Diffusion of Peptides and Small Molecules Encapsulated in Lipidic Cubic Phases by Band-Selective Excitation Short Transient 1H PGSE NMR.

In model membrane systems, such as lipidic cubic phases (LCPs), 1H NMR spectra are dominated by resonances from water and lipid molecules. The measurement of translational diffusion of peptides/molecules encapsulated in LCPs using conventional 1H pulsed-gradient spin-echo (PGSE) NMR is, therefore, immensely difficult due to the dynamic range caused by the intense resonance(s) from the surrounding environment. The present study reports the use of a band-selective short transient PGSE sequence, avoiding the perturbation of both hydration water and lipids, for measuring the diffusion of molecules encapsulated within the lipid bilayer and the aqueous channels of LCPs.

Physiochemical changes, metabolite discrepancies of brown seaweed-derived sulphated polysaccharides in the upper gastrointestinal tract and their effects on bioactive expression.

Brown seaweed-derived polysaccharides, notably fucoidan and laminarin, are known for their extensive array of bioactivities and physicochemical properties. However, the effects of upper digestive tract modification on the bioactive performance of fucoidan and laminarin fractions (FLFs) sourced from Australian native species are largely unknown. Here, the digestibility and bioaccessibility of FLFs were evaluated by tracking the dynamic changes in reducing sugar content (CR), profiling the free monosaccharide composition using LC-MS, and comparing high-performance gel permeation chromatography profile variation via LC-SEC-RI. The effects of digestive progression on bioactive performance were assessed by comparing the antioxidant and antidiabetic potential of FLFs and FLF digesta. We observed that molecular weight (Mw) decreased during gastric digestion indicating that FLF aggregates were disrupted in the stomach. During intestinal digestion, Mw gradually decreased and CR increased indicating cleavage of glycosidic bonds releasing free sugars. Although the antioxidant and antidiabetic capacities were not eliminated by the digestion progression, the bioactive performance of FLFs under a digestive environment was reduced contrasting with the same concentration level of the undigested FLFs. These data provide comprehensive information on the digestibility and bioaccessibility of FLFs, and shed light on the effects of digestive progression on bioactive expression.

Insights into dynamic properties of water in lipidic cubic phases by 2D nuclear Overhauser effect (NOE) NMR spectroscopy.

Two-dimensional NOE (nuclear Overhauser effect) NMR spectroscopy was employed to investigate the dynamic properties of water within lyotropic bicontinuous lipidic cubic phases (LCPs) formed by monoolein (MO). Experiments observed categorically different effective residence times of water molecules: (i) in proximity to the glycerol moiety of MO, and (ii) adjacent to the hydrophobic chain towards the hydrocarbon tail of MO, as evidenced by the opposite signs of intermolecular NOE cross peaks between protons of water and those of MO in 2D 1H-1H NOESY spectra. Spectroscopic data delineating the different effective residence times of water molecules within both the gyroid (QIIG) and diamond (QIID) phase groups corresponding to hydration levels of 35 and 40 wt%, respectively, are presented. Additionally, an increase in effective residence time of water molecules in proximity to the glycerol moiety of MO in LCPs was observed upon storage at ambient temperature and in the presence of an additive lipid, cholesterol. Atom-specific NOE build-up curves for protons of water and those of MO are also given. The results presented herein provide new insight into the physicochemical properties and behaviour of water in LCPs, and demonstrate an additional avenue for experimental study of water-lipid interactions and hydration dynamics in model membranes and nanomaterials using 2D NOE NMR spectroscopy.

NMR measurement of biomolecular translational and rotational motion for evaluating changes of protein oligomeric state in solution.

