In the present study we examine the thermodynamics of binding of two related pyrazine-derived ligands to the major urinary protein, MUP-I, using a combination of isothermal titration calorimetry (ITC), X-ray crystallography, and NMR backbone (15)N and methyl side-chain (2)H relaxation measurements. Global thermodynamics data derived from ITC indicate that binding is driven by favorable enthalpic contributions, rather than the classical entropy-driven hydrophobic effect. Unfavorable entropic contributions from the protein backbone and side-chain residues in the vicinity of the binding pocket are partially offset by favorable entropic contributions at adjacent positions, suggesting a "conformational relay" mechanism whereby increased rigidity of residues on ligand binding are accompanied by increased conformational freedom of side chains in adjacent positions. The principal driving force governing ligand affinity and specificity can be attributed to solvent-driven enthalpic effects from desolvation of the protein binding pocket.

Lithium is widely used in psychotherapy. The 6Li isotope has a long intrinsic longitudinal relaxation time T1 on the order of minutes, making it an ideal candidate for hyperpolarization experiments. In the present study, we demonstrated that lithium-6 can be readily hyperpolarized within 30 min, while retaining a long polarization decay time on the order of a minute. We used the intrinsically long relaxation time for the detection of 500 nM contrast agent in vitro. Hyperpolarized lithium-6 was administered to the rat and its signal retained a decay time on the order of 70 s in vivo. Localization experiments imply that the lithium signal originated from within the brain and that it was detectable up to 5 min after administration. We conclude that the detection of sub-micromolar contrast agents using hyperpolarized NMR nuclei such as 6Li may provide a novel avenue for molecular imaging.

Major urinary protein (MUP) is a pheromone-carrying protein of the lipocalin family. Previous studies by isothermal titration calorimetry (ITC) show that the affinity of MUP for the pheromone 2-methoxy-3-isobutylpyrazine (IBMP) is mainly driven by enthalpy, with a small unfavourable entropic contribution. Entropic terms can be attributed in part to changes in internal motions of the protein upon binding. Slow internal motions can lead to correlated or anti-correlated modulations of the isotropic chemical shifts of carbonyl C' and amide N nuclei. Correlated chemical shift modulations (CSM/CSM) in MUP have been determined by measuring differences of the transverse relaxation rates of zero- and double-quantum coherences ZQC{C'N} and DQC{C'N}, and by accounting for the effects of correlated fluctuations of dipole-dipole couplings (DD/DD) and chemical shift anisotropies (CSA/CSA). The latter can be predicted from tensor parameters of C' and N nuclei that have been determined in earlier work. The effects of complexation on slow time-scale protein dynamics can be determined by comparing the temperature dependence of the relaxation rates of APO-MUP (i.e., without ligand) and HOLO-MUP (i.e., with IBMP as a ligand).

Glycine is an amino acid present in mammalian brain, where it acts as an inhibitory and excitatory neurotransmitter. The two detectable protons of glycine give rise to a singlet at 3.55 ppm that overlaps with the more intense myo-inositol resonances, and its measurement has traditionally required specific editing efforts. The aim of the current study was to reduce the signal intensity of myo-inositol relative to that of glycine by exploiting the fast signal J-evolution of the myo-inositol spin system when using a single spin-echo localization method we recently introduced. Glycine was detected at TE ‫؍‬ 20 ms with an average Cramé r-Rao lower bound (CRLB) of 8.6% ؎ 1.5% in rat brain (N ‫؍‬ 5), at 9.4 T. The concentration of glycine was determined using LCModel analysis at 1.1 ؎ 0.1 mM, in good agreement with biochemical measurements previously reported. We conclude that at high magnetic fields, glycine can be measured at a relatively short echo time ( Key words: glycine; strongly coupled spin system; NMR spectroscopy; density matrix simulations; rat brain Glycine is an amino acid present in mammalian brain, where it acts as an inhibitory and excitatory neurotransmitter (1). Because of its critical role in N-methyl-D-aspartic acid (NMDA) transmission, altered levels of glycine are involved in a number of pathologies. In particular, elevated levels of glycine have been observed in the brain tissues of hyperglycinemia patients and in tumors (2,3). In a number of studies, glycine has been administered to schizophrenia patients to improve the NMDA receptor function, with the result of a significant decrease in negative and cognitive symptoms (4,5). Given its importance in mediating the action of the major excitatory neurotransmitter glutamate, a direct measurement of brain glycine concentration is desirable. The noninvasive measurement of glycine in brain is hampered by the fact that its twoproton singlet resonance at 3.55 ppm overlaps with the resonances of myo-inositol, which is present in the brain at much higher concentrations. Not surprisingly, glycine detection has only been reported in the human brain, using long TE when glycine was elevated or using additional editing techniques, such as TE-averaged point-resolved spectroscopy (PRESS) or 2D J-PRESS (6,7). Despite the fact that animal studies are typically performed at high field strengths (Ն7 T), where the substantial increase in SNR and spectral resolution could allow glycine detection without editing or TE-averaging, we are not aware of any such report to date.At very short TE (ϳ1-2 ms), multiplet resonances from coupled spin systems display virtually no dephasing induced by J-modulation. As TE increases, a loss of signal intensity occurs due to J-modulation. In particular, when using a single spin echo coherence generation at high field, myo-inositol undergoes rapid J-modulation due to its large J-coupling of ϳ10 Hz. In this study we sought to reduce the signal intensity of myo-inositol relative to that of glycine by exploiting its J-evolution when using a...

