The role of conventions in the running of farm shops in the UK

UK farm shops have received little attention in the alternative food network (AFN) literature. Farm shop environmental context was interrogated to establish its influence on the consumption experience. A holistic multi-methods approach was adopted drawing on autoethnography, an exploratory survey, G.I.S, an open-ended customer questionnaire, customer e-WOM reviews, online space rhetoric and imagery, and interviews with owners and farmers partners. Braun and Clarke’s formalised approach to thematic analysis was employed. A negotiated ‘transference’ of trust was identified between vendor/producers and consumers, dependent on primary producers adhering to their production ethos when sourcing brought-in products. Negotiated transference of trust was identified as connected to institutional proximity, afforded to farm-sited farm shops, facilitating customers’ acceptance of AFN hybridity. Farm shops met Pine and Gilmore’s (1998) five factors that aid consumption experiences. These enterprises were revealed to be places of nostalgia-evoking experiential consumption (Hamilton & Wagner, 2014) realised through owner intention (e.g., retroscaping) and incidental associations (i.e., multi-sensory on-farm location). Farm shops facilitated customer escapism through a positive themed event, revealing how the materialities of the farm shop and its working farm environ establish experiential consumption; identified as a source of resistance to supermarket co-optation. Farmers’ partners exhibited an entrepreneur identity in the development and running of the enterprise. Reporting positive rewards of status, emotional pride, and economic contribution to the farm business. These benefits appeared compartmentalised to the enterprise with renegotiation of reproductive work allocation unrealised. The multi-disciplinary approach employed a conceptually-driven lens, utilising convention theory and Brown’s (1999) retroscape, to explore the practices and representations of alterity, experiential consumption, and patriarchy in the formation of the farm shop. The findings provide knowledge progression and promise new ways of understanding AFNs.</p

¹⁷O NMR Investigation of Water Structure and Dynamics

The structure and dynamics of the bound water in barium chlorate monohydrate were studied with ¹⁷O nuclear magnetic resonance (NMR) spectroscopy in samples that are stationary and spinning at the magic-angle in magnetic fields ranging from 14.1 to 21.1 T. ¹⁷O NMR parameters of the water were determined, and the effects of torsional oscillations of the water molecule on the ¹⁷O quadrupolar coupling constant (C_Q) were delineated with variable temperature MAS NMR. With decreasing temperature and reduction of the librational motion, we observe an increase in the experimentally measured <i>C</i>_Q explaining the discrepancy between experiments and predictions from density functional theory. In addition, at low temperatures and in the absence of ¹H decoupling, we observe a well-resolved ¹H–¹⁷O dipole splitting in the spectra, which provides information on the structure of the H₂O molecule. The splitting arises because of the homogeneous nature of the coupling between the two ¹H–¹⁷O dipoles and the ¹H–¹H dipole

¹H Dynamic Nuclear Polarization Based on an Endogenous Radical

We demonstrate a 15-fold enhancement of solid-state NMR signals via dynamic nuclear polarization (DNP) based on a stable, naturally occurring radical in a protein: the flavin mononucleotide (FMN) semiquinone of flavodoxin. The line width of flavodoxin’s EPR signal suggests that the dominant DNP mechanism is the solid effect, consistent with the field-dependent DNP enhancement profile. The magnitude of the enhancement as well as the bulk-polarization build-up time constant (τ_B) with which it develops are dependent on the isotopic composition of the protein. Deuteration of the protein to 85% increased the nuclear longitudinal relaxation time <i>T</i>_1n and τ_B by factors of five and seven, respectively. Slowed dissipation of polarization can explain the 2-fold higher maximal enhancement than that obtained in proteated protein, based on the endogenous semiquinone. In contrast, the long τ_B of TOTAPOL-based DNP in nonglassy samples was not accompanied by a similarly important long <i>T</i>_1n, and in this case the enhancement was greatly reduced. The low concentrations of radicals occurring naturally in biological systems limit the magnitude of DNP enhancement that is attainable by this means. However, our enhancement factors of up to 15 can nonetheless make an important difference to the feasibility of applying solid-state NMR to biochemical systems. We speculate that DNP based on endogenous radicals may facilitate MAS NMR characterization of biochemical complexes and even organelles, and could also serve as a source of additional structural and physiological information

