Thematic Review Series: Living History of Lipids: Discovery of essential fatty acids

Dietary fat was recognized as a good source of energy and fat-soluble vitamins by the first part of the 20th century, but fatty acids were not considered to be essential nutrients because they could be synthesized from dietary carbohydrate. This well-established view was challenged in 1929 by George and Mildred Burr who reported that dietary fatty acid was required to prevent a deficiency disease that occurred in rats fed a fat-free diet. They concluded that fatty acids were essential nutrients and showed that linoleic acid prevented the disease and is an essential fatty acid. The Burrs surmised that other unsaturated fatty acids were essential and subsequently demonstrated that linolenic acid, the omega-3 fatty acid analog of linoleic acid, is also an essential fatty acid. The discovery of essential fatty acids was a paradigm-changing finding, and it is now considered to be one of the landmark discoveries in lipid research.     

Lipogenesis Is Decreased by Grape Seed Proanthocyanidins According to Liver Proteomics of Rats Fed a High Fat Diet

Bioactive proanthocyanidins have been reported to have several beneficial effects on health in relation to metabolic syndrome, type 2 diabetes, and cardiovascular disease. We studied the effect of grape seed proanthocyanidin extract (GSPE) in rats fed a high fat diet (HFD). This is the first study of the effects of flavonoids on the liver proteome of rats suffering from metabolic syndrome. Three groups of rats were fed over a period of 13 weeks either a chow diet (control), an HFD, or a high fat diet supplemented for the last 10 days with GSPE (HFD + GSPE). The liver proteome was fractionated, using a Triton X-114-based two-phase separation, into soluble and membrane protein fractions so that total proteome coverage was considerably improved. The data from isobaric tag for relative and absolute quantitation (iTRAQ)-based nano-LC-MS/MS analysis revealed 90 proteins with a significant (p < 0.05) minimal expression difference of 20% due to metabolic syndrome (HFD versus control) and 75 proteins due to GSPE treatment (HFD + GSPE versus HFD). The same animals have previously been studied (Quesada, H., del Bas, J. M., Pajuelo, D., Díaz, S., Fernandez-Larrea, J., Pinent, M., Arola, L., Salvadó, M. J., and Bladé, C. (2009) Grape seed proanthocyanidins correct dyslipidemia associated with a high-fat diet in rats and repress genes controlling lipogenesis and VLDL assembling in liver. Int. J. Obes. 33, 1007–1012), and GSPE was shown to correct dyslipidemia observed in HFD-fed rats probably through the repression of hepatic lipogenesis. Our data corroborate those findings with an extensive list of proteins describing the induction of hepatic glycogenesis, glycolysis, and fatty acid and triglyceride synthesis in HFD, whereas the opposite pattern was observed to a large extent in GSPE-treated animals. GSPE was shown to have a wider effect than previously thought, and putative targets of GSPE involved in the reversal of the symptoms of metabolic syndrome were revealed. Some of these novel candidate proteins such as GFPT1, CD36, PLAA (phospholipase A₂-activating protein), METTL7B, SLC30A1, several G signaling proteins, and the sulfide-metabolizing ETHE1 and SQRDL (sulfide-quinone reductase-like) might be considered as drug targets for the treatment of metabolic syndrome.An increase in high calorie diets and a sedentary lifestyle are considered the key factors in explaining the epidemic rise in obesity in developed countries (1). Obese patients, especially those with abdominal obesity due to visceral adipose tissue accumulation, run a higher risk of impaired glucose tolerance, which frequently evolves into insulin resistance (2). Obesity and insulin resistance are frequently associated with hypertension, proatherogenic dyslipidemia, chronic inflammation, a prothrombotic state, and recently also fatty liver (3), conditions that together make up what is known as metabolic syndrome and lead to an increased risk of developing cardiovascular disease (CVD)¹ and type 2 diabetes (4). Conversely, some dietary patterns and specific food components have been associated with a lower prevalence of obesity, type 2 diabetes, and CVD. In this sense, the traditional Mediterranean diet (characterized by a high fiber content, low glycemic index carbohydrates, unsaturated fats, vitamins, and antioxidant polyphenols) has been linked to a lower incidence of CVD, obesity, and type 2 diabetes (5–8). Moreover, the French population presents a very low prevalence of death due to CVD despite consuming a diet rich in saturated fats and cholesterol. This phenomenon, known as “the French paradox” (9), has been ascribed to the moderate consumption of red wine and specifically to its content of polyphenols (10–12).Polyphenols include flavonoids of which flavan-3-ols and their oligomeric forms (proanthocyanidins) have been reported to exhibit several beneficial health effects by acting as antioxidant, anticarcinogen, cardioprotective, antimicrobial, antiviral, and neuroprotective agents (for a review, see Ref. 13). Specifically, grape and wine proanthocyanidins have a cardioprotective effect through increasing plasma high density lipoprotein cholesterol, decreasing low density lipoprotein-derived atherosclerotic foam cell lesions, attenuating oxidant formation by quenching harmful radicals, increasing endothelium-dependent vasorelaxation, etc. (13). In this context, our group has been working for years on the effect of a grape seed proanthocyanidin extract (GSPE) (containing monomers and oligomers of flavan-3-ols) in relation to metabolic syndrome. In previous works, we have found that GSPE prevents oxidative injury (14), has an insulinomimetic effect on adipocytes and adipose tissue (15), modulates glucose homeostasis (16), decreases plasma levels of triglycerides (TGs) and apolipoprotein B in normolipidemic rats (17), and acts as an in vitro (18, 19) and in vivo (20) anti-inflammatory. We have also shown that GSPE decreases postprandial plasma TG and apolipoprotein B in mice through a hepatic induction of a farnesoid X receptor (FXR) and the small heterodimer partner (SHP) that in turn down-regulates SREBP1c and other lipogenic genes in the liver (21, 22). Furthermore, we have demonstrated that the molecules responsible for the reduced TG synthesis in HepG2 cells treated with GSPE are the sum of a proanthocyanidins trimer and a dimer gallate because they reproduce the GSPE effect (23).The effect of GSPE on metabolic syndrome has been studied in our laboratory by feeding rats a “cafeteria diet.” This diet is an experimental model of a western high sugar and high fat diet extensively used to produce obesity in rats because its palatability induces the animals to increase their energy intake (24). In a recent study conducted by our group (25) as well as this study, the rats were fed a high fat diet (HFD) (cafeteria diet) for 13 weeks, and one group of the animals was treated with a daily dose of GSPE (25 mg/kg of body weight) for the last 10 days (HFD + GSPE). In that study, HFD was shown to cause the animals to be overweight and to suffer from fatty liver, dyslipidemia, and hepatic overexpression of key genes involved in lipogenesis and VLDL assembly, whereas GSPE treatment corrected dyslipidemia and down-regulated some of the genes up-regulated by HFD (25).To better investigate the mechanism behind the changes observed in HFD- and HFD + GSPE-fed rats, we analyzed protein expression in the liver. Because GSPE treatment and obesity have multiple effects, a proteome-wide approach is needed to map proteins from different pathways. Proteomics studies related to obesity, metabolic syndrome, fatty liver, or insulin resistance have previously been performed on the liver (26–32). Two such studies looked into the effects of flavonoids in mouse livers (33, 34), but to our knowledge, this is the first hepatic proteome analysis of the effect of flavonoids in rats suffering from metabolic syndrome. To improve the proteome coverage of the complex liver samples, we performed a proteome fractionation according to protein solubility using a two-phase detergent protocol (35). This strategy was advantageous because it captured membrane proteins that otherwise would have been difficult to detect. The resulting soluble and membrane protein fractions were digested, iTRAQ-labeled, fractionated according to isoelectric point, and analyzed by nano-LC-MS/MS. The proteomics study presented here reports a differential expression due to HFD or HFD + GSPE for approximately 140 proteins, indicating that both conditions were potent modifiers of the liver proteome. We have focused on the sugar and lipid metabolism data, which confirmed the repression of hepatic lipogenesis in HFD + GSPE rats. Additionally, new proteins have been revealed as putative GSPE targets.     

Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans

The N-glycosylation of the model nematode Caenorhabditis elegans has proven to be highly variable and rather complex; it is an example to contradict the existing impression that “simple” organisms possess also a rather simple glycomic capacity. In previous studies in a number of laboratories, N-glycans with up to four fucose residues have been detected. However, although the linkage of three fucose residues to the N,N′-diacetylchitobiosyl core has been proven by structural and enzymatic analyses, the nature of the fourth fucose has remained uncertain. By constructing a triple mutant with deletions in the three genes responsible for core fucosylation (fut-1, fut-6 and fut-8), we have produced a nematode strain lacking products of these enzymes, but still retaining maximally one fucose residue on its N-glycans. Using mass spectrometry and HPLC in conjunction with chemical and enzymatic treatments as well as NMR, we examined a set of α-mannosidase-resistant N-glycans. Within this glycomic subpool, we can reveal that the core β-mannose can be trisubstituted and so carries not only the ubiquitous α1,3- and α1,6-mannose residues, but also a “bisecting” β-galactose, which is substoichiometrically modified with fucose or methylfucose. In addition, the α1,3-mannose can also be α-galactosylated. Our data, showing the presence of novel N-glycan modifications, will enable more targeted studies to understand the biological functions and interactions of nematode glycans.Nematodes represent, along with arthropods, one of the largest groups of animals to exist on the planet; 25.000 species are described, but the existence of up to one million has been estimated (1, 2). They have various ecological niches and include free-living “worms” in the soil, fungivorous, entomopathogenic, and necromenic species as well as parasites of plants and mammals, which share the basic conserved body plan (more-or-less a digestive tube surrounded with muscle, whether larger or smaller). There are five major clades (Rhabditina, Enoplia, Spirurina, Tylenchina, and Dorylaimia) (2), yet the glycosylation of only a few nematode species has been studied with an inevitable focus on the model nematode Caenorhabditis elegans and parasitic species (3). Thereby, the use of C. elegans mutants has been highly valuable in dissecting aspects of nematode N-glycan biosynthesis and revealing the in vivo substrates for certain glycosyltransferases (4).As many nematodes are parasites, their interactions with the immune systems of their hosts have attracted attention; particularly, there are relationships between autoimmunity, allergy, vaccination, and helminth infections. The “old friends” hypothesis seeks to understand the evolutionary factors that have shaped the immune system and to explain correlations between lifestyles in the developed world and modern “epidemics,” which are due to immunological misbalance (5–7). Promising data have suggested that “worm therapy” may bring advantages to some patients with Crohn''s disease or allergies (8, 9); however, such approaches are controversial. Nevertheless, crude extracts even of Caenorhabditis elegans were shown to induce a glycan-dependent Th2 response (10), whereas the excretory-secretory products of some nematodes also have immunomodulatory activity (11). Furthermore, the native glycoproteins of some nematodes have proven effective in vaccination trials, whereas recombinant forms are not, which is suggestive that post-translational modifications may have a role in an efficacious immune response (12).As at least some of the molecules relevant to nematode immunomodulation or vaccination are glycoproteins, a proper understanding of nematode glycosylation is of biomedical and veterinary relevance. Over the years, it has become apparent that the core chitobiosyl region of nematode N-glycans is subject to a range of modifications, with up to three core fucose residues being present (α1,3- and α1,6-linked on the reducing-terminal “proximal” GlcNAc and α1,3-linked on the second “distal” GlcNAc). However, up to four fucose residues have been detected on C. elegans N-glycans and the exact nature of the linkage of the fourth fucose has remained obscure despite work in our own and other laboratories (3, 13–15). Combined with the latest knowledge regarding the specificity of C. elegans core fucosyltransferases (13, 16, 17) as well as our recent data regarding the exact structures of N-glycans from the C. elegans double hexosaminidase mutant and other nematodes (18–20), we concluded that some models for the tri- and tetrafucosylated N-glycans were incorrect. By preparing a triple mutant unable to core fucosylate its N-glycans, we generated a C. elegans strain containing maximally one fucose residue on the N-linked oligosaccharides. Thereby a pool of unusual mannosidase-resistant N-glycans was identified and, using mass spectrometry (MS) and NMR, we reveal their modification with bisecting galactose frequently capped with fucose or methylfucose.     

Biocatalyst Engineering by Assembly of Fatty Acid Transport and Oxidation Activities for In Vivo Application of Cytochrome P-450BM-3 Monooxygenase