Defining protein oligomeric state and/or its changes in solution is of significant interest for many biophysical studies carried out in vitro, especially when the nature of the oligomeric state is crucial in the subsequent interpretation of experimental results and their biological relevance. Nuclear magnetic resonance (NMR) is a well-established methodology for the characterization of protein structure, dynamics, and interactions at the atomic level. As a spectroscopic method, NMR also provides a compelling means for probing both molecular translational and rotational motion, two predominant measures of effective molecular size in solution, under identical conditions as employed for structural, dynamic and interaction studies. Protein translational diffusion is readily measurable by pulse gradient spin echo (PGSE) NMR, whereas its rotational correlation time, or rotational diffusion tensor when its 3D structure is known, can also be quantified from NMR relaxation parameters, such as 15N relaxation parameters of backbone amides which are frequently employed for probing residue-specific protein backbone dynamics. In this article, we present an introductory overview to the NMR measurement of bimolecular translational and rotational motion for assessing changes of protein oligomeric state in aqueous solution, via translational diffusion coefficients measured by PGSE NMR and rotational correlation times derived from composite 15N relaxation parameters of backbone amides, without need for the protein structure being available.

NMR spectroscopy of lipidic cubic phases.

Lipidic cubic phase (LCP) structures have been used for stabilisation and crystallisation of membrane proteins and show promising properties as drug carriers. In this mini-review, we present how NMR spectroscopy has played a major role in understanding the physico-chemical properties of LCPs and how recent advances in pulsed field gradient NMR techniques open new perspectives in characterising encapsulated molecules.

A solution NMR view of lipidic cubic phases: Structure, dynamics, and beyond.

Nuclear magnetic resonance (NMR) spectroscopy is well-established nowadays for the elucidation of the 3D structures of proteins and protein complexes, the evaluation of biomolecular dynamics with atomistic resolution across a range of time scales, the screening of drug candidates with site specificity, and for the quantitation of molecular translational diffusion. Lyotropic lipidic cubic phases (LCPs) are lipid bilayer-based materials with a complex geometry, formed via the spontaneous self-assembly of certain lipids in an aqueous environment at specific temperature ranges. LCPs have been successfully applied to the in meso crystallization of membrane proteins for structural studies by X-ray crystallography, and have also shown promising potential for serving as matrices for drug and nutrient delivery/release in vivo. The characterization of the structural and dynamics properties of LCPs is of significant interest for the application of these materials. Here we present a systematic review detailing the characterization of LCPs by solution NMR. Using LCPs formed by monoolein (MO) as an example, various aspects of LCPs readily accessible by solution NMR are covered, including spectral perturbation in the presence of additives, quantification of hydration levels, 13C relaxation-based measurements for studying atom-specific dynamics along the MO hydrocarbon chain, PGSE NMR measurement of translational diffusion and its correlation with release profiles, and the encapsulation of soluble proteins in LCPs. A brief discussion of future perspectives for the characterization of LCPs by solution NMR is also presented.

Water diffusion in complex systems measured by PGSE NMR using chemical shift selective stimulated echo: Elimination of magnetization exchange effects.

The interpretation of molecular translational diffusion as measured by pulsed gradient spin-echo NMR (PGSE NMR) can be complicated by the presence of chemical exchange and/or dipolar cross-relaxation (including relayed cross-relaxation via spin diffusion). The magnitude of influence depends on the kinetics of exchange and/or dipolar cross-relaxation present within the system as well as the PGSE NMR sequences chosen for measurements. First, we present an exchange induced zero-crossing phenomenon for signal attenuation of water in lipidic cubic phases (formed by a mixture of monoolein and water) in the presence of pulsed gradients observed using a standard STimulated Echo (STE) sequence. This magnetization exchange induced zero-crossing phenomenon, a pseudo-negative diffraction-like feature, resembles that reported previously for restricted diffusion when locally anisotropic pores are polydisperse or randomly oriented. We then demonstrate the elimination of these exchange and/or dipolar cross-relaxation induced effects with the use of a chemical shift selective STE (CHESS-STE) sequence, adapted from the previously reported band-selective short transient STE sequence, along with results obtained from the bipolar pulse pair STE sequence for comparison. The CHESS-STE sequence introduced here represents a generic form of PGSE NMR sequences for obtaining water diffusion coefficients free from the influence of exchange and/or dipolar cross-relaxation in complex systems. It has potential applications in measuring translational diffusion of water in biopolymer mixtures as well as probing the microscopic structure in materials via water restricted diffusion measured by PGSE NMR, particularly when the potential presence of exchange/cross-relaxation is of concern.

