurements and H/H exchange experiments further complement the CHESPA data by probing the dynamical changes within the CBD-D of PfPKG that occur upon inhibition.
Neurotoxic on-pathway pre-fibrillar soluble oligomers have been linked to Dominantly Inherited Alzheimer’s Disease (DIAD). However, the current knowledge about the molecular determinants of Aβ oligomer toxicity is at best scant. Here, we comparatively analyze Aβ oligomers prepared in the absence or presence of a catechin library that modulates their cellular toxicity. Our comparative analyses rely on a combination of solution-state NMR with dynamic light scattering, fluorescence spectroscopy, electron microscopy and X-ray diffraction, revealing unique molecular signatures that distinguish toxic vs. non-toxic Aβ oligomers. These include increased β-sheet content and surface hydrophobicity as well as a concomitant decrease in surface coating by catechins and in catechin-induced self-association. These molecular features enhance the contacts of the Aβ central hydrophobic core and C-terminus with synaptic vesicle mimics. Overall, our results offer a foundation for understanding the molecular mechanism of Aβ oligomer toxicity.
In the last decade, X-ray crystallography and cryo-electron microscopy have brought about a renaissance in structural biology with nearly 150,000 structures deposited in the protein database. However, the “structure-function” perspective ignores the reality of protein dynamics and the fact that most proteins adopt a fluid ensemble of functional conformers (states). We are interested in the role of this ensemble along the reaction coordinate pathway. NMR allows us to peer into structures of excited states and dynamics over timescales of nanoseconds to seconds. This talk will review studies associated with a homodimeric enzyme, fluroacetate dehalogenase, and the role of the ensemble in accomplishing catalysis. New data will be presented regarding allostery and substrate inhibition in the enzyme and a mechanistic perspective based on NMR. Finally I will give a brief overview of NMR approaches that uniquely address mechanistic questions of function associated with a key class of membrane receptors called GPCRs.
Hydrophobins are low molecular weight (5-20 kDa) self-assembling proteins secreted by fungi that accumulate at hydrophobic-hydrophilic interfaces and are extremely surface-active. Hydrophobins may undergo structural rearrangement and oligomerize to form rodlets, which are an insoluble functional amyloid that coats fungal spores to act as a water repellent, facilitate dispersal into the air, and prevent immune recognition. Due to their biochemical properties hydrophobins are a target for commercial applications, where they could be incorporated as biodegradable foam stabilizers or emulsifiers. To better understand which sequence characteristics determine hydrophobin properties, we are characterizing the structure and properties of several class IB hydrophobins. Target class IB hydrophobins from Serpula lacrymans (SL1), Wallemia ichthyophaga (WI1), and Phanerochaete carnosa (PC1) were chosen for study due to their different sequence composition. We expressed uniformly 13C/15N-labeled protein in E. coli and then purified it to homogeneity using Ni2+ affinity and reverse phase high performance liquid chromatography. We then determined the high-resolution structure of each hydrophobin using NMR spectroscopy. We discovered that each hydrophobin contains shared structural features despite their dissimilar sequence compositions. The core feature is a four strand anti-parallel β-sheet that is connected by three loop sequences (L1-L3). In all hydrophobins the β-sheet folds upon itself to form a β-barrel-like structure, however the first loop is disordered in WI1 while it is an a-helix in SL1 and PC1. Spectroscopic amyloid formation assays indicate that SL1, WI1, and PC1 have differing propensities to form rodlets. Overall, these structures highlight the similarities and differences that exist in hydrophobins. This work is the basis of future experiments investigating the mechanism of rodlet formation by hydrophobins.
The University of Guelph NMR Centre supports academic and industrial researchers across the region in the disciplines of biophysics, chemistry, food science, environmental science, and biology. The NMR Centre comprises six spectrometers spanning 400 MHz to 800 MHz, including a 600 MHz Dynamic Nuclear Polarization spectrometer and over 30 probes.
The first part of this presentation will describe recent events surrounding the Centre's actively cooled 800 MHz magnet. In late 2017, the magnet began to warm up from its nominal coil temperature, necessitating an increase in helium consumption to maintain the magnet. The diagnosis of the problem, the exciting and harrowing repair process, and the subsequent reduction in helium consumption (and operational costs) will be presented.
