Accurate Mimic Research Articles

And the Oscar goes to… – Species‐specific ant behaviour related to predator defence in ant‐mimicking spiders

AbstractMimicry, the superficial resemblance between organisms of two or more species, is considered a textbook example for natural selection, for which it is assumed that predators are important selective agents. Ant mimicking spiders have received considerable attention in studies on mimicry because they include examples with remarkable, species‐specific morphological adaptations. Ant‐like behaviours such as erratic locomotory patterns, abdomen bobbing and vertical movements of the first or second pair of legs to imitate antennal movements, are assumed to have evolved before morphological mimicry and may be considered adaptations to general ant resemblance. Species‐specific behavioural ant mimicry (behaviour that is only observed in specific ants and imitated by their mimics) was very rarely documented and ant resembling behaviour that repels predator attacks has not been confirmed yet. In this study we report and discuss such species‐specific behaviour, a spray display (SD), in the castianeirine spiders Myrmecotypus iguazu (a morphologically accurate mimic of the carpenter ant Camponotus sericeiventris), and M. tahyinandu (a morphologically accurate mimic of C. crassus). The SD consisted of the raising of the cephalothorax and moving of the abdomen ventrally under the cephalothorax, pointing the apex forward, and holding the antennae (ant) or first pair of legs (spiders) at an angle of about 45°–120°. The morphological adaptations that are required to perform the SD and the lack of an alternative explanation for the purpose of this display suggest that the SD has evolved to enhance both general behavioural and morphologically accurate ant resemblance. The two observed Myrmecotypus species may be considered the behaviorally most accurate ant‐resembling spiders known to date, as the SD may provide protection against spider‐ and ant‐eating predators at a point in predator interactions where other myrmecomorph spiders may abandon their ant‐resembling behaviour.

NIR-II Fluorescence Imaging for the Detection and Resection of Cancerous Foci and Lymph Nodes in Early-Stage Orthotopic and Advanced-Stage Metastatic Ovarian Cancer Models.

The high mortality rate of ovarian cancer can be primarily attributed to late diagnosis and early lymph node (LN) metastasis. The anatomically deep-located ovaries own intricate anatomical structures and lymphatic drainages that compromise the resolution and sensitivity of near-infrared first-window (NIR-I) fluorescence imaging. Reported NIR-II imaging studies of ovarian cancer focused on late-stage metastasis detection via the intraperitoneal xenograft model. However, given the significant improvement in patient survival associated with early-stage cancer detection, locating tumors that are restricted within the ovary is equally crucial. We obtained the polymer nanoparticles with bright near-infrared-II fluorescence (NIR-II NPs) by nanoprecipitation of DSPE-PEG, one of the ingredients of FDA-approved nanoparticle products, and benzobisthiadiazole, an organic NIR-II dye. The one-step synthesis and safe component lay the groundwork for its clinical translation. Benefiting from the NIR-II emission (∼1060 nm), NIR-II NPs enabled a high signal-to-noise (S/N) ratio (13.4) visualization of early-stage orthotopic ovarian tumors with NIR-II fluorescence imaging for the first time. Imaging with orthotopic xenograft allows a more accurate mimic of human ovarian cancer origin, thereby addressing the dilemma of translating existing nanoprobe preclinical research by providing the nano-bio interactions with early local tumor environments. After PEGylation, the desirable-sized probe (∼80 nm) exhibited high lymphophilicity and relatively extended circulation. NIR-II NPs maintained their accurate detection of orthotopic tumors, tumor-regional LNs, and minuscule (<1 mm) disseminated peritoneal metastases simultaneously (with S/N ratios all above 5) in mice with advanced-stage cancer in real time ∼36 h after systematic delivery. With NIR-II fluorescence guidance, we achieved accurate surgical staging in tumor-bearing mice and complete tumor removal comparable to clinical practice, which provides preclinical data for translating NIR-II fluorescence image-guided surgery.

Increased speed of movement reduced identification of Batesian ant-mimicking spiders by surrogate predators.

In Batesian mimicry, the mimic gains protection from predators by imitating a noxious model. Some myrmecomorphic species use ants as models as ants have strong defensive capabilities. Ants are highly mobile models, and besides colour, shape, and size, mimics also imitate their movement. Yet, former studies focused mainly on static traits. Here, I tested the hypothesis that artificially increased speed of movement reduces the probability of the mimic being identified. First, images of 14 myrmecomorphic spider species and their models were used for humans to rank their mimetic accuracy. Humans were used as surrogate predators to obtain scores for each mimetic pair. In the second experiment, the effect of movement playback speed on the probability of identification was investigated, again using humans. Videos of mimics were played at different speeds, and the identification probability was recorded. While ants were correctly identified at any playback speed, the identification of myrmecomorphic spiders declined with increasing playback speed. In other words, the latency to correct identification increased with playback speed. Overall, mimics with higher accuracy scores were more difficult to identify while moving. The natural speed of movement of accurate mimics was similar to that of inaccurate ones. Movement is thus an important trait for myrmecomorphic species.

Contributions to the knowledge of Neotropical ant-like spiders: Myrmecotypus tahyinandu sp. n. from Bolivian Chiquitano forest, a new country record for M. niger, and indirect evidence for species-specific mimicry (Araneae: Corinnidae: Castianeirinae).