Chemical Exchange of Hydroxyl Groups in Lipidic Cubic Phases Characterized by NMR.

Proton transportation in proximity to the lipid bilayer membrane surface, where chemical exchange represents a primary pathway, is of significant interest in many applications including cellular energy turnover underlying ATP synthesis, transmembrane mobility, and transport. Lipidic inverse bicontinuous cubic phases (LCPs) are unique membrane structures formed via the spontaneous self-assembly of certain lipids in an aqueous environment. They feature two networks of water channels, separated by a single lipid bilayer which approximates the geometry of a triply periodic minimal surface. When composed of monoolein, the LCP bilayer features two glycerol hydroxyl groups at the lipid-water interface which undergo exchange with water. Depending on the conditions of the aqueous solution used in the formation of LCPs, both resonances of the glycerol hydroxyl groups may be observed by solution 1H NMR. In this study, PFG-NMR and 1D EXSY were employed to gain insight into chemical exchange between the monoolein hydroxyl groups and water in LCPs. Results including the relative population of hydroxyl protons in exchange with water for a number of LCPs at different hydration levels and the exchange rate constants at 35 wt % hydration are reported. Several technical aspects of PFG-NMR and EXSY-NMR for the characterization of chemical exchange in LCPs are discussed, including an alternative way to analyze PFG-NMR data of exchange systems which overcomes the inherent low sensitivity at high diffusion encoding.

Physiochemical Characterization and Stability of Lipidic Cubic Phases by Solution NMR.

Lipidic inverse bicontinuous cubic phases (LCPs), formed via the spontaneous self-assembly of lipids such as monoolein, have found increasing applications in the stabilization and crystallization of integral membrane proteins for structural characterization using X-ray crystallography. Their use as effective drug release matrices has also been demonstrated. Nuclear magnetic resonance (NMR) spectroscopy, both solution and solid state, has previously been employed for the characterization of LCPs and related systems. Herein, we report a number of novel features of solution NMR for probing the fundamental composition and structural properties of monoolein-based LCPs. These include (1) more complete assignments of both 1H and 13C chemical shifts, (2) direct quantification of hydration level in LCPs using one-dimensional (1D) 1H NMR, and (3) monitoring longer-term stability of LCPs and evaluating alterations introduced into standard LCPs at the submolecular level.

Heteronuclear NMR spectroscopy of proteins encapsulated in cubic phase lipids.

Lipidic cubic phases, which form spontaneously via the self-assembly of certain lipids in an aqueous environment, are highly prospective nanomaterials with applications in membrane protein X-ray crystallography and drug delivery. Here we report 1H-15N heteronuclear single/multiple quantum coherence (HSQC, HMQC) spectra of 15N-enriched proteins encapsulated in inverse bicontinuous lipidic cubic phases obtained on a standard commercial high resolution NMR spectrometer at ambient temperature. 15N-enriched proteins encapsulated in this lipidic cubic phase show: (i) no significant changes in tertiary structure, (ii) significantly reduced solvent chemical exchange of backbone amides, which potentially provides a novel concept for quantifying residue-specific hydration; and (iii) improved spectral sensitivity achieved with band-selective excitation short-transient (BEST) spectroscopy, which is attributed to the presence of an abundant source of 1H nuclear spins originating from the lipid component of the cubic phase.

Structural insights into BCL2 pro-survival protein interactions with the key autophagy regulator BECN1 following phosphorylation by STK4/MST1.

