The visualization of complex cellular processes involving multiple proteins requires the use of spectroscopically distinguishable fluorescent reporters. We have previously introduced the SNAP-tag as a general tool for the specific labeling of SNAP-tag fusion proteins in living cells. The SNAP-tag is derived from the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and can be covalently labeled in living cells using O6-benzylguanine derivatives bearing a chemical probe. Here we report the generation of an AGT-based tag, named CLIP-tag, which reacts specifically with O2-benzylcytosine derivatives. Because SNAP-tag and CLIP-tag possess orthogonal substrate specificities, SNAP and CLIP fusion proteins can be labeled simultaneously and specifically with different molecular probes in living cells. We furthermore show simultaneous pulse-chase experiments to visualize different generations of two different proteins in one sample.

A quarter of all anthropogenic methane emissions in the United States are from enteric fermentation, primarily from ruminant livestock. This study was undertaken to test the effect of a methane inhibitor, 3-nitrooxypropanol (3NOP), on enteric methane emission in lactating Holstein cows. An experiment was conducted using 48 cows in a randomized block design with a 2-wk covariate period and a 12-wk data collection period. Feed intake, milk production, and fiber digestibility were not affected by the inhibitor. Milk protein and lactose yields were increased by 3NOP. Rumen methane emission was linearly decreased by 3NOP, averaging about 30% lower than the control. Methane emission per unit of feed dry matter intake or per unit of energy-corrected milk were also about 30% less for the 3NOP-treated cows. On average, the body weight gain of 3NOP-treated cows was 80% greater than control cows during the 12-wk experiment. The experiment demonstrated that the methane inhibitor 3NOP, applied at 40 to 80 mg/kg feed dry matter, decreased methane emissions from high-producing dairy cows by 30% and increased body weight gain without negatively affecting feed intake or milk production and composition. The inhibitory effect persisted over 12 wk of treatment, thus offering an effective methane mitigation practice for the livestock industries.

The objective was to measure effects of 3-nitrooxypropanol (3 NP) on methane production of lactating dairy cows and any associated changes in digestion and energy and N metabolism. Six Holstein-Friesian dairy cows in mid-lactation were fed twice daily a total mixed ration with maize silage as the primary forage source. Cows received 1 of 3 treatments using an experimental design based on two 3 × 3 Latin squares with 5-wk periods. Treatments were a control placebo or 500 or 2,500 mg/d of 3 NP delivered directly into the rumen, via the rumen fistula, in equal doses before each feeding. Measurements of methane production and energy and N balance were obtained during wk 5 of each period using respiration calorimeters and digestion trials. Measurements of rumen pH (48 h) and postprandial volatile fatty acid and ammonia concentrations were made at the end of wk 4. Daily methane production was reduced by 3 NP, but the effects were not dose dependent (reductions of 6.6 and 9.8% for 500 and 2,500 mg/d, respectively). Dosing 3 NP had a transitory inhibitory effect on methane production, which may have been due to the product leaving the rumen in liquid outflow or through absorption or metabolism. Changes in rumen concentrations of volatile fatty acids indicated that the pattern of rumen fermentation was affected by both doses of the product, with a decrease in acetate:propionate ratio observed, but that acetate production was inhibited by the higher dose. Dry matter, organic matter, acid detergent fiber, N, and energy digestibility were reduced at the higher dose of the product. The decrease in digestible energy supply was not completely countered by the decrease in methane excretion such that metabolizable energy supply, metabolizable energy concentration of the diet, and net energy balance (milk plus tissue energy) were reduced by the highest dose of 3 NP. Similarly, the decrease in N digestibility at the higher dose of the product was associated with a decrease in body N balance that was not observed for the lower dose. Milk yield and milk fat concentration and fatty acid composition were not affected but milk protein concentration was greater for the higher dose of 3 NP. Twice-daily rumen dosing of 3 NP reduced methane production by lactating dairy cows, but the dose of 2,500 mg/d reduced rumen acetate concentration, diet digestibility, and energy supply. Further research is warranted to determine the optimal dose and delivery method of the product.