The second part of this poster will provide a general overview of the facility, along with recent experiments of interest to the NMR community in the fields of chemistry, protein biochemistry, and metabolomics.
When characterizing perturbations to biomolecules, a typical approach is to probe structural changes using multi-dimensional NMR experiments that rely on 13C and/or 15N. Alternatively, the lack of fluorination in natural biomolecules means that 19F NMR provides a highly sensitive means of using one-dimensional experiments to monitor biomolecules with almost no background. This is being applied to the apelinergic system: a class A (rhodopsin-like) G-protein-coupled receptor, and two families of peptide ligands, the apelin and apela (ELABELA/toddler) ligands (55, 36, 17, or 13 and 32, 22, 21, or 11 residue isoforms respectively). Apelin receptor segments containing either the N-terminal and first transmembrane α-helix (AR55) or segments containing the N-terminal and first three transmembrane α-helices (TM1-3) have been biosynthetically labelled with 4-,6-, or 7-fluorotryptophan, and are being titrated against a high binding affinity, 13-residue long apelin analogue containing a 2,4,5-trifluorophenylalanine C-terminal substitution. Binding to W51 and W24 was observed with TM1-3, whereas binding only to W51 was observed with the shorter AR55, indicating that the presence of both the N-terminal tail and extracellular loop 1 lead to more favorable binding possibilities. Based on peak and density profile shifting, at similar molar ratios of ligand and TM1-3, a potential intermediate binding state was observed. At increased receptor to ligand ratios, site-specific perturbation to the C-terminal phenylalanine of the apelin analog were observed as a function of 19F substitution site, indicating an orientational preference in binding to the receptor. Overall, the strategy employed here provides a relatively quick, clean method to sensitively and simultaneously track both ligand and receptor perturbations using one-dimensional NMR experiments.
QANUC, a solution state NMR facility located at McGill University, in Montreal, Québec, will be presented. The centre provides academic, government and industrial researchers with access to high field NMR spectroscopy at 800 MHz and 500 MHz. A brand new Bruker 800 MHz spectrometer was installed in June 2017. This instrument is equipped with an HCN cryoprobe with cooled 1H and 13C preamps, offering excellent signal to noise and high resolution. The 500 MHz Varian instrument normally runs with an RT HCN probe, and a 19F probe is also available. It remains an excellent choice for small proteins (≥ 0.5mM), peptides and small molecules. An overview of facility operation will be provided, along with highlights of recent user projects, demonstrating the range of experiments available to researchers.
Rapid detection of nosocomial outbreaks is critical to implement appropriate infection control protocols in hospitals. Common pathogens associated with outbreaks include vancomycin resistant Enterococcus faecium (VRE), methicillin resistant Staphylococcus aureus, and multi-resistant Gram-negative strains, which are all associated with high mortality and morbidity rates. Despite the need for rapid outbreak detection, the current gold standard for monitoring outbreaks remains to be pulsed field gel electrophoresis (PFGE), a time-consuming, and expensive technique, with low throughput, often requiring up to 5 days until results are produced. Although recognized for its high discriminatory capabilities, PFGE is also known to be laborious, and interpretation of the data is subjective, making it difficult for inter-laboratory comparison for large scale epidemiological studies. Whole-organism fingerprinting techniques such as High Resolution Magic Angle Spinning (HR-MAS) NMR and attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy are rapid and reagent-free methods, that can analyze microorganisms in their native state. In our work, ATR-FTIR spectroscopy has been used for bacterial identification and classification to the subspecies-level, including the discrimination between antibiotic-resistant and susceptible strains in the absence of antibiotics. HR-MAS NMR spectroscopy recently gained popularity to investigate the metabolomics of clinical specimens, including live microorganisms. The present study examined the possibility of employing ATR-FTIR, 1H and 31P HR-MAS NMR spectroscopy in combination with multivariate statistical analyses, as rapid methods for discriminating among VRE clonal strains related to hospital outbreaks. A set of 135 clinical isolates of VRE previously typed by PFGE as one of two pulsotypes (AA and CC) was employed in the ATR-FTIR portion of the study. A subset of 10 isolates per pulsotype were used to evaluate the potential use of 1H and 31P HRMAS NMR spectroscopy in strain typing. Spectral data analyses were performed by hierarchical cluster analysis and principal component analysis (PCA) in conjunction with the use of a feature selection algorithms developed in our laboratory. The spectra of VRE isolates were correctly assigned to their respective pulsotypes (AA or CC) yielding 99% concordance with PFGE results. This study demonstrates that the spectrum obtained from ATR-FTIR, 1H or 31P HR-MAS NMR techniques can be used for near real-time prospective tracking of hospital outbreaks. The spectral differences observed in the 31P HR-MAS NMR spectra suggest differences in phosphorous containing biomolecules. 2D heteronuclear NMR spectroscopy and mass spectrometry are underway to identify the compounds responsible for the discrimination between VRE pulsotypes.