BECN1/Beclin 1 is a critical protein in the initiation of autophagosome formation. Recent studies have shown that phosphorylation of BECN1 by STK4/MST1 at threonine 108 (T108) within its BH3 domain blocks macroautophagy/autophagy by increasing BECN1 affinity for its negative regulators, the anti-apoptotic proteins BCL2/Bcl-2 and BCL2L1/Bcl-xL. It was proposed that this increased binding is due to formation of an electrostatic interaction with a conserved histidine residue on the anti-apoptotic molecules. Here, we performed biophysical studies which demonstrated that a peptide corresponding to the BECN1 BH3 domain in which T108 is phosphorylated (p-T108) does show increased affinity for anti-apoptotic proteins that is significant, though only minor (<2-fold). We also determined X-ray crystal structures of BCL2 and BCL2L1 with T108-modified BECN1 BH3 peptides, but only showed evidence of an interaction between the BH3 peptide and the conserved histidine residue when the histidine flexibility was restrained due to crystal contacts. These data, together with molecular dynamics studies, indicate that the histidine is highly flexible, even when complexed with BECN1 BH3. Binding studies also showed that detergent can increase the affinity of the interaction. Although this increase was similar for both the phosphorylated and non-phosphorylated peptides, it suggests factors such as membranes could impact on the interaction between BECN1 and BCL2 proteins, and therefore, on the regulation of autophagy. Hence, we propose that phosphorylation of BECN1 by STK4/MST1 can increase the affinity of the interaction between BECN1 and anti-apoptotic proteins and this interaction can be stabilized by local environmental factors. Abbreviations: asu: asymmetric unit; BH3: BCL2/Bcl-2 homology 3; DAPK: death associated protein kinase; MD: molecular dynamics; MST: microscale thermophoresis; NMR: nuclear magnetic resonance; PDB: protein data bank; p-T: phosphothreonine; SPR: surface plasmon resonance; STK4/MST1: serine/threonine kinase 4.

Measuring translational diffusion of 15N-enriched biomolecules in complex solutions with a simplified 1H-15N HMQC-filtered BEST sequence.

Pulsed-field gradient nuclear magnetic resonance has seen an increase in applications spanning a broad range of disciplines where molecular translational diffusion properties are of interest. The current study introduces and experimentally evaluates the measurement of translational diffusion coefficients of 15N-enriched biomolecules using a 1H-15N HMQC-filtered band-selective excitation short transient (BEST) sequence as an alternative to the previously described SOFAST-XSTE sequence. The results demonstrate that accurate translational diffusion coefficients of 15N-labelled peptides and proteins can be obtained using this alternative 1H-15N HMQC-filtered BEST sequence which is implementable on NMR spectrometers equipped with probes fitted with a single-axis field gradient, including most cryoprobes dedicated to bio-NMR. The sequence is of potential use for direct quantification of protein or peptide translational diffusion within complex systems, such as in mixtures of macromolecules, crowded solutions, membrane-mimicking media and in bicontinuous cubic phases, where conventional sequences may not be readily applicable due to the presence of intense signals arising from sources other than the protein or peptide under investigation.

The molecular basis of JAK/STAT inhibition by SOCS1.

The SOCS family of proteins are negative-feedback inhibitors of signalling induced by cytokines that act via the JAK/STAT pathway. SOCS proteins can act as ubiquitin ligases by recruiting Cullin5 to ubiquitinate signalling components; however, SOCS1, the most potent member of the family, can also inhibit JAK directly. Here we determine the structural basis of both these modes of inhibition. Due to alterations within the SOCS box domain, SOCS1 has a compromised ability to recruit Cullin5; however, it is a direct, potent and selective inhibitor of JAK catalytic activity. The kinase inhibitory region of SOCS1 targets the substrate binding groove of JAK with high specificity and thereby blocks any subsequent phosphorylation. SOCS1 is a potent inhibitor of the interferon gamma (IFNγ) pathway, however, it does not bind the IFNγ receptor, making its mode-of-action distinct from SOCS3. These findings reveal the mechanism used by SOCS1 to inhibit signalling by inflammatory cytokines.

Discovery and characterization of a sulfoquinovose mutarotase using kinetic analysis at equilibrium by exchange spectroscopy.