Ruminants, such as cows, sheep, and goats, predominantly ferment in their rumen plant material to acetate, propionate, butyrate, CO2, and methane. Whereas the short fatty acids are absorbed and metabolized by the animals, the greenhouse gas methane escapes via eructation and breathing of the animals into the atmosphere. Along with the methane, up to 12% of the gross energy content of the feedstock is lost. Therefore, our recent report has raised interest in 3-nitrooxypropanol (3-NOP), which when added to the feed of ruminants in milligram amounts persistently reduces enteric methane emissions from livestock without apparent negative side effects [Hristov AN, et al. (2015) Proc Natl Acad Sci USA 112(34):10663–10668]. We now show with the aid of in silico, in vitro, and in vivo experiments that 3-NOP specifically targets methyl-coenzyme M reductase (MCR). The nickel enzyme, which is only active when its Ni ion is in the +1 oxidation state, catalyzes the methane-forming step in the rumen fermentation. Molecular docking suggested that 3-NOP preferably binds into the active site of MCR in a pose that places its reducible nitrate group in electron transfer distance to Ni(I). With purified MCR, we found that 3-NOP indeed inactivates MCR at micromolar concentrations by oxidation of its active site Ni(I). Concomitantly, the nitrate ester is reduced to nitrite, which also inactivates MCR at micromolar concentrations by oxidation of Ni(I). Using pure cultures, 3-NOP is demonstrated to inhibit growth of methanogenic archaea at concentrations that do not affect the growth of nonmethanogenic bacteria in the rumen.

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We present a comparative study of the ultrafast photophysics of all-trans retinal in the protonated Schiff base form in solvents with different polarities and viscosities. Steady-state spectra of retinal in the protonated Schiff base form show large absorption-emission Stokes shifts (6500-8100 cm(-1)) for both polar and nonpolar solvents. Using a broadband fluorescence up-conversion experiment, the relaxation kinetics of fluorescence is investigated with 120 fs time resolution. The time-zero spectra already exhibit a Stokes-shift of approximately 6000 cm(-1), indicating depopulation of the Franck-Condon region in < or =100 fs. We attribute it to relaxation along skeletal stretching. A dramatic spectral narrowing is observed on a 150 fs timescale, which we assign to relaxation from the S(2) to the S(1) state. Along with the direct excitation of S(1), this relaxation populates different quasistationary states in S(1), as suggested from the existence of three distinct fluorescence decay times with different decay associated spectra. A 0.5-0.65 ps decay component is observed, which may reflect the direct repopulation of the ground state, in line with the small isomerization yield in solvents. Two longer decay components are observed and are attributed to torsional motion leading to photo-isomerization. The various decay channels show little or no dependence with respect to the viscosity or dielectric constant of the solvents. This suggests that in the protein, the bond selectivity of isomerization is mainly governed by steric effects.

A general method for the covalent immobilization of fusion proteins is presented. The approach is based on the unusual mechanism of the human O6-alkylguanine-DNA alkyltransferase, which irreversibly transfers the alkyl group from its substrate, alkylated or benzylated guanine, to a reactive cysteine residue. By attaching the benzyl group to a surface, hAGT fusion proteins immobilize themselves in a specific and covalent manner. The specificity of the reaction of hAGT with its substrate even allows the specific immobilization of hAGT fusion proteins directly out of cell extracts, making the approach an attractive alternative to currently used immobilization procedures.

Interpretative Summary: Effect of 3-nitrooxypropanol on ruminal fermentation, 1 methane and hydrogen emissions, and methane isotopic signature in dairy cows. By 2 Lopes et al. Page 0000. An inhibitor, 3-nitrooxypropanol, decreased by 31% enteric 3 methane and increased hydrogen emissions in lactating dairy cows, but had no effect on 4 rumen archaeal community composition nor a significant change in the isotope 5 composition of methane. Methanomicrobium) was not affected by 3NOP, but the proportion of methanogens in the 42 total cell counts tended to be decreased by 3NOP. Prevotella spp., the predominant 43 bacterial genus in ruminal contents in this experiment, was also not affected by 3NOP. 44Compared with the control, Ruminococcus and Clostridium spp. were decreased and 45Butyrivibrio spp. was increased by 3NOP. This experiment demonstrated that a 46 substantial inhibition of enteric methane emission by 3NOP in dairy cows was 47 3 accompanied with increased hydrogen emission and decreased acetate:propionate ratio, 48 but there was no effect on rumen archaeal community composition nor a significant 49 change in the isotope composition of methane. 50Key Words: methane, 3-nitrooxypropanol, rumen fermentation, dairy cow 51 52 4 INTRODUCTION 53In the rumen, methane (CH 4 ) is an end product of microbial fermentation of 54 carbohydrates and amino acids. Methanogenesis is the major sink for hydrogen in the 55 rumen, but enteric CH 4 represents also a net feed energy loss for the animal (Johnson and 56