Proton-Assisted Recoupling (PAR) in Peptides and Proteins

Proton-assisted recoupling (PAR) is examined by exploring optimal experimental conditions and magnetization transfer rates in a variety of biologically relevant nuclear spin-systems, including simple amino acids, model peptides, and two proteins–nanocrystalline protein G (GB1), and importantly amyloid beta 1–42 (M₀Aβ_1–42) fibrils. A selective PAR protocol, SUBPAR (setting up better proton assisted recoupling), is described to observe magnetization transfer in one-dimensional spectra, which minimizes experiment time (in comparison to two-dimensional experiments) and thereby enables an efficient assessment of optimal PAR conditions for a desired magnetization transfer. In the case of the peptide spin systems, experimental and simulated PAR data sets are compared on a semiquantitative level, thereby elucidating the interactions influencing PAR magnetization transfer and their manifestations in different spin transfer networks. Using the optimum Rabi frequencies determined by SUBPAR, PAR magnetization transfer trajectories (or buildup curves) were recorded and compared to simulated results for short peptides. PAR buildup curves were also recorded for M₀Aβ_1–42 and examined conjointly with a recent structural model. The majority of salient cross-peak intensities observed in the M₀Aβ_1–42 PAR spectra are well-modeled with a simple biexponential equation, although the fitting parameters do not show any strong correlation to internuclear distances. Nevertheless, these parameters provide a wealth of invaluable semiquantitative structural constraints for the M₀Aβ_1–42. The results presented here offer a complete protocol for recording PAR ¹³C–¹³C correlation spectra with high-efficiency and using the resulting information in protein structural studies

Proton Association Constants of His 37 in the Influenza‑A M2_18–60 Dimer-of-Dimers

The membrane protein M2 from influenza-A forms a single-pass transmembrane helix that assembles in lipid membrane as homotetramers whose primary function is to act as a proton transporter for viral acidification. A single residue, histidine 37 (His 37), is known to be responsible for selectivity and plays an integral role in the protein’s function. We report pH-dependent ¹⁵N MAS NMR spectra of His 37 within the influenza-A proton conduction domain of M2, M2_18–60, which has been previously shown to be a fully functional construct and was recently determined to adopt a dimer-of-dimers structure in lipids. By extracting the ratio of [His]/[HisH⁺] as a function of pH, we obtained two doubly degenerate proton disassociation constants, 7.63 ± 0.15 and 4.52 ± 0.15, despite a possible maximum of four. We also report the ¹H_Nε chemical shifts at pH 6.5 recorded at 60 kHz MAS in a CP-based ¹H–¹⁵N spectrum. We were unable to detect resonances indicative of direct proton sharing among His 37 side chains when the tetramer is in the +2 state. In the neutral state, His 37 is exclusively in the τ tautomer, indicating that the δ nitrogen is protonated solely as a function of pH. We also found that the plot of [HisH⁺]/[His] as a function of pH is qualitatively similar to previously reported proton conduction rates, indicating that proton conduction rate is proportional to the level of histidine protonation within the channel. Two-dimensional ¹³C–¹³C and ¹³C–¹⁵N correlations suggest that at low pH multiple conformations are populated as the spectra broaden and eventually disappear as the acidity is increased. A second highly resolved state at low pH was not observed