This poster reports on the synthesis and solid-state 17O NMR characterization of six site-specifically 17O-labelled D-glucose compounds: [1-17O]-D-glucose, [2-17O]-D-glucose, [3-17O]-D-glucose, [4-17O]-D-glucose, [5-17O]-D-glucose, and [6-17O]-D-glucose. Static and magic-angle-spinning 17O NMR spectra were recorded at 21.1 T for these compounds, from which the 17O chemical shift and nuclear quadrupolar coupling tensors were determined for each oxygen site. This is the first time that 17O NMR tensors are measured for all oxygen atoms in D-glucose.
Growing energy demands have necessitated the development of highly efficient energy storage devices. Consequently, it has become crucial to research electrochemical energy storage via batteries. Presently, the most commercialized type of batteries are lithium ion batteries. However, concerns about the relatively limited global lithium supply have led to the development of sodium ion batteries (SIBs), which are capable of producing energy densities comparable to that of Li-ion batteries. Because much of a battery’s performance depends on the characteristics of the cathode material, this work focuses on a promising cathode material for SIBs, NVPF (Na3V2(PO4)2F3). While NVPF is well understood from a structural standpoint, ion dynamics within the material are still unknown. The presence of the paramagnetic V3+ ion in the sample has also made it challenging to analyze by solid-state nuclear magnetic resonance (ssNMR) spectroscopy because it causes peak broadening and an extremely fast relaxation time (T1) for Na. Synthesizing a less paramagnetic species, in this case NVGPF (Na3VGa(PO4)2F3), through Ga-substitution has allowed for the exploration of ionic site exchange through various 23Na ssNMR techniques. 1D Selective Inversion (SI) experiments have revealed ion dynamics between distinct regions of the NVGPF material, while 2D Exchange Spectroscopy (EXSY) was also applied to determine the rate of ionic exchange within regions of the material. DFT calculations, like those previously done by our group on the Na2FePO4F cathode material,1 are also being performed that will aid in assigning the observed chemical shift regions in the NVGPF spectra. The work presented here demonstrates the value of using ssNMR as the primary tool for studying dynamics in paramagnetic and inorganic systems.
The research team of Pr. Xavier Roucou developed a new database which considers the non-coding regions of the genome. This new database led to the discovery of multiple new proteins from overlooked regions, namely 5’ & 3’ UTR, alternate reading frames, and pseudogenes. This new database, coupled with the re-analysis of high through-put mass spectrometry, led to the discovery of UbKEKS, a ubiquitin isoform encoded in the pseudogene UBBP4. Until now, only one ubiquitin was known and expressed in every cellular type.
Ubiquitin is a 76 amino acid protein use as a post-translational modification by covalently linking the c-terminal glycine to the amine group of a lysine. This protein serves as a signal in a variety of cellular pathways, notably the TGFβ, MAP kinase and degradation pathway. The regulation of these pathways being affected in most cancers, their study is of the utmost importance.
Preliminary results suggest that this new ubiquitin act as a post-translational modification but doesn’t interact with the proteasome as opposed to its canonical counterpart.
In this study, we will investigate the structural and dynamic properties of this newly identified ubiquitin and eventually characterize its interaction with different ubiquitin binding domains.