The application of whole cells containing cytochrome P-450_BM-3 monooxygenase [EC 1.14.14.1] for the bioconversion of long-chain saturated fatty acids to ω-1, ω-2, and ω-3 hydroxy fatty acids was investigated. We utilized pentadecanoic acid and studied its conversion to a mixture of 12-, 13-, and 14-hydroxypentadecanoic acids by this monooxygenase. For this purpose, Escherichia coli recombinants containing plasmid pCYP102 producing the fatty acid monooxygenase cytochrome P-450_BM-3 were used. To overcome inefficient uptake of pentadecanoic acid by intact E. coli cells, we made use of a cloned fatty acid uptake system from Pseudomonas oleovorans which, in contrast to the common FadL fatty acid uptake system of E. coli, does not require coupling by FadD (acyl-coenzyme A synthetase) of the imported fatty acid to coenzyme A. This system from P. oleovorans is encoded by a gene carried by plasmid pGEc47, which has been shown to effect facilitated uptake of oleic acid in E. coli W3110 (M. Nieboer, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1996). By using a double recombinant of E. coli K27, which is a fadD mutant and therefore unable to consume substrates or products via the β-oxidation cycle, a twofold increase in productivity was achieved. Applying cytochrome P-450_BM-3 monooxygenase as a biocatalyst in whole cells does not require the exogenous addition of the costly cofactor NADPH. In combination with the coenzyme A-independent fatty acid uptake system from P. oleovorans, cytochrome P-450_BM-3 recombinants appear to be useful alternatives to the enzymatic approach for the bioconversion of long-chain fatty acids to subterminal hydroxylated fatty acids.Cytochrome P-450_BM-3 monooxygenase (CytP450_BM-3) is a soluble NADPH-dependent monooxygenase from Bacillus megaterium ATCC 14581 (13). It is a class II P-450 enzyme that contains flavin adenine dinucleotide, flavin mononucleotide, and a heme moiety (17). Unlike most CytP450 monooxygenases, which consist of a distinct monooxygenase and a reductase, CytP450_BM-3 contains these functionalities in a single polypeptide (3, 15, 18).The enzyme hydroxylates a variety of long-chain aliphatic substrates, such as fatty acids, alkanols, and alkylamides at the ω-1, ω-2, and ω-3 positions (4, 17), and oxidizes unsaturated fatty acids to epoxides in vitro (17, 23) with high enantioselectivity. Oxidation of eicosapentenoic acid (C_20:5) and arachidonic acid (C_20:4) yielded 17(S),18(R)-epoxyeicosatetraenoic acid (94% enantiomeric excess [e.e.]) for the former and a mixture of 18-(R)-hydroxyarachidonic acid (92% e.e.) and 14(S),15(R)-epoxyeicosatrienoic acid at 98% e.e. for the latter substrate (8). Recently, it has been demonstrated that the enzyme also produces α,ω diacids from ω-oxo fatty acids by oxidation of the terminal aldehyde functionality (9). The catalytic constant (k_cat) of CytP450_BM-3 is among the highest found for P-450 monooxygenases, ranging from 15 s⁻¹ for laureate to 75 s⁻¹ for pentadecanoic acid (11). For comparison, a typical microsomal P-450 monooxygenase from human liver (CYP2J2) had a k_cat of 10⁻³ s⁻¹ for arachidonic acid (32), compared to a k_cat of 55 s⁻¹ for CytP450_BM-3 for the same substrate (8).This high catalytic efficiency prompted us to investigate the applicability of CytP450_BM-3 as a biocatalyst for the subterminal hydroxylation of long-chain fatty acids (LCFAs). Since these subterminal hydroxy LCFAs are chiral molecules, their application in the production of enantiopure synthetic building blocks, especially for pharmaceutical agents, could be envisioned. Further, long-chain hydroxy acids find applications as precursors for polymers or cyclic lactones, which are used as components of fragrances and as antibiotics. Although chemical syntheses have been developed for ω-1 hydroxy fatty acids (from C₁₂ to C₁₈) (26, 28, 29) and for ω-2 and ω-3 hydroxyoctadecanoic acids (2), they require expensive functionalized substrates and are in general complicated, multistep processes (26, 28, 29) which cannot be carried out with unmodified fatty acids as inexpensive starting material. In principle, such inexpensive substrates can be oxidized to hydroxy fatty acids by biocatalysts, either in vitro or in vivo. The latter is preferred, since whole cells actively regenerate the NADPH required for fatty acid oxidation with monooxygenases such as CytP450_BM-3. In designing a suitable whole-cell biocatalyst, several additional points had to be considered.First, uptake must be efficient. Second, degradation of substrate or product must be avoided. In fact, biotransformations of fatty acids with whole cells are usually inefficient due to limited uptake of these compounds at neutral pH, and when taken up, they are degraded via β-oxidation. The transport of LCFAs in Escherichia coli is mediated via the fadL and fadD gene products. FadL is the transporter that carries LCFAs across the outer membrane and is absolutely required for LCFA transport (20). FadD, the acyl coenzyme A (CoA) synthetase, is located at the inner side of the cytoplasmic membrane and is required for formation of the acyl coenzyme A thioester, after which the activated fatty acids are channeled into the β-oxidation cycle for fatty acid degradation (21, 22). Thus, we used a FadD mutant, E. coli K27, as a suitable host for the production of subterminal hydroxyalkanoic acids (20). E. coli K27 cannot couple free fatty acids to coenzyme A, thus preventing substrate or product degradation by the host. Such fadD mutants are, however, also impaired in efficient uptake of fatty acids (20). We circumvented this by introducing a fatty acid uptake system from Pseudomonas oleovorans encoded on pGEc47. Finally, we introduced the P-450_BM-3 monooxygenase on pCYP102 into the fadD mutant E. coli. The resulting recombinant, E. coli K27(pCYP102, pGEc47), is a promising tailored biocatalyst for the oxidation of saturated LCFAs to ω-1, ω-2, and ω-3 hydroxy fatty acids.     

Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL)

Tetrahydrolipstatin (THL) is bactericidal but its precise target spectrum is poorly characterized. Here, we used a THL analog and activity-based protein profiling to identify target proteins after enrichment from whole cell lysates of Mycobacterium bovis Bacillus Calmette-Guérin cultured under replicating and non-replicating conditions. THL targets α/β-hydrolases, including many lipid esterases (LipD, G, H, I, M, N, O, V, W, and TesA). Target protein concentrations and total esterase activity correlated inversely with cellular triacylglycerol upon entry into and exit from non-replicating conditions. Cellular overexpression of lipH and tesA led to decreased THL susceptibility thus providing functional validation. Our results define the target spectrum of THL in a biological species with particularly diverse lipid metabolic pathways. We furthermore derive a conceptual approach that demonstrates the use of such THL probes for the characterization of substrate recognition by lipases and related enzymes.Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is responsible for nearly 2 million deaths each year. The host immune response toward aerosol infection is to quarantine tubercle bacilli in a granulomatous structure (1, 2). However, granuloma-associated mycobacteria can switch to a non-replicative, “dormant” state and successfully evade immune response for decades after infection (3, 4). The metabolic events that permit tubercle bacilli to enter host cells and revive from states of persistence suggest that lipids are utilized as a carbon source (5–7). During times of oxygen deprivation and in the absence of host cells, cultivated mycobacteria store fatty acids (FAs) in the form of triacylglycerol (TAG)¹-enriched lipid droplets (8–10). Upon resuscitation (by the re-introduction of oxygen), these lipid droplets vanish and TAGs are hydrolyzed (11). Unfortunately, the molecular mechanisms for TAG build-up and breakdown are far less well understood in bacteria when compared with those processes in eukaryotes.Comparative sequence analysis of the Mtb genome has revealed that it contains 250 genes encoding enzymes involved in lipid metabolism compared with only 50 enzymes in Escherichia coli, which has a genome of comparable size. Among these genes, 150 are predicted to encode proteins involved in lipid catabolism (12, 13). A family of 24 carboxyl ester hydrolases called “lip” genes (lipC to Z, except K and S) has been predicted to play a role in lipid catabolism (14). Among these, only a few have been functionally characterized and related to mycobacterial dormancy and resuscitation (15–18).Tetrahydrolipstatin, a serine esterase inhibitor, covalently binds to and inhibits mammalian lipases and fatty acid synthase (FAS) and is marketed as “Orlistat” for the treatment of severe forms of obesity (19). THL was previously shown to inhibit both active and latent forms of mycobacteria (11, 20–22) but the bacterial target spectrum remains poorly characterized. Therefore, to (1) define the THL target spectrum in a mycobacterial species and (2) to obtain biochemical insights into regulation of lipases and esterases in different metabolic states, we employed a chemical-proteomics approach using activity-based protein profiling (ABPP) with a bait that has been described to bind to lipolytic enzymes (23–25). We identified several known lipases (as anticipated), putative lipase and esterases, and hypothetical proteins of unknown functions, thereby providing a comprehensive resource of experimentally determined THL targets in mycobacteria. Importantly, we systematically compared readouts of fluorescently tagged THL-proteins (7 bands on one-dimensional SDS-PAGE) with those of mass spectrometry-based peptide identification of enriched protein fractions (247 in growing cells). This comparison led to the identification of 14 THL targets, two of which were further validated experimentally. We furthermore provide a conceptual framework for the evaluation of this target list using both experimental as well as bioinformatics approaches in two examples, lipH and tesA. Overall, our data indicate that THL is an anti-mycobacterial drug because of its potential to (1) bind to a relatively wide range of lipolytic enzymes and (2) prevent bacilli from resuscitating from a nonreplicating persistent (NRP) state when lipid metabolism is particularly important.     