Community Supported Agriculture (CSA) is a direct partnership between producer(s) and a group of consumers/members to share the risks and responsibilities of farming activities. CSA aims at producing and providing environmentally, socially, economically, and nutritionally sustainable food. Past research has focused on CSA members’ motivations. This research aims to gain a better understanding of CSA farmers’ perceived benefits and drawbacks in managing a CSA farm, and whether CSA management perception varies in different countries. The research collected data from 35 farmers that were based in the United States (US) and Hungary (HU). Data elaboration includes a one-way Anova test, Chi-square test, principal component analysis, and multiple multivariate linear regressions. The results support that US and HU farmers have similar positive perceptions of CSA farming management, especially in food quality, nutritional value products, environmental, and community benefits. The main differences concentrate on economic, financial, and management perceptions. CSA success as an alternative agro-food production and distribution system relies on the capability to involve CSA members. Therefore, CSA farmers’ management skills may evolve to ensure the performance of communication and community engaging practices. The main CSA concern is ensuring a fair income and living wage for the farmers and labor force. There is a need for better balancing non-monetary and monetary benefits for the farmers.

Abstract.A simple method designed to measure autorelaxation rates of double-and zero-quantum coherences DQC/ZQC{C'N} involving a carbonyl C' and the neighboring amide N nucleus in protein backbones provides valuable insight into slow motions in spite of interference both from the attached amide proton H s and from remote protons such as H ~ in nondeuterated proteins. The method has been applied to human ubiquitin.

Cross-correlated fluctuations of isotropic chemical shifts can provide evidence for slow motions in biomolecules. Slow side-chain dynamics have been investigated in (15)N and (13)C enriched ubiquitin by monitoring the relaxation of C(alpha)-C(beta) two-spin coherences (Frueh et al., 2001). This method, which had hitherto been demonstrated only for protonated ubiquitin, has now been applied to both protonated and deuterated proteins. Deuteration reduces the dipole-dipole contributions to the DD/DD cross-correlation, thus facilitating the observation of subtle effects due to cross-correlation of the fluctuations of the isotropic (13)C chemical shifts. The decays of double- and zero-quantum coherences are significantly slower in the deuterated protein than in the protonated sample. Slow motions are found both in loops and in secondary structure elements.

The internal dynamics of recombinant Major Urinary Protein (rMUP) have been investigated by monitoring transverse nitrogen-15 relaxation using multiple-echo Carr-Purcell-Meiboom-Gill (CPMG) experiments. While the ligand-free protein (APO-rMUP) features extensive evidence of motions on the milliseconds time scale, the complex with 2-methoxy-3-isobutylpyrazine (HOLO-rMUP) appears to be much less mobile on this time scale. At 308 K, exchange rates k (ex) = 500-2000 s(-1) were typically observed in APO-rMUP for residues located adjacent to a beta-turn comprising residues 83-87. These residues occlude an entry to the binding pocket and have been proposed to be a portal for ligand entry in other members of the lipocalin family, such as the retinol binding protein and the human fatty-acid binding protein. Exchange rates and populations are largely uncorrelated, suggesting local 'breathing' motions rather than a concerted global conformational change.