The study of the spatial organization of biological molecules is at the heart of the research in structural biology. This approach allows scientists to better understand structure-function relationships involved in a plethora of biological processes. This includes the investigation of mechanisms responsible for the onset and development of various pathologies and the design of novel molecules capable of disrupting such mechanisms. The creation of the Structural Biology Platform at the Université de Montréal (PSB-UdeM) was completed in 2018 following substantial funding from the Canadian Foundation for Innovation (CFI). It now supplies state-of-the-art research equipment to answer structural biology questions for the scientific community. The platform is equipped with three high-field NMR spectrometers with Bruker NEO consoles, including a 500 MHz instrument, a 600 MHz magnet equipped with a cryoprobe and a new 700 MHz instrument. The platform also includes a biological small-angle X-ray scattering (SAXS) system designed by SAXSLAB/Xenocs and coupled to a Excillum Metaljet X-ray source. This SAXS system uses liquid handling robotics and automated sample loading to allow rapid and high-throughput evaluation of macromolecular shapes and dynamics through the analysis of X-ray scattering in solution. A structural bioinformatics platform is also in place to enable users to efficiently analyze their data and accomplish resource-intensive computational tasks including structure calculations from NMR, X-ray crystallography and SAXS data. Other biophysical tools complement the instruments offered through the structural biology platform. These include a size exclusion chromatography system combined with a multi-angle light scattering detector (SEC-MALS) for absolute molar mass determination, robots for preparing crystal trays, an isothermal titration calorimetry system for thermodynamics studies and a fluorometer for fluorescence spectroscopy.
The capsular polysaccharide (CPS) represents a key virulence factor for most encapsulated streptococci. Streptococcus suis and Group B Streptococcus (GBS) are both well-encapsulated pathogens of clinical importance in veterinary and/or human medicine and responsible for invasive systemic diseases. CPS differences are the basis for serological differentiation of the species into serotypes.
S. suis serotypes 2 and 1/2, which possess identical gene content in their cps loci, express CPSs that differ only by substitution of galactose (Gal) by N‑acetylgalactosamine (GalNAc) in the CPS side chain. The same sugar substitution differentiates the CPS of serotypes 14 and 1, whose cps loci are also identical in gene content. Using mutagenesis and CPS structural analysis, it was found that a single amino acid polymorphism in the glycosyltransferase CpsK defines the enzyme substrate predilection for Gal or GalNAc and therefore determines CPS composition, structure, and strain serotype.1
S. suis and GBS are the only Gram-positive bacteria which express a sialylated CPS at their surface. An important difference between these two sialylated CPSs is the linkage between the side-chain galactose and sialic acid, being α‑2,6 for S. suis but α‑2,3 for GBS. It is still unclear how sialic acid may affect CPS production and, consequently, the pathogenesis of the disease caused by these two bacterial pathogens. The role of sialic acid and the putative effect of sialic acid linkage modification in CPS synthesis were investigated using inter-species allelic exchange mutagenesis. It was shown that sialic acid (and its α‑2,6 linkage) is crucial for S. suis CPS synthesis, whereas for GBS, CPS synthesis may occur in presence of an α‑2,6 sialyltransferase or in absence of sialic acid moiety. To evaluate the effect of the CPS composition/structure on sialyltransferase activity, two distinct capsular serotypes within each bacterial species were compared (S. suis serotypes 2 and 14 and GBS serotypes III and V). In spite of common CPS structural characteristics and similarities in the cps loci, sialic acid exerts differential control of CPS expression by S. suis and GBS.2