The Role of Binding Energy in Catalysis: the Work of William P. Jencks

Two Functional Domains of Coenzyme A Activate Catalysis by Coenzyme A Transferase. Pantetheine and Adenosine 3′-Phosphate 5′-Diphosphate (Fierke, C. A., and Jencks, W. P. (1986) J. Biol. Chem. 261, 7603–7606)William Platt Jencks (1927–2007) was born in Bar Harbor, Maine. He became interested in chemistry when he received a chemistry set for Christmas in 1934. He immediately carried out one of the experiments described in the instructions, the addition of dilute acid to a sulfide salt to produce H₂S. The experiment was so successful that his house had to be evacuated due to the smell of rotten eggs. According to Jencks, “My family and I did not find it necessary to replicate this experiment” (1).Open in a separate windowWilliam P. JencksJencks enrolled at Harvard College, intending to study chemistry. However, after taking a first year course in chemistry that “described a large number of chemical reactions, one after the other, with no indication of what was interesting about any of them” (1), he switched his major to English. Despite this change in the direction of his studies, Jencks ended up entering Harvard Medical School after his junior year because he wasn''t sure what else to do.After completing his first year of medical school, Jencks spent a summer at the Marine Biological Laboratory in Woods Hole, taking courses and doing research on lobster shell pigments with Journal of Biological Chemistry (JBC) Classic author George Wald (2). He received his M.D. in 1951 and then interned at Peter Bent Brigham Hospital in Boston. However, after a while, Jencks found medicine to be “a very broad field in which it would be difficult to obtain definitive answers to fundamental problems” (1). Wald suggested Jencks try doing research at Massachusetts General Hospital with Nobel laureate Fritz Lipmann (who was featured in a previous JBC Classic (3)). Jencks ended up spending 2 years with Lipmann, studying coenzyme A transferase, which led to his longtime interest in the physical organic chemistry of acyl transfer reactions. After leaving Massachusetts General Hospital, Jencks spent a year doing postdoctoral studies at Harvard University with Nobel laureate Robert Woodward before joining the faculty at Brandeis University in 1957, serving as assistant, associate, and then full professor of biochemistry. He retired in 1996 as professor emeritus of biochemistry.During his 39 years at Brandeis University, Jencks studied the mechanisms by which enzymes facilitate chemical reactions of molecules that are not otherwise inclined to react at a useful rate.The JBC Classic reprinted here looks at the noncovalent interactions between succinyl-CoA 3-ketoacid coenzyme A transferase and coenzyme A. In the paper, Jencks and Carol A. Fierke used a small coenzyme A analog, methylmercaptopropionate, to show that noncovalent interactions between the enzyme and the side chain of CoA are responsible for the reaction rate increase brought about by the enzyme. They report that interaction between the enzyme and the pantetheine moiety of CoA provides the majority of substrate destabilization and rate acceleration, whereas the interaction with the 3′-phospho-ADP1 moiety provides binding energy that overcomes this destabilization and permits significant binding of acyl-CoA substrates to the enzyme. This paper helped to illuminate a striking example of the role of binding energy in catalysis.Jencks received many honors and awards for his contributions to science, including memberships in the National Academy of Sciences (1971) and the American Philosophical Society (1995) and foreign membership in the Royal Society. He also received the 1962 American Chemical Society (ACS) Award in Biological Chemistry, the 1993 American Society of Biological Chemists Award, the 1995 ACS James Flack Norris Award in Physical Organic Chemistry, and the 1996 ACS Repligen Award for Chemistry of Biological Processes.¹     

Global Analysis of Myocardial Peptides Containing Cysteines With Irreversible Sulfinic and Sulfonic Acid Post-Translational Modifications