Myrmecotypus tahyinandu sp. n. is described from the Bolivian Chiquitano forest, and M. niger Chickering, 1937 is recorded from Bolivia for the first time. The morphological differentiation among the closely related M. tahyinandu sp. n. and M. iguazu Rubio Arbino, 2009 is likely attributable to the selection for specific ant mimicry. Adults of M. tahyinandu sp. n. are accurate mimics of the ant Camponotus crassus Mayr, 1862, M. iguazu of C. sericeiventris (Guérin-Méneville, 1838), and M. niger of Dolichoderus bispinosus (Olivier, 1792). This study is the first to identify ant models for morphologically closely related Castianeirinae, providing a promising starting point for future research on ant mimicry.

Transformational Mimicry in a Myrmecomorphic Spider.

Species that are Batesian mimics during postembryonic development shift between mimetic models as they grow in size. However, it has not yet been tested whether these successive mimetic phenotypes are similarly protected from predators. Early-instar phenotypes could represent an inaccurate phenotype or an accurate phenotype because of selection from different predators. Here, we tested the hypotheses of transformational Batesian mimicry in the ant-mimicking jumping spider Leptochestes berolinensis. We quantified the mimetic accuracy of different ontogenetic stages to potential ant models by using a multitrait approach. We measured movement, body profile, body size, and coloration. Analysis revealed adults to be more accurate mimics than juveniles. Adults were similar to smaller morphs of Camponotus or Lasius ants, whereas juveniles were more similar to Lasius and Colobopsis ants. We tested whether predators, mantises, and Pisaura spiders were deceived by mimics after having experience with ant models. These predators never captured any ant or a mimic but always captured the nonmyrmecomorphic spider. We conclude that L. berolinensis is a Batesian mimic of ants undergoing transformational mimicry, with all stages being accurate mimics.

Cyclic loading and a modified ASTM G65 abrasion test

Wear is a multi-faceted phenomenon that occurs through a number of mechanisms. Surface mining operations are principally impacted by abrasive wear during the excavation process. Equipment downtime due to ground engaging tool (GET) abrasive wear significantly affects production of a mining operation. In comparison to the ASTM G65 constant load abrasion test, an equally simple cyclic load test has been designed and trialed to better mimic and predict wear conditions in an oil sand mining scenario.In the modified ASTM G65 test, bitumen stripped oil sand quartz media replaced Ottawa Sand. Coupon specimens, representing GET surfaces, were hardfaced with chromium carbide weld overlay. An actuator controlling applied loads facilitated a more accurate mimic of a dig cycle for an ultra-class electric rope shovel.Shovel kinematics were analyzed to determine resistive forces experienced at the shovel bucket teeth. Recorded field data was scaled to appropriate laboratory testing magnitudes. Wear scar inspection suggested that cyclic loading scenarios potentially generate more severe wear damage versus constant loading. Field tooth life was predicted using a specific energy technique. Test results indicated that a cyclic loading approach provided closer correlation to measured shovel tooth life for an oil sands operation than a constant load approach.

Why many Batesian mimics are inaccurate: evidence from hoverfly colour patterns.

Mimicry is considered a classic example of the elaborate adaptations that natural selection can produce, yet often similarity between Batesian (harmless) mimics and their unpalatable models is far from perfect. Variation in mimetic accuracy is a puzzle, as natural selection should favour mimics that are hardest to distinguish from their models. Numerous hypotheses exist to explain the persistence of inaccurate mimics, but most have rarely or never been tested against empirical observations from wild populations. One reason for this is the difficulty in measuring pattern similarity, a key aspect of mimicry. Here, we use a recently developed method, based on the distance transform of binary images, to quantify pattern similarity both within and among species for a group of hoverflies and their hymenopteran models. This allowed us to test three key hypotheses regarding inaccurate mimicry. Firstly, we tested the prediction that selection should be more relaxed in less accurate mimics, but found that levels of phenotypic variation are similar across most hoverfly species. Secondly, we found no evidence that mimics have to compromise between accuracy to multiple model species. However, we did find that darker-coloured hoverflies are less accurate mimics, which could lead to a trade-off between mimicry and thermoregulation in temperate regions. Our results shed light on a classic problem concerning the limitations of natural selection.

Pollinators can prefer rewarding models to mimics: consequences for the assumptions of Batesian floral mimicry

Many members of the Orchidaceae offer no rewards to pollinators and attract them through deception. One common approach is to mimic the specific floral signals of co-flowering species that are rewarding (Batesian floral mimicry) to exploit their pollinators. We investigated two putative Batesian floral mimics, Disa gladioliflora subsp. capricornis and Disa ferruginea, within a single community to determine whether their pollinators are able to discriminate them from their models. Despite mimics being indistinguishable from models in terms of floral colour as perceived by pollinators, strong preference for model flowers was observed in both systems. We nevertheless recorded frequent pollen transfer in D. gladioliflora subsp. capricornis, suggesting pollination still occurs in the face of pollinator discrimination. Although the inability of pollinators to discriminate between models and mimics is often cited as a requirement for Batesian floral mimicry, we suggest that this