Spider silks are proteinaceous materials employed in the architecture of webs or nests, cocoons or to wrap prey. Unlike silkworms that usually produce one type of silk, a single spider may produce several variants of silk. Each silk is distinct in its protein domain architecture, anatomical localization, and biological role, with mechanical properties matching to its function. Aciniform silk is employed to restrain prey and in egg-case architecture and, hence, is popularly known as wrapping silk. This silk is formed from a protein with unique domain architecture and mechanical behaviour. Unlike other silk variants which have short (~several amino acid) motifs repeated numerous times, the core repetitive domain of aciniform silk of Argiope trifasciata comprises a relatively small number (≥14) of identical 200 amino acid-long units (‘W units’). Previous NMR spectroscopy-studies and hydrodynamic measurements suggest a compact ‘beads-on-a-string’ structure, rich in a-helical character for the soluble W unit. Notably, one a-helix was found to be less stable than the others in the soluble state. Aciniform silk fibres also retain significant a-helical character, unlike many other silks. This led to the hypothesis that localized unfolding of the less stable helix of the W unit will serve to lengthen the linker and decrease protein compactness, thus favouring protein entanglement and increasing intermolecular interactions, in turn triggering β-sheet formation. To test this hypothesis, we introduced two W unit serine-to-cysteine mutations: one in this unstable helix and another in the globular core, engineered to form a disulfide staple linking the helix to the core. Compellingly, in the oxidized state, the mutant cannot fibrillize, while the reduced state is fully functional. Through comparative analysis of stapled to the reduced and native forms of the W unit in the solution-state, we are testing the hypothesis that denaturation and decompaction of this helix is key in initiation of fibrillogenesis in aciniform silk. This, in turn, will facilitate engineering of W units with modified mechanical behaviour.
The DDP is a state-of-the-art facility based in Montréal, QC providing structure-based drug design, and metabolomics analysis by solution and High Resolution Magic Angle Spinning (HR‑MAS) NMR spectroscopy, together with liquid-chromatography mass spectrometry (LC‑MS). Our services are offered to local and international academic and industrial researchers. With drug discovery expertise and analytical instrument facilities, the platform identifies and validates promising new biological and chemical entities (including biomarkers) for life-threatening diseases, and provides access and training to users on a broad range of cutting-edge technologies such as NMR spectroscopy (600 and 400 MHz) equipped with HR-MAS and CryoProbes, along with LC-MS and MALDI-TOF MS & IMS. These technologies which are available at the platform will be presented in this poster.
The microphthalmia-associated transcription factor MITF is a master regulator of development and differentiation within the melanocyte lineage. While essential to the regulation of key genes in the melanocyte cell cycle, aberrant MITF activity can lead to multiple malignancies including melanoma skin cancer. MITF recruits transcriptional co-activators, such as the histone acetyltransferase CREB-binding protein and its close homologue p300 (CBP/p300), through two transactivation domains (TAD) located on either side of a central basic helix-loop-helix leucine zipper DNA binding domain. The structural and mechanistic details of MITF TAD binding to CREB-binding protein (CBP/p300) was explored through biophysical and in-vitro techniques. Pulldown experiments and 1H-15N HSQC NMR-based titrationsdemonstrate the disordered N-terminal activation domain of MITF interacts with high affinity to the TAZ2 domain of CBP/p300. A reduction in chemical shift perturbations upon the addition of E1A, which is a known inhibitor of MITF function, indicates that E1A and MITF compete for the same TAZ2 binding site. Furthermore, in mammalian-one-hybrid assays the transcriptional activity of MITF was enhanced in the presence of co-transfected CBP/p300 and abolished upon deletion of the N-terminal MITF TAD. Understanding atomic-level details of how MITF interacts with its transcriptional co-activators at gene promoters may outline new strategies to disrupt MITF function and enable it to be a new target for melanoma therapy.