Cysteine (Cys) oxidation is a crucial post-translational modification (PTM) associated with redox signaling and oxidative stress. As Cys is highly reactive to oxidants it forms a range of post-translational modifications, some that are biologically reversible (e.g. disulfides, Cys sulfenic acid) and others (Cys sulfinic [Cys-SO₂H] and sulfonic [Cys-SO₃H] acids) that are considered “irreversible.” We developed an enrichment method to isolate Cys-SO₂H/SO₃H-containing peptides from complex tissue lysates that is compatible with tandem mass spectrometry (MS/MS). The acidity of these post-translational modification (pK_a Cys-SO₃H < 0) creates a unique charge distribution when localized on tryptic peptides at acidic pH that can be utilized for their purification. The method is based on electrostatic repulsion of Cys-SO₂H/SO₃H-containing peptides from cationic resins (i.e. “negative” selection) followed by “positive” selection using hydrophilic interaction liquid chromatography. Modification of strong cation exchange protocols decreased the complexity of initial flowthrough fractions by allowing for hydrophobic retention of neutral peptides. Coupling of strong cation exchange and hydrophilic interaction liquid chromatography allowed for increased enrichment of Cys-SO₂H/SO₃H (up to 80%) from other modified peptides. We identified 181 Cys-SO₂H/SO₃H sites from rat myocardial tissue subjected to physiologically relevant concentrations of H₂O₂ (<100 μm) or to ischemia/reperfusion (I/R) injury via Langendorff perfusion. I/R significantly increased Cys-SO₂H/SO₃H-modified peptides from proteins involved in energy utilization and contractility, as well as those involved in oxidative damage and repair.Cysteine (Cys)¹ is an integral site for protein post-translational modification (PTM) in response to physiological and pathological stimuli. Numerous studies have identified roles for biologically reversible Cys PTM, including disulfides, S-nitrosothiols, and sulfenic acids (Cys-SOH), in the regulation of protein function during redox signaling (reviewed in (1, 2)). Additionally, Cys can be oxidized in pathologies associated with oxidative stress (e.g. neurodegeneration, cancer, and cardiovascular disease (2)). Various redox proteomics methods exist for enrichment of these reversibly oxidized Cys, based on reduction to the thiol and then capture by: 1) alkylation with a chemical tag (e.g. isotope coded affinity tags) (3–6); 2) thiol-disulfide exchange (7–10); or 3) heavy metal ion chelation (11, 12). Oxidative Cys PTMs with predominantly no known means of enzymatic reduction have also been identified. These “over” or “irreversibly” oxidized Cys PTM (sulfinic [Cys-SO₂H] and sulfonic [Cys-SO₃H] acids) are primarily associated with oxidative stress. Only one example of reversible Cys-SO₂H modification has been characterized—in peroxiredoxins (Prx) by the ATP-dependent sulfiredoxin (Srx)(13); however, Srx is not thought to reduce Cys-SO₂H in other proteins, and no mechanism has yet been found for Cys-SO₃H reduction. At basal levels, ∼1–2% of Cys exist as Cys-SO₂H/SO₃H (14), and the RSO₂H modification has functional significance in some proteins (e.g. DJ-1 is activated in Alzheimer''s disease by Cys-SO₂H at Cys-106) (15).Cys-SO₂H/SO₃H are produced via sequential oxidation of Cys-SOH, which itself is formed because of Cys thiol oxidation by reactive oxygen and nitrogen species (ROS/RNS), such as hydrogen peroxide (H₂O₂) or peroxynitrite. This reaction is relatively inefficient and requires three equivalents of oxidant, as well as the protection of the initial Cys-SOH from nucleophilic attack. Therefore, Cys forming these PTM, particularly at biologically relevant concentrations of oxidant, are likely to be highly reactive or located in a unique microenvironment that accommodates their production without prior reduction of the Cys-SOH (e.g. by thiol or amine attack). Such sites may thus be candidates as redox or regulatory sensors (reviewed in (16)). Alternatively, over-oxidation to Cys-SO₂H/SO₃H during elevated oxidative stress may serve as a marker of oxidative damage, and target proteins for degradation.Information on Cys-SO₂H/SO₃H PTM in complex samples has thus far been generated only by amino acid analysis (hydrolyzed lysates) (14) or two-dimensional gel electrophoresis (2-DE), where these PTM cause an acidic shift (17, 18). The former provides no information on specific proteins, whereas the latter relies on the modified population being of sufficient intensity for observation and/or the availability of antibodies against a protein-of-interest. A recent study identified 44 Cys-SO₂H/SO₃H-modified peptides in nonphysiologically H₂O₂ oxidized (440 μm) cells utilizing long column ultra-high pressure liquid chromatography (LC) (19). Global analysis of irreversible Cys-PTM thus requires enrichment that considers: (1) Cys is the second least abundant amino acid in proteins (∼1.5%) (20), and (2) Cys-SO₂H/SO₃H are expected to occupy only 1–2% of these Cys sites, under physiological (and perhaps even pathological) conditions.Specific peptide enrichment by LC followed by bottom-up proteomics is a common approach used successfully for many PTMs (21, 22). Limited studies, however, have explored such techniques for Cys-SO₂H/SO₃H-containing peptides, and none have examined complex lysates—only single purified proteins (23, 24). Given that these PTM are among the most acidic modifications, with an average pK_a of RSO₂H < 2 and RSO₃H ∼−3, it is pertinent to isolate these peptides by exploiting their unique charge distribution. At acidic pH, where nonmodified tryptic peptides will have an average in-solution charge state between one and two (depending on pK_a of acidic residues and the C terminus), Cys-SO₂H/SO₃H-containing peptides will have an added negative charge, and, thus, have average charge distribution ≤ 1. Selection can therefore be performed on either positively or negatively charged resins with the former being a “positive” selection for Cys-SO₂H/SO₃H-containing peptides (retained by the resin), whereas the latter is a “negative” selection (Cys-SO₂H/SO₃H-containing peptides will not be retained by the resin). Both approaches have been used (23, 24) to capture peptides from bovine serum albumin (BSA) oxidized by performic acid – causing scission of disulfide bonds and conversion of Cys to Cys-SO₃H, and methionine (Met) to the sulfone Met(O₂). The studies gave comparative results, with positive selection (24) increasing Cys coverage in comparison to negative selection (23) (60% versus 45%) at the expense of specificity, with more non-Cys peptides observed in the elution.Ultimately, any enrichment approach must be able to purify Cys-SO₂H/SO₃H-containing peptides from cells and tissues under physiological and/or pathological conditions, both of which will generate considerably lower levels of Cys-SO₂H/SO₃H than performic acid. Myocardial ischemia and reperfusion (I/R) injury is characterized by a “burst” of ROS/RNS that is observed upon reperfusion (25, 26). These ROS/RNS overwhelm the natural antioxidant defenses of the heart (27) and lead to oxidative stress that contributes to contractile dysfunction (28–30). Several studies have observed an increase in reversible Cys PTM following I/R (31–36), and an increase in Cys-SO₂H/SO₃H may also contribute to cellular dysfunction that ultimately leads to apoptosis and necrosis that follows prolonged I/R (myocardial infarction). Given the common practice of peptide fractionation with strong cation exchange (SCX) as a first dimension during bottom-up proteomics, we wished to explore its utility in identifying Cys-SO₂H/SO₃H sites in complex samples. Performic oxidized BSA and myocardial protein extracts were utilized to study the interactions occurring at each step of the method, and then the method was applied to myocardial protein extract that had been exposed to a high concentration of a less efficient oxidant (H₂O₂). Finally, the method was used to identify Cys-SO₂H/SO₃H-containing peptides derived from either physiologically relevant concentrations of H₂O₂ (i.e. ≤100 μm, an estimate of the likely pathological H₂O₂ levels (37, 38)) or from rat myocardial tissue subjected to I/R injury.     

Reverse-Polynomial Dilution Calibration Methodology Extends Lower Limit of Quantification and Reduces Relative Residual Error in Targeted Peptide Measurements in Blood Plasma