Dynamic Nuclear Polarization of ¹⁷O: Direct Polarization

Dynamic nuclear polarization of ¹⁷O was studied using four different polarizing agents: the biradical TOTAPOL and the monoradicals trityl and SA-BDPA, as well as a mixture of the latter two. Field profiles, DNP mechanisms, and enhancements were measured to better understand and optimize directly polarizing this low-gamma quadrupolar nucleus using both mono- and biradical polarizing agents. Enhancements were recorded at <88 K and were >100 using the trityl (OX063) radical and <10 with the other polarizing agents. The >10 000-fold savings in acquisition time enabled a series of biologically relevant small molecules to be studied with small sample sizes and the measurement of various quadrupolar parameters. The results are discussed with comparison to room temperature studies and GIPAW quantum chemical calculations. These experimental results illustrate the strength of high field DNP and the importance of radical selection for studying low-gamma nuclei

Dynamic Nuclear Polarization of Oxygen-17

Oxygen-17-detected DNP NMR of a water/glycerol glass enabled an 80-fold enhancement of signal intensity at 82 K, using the biradical TOTAPOL. The >6000-fold savings in acquisition time enable ¹⁷O–¹H distance measurements and heteronuclear correlation experiments. These experiments are the initial demonstration of the feasibility of DNP NMR on quadrupolar ¹⁷O

Magic-Angle-Spinning NMR of the Drug Resistant S31N M2 Proton Transporter from Influenza A

We report chemical shift assignments of the drug-resistant S31N mutant of M2_18–60 determined using 3D magic-angle-spinning (MAS) NMR spectra acquired with a ¹⁵N–¹³C ZF-TEDOR transfer followed by ¹³C–¹³C mixing by RFDR. The MAS spectra reveal two sets of resonances, indicating that the tetramer assembles as a dimer of dimers, similar to the wild-type channel. Helicies from the two sets of chemical shifts are shown to be in close proximity at residue H37, and the assignments reveal a difference in the helix torsion angles, as predicted by TALOS+, for the key resistance residue N31. In contrast to wild-type M2_18–60, chemical shift changes are minimal upon addition of the inhibitor rimantadine, suggesting that the drug does not bind to S31N M2

Equilibration of Tyrosyl Radicals (Y₃₅₆^•, Y₇₃₁^•, Y₇₃₀^•) in the Radical Propagation Pathway of the Escherichia coli Class Ia Ribonucleotide Reductase

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric tyrosyl radical (Y₁₂₂^•) cofactor in β2 to initiate catalysis in α2. Each turnover requires reversible long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y₁₂₂^• ⇆ [W₄₈?] ⇆ Y₃₅₆ within β to Y₇₃₁ ⇆ Y₇₃₀ ⇆ C₄₃₉ within α). Previously, we reported that a β2 mutant with 3-nitrotyrosyl radical (NO₂Y^•; 1.2 radicals/β2) in place of Y₁₂₂^• in the presence of α2, CDP, and ATP catalyzes formation of 0.6 equiv of dCDP and accumulates 0.6 equiv of a new Y^• proposed to be located on Y₃₅₆ in β2. We now report three independent methods that establish that Y₃₅₆ is the predominant location (85–90%) of the radical, with the remaining 10–15% delocalized onto Y₇₃₁ and Y₇₃₀ in α2. Pulsed electron–electron double-resonance spectroscopy on samples prepared by rapid freeze quench (RFQ) methods identified three distances: 30 ± 0.4 Å (88% ± 3%) and 33 ± 0.4 and 38 ± 0.5 Å (12% ± 3%) indicative of NO₂Y₁₂₂^•–Y₃₅₆^•, NO₂Y₁₂₂^•–NO₂Y₁₂₂^•, and NO₂Y₁₂₂^•–Y_731(730)^•, respectively. Radical distribution in α2 was supported by RFQ electron paramagnetic resonance (EPR) studies using Y₇₃₁(3,5-F₂Y) or Y₇₃₀(3,5-F₂Y)-α2, which revealed F₂Y^•, studies using globally incorporated [β-²H₂]Y-α2, and analysis using parameters obtained from 140 GHz EPR spectroscopy. The amount of Y^• delocalized in α2 from these two studies varied from 6% to 15%. The studies together give the first insight into the relative redox potentials of the three transient Y^• radicals in the PCET pathway and their conformations