Bacterial sulfoglycolytic pathways catabolize sulfoquinovose (SQ), or glycosides thereof, to generate a three-carbon metabolite for primary cellular metabolism and a three-carbon sulfonate that is expelled from the cell. Sulfoglycolytic operons encoding an Embden-Meyerhof-Parnas-like or Entner-Doudoroff (ED)-like pathway harbor an uncharacterized gene (yihR in Escherichia coli; PpSQ1_00415 in Pseudomonas putida) that is up-regulated in the presence of SQ, has been annotated as an aldose-1-epimerase and which may encode an SQ mutarotase. Our sequence analyses and structural modeling confirmed that these proteins possess mutarotase-like active sites with conserved catalytic residues. We overexpressed the homolog from the sulfo-ED operon of Herbaspirillum seropedicaea (HsSQM) and used it to demonstrate SQ mutarotase activity for the first time. This was accomplished using nuclear magnetic resonance exchange spectroscopy, a method that allows the chemical exchange of magnetization between the two SQ anomers at equilibrium. HsSQM also catalyzed the mutarotation of various aldohexoses with an equatorial 2-hydroxy group, including d-galactose, d-glucose, d-glucose-6-phosphate (Glc-6-P), and d-glucuronic acid, but not d-mannose. HsSQM displayed only 5-fold selectivity in terms of efficiency (kcat/KM) for SQ versus the glycolysis intermediate Glc-6-P; however, its proficiency [kuncat/(kcat/KM)] for SQ was 17 000-fold better than for Glc-6-P, revealing that HsSQM preferentially stabilizes the SQ transition state.

Predicting the release profile of small molecules from within the ordered nanostructured lipidic bicontinuous cubic phase using translational diffusion coefficients determined by PFG-NMR.

The ordered nanostructured lipidic bicontinuous cubic phase has demonstrated potential as a drug release material, due to its ability to encapsulate a wide variety of compounds, which may undergo sustained, diffusion controlled release over time. Control of drug release has been shown to depend on the nanostructural parameters of the lipid mesophase. Herein, the diffusion and release of two amino acids, encapsulated within a range of different lipidic cubic mesophases are investigated. Pulsed-field gradient NMR was used to determine the diffusion coefficient of the encapsulated amino acid, which was found to be correlated with the nanoscale diameter of the water channels within the cubic mesophase. This information was used to predict the release profiles of encapsulated compounds from within the cubic mesophase, which was verified by directly measuring the release of each amino acid in vitro. Predicted release profiles tracked reasonably close to the measured release profiles, indicating that NMR determined diffusion measurements can be used to predict release profiles.

C-Terminal Modification and Multimerization Increase the Efficacy of a Proline-Rich Antimicrobial Peptide.

Two series of branched tetramers of the proline-rich antimicrobial peptide (PrAMP), Chex1-Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram-negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers that also incorporated C-terminal peptide chemical modification. DTM-linked tetrameric peptides containing a C-terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49-2.33 µm. A second series of tetrameric analogues with C-terminal hydrazide modification was prepared by using alternative conjugation linkers including trans-1,4-dibromo-2-butene, α,α'-dibromo-p-xylene, or 6-bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram-negative nosocomial pathogens, particularly the butene-linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 µm/8 µg mL-1 ) where the monomer is inactive. None of these peptides showed any cytotoxicity to mammalian cells up to 25 times the MIC. A diffusion NMR study of the tetrameric hydrazides showed that the more active antibacterial analogues were those with a more compact structure having smaller hydrodynamic radii. The results show that C-terminal PrAMP hydrazidation together with its rational tetramerization is an effective means for increasing both diversity and potency of PrAMP action.

Characterisation of the conformational preference and dynamics of the intrinsically disordered N-terminal region of Beclin 1 by NMR spectroscopy.

Beclin 1 is a 450 amino acid protein that plays critical roles in the early stages of autophagosome formation. We recently reported the successful expression, purification and structural characterisation of the entire N-terminal region of Beclin 1 (residues 1-150), including its backbone NMR chemical shift assignments. Based on assigned backbone NMR chemical shifts, it has been established that the N-terminal region of Beclin 1 (1-150), including the BH3 domain (112-123), is intrinsically disordered in the absence of its interaction partners. Here, a detailed study of its conformational preference and backbone dynamics obtained from an analysis of its secondary structure populations using the δ2D method, and the measurements of effective hydrodynamic radius as well as (1)H temperature coefficients, (1)H solvent exchange rates, and (15)N relaxation parameters of backbone amides using NMR spectroscopy is reported. These data provide further evidence for the intrinsically disordered nature of the N-terminal region of Beclin 1 and support the view that the helical conformation adopted by the Beclin 1 BH3 domain upon interaction with binding partners such as BCL-2 pro-survival proteins is likely induced rather than pre-existing.