Matrix effect is the alteration of an analyte''s concentration-signal response caused by co-existing ion components. With electrospray ionization (ESI), matrix effects are believed to be a function of the relative concentrations, ionization efficiency, and solvation energies of the analytes within the electrospray ionization droplet. For biological matrices such as plasma, the interactions between droplet components is immensely complex and the effect on analyte signal response not well elucidated. This study comprised of three sequential quantitative analyses: we investigated whether there is a generalizable correlation between the range of unique ions in a sample matrix (complexity); the amount of matrix components (concentration); and matrix effect, by comparing an E. coli digest matrix (∼2600 protein proteome) with phospholipid depleted human blood plasma, and unfractionated, nondepleted human plasma matrices (∼10⁷ proteome) for six human plasma peptide multiple reaction monitoring assays. Our data set demonstrated analyte-specific interactions with matrix complexity and concentration properties resulting in significant ion suppression for all peptides (p < 0.01), with nonuniform effects on the ion signals of the analytes and their stable-isotope analogs. These matrix effects were then assessed for translation into relative residual error and precision effects in a low concentration (∼0–250 ng/ml) range across no-matrix, complex matrix, and highly complex matrix, when a standard addition stable isotope dilution calibration method was used. Relative residual error (%) and precision (CV%) by stable isotope dilution were within <20%; however, error in phospholipid-depleted and nondepleted plasma matrices were significantly higher compared with no-matrix (p = 0.006). Finally a novel reverse-polynomial dilution calibration method with and without phospholipid-depletion was compared with stable isotope dilution for relative residual error and precision. Reverse-polynomial dilution techniques extend the Lower Limit of Quantification and reduce error (p = 0.005) in low-concentration plasma peptide assays and is broadly applicable for verification phase Tier 2 multiplexed multiple reaction monitoring assay development within the FDA-National Cancer Institute (NCI) biomarker development pipeline.Plasma is the overriding human medium sampled for established and novel protein biomarkers (1, 2). As of 2011, 1929 high-confidence proteins have been cataloged by the Human Plasma Proteome Project, with estimates that there are up to 10⁷ unique protein sequences in plasma that span a concentration range across 10 orders of magnitude (1, 3). 99% of the protein mass in plasma is made up of 22 proteins including Albumin, Fibrinogen, and a range of immunoglobulins, leaving more than 1900 known small proteins and essentially the entirety of the projected plasma proteome in the remaining 1% (4). It is these low-mass, low abundance proteins such as the Interleukins, C-Reactive Protein, and Carcinoma Antigen 125 (CA125), that are indicative of many important physiological and pathological processes, and proteomic scientists and clinicians have thus focused their efforts in qualitatively and quantitatively defining this fraction for novel biomarkers (4–6).The development of plasma biomarkers is a large-scale undertaking that spans discovery, verification, and validation phases in a multistage pipeline: Thousands of “discovered” differentiated proteins are evaluated for probability of effect, from which 10–100s of proteins are then selected for targeted quantification in verification phase to evaluate sensitivity and specificity for its intended indication (2, 7). Finally a panel of the strongest marker candidates is progressed to validation phase, and FDA-level validated quantitative assays are used to test the clinical utility of the biomarker panel. Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS/MS)¹ is the most robust analytical method available for proteomic scientists in this pipeline, able to separate complex mixtures and specifically and sensitively identify and quantify its components (2, 7–10), The ability to ionize and evaporate the contents of a liquid sample (coupling LC to MS/MS) is the basis that allows this to happen (9). Electrospray Ionization (ESI) is the most widely used ionization apparatus in LC-MS/MS bioanalysis because of its ionization efficiency and stability and low chemical specificity (9, 10). Although these properties make ESI very robust, the complexity of biological matrices poses a significant challenge for LC-ESI-MS/MS-based quantitation; despite chromatography and nanospray technology, the ESI droplet of a plasma peptide-digest sample (given its immense range of unique protein/peptide sequences and concentrations) can contain an unknown multitude of co-eluting components that “compete” to dissolve from the droplet and reach gas phase, suppressing and varying the signal intensity responses for a given analyte concentration (9–13). These ionization competing elements can also go on to produce isobaric signals in the third quadrupole that interfere with an analyte''s transition signals (14). Termed “matrix effects,” these phenomena of complex sample matrices can significantly impede quantitative accuracy (15). For high-throughput clinical assays, matrix effects are controlled for by preparing calibration standards in the same biological matrix to mimic the conditions of the samples intended for study as per FDA bioanalytical method validation guidelines (16). The catch to this technique is that the signal from the endogenous analyte in the background matrix hinders accuracy when the nominal concentration is close to or below the endogenous signal (14, 17). There is a need for broadly applicable methods of controlling matrix effects and increasing accuracy in low concentration MRM peptide assays for nondepleted, unfractionated plasma that can be adopted for the highly multiplexed, high throughput, “Tier 2” MS assays required in verification phase of the biomarker development pipeline (2, 8). Several simple methods have independently demonstrated the ability to increase accuracy in various hyphenated-MS assays in complex matrices: “Reverse” curves utilize the stable-isotope analog not as an internal standard but as a surrogate calibration analyte to circumvent interference from the endogenous analyte signal and extend assay Lower Limit(s) of Quantification (LLOQ), and nonlinear calibration techniques have proven to more accurately reflect the concentration-MS detector response at the low and high end of concentration gradients (8, 14, 18–21). Specifically in the case of biological matrices, phospholipids are particularly deleterious ion suppressing elements because of their easily ionizable, polar, and hydrophobic moieties that can have complex interactions with co-eluting analytes as well as the chromatography stationary and mobile phases required for most other analytes (22–25). Combination solid-phase extraction (SPE) and phospholipid removal techniques have proved to effectively minimize ion suppression effects in ESI-MS assays (22–25).In this study, we investigated whether there is a generalizable linear correlation between the number of unique ions (complexity) in a biological sample matrix, the amount of ionizable matrix content (concentration), and matrix effects, for six human plasma peptides comparing serial dilutions of an Escherichia Coli (E. coli) peptide-digest against phospholipid-depleted and nondepleted unfractionated human plasma peptide-digest (highly complex) matrices. We examined the influence of matrix effects on relative residual error in a low-concentration (∼0–250 ng/ml) plasma peptide range, and compared the utility of a reverse-polynomial dilution (RPD) calibration method versus standard addition stable-isotope dilution (SID) in phospholipid-depleted and nondepleted unfractionated human plasma. A peptide-centric matrix effect is reported and the effect of the endogenous analyte signal on relative residual error in low-concentration (∼0–250 ng/ml) plasma peptide assays is established. A RPD calibration technique that extends LLOQ and reduces relative residual error in low-concentration plasma peptide MRM assays is presented.     

Naegleria gruberi De Novo Basal Body Assembly Occurs via Stepwise Incorporation of Conserved Proteins

Centrioles and basal bodies are discrete structures composed of a cylinder of nine microtubule triplets and associated proteins. Metazoan centrioles can be found at mitotic spindle poles and are called basal bodies when used to organize microtubules to form the core structure of flagella. Naegleria gruberi, a unicellular eukaryote, grows as an amoeba that lacks a cytoplasmic microtubule cytoskeleton. When stressed, Naegleria rapidly (and synchronously) differentiates into a flagellate, forming a complete cytoplasmic cytoskeleton de novo, including two basal bodies and flagella. Here, we show that Naegleria has genes encoding conserved centriole proteins. Using novel antibodies, we describe the localization of three centrosomal protein homologs (SAS-6, γ-tubulin, and centrin-1) during the assembly of the flagellate microtubule cytoskeleton. We also used these antibodies to show that Naegleria expresses the proteins in the same order as their incorporation into basal bodies, with SAS-6 localizing first, followed by centrin and finally γ-tubulin. The similarities between basal body assembly in Naegleria and centriole assembly in animals indicate that mechanisms of assembly, as well as structure, have been conserved throughout eukaryotic evolution.The beautiful and enigmatic pinwheel structures of centrioles and basal bodies have captured the imaginations of cell biologists for over a century. These small (∼1-μm) organelles are composed largely of a cylinder of nine microtubule triplets (11). The surrounding amorphous material harbors the microtubule-organizing activities of the centrosome, placing centrioles at the hub of the microtubule cytoskeleton. Metazoan centrosomes define mitotic spindle poles, and their centrioles are called basal bodies when used to form cilia (29). Moreover, in 1900 Meeves showed in a series of classical experiments that centrioles and basal bodies are interconvertible structures (34). Centrioles must replicate exactly once per cell cycle, as duplication errors can lead to problems with chromosome segregation and cell morphology (17).Virtually all animal cells have a pair of centrosomal centrioles that duplicate via “templated” assembly, with the new centriole developing perpendicular and attached to a preexisting centriole (4). Centrioles can also be formed “de novo” in cytosol devoid of preexisting centrioles and basal bodies (20). In addition to many in vivo examples (20), terminally differentiated fibroblasts held in S phase can assemble centrioles de novo after removal of preexisting centrioles by laser microsurgery (15).The amoeboflagellate Naegleria gruberi grows as an amoeba that completely lacks a cytoplasmic microtubule cytoskeleton. However, when exposed to stressors such as temperature, osmotic, or pH changes, Naegleria rapidly differentiates into a flagellate, forming a complete cytoplasmic cytoskeleton from scratch, including two basal bodies and flagella (8). This differentiation occurs synchronously, with approximately 90% of cells growing visible flagella in a 15-min window (T₅₀ = 65 min after initiation of differentiation). As part of this differentiation, Naegleria has been shown to assemble the pinwheel structure of the basal bodies de novo, about 10 min before flagella are seen (11).Two centrosomal proteins that have been studied during Naegleria differentiation are centrin and γ-tubulin. Centrin is a calcium-binding phosphoprotein that is an integral component of the wall and lumen of basal bodies and of the pericentriolar lattice in many organisms (4, 19). During differentiation, Naegleria induces synthesis of centrin protein, which then localizes specifically to basal body structures throughout differentiation (18). γ-Tubulin is a general microtubule nucleation factor that localizes to microtubule-organizing centers (MTOCs) of many types. Surprisingly, Naegleria''s γ-tubulin homolog has been reported to localize to basal body precursor complexes and then move to the other end of the cell before disappearing completely (32).A third protein that has come under recent scrutiny for its role in centriole duplication is SAS-6, a functionally conserved coiled-coil protein required for the formation of diverse basal body precursor structures (7, 21,–23, 31). In Caenorhabditis elegans and Drosophila melanogaster, SAS-6 is recruited at S phase to form the “central tube,” a cylindrical basal body precursor that lacks microtubules (22, 23). SAS-6 is also required for the formation of the flat ring or cartwheel with nine radiating spokes, which is the first structure to be formed in the Chlamydomonas basal body (21).To determine if Naegleria is likely to have typical basal body components, we identified conserved basal body genes in the Naegleria genome. We also made antibodies to and localized Naegleria''s homologs of SAS-6 and γ-tubulin. Finally, we have determined the order of expression and incorporation of these proteins, as well as centrin, during Naegleria de novo basal body assembly.     

