Nitric oxide ( ⅐ NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon ␤ to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9-and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC 50 of ϳ3 M M for both nitroalkenes, an IC 50 equivalent to the potent thiol oxidant peroxynitrite (ONOO ؊ ) and an IC 50 30-fold less than H 2 O 2 , indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that this reversible post-translational protein modification represents a new pathway for redox regulation of enzyme function, cell signaling, and protein trafficking. Nitric oxide ( ⅐ NO)5 exerts a broad influence on cell and inflammatory signaling via both cGMP-dependent and -independent oxidative, nitrosative, and nitrative reactions (1, 2). The nitration of polyunsaturated fatty acids present in both membranes and lipoproteins is now emerging as a novel mechanism for transducing ⅐ NO-dependent redox signaling (3, 4). Recent evidence indicates that all major unsaturated fatty acids present in human blood contain some proportion of alkenyl nitro derivatives (R 1 HCϭC(NO 2 )R 2 ), also termed nitroalkenes. Because of the prevalence of fatty acid nitroalkenes in healthy humans, these species are now appreciated as an abundant pool of bioactive oxides of nitrogen in the vasculature (5). The two most clinically abundant nitroalkene fatty acid derivatives, nitro-oleic acid (9-and 10-nitro-9-cis-octadeca...

Mass spectrometric analysis of human plasma and urine revealed abundant nitrated derivatives of all principal unsaturated fatty acids. Nitrated palmitoleic, oleic, linoleic, linolenic, arachidonic and eicosapentaenoic acids were detected in concert with their nitrohydroxy derivatives. Two nitroalkene derivatives of the most prevalent fatty acid, oleic acid, were synthesized (9-and 10-nitro-9-cis-octadecenoic acid; OA-NO 2 ), structurally characterized and determined to be identical to OA-NO 2 found in plasma, red cells, and urine of healthy humans. These regioisomers of OA-NO 2 were quantified in clinical samples using 13 C isotope dilution. Plasma free and esterified OA-NO 2 concentrations were 619 ؎ 52 and 302 ؎ 369 nM, respectively, and packed red blood cell free and esterified OA-NO 2 was 59 ؎ 11 and 155 ؎ 65 nM. The OA-NO 2 concentration of blood is ϳ50% greater than that of nitrated linoleic acid, with the combined free and esterified blood levels of these two fatty acid derivatives exceeding 1 M. OA-NO 2 is a potent ligand for peroxisome proliferator activated receptors at physiological concentrations. CV-1 cells co-transfected with the luciferase gene under peroxisome proliferator-activated receptor (PPAR) response element regulation, in concert with PPAR␥, PPAR␣, or PPAR␦ expression plasmids, showed dose-dependent activation of all PPARs by OA-NO 2 . PPAR␥ showed the greatest response, with significant activation at 100 nM, while PPAR␣ and PPAR␦ were activated at ϳ300 nM OA-NO 2 . OA-NO 2 also induced PPAR␥-dependent adipogenesis and deoxyglucose uptake in 3T3-L1 preadipocytes at a potency exceeding nitrolinoleic acid and rivaling synthetic thiazolidinediones. These data reveal that nitrated fatty acids comprise a class of nitric oxide-derived, receptor-dependent, cell signaling mediators that act within physiological concentration ranges.The oxidation of unsaturated fatty acids converts lipids, otherwise serving as cellular metabolic precursors and structural components, into potent signaling molecules including prostaglandins, leukotrienes, isoprostanes, and hydroxy-and hydroperoxyeicosatetraenoates. These enzymatic and auto-catalytic oxidation reactions yield products that orchestrate immune responses, neurotransmission, and the regulation of cell growth. For example, prostaglandins are cyclooxygenase-derived lipid mediators that induce receptor-dependent regulation of inflammatory responses, vascular function, initiation of parturition, cell survival, and angiogenesis (1). In contrast, the various isoprostane products of arachidonic acid auto-oxidation exert vasoconstrictive and pro-inflammatory signaling actions via receptor-dependent and -independent mechanisms (2). A common element of these diverse lipid signaling reactions is that nitric oxide ( ⅐ NO) 6 and other oxides of nitrogen significantly impact lipid mediator formation and bioactivities.The ability of ⅐ NO and ⅐ NO-derived species to oxidize, nitrosate, and nitrate biomolecules serves as the molecular basis for how ⅐ NO influences the sy...

The crystal structure of the ternary complex citrate synthase-oxaloacetate-carboxymethyl coenzyme A has been solved to a resolution of 1.9 A and refined to a conventional crystallographic R factor of 0.185. The structure resembles a proposed transition state of the condensation reaction and suggests that the condensation reaction proceeds through a neutral enol rather than an enolate intermediate. A mechanism for the condensation reaction is proposed which involves the participation of three key catalytic groups (Asp 375, His 274, and His 320) in two distinct steps. The proposed mechanism invokes concerted general acid-base catalysis twice to explain both the energetics of the reaction and the experimentally observed inversion of stereochemistry at the attacking carbon atom.

Crystal structures of the tetrameric yellow-fluorescent protein zFP538 from the button polyp Zoanthus sp. and a green-emitting mutant (K66M) are presented. The atomic models have been refined at 2.7 and 2.5 A resolution, with final crystallographic R factors of 0.206 (R(free) = 0.255) and 0.190 (R(free) = 0.295), respectively, and have excellent stereochemistry. The fold of the protomer is very similar to that of green (GFP) and red (DsRed) fluorescent proteins; however, evidence from crystallography and mass spectrometry suggests that zFP538 contains a three-ring chromophore derived from that of GFP. The yellow-emitting species (lambda(em)(max) = 538 nm) is proposed to result from a transimination reaction in which a transiently appearing DsRed-like acylimine is attacked by the terminal amino group of lysine 66 to form a new six-membered ring, cleaving the polypeptide backbone at the 65-66 position. This extends the chromophore conjugation by an additional double bond compared to GFP, lowering the absorption and emission frequencies. Substitution of lysine 66 with aspartate or glutamate partially converts zFP538 into a red-fluorescent protein, providing additional support for an acylimine intermediate. The diverse and unexpected roles of the side chain at position 66 give new insight into the chemistry of chromophore maturation in the extended family of GFP-like proteins.

The design and optimization of fluorescent molecules has driven the ability to interrogate complex biological events in real time. Notably, most advances in bioimaging fluorophores are based on optimization of core structures that have been known for over a century. Recently, new synthetic methods have resulted in an explosion of nonplanar conjugated macrocyclic molecules with unique optical properties yet to be harnessed in a biological context. Herein we report the synthesis of the first aqueous-soluble carbon nanohoop (i.e., a macrocyclic slice of a carbon nanotube prepared via organic synthesis) and demonstrate its bioimaging capabilities in live cells. Moreover, we illustrate that these scaffolds can be easily modified by well-established “click” chemistry to enable targeted live cell imaging. This work establishes the nanohoops as an exciting new class of macrocyclic fluorophores poised for further development as novel bioimaging tools.