c-Myc is a b-HLH-LZ (basic-region-Helix-Loop-helix-Leucine Zipper) transcription factor. It is present at supra-physiological and oncogenic levels in most tumor cells. Such supra-physiological levels are considered oncogenic because they instigate the persistent transcription of metabolic, proliferative and anti-apoptotic gene networks to which tumor cells become addicted. However, addiction of tumor cells to c-Myc makes them more sensitive to its inhibition compared to normal cells. The privileged strategy to develop c-Myc inhibitors is the development of small molecules that specifically target its b-HLH-LZ domain in order to prevent heterodimerization with Max, its “obligate partner” and binding to an array of DNA sequence at transcriptional start sites. Although small molecule inhibitors have been discovered through screening, they reportedly suffer from poor affinity and specificity. The main obstacle to the development of more portent and specific inhibitors resides in the fact that no 3D structure of the b-HLH-LZ of c-Myc in an inactive form and bound by an inhibitor is available yet. We recently discovered that the b-HLH-LZ of c-Myc (c-Myc*) forms a thermodynamically stable and specific complex with a sub-domain (dubbed Mid2s by us) of the Myc interacting region of Miz-1 (Myc Interacting Zinc finger protein-1). Earlier research using yeast two hybrid protocols had demonstrated that the Myc interacting region of Miz-1 could interact with the HLH of c-Myc. We have recently shown that Mid2s directly competes with the b-HLH-LZ of Max (Max*) to form a mutually exclusive complex with c-Myc*. Titration of 15N-labeled c-Myc* with Mid2s leads the a gradual and profound perturbation (in the slow exchange regime) in the position of the amide cross-peaks (corresponding to amides in the HLH) on the 1H-15N HSQC. This clearly demonstrates that Mid2s stabilizes c-Myc* in a specific and stable structure and that it contains sought-after, structural and chemical information to yield a highly pertinent lead compound. In this poster we will present our most recent progress in the structural and dynamical characterization of this complex by solution-sate NMR. Our research project will generate the missing structural and chemical information to yield lead compounds with unprecedented affinity and specificity to inhibit oncogenic c-Myc.
Starch is the most abundant energy storage molecule in plants and is, with cellulose, the most abundant polysaccharide in nature. This glucose polymer consists of alternating amorphous and crystalline domains which can be found into two different structures, i.e., A and B-types with specific physicochemical properties and valued differently in the food industry, for example. Using two-dimensional (2D) high resolution solid-state NMR on 13C labelled starch obtained from C. reinhardtii microalgae, we established complete and unambiguous assignments for starch and its constituents (amylopectin and amylose) in the two crystalline forms and in the amorphous state. The 2D-INADEQUATE experiments under magic-angle spinning (MAS) offer high resolution and spectral dispersion enabling assignment of hereto unreported non-reducing end groups and the assessment of starch chain length, crystallinity, hydration and amylose content. Polarization transfer efficiency, as well as linewidths and intensity distributions in the single- and double-quantum dimensions are analyzed in terms of dynamics and conformational disorder, in particular in the amorphous state. This work illustrates how the INADEQUATE experiment can be used to garner information on organic disordered solids. Furthermore, we show how these NMR methods enable the detection and identification of starch in situ in intact cells, as well as by-products, therefore eliminating time consuming and potentially altering purification steps. We thus provide a solid basis for the NMR study of starch structure, its chemical modifications or biosynthesis in living microorganisms, making in situ NMR a powerful tool to study molecules directly in the cell.
Keywords: whole cell NMR, Magic-angle spinning, 2D INADEQUATE, A/ B and amorphous starches.
cAMP is an essential second messenger for transducing downstream effects of hormones and neurotransmitters in mammalian cells. The main receptor for cAMP is the regulatory subunit of protein kinase A (PKA). PKA includes two components, a catalytic (C) subunit and a regulatory (R) subunit spanning two homologous cAMP binding domains, which are known to bind cAMP with high affinity (KD ~ nM). The molecular mechanism underlying the activation of PKA by cAMP is well-understood. However, much less is known about how the termination of the cAMP signal occurs in PKA. Though it is known that cAMP phosphodiesterases (PDEs) catalyze the hydrolysis of the 3’-5’ phosphodiester bond in cAMP to generate 5’-AMP, signal termination through PDEs is expected to be kinetically limited by the very slow off-rate for the dissociation of cAMP from PKA R subunit in the absence of PDE-PKA R interactions. Recently, the Anand group reported that the termination of cAMP signaling is initiated through the formation of a PDE-PKA R subunit complex. To further investigate the PDE-PKA R subunit communication, we analyzed by NMR the RIa subunit of PKA in its apo and cAMP-bound state with and without PDE. The observed changes suggest a direct and specific interaction between the PKA RIa subunit and PDE. The atomic resolution provided by NMR allowed us to analyze the residues perturbed by the PDE:PKA complex formation.