Effects of the Dietary ω3:ω6 Fatty Acid Ratio on Body Fat and Inflammation in Zebrafish (Danio rerio)

The diets of populations in industrialized nations have shifted to dramatically increased consumption of ω6 polyunsaturated fatty acids (PUFA), with a corresponding decrease in the consumption of ω3 PUFA. This dietary shift may be related to observed increases in obesity, chronic inflammation, and comorbidities in the human population. We examined the effects of ω3:ω6 fatty acid ratios in the context of constant total dietary lipid on the growth, total body fat, and responses of key inflammatory markers in adult zebrafish (Danio rerio). Zebrafish were fed diets in which the ω3:ω6 PUFA ratios were representative of those in a purported ancestral diet (1:2) and more contemporary Western diets (1:5 and 1:8). After 5 mo, weight gain (fat free mass) of zebrafish was highest for those that received the 1:8 ratio treatment, but total body fat was lowest at this ratio. Measured by quantitative real-time RT–PCR, mRNA levels from liver samples of 3 chronic inflammatory response genes (C-reactive protein, serum amyloid A, and vitellogenin) were lowest at the 1:8 ratio. These data provide evidence of the ability to alter zebrafish growth and body composition through the quality of dietary lipid and support the application of this model to investigations of human health and disease related to fat metabolism.Abbreviations: LC-PUFA, long-chain PUFA; PUFA, polyunsaturated fatty acidsMost animals require specific (essential) dietary fatty acids, and deficiencies in these fatty acids typically exert a negative effect on their health at some level. The ω3 and ω6 families of fatty acids are essential polyunsaturated fatty acids (PUFA) or long-chain PUFA (LC-PUFA) for many animals, including humans; however, consensus regarding the recommended dietary levels of these PUFA has not been achieved for any species, including humans. Several studies have proposed that a disproportionately high intake of ω6 PUFA and LC-PUFA promotes inflammation, resulting in chronic inflammatory diseases associated with metabolic syndrome.^10,22 This ‘high’ intake is difficult to describe accurately because both individual as well as regional diversity in the dietary intake of ω3 and ω6 fatty acids exist globally. Over the last century, diets in the western hemisphere have shifted to a dramatically increased consumption of total lipids. This increase in total fat consumption is associated with increases in ω6 PUFA and ω6 LC-PUFA intakes and corresponding decreases in ω3 PUFA and ω3 LC-PUFA.¹⁶ The shift in the dietary ω3:ω6 ratio, toward ω6 and away from ω3 fatty acids, in industrialized societies has been proposed to be the major factor contributing to inflammatory diseases.²² This proinflammatory effect is often attributed to the production of arachidonic acid metabolites, which act as potent proinflammatory and plaque forming molecules, from ω6 fatty acids, like linoleic acid.⁷ However, many antiinflammatory mediators also are produced during the metabolism of ω6. Several studies support a possible association between a reduced risk of coronary heart disease and increased dietary ω6 PUFA.⁷ The American Heart Association Science Advisory Panel has stated, “At present, there is little direct evidence that supports a net proinflammatory, proatherogenic effect of linoleic acid (18:2 ω6) in humans.”¹¹ The authors of a recent review¹⁹ concluded that reducing the intake of dietary ω6 fatty acid did not change the levels of arachidonic acid in the plasma, serum, or erythrocytes of adults who consumed western-type, high-fat diets. Other scientists¹⁸ have suggested that specific proportional combinations of ω3 and ω6 fatty acids may actually decrease the concentrations of proinflammatory cytokines.Zebrafish continue to gain popularity as an animal model for cardiovascular disease.⁴ For example, blood vessel plaques formed in zebrafish that consumed a high-cholesterol (4%) diet, mimicking atherosclerosis in humans.²⁴ Recent advances in the area of zebrafish nutrition²⁵ allow the use of formulated diets, wherein the levels of specific nutrients, such as fatty acids, can be modified to evaluate response. The current study evaluated the effects of different dietary ω3:ω6 fatty acid ratios on weight gain, body composition, and inflammatory response proteins in the zebrafish.     

Tomographic Three-dimensional Reconstruction of Insect Flight Muscle Partially Relaxed by AMPPNP and Ethylene Glycol