The BECN1 N-terminal domain is intrinsically disordered.

BECN1/Beclin 1 has a critical role in the early stages of autophagosome formation. Recently, structures of its central and C-terminal domains were reported, however, little structural information is available on the N-terminal domain, comprising a third of the protein. This lack of structural information largely stems from the inability to produce this region in a purified form. Here, we describe the expression and purification of the N-terminal domain of BECN1 (residues 1 to 150) and detailed biophysical characterization, including NMR spectroscopy. Combined, our studies demonstrated at the atomic level that the BECN1 N-terminal domain is intrinsically disordered, and apart from the BH3 subdomain, remains disordered following interaction with a binding partner, BCL2L1/BCL-XL. In addition, the BH3 domain α-helix induced upon interaction with BCL2L1 reverts to a disordered state when the complex is dissociated by exposure to a competitive inhibitor. No significant interactions between N- and C-terminal domains were detected.

Characterization of the Lipid-Binding Site of Equinatoxin II by NMR and Molecular Dynamics Simulation.

Equinatoxin II (EqtII) is a soluble, 20 kDa pore-forming protein toxin isolated from the sea anemone Actinia equina. Although pore formation has long been known to occur in distinct stages, including monomeric attachment to phospholipid membranes followed by detachment of the N-terminal helical domain and oligomerization into the final pore assembly, atomistic-level detail of the protein-lipid interactions underlying these events remains elusive. Using high-resolution solution state NMR of uniformly-(15)N-labeled EqtII at the critical micelle concentration of dodecylphosphocholine, we have mapped the lipid-binding site through chemical shift perturbations. Subsequent docking of an EqtII monomer onto a dodecylphosphocholine micelle, followed by 400 ns of all-atom molecular dynamics simulation, saw several high-occupancy lipid-binding pockets stabilized by cation-π, hydrogen bonding, and hydrophobic interactions; and stabilization of the loop housing the conserved arginine-glycine-aspartate motif. Additional simulation of EqtII with an N-acetyl sphingomyelin micelle, for which high-resolution NMR data cannot be obtained due to aggregate formation, revealed that sphingomyelin specificity might occur via hydrogen bonding to the 3-OH and 2-NH groups unique to the ceramide backbone by side chains of D109 and Y113; and main chains of P81 and W112. Furthermore, a binding pocket formed by K30, K77, and P81, proximate to the hinge region of the N-terminal helix, was identified and may be implicated in triggering pore formation.

Measuring translational diffusion coefficients of peptides and proteins by PFG-NMR using band-selective RF pulses.

Molecular translational self-diffusion, a measure of diffusive motion, provides information on the effective molecular hydrodynamic radius, as well as information on the properties of media or solution through which the molecule diffuses. Protein translational diffusion measured by pulsed-field gradient nuclear magnetic resonance (PFG-NMR) has seen increased application in structure and interaction studies, as structural changes or protein-protein interactions are often accompanied by alteration of their effective hydrodynamic radii. Unlike the analysis of complex mixtures by PFG-NMR, for monitoring changes of protein translational diffusion under various conditions, such as different stages of folding/unfolding, a partial region of the spectrum or even a single resonance is sufficient. We report translational diffusion coefficients measured by PFG-NMR with a modified stimulated echo (STE) sequence where band-selective pulses are employed for all three (1)H RF pulses. Compared with conventional non-selective sequence, e.g. the BPP-LED sequence, the advantage of this modified band-selective excitation short transient (BEST) version of STE (BEST-STE) sequence is multi-fold, namely: (1) potential sensitivity gain as in generalized BEST-based sequences, (2) water suppression is no longer required as the magnetization of solvent water is not perturbed during the measurement, and (3) dynamic range problems due to the presence of intense resonances from molecules other than the protein or peptide of interest, such as non-deuterated detergent micelles, are avoided.