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The understanding of protein–protein interaction mechanisms is key to the atomistic description of cell signaling pathways and for the development of new drugs. In this context, the mechanism of intrinsically disordered proteins folding upon binding has attracted attention. The VirB9 C-terminal domain (VirB9Ct) and the VirB7 N-terminal motif (VirB7Nt) associate with VirB10 to form the outer membrane core complex of the Type IV Secretion System injectisome. Despite forming a stable and rigid complex, VirB7Ntbehaves as a random coil, while VirB9Ctis intrinsically dynamic in the free state. Here we combined NMR, stopped-flow fluorescence, and computer simulations using structure-based models to characterize the VirB9Ct-VirB7Ntcoupled folding and binding mechanism. Qualitative data analysis suggested that VirB9Ctpreferentially binds to VirB7Ntby way of a conformational selection mechanism at lower temperatures. However, at higher temperatures, energy barriers between different VirB9Ctconformations are more easily surpassed. Under these conditions the formation of non-native initial encounter complexes may provide alternative pathways toward the native complex conformation. These observations highlight the intimate relationship between folding and binding, calling attention to the fact that the two molecular partners must search for the most favored intramolecular and intermolecular interactions on a rugged and funnelled conformational energy landscape, along which multiple intermediates may lead to the final native state.

This study evaluated the effects of insufficient (WET) or ample (CUR) curing on red clover silage (29.4 and 45.3% DM, respectively) and hay (65.1 and 89.1% DM, respectively) in vitro fermentation measures. Measurements were taken at the start of storage (STRT), after 14 d (MicA), and once storage processes had stabilized for hay and silage (50 and 78 d, respectively; LATE). The red clover field was divided into 5 blocks and each block was divided into 2 plots and then assigned randomly to silage and hay (storage methods). Hay was packed into mini-bales (~500 fresh g; ~232 kg DM/m3) and silage into mini-silos (19.5 L; ~214 kg DM/m3). Samples at each storage stage were analyzed for in vitro gas production (48 h). Gas kinetics were recorded using the Ankom RF Gas Production System. Data were analyzed as a randomized complete block design (5 blocks) with a 2 (curing extents) x 2 (storage methods) x 3 (storage phases) factorial. Differences were declared at P ≤ 0.05. The data was analyzed using the GLIMMIX procedure of SAS 9.4. At STRT, the Asymptotic maximal gas production (M) and fermentation rate (k) were not different between CUR and WET hay (x̅ = 240.5 ± 7.33 mL/g incubated DM and x̅ = 13.2 ± 0.56% of gas production/h, respectively). However, at MicA, CUR hay had a greater M and k than WET hay (232.7 vs. 192.6 and 13.9 vs. 9.33, respectively). The same was observed at LATE (237.6 vs. 197.6 and 13 vs. 8.36, respectively). At STRT, the CUR and WET silage was not different for M and k (x̅ = 233.4 ± 7.33 mL/g incubated DM and x̅ = 14.06 ± 0.56% of gas production/h, respectively). A similar trend was observed at MicA (x̅ = 220 and 13.6, respectively) and LATE (x̅ = 219.7 and 14.7). In conclusion, ample curing of red clover hay preserves the fermentation kinetics relative to insufficient curing, especially after 14 d of storage. In the case of red clover silage, the fermentation kinetics was not affected by the curing extent across the storage period.

The Na+/Ca2+exchanger of Drosophila melanogaster, CALX, is the main Ca2+-extrusion mechanism in olfactory sensory neurons and photoreceptor cells. Na+/Ca2+exchangers have two Ca2+sensor domains, CBD1 and CBD2. In contrast to the mammalian homologs, CALX is inhibited by Ca2+binding to CALX-CBD1, whereas CALX-CBD2 does not bind Ca2+at physiological concentrations. CALX-CBD1 consists of a β-sandwich and displays four Ca2+-binding sites at the tip of the domain. In this study, we used NMR spectroscopy and isothermal titration calorimetry (ITC) to investigate the cooperativity of Ca2+binding to CALX-CBD1. We observed that this domain binds Ca2+in the slow exchange regime at the NMR chemical shift timescale. Ca2+binding restricts the dynamics in the Ca2+-binding region. Experiments of 15N chemical exchange saturation transfer and 15N R2dispersion allowed the determination of Ca2+dissociation rates (∼30 s−1). NMR titration curves of residues in the Ca2+-binding region were sigmoidal because of the contribution of chemical exchange to transverse magnetization relaxation rates, R2. Hence, a novel, to our knowledge, approach to analyze NMR titration curves was proposed. Ca2+-binding cooperativity was examined assuming two different stoichiometric binding models and using a Bayesian approach for data analysis. Fittings of NMR and ITC binding curves to the Hill model yielded nHill∼2.9, near maximal cooperativity (nHill = 4). By assuming a stepwise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca2+is approximately three orders of magnitude higher than that of binding a single Ca2+. Hence, four Ca2+ions bind almost simultaneously to CALX-CBD1. Cooperative Ca2+binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca2+concentration in sensory neuronal cells.

This objective was to evaluate the effects of insufficient (WET) or ample (CUR) curing on red clover silage (29.4 and 45.3% DM, respectively) and hay (65.1 and 89.1% DM, respectively) ruminal degradation. The red clover field was divided into 5 blocks and each block was divided into 2 plots and then assigned randomly to silage and hay. Hay was packed into mini-bales (~500 fresh g; ~232 kg DM/m3) and silage into mini-silos (19.5 L; ~214 kg DM/m3). Measurements were taken once storage processes had stabilized for hay and silage (50 and 78 d, respectively). Samples were ground to pass 4-mm screen and weighed (5 g DM) into 10 x 20 cm ANKOM R1020 in situ bags. Bags were hooked to a rope, placed in the ventral sac of the rumen of 2 dairy cows for 0, 3, 6, 12, 24, 48, 72, and 96 h. All bags were removed simultaneously, washed, dried, and weighed. This procedure was conducted separately for each block. Data were analyzed as a randomized complete block design (5 blocks) with a 2 (curing extents) x 2 (storage methods) factorial. Differences were declared at P≤ 0.05. Discrete lag times were higher for WET than CUR (0.23 vs. 0 ± 0.08 h, respectively). The potentially degradable fraction was greater in hay than in silage (50.2 vs. 45.1 ± 1.32% of DM, respectively). Moreover, WET had a greater potentially degradable fraction than CUR (49.6 vs. 45.7 ± 1.32% of DM, respectively). The degradation rate was less in hay than silage (5 vs. 8 ± 0.4%/h, respectively). The WET treatment had a decreased degradation rate than CUR (5 vs. 7, respectively). The undegradable fraction was not different between WET and CUR hay (x̅ = 23.3 ± 1.13% of DM). In contrast, WET silage had a decreased undegradable fraction than CUR silage (17.1 vs. 20, respectively). In conclusion, haymaking increased the potentially degradable fraction relative to ensiling but reduced the rate of digestion. For both storage methods, curing reduced the potentially degradable fraction but increased the rate of digestion.

Our objective was to assess the effects of insufficient (WET) or ample (CUR) curing on the phytoestrogen concentrations of red clover silage (29.4 and 45.3% DM) and hay (65.1 and 89.1, respectively). Measurements were taken at the start of storage (STRT), after 14 d (MicA), and once storage processes had stabilized for hay and silage (50 and 78 d, respectively; LATE). Hay was packed into mini-bales (~500 fresh g; ~232 kg DM/m3) and silage into mini-silos (19.5 L; ~214 kg DM/m3). Data were analyzed as a randomized complete block design (5 blocks) with a 2 (curing extents) x 2 (storage methods) x 3 (storage phases) factorial. Differences were declared at P≤ 0.05. The concentration of formononetin was greater in silage vs. hay at STRT (4,624 vs. 2,893 ± 175 mg/kg of DM, respectively), MicA (5,383 vs. 2,110), and LATE (5,666 vs. 2,569). Also, more formononetin was found in WET vs. CUR silage (5,841 vs. 4,608.1± 154 mg/kg of DM, respectively). However, no differences were observed between WET vs. CUR hay (x̅ = 2,524). At STRT, biochanin A was greater in silage vs. hay (3,105 vs. 2,012 ± 139 mg/kg of DM, respectively). A similar trend was observed at MicA (3,233 vs. 1,057), and LATE (3,295 vs. 1,228). In addition, more biochanin A was observed in WET vs. CUR silage (3,793 vs. 2,628.9 ± 127 mg/kg of DM, respectively). The opposite was observed in WET vs. CUR hay (1,219.5 vs. 1,644.9). The genistein concentration was greater in silage vs. hay at STRT (149 vs. 120 ± 8.36 mg/kg of DM, respectively), MicA (246 vs. 62.6), and LATE (264 vs. 72.9). Also, the concentration of genistein was greater in WET vs. CUR silage (236 vs. 202.3 ± 7.21 mg/kg of DM, respectively) but the opposite was observed in WET vs. CUR hay (72.8 vs. 97.3). In the case of daidzein, hay had a greater concentration of this phytoestrogen than silage (12.8 vs. 6.67 ± 0.43 mg/kg of DM, respectively). In conclusion, red clover silage maintains greater concentrations of formononetin, biochanin A, and genistein than hay during curing period and across the storage phase. Also, adequate curing is more beneficial for silage than hay in terms of decreasing the phytoestrogen concentrations.