Rigor insect flight muscle (IFM) can be relaxed without ATP by increasing ethylene glycol concentration in the presence of adenosine 5′-[β′γ- imido]triphosphate (AMPPNP). Fibers poised at a critical glycol concentration retain rigor stiffness but support no sustained tension (“glycol-stiff state”). This suggests that many crossbridges are weakly attached to actin, possibly at the beginning of the power stroke. Unaveraged three-dimensional tomograms of “glycol-stiff” sarcomeres show crossbridges large enough to contain only a single myosin head, originating from dense collars every 14.5 nm. Crossbridges with an average 90° axial angle contact actin midway between troponin subunits, which identifies the actin azimuth in each 38.7-nm period, in the same region as the actin target zone of the 45° angled rigor lead bridges. These 90° “target zone” bridges originate from the thick filament and approach actin at azimuthal angles similar to rigor lead bridges. Another class of glycol-PNP crossbridge binds outside the rigor actin target zone. These “nontarget zone” bridges display irregular forms and vary widely in axial and azimuthal attachment angles. Fitting the acto-myosin subfragment 1 atomic structure into the tomogram reveals that 90° target zone bridges share with rigor a similar contact interface with actin, while nontarget crossbridges have variable contact interfaces. This suggests that target zone bridges interact specifically with actin, while nontarget zone bridges may not. Target zone bridges constitute only ∼25% of the myosin heads, implying that both specific and nonspecific attachments contribute to the high stiffness. The 90° target zone bridges may represent a preforce attachment that produces force by rotation of the motor domain over actin, possibly independent of the regulatory domain movements. Force production by myosin heads during muscle contraction has long been modeled as a transition of attached crossbridges from a 90° to a 45° axial angle. Efforts to image crossbridge forms and angles intermediate between 90° heads in ATP-relaxed insect flight muscle (IFM)¹ and the 45° angled bridges in rigor have used nucleotide analogs such as adenosine 5′-[β′γ-imido] triphosphate (AMPPNP) in stable equilibrium states to drive the crossbridges backwards from the 45° angle in rigor to an attached 90° preforce form, otherwise similar to myosin heads in ATP-relaxed fibers (Reedy et al., 1988; Tregear et al., 1990). However, AMPPNP alone will not fully relax IFM, and crossbridges binding AMPPNP retain many rigor-like features (Schmitz et al., 1996; Winkler et al., 1996). On the other hand, AMPPNP in combination with ethylene glycol will relax IFM. When poised at a critical glycol concentration, muscle stiffness is as high as rigor, suggesting crossbridge attachment, but fibers will not bear sustained tension (Clarke et al., 1984; Tregear et al., 1984). Two-dimensional (2-D) analysis of electron micrographs showed that this stiff glycol-PNP state resembled ATP-relaxed fibers in having dense collars every 14.5 nm along the thick filament and thin crossbridges originating from these collars at various axial angles around 90°. However, unlike relaxed muscle, stiff glycol-PNP fibers showed both 90° angled bridges that were regularly spaced every 38.7 nm and more intensity on the 19.3-nm layer line in optical and x-ray diffraction patterns (Reedy et al., 1988; Tregear et al., 1990). Crossbridges in this partially relaxed, glycol-PNP state are important because they may represent the form of the initial attachment of myosin with bound nucleotide preceding force generation (Marston and Tregear, 1984; Tregear et al., 1984; Reedy et al., 1988). This putative preforce 90° crossbridge could not be characterized in 3-D because its variable form and lattice arrangement precluded imaging by averaging methods of 3-D reconstruction. Recently, nonaveraging tomographic methods have been developed and successfully applied to rigor and aqueous-PNP, facilitating characterization of variable crossbridge forms that occur in situ (Taylor and Winkler, 1995, 1996; Schmitz et al., 1996; Winkler and Taylor, 1996). IFM is superb for structural study because the symmetry and spatial arrangement of filaments results in paired crossbridges on opposite sides of the actin filament. This in turn has given rise to a unique shorthand terminology. The individual crossbridge forms are not unique to IFM, only their symmetrical placement about the thin filament. The filament arrangement also facilitates the microtomy of a type of thin section with coplanar filaments that provide views of the entire crossbridge. The best of these, the myac layer, is a 25-nm-thick longitudinal section containing alternating myosin and actin filaments. In rigor, the maximum number of myosin heads attach to actin, forming doublet pairs every 38.7 nm, the “double chevrons” (Reedy, 1968). “Lead bridges,” which form the pair proximal to the M-band, consist of both heads of a myosin molecule and show an overall axial angle of 45° (Taylor et al., 1984). “Rear bridges,” which form the pair proximal to the Z-disk, consist of a single myosin head angled closer to 90°. Crossbridges originate from the thick filament along helical tracks so the azimuths of their origins follow a regular pattern. Relative to the thin filament in the myac layer, the lead bridges originate from the left-front and back-right of the adjacent thick filaments, while rear bridges originate from the left-back and right-front. At their actin ends, the crossbridge attachments follow the changing rotation of the actin protomers along the actin helix. The combination of the azimuth of the origin and the azimuth of the crossbridge contact to actin define the azimuthal angle of the crossbridge.Target zone is the name given to the region of the thin filament where crossbridges bind (Reedy, 1968); by implication this is the region of the thin filament where actin monomers are most favorably placed for actomyosin interaction. In our previous 3-D reconstructions of rigor and aqueous-PNP (Schmitz et al., 1996; Winkler et al., 1996), it was recognized that troponin maintained a constant position with respect to the most regularly positioned crossbridges, the lead bridges, and could thus be used as a landmark to determine the actin dyad orientation in the lead bridge target zone. The most sterically favorable actin position for crossbridge binding in the IFM lattice is midway between troponin densities, where lead bridges bind. The strained structure of the rigor rear bridges suggests that they bind at the very edge of the target zone (Schmitz et al., 1996; Winkler et al., 1996). The target zone defined by lead bridges alone is narrower than target zones previously considered for rigor muscle (Reedy, 1968) because it does not include rear bridge targets. When aqueous AMPPNP was added to rigor IFM, the tension dropped by two thirds, but the stiffness remained as high as rigor. This initially suggested a reversal of the power stroke, but 3-D reconstructions revealed that the lead bridges remained attached, midway between troponin densities, at axial and azimuthal angles close to rigor. The drop in tension without a large change in axial angle seemed to contradict the lever arm hypothesis for motion producing force. However, a cause for the loss of tension was found in tomograms, which showed that rear bridges detached and were replaced by nonrigor bridges bound to actins outside of the rigor target zone, to sites not selected by crossbridges even under the high-affinity conditions of rigor. These nontarget bridges in aqueous-PNP had variable axial and azimuthal angles and appeared to bind actin with variable contact interfaces. This suggested that they were nonspecifically bound to actin. Moreover, their variable structure did not suggest how a simple axial angle change could convert them to a familiar form, such as an angled rigor bridge. However, an intriguing doublet crossbridge group with a consistent structure was recognized in aqueous-PNP. Immediately M-ward of the “lead” rigor-like bridge was a “nonrigor” bridge bound at a 90° or antirigor angle. In this doublet, called a mask motif, both lead and M-ward nonrigor bridge pairs had similar azimuths and contact interfaces with actin and bound within the lead bridge target zone. A simple angle change could convert the M-ward, nonrigor bridge in a mask motif to a single headed lead bridge. Thus, in the mask motif, the lead bridge could be at the end of the power stroke, with the M-ward, nonrigor bridge near the beginning. The pairing of rigor and antirigor angled crossbridges bound to the same target zone suggests that crossbridges might act as a relay during muscle contraction (Schmitz et al., 1996). The affinity of myosin for actin in aqueous-PNP is high compared with weak binding intermediates thought to represent the beginning of the power stroke (Green and Eisenberg, 1980; Biosca et al., 1990). Therefore, the M-ward crossbridge in the mask motif may not represent the best candidate for a preforce crossbridge. Thus, it is important to characterize crossbridge structure in a state with lower actomyosin affinity, such as the stiff glycol-PNP state, where earlier 2-D analysis indicated that weakly attached 90° bridges are prevalent (Reedy et al., 1988). In this work, we have used two spatially invariant features, troponin position and lead crossbridge origins, to identify distinct classes of crossbridges. The invariant position of troponin recognized in 3-D reconstructions allows us to identify the lead bridge target zone and the actin dyad orientation relative to the bound crossbridges. In addition, the “front-back” rule for the azimuth of the origins of the lead target zone bridges distinguishes crossbridges that bind actin with the correct azimuth for specific binding from those that bind nonspecifically. By fitting the myosin subfragment 1 (S1) atomic structure to the in situ bridges, we can compare the positions of the motor and regulatory domains. Previous results and models have introduced the idea that during a power stroke, the crossbridge rotates over the actin binding site while acting as a long, relatively rigid lever arm (Huxley and Simmons, 1971), while others propose that the motor domain position remains constant and light chain domain movements provide a shorter lever arm (Rayment et al., 1993b ; Whittaker et al., 1995). Our previous results (Reedy et al., 1987, 1988; Schmitz et al., 1996; Winkler et al., 1996) and the present work show (a) that regulatory domain position can vary significantly while motor domain position remains constant and (b) that the motor domain can bind actin with varying orientations. This work supports the possibility that both rotation of the motor domain on actin and movements of the regulatory domain could contribute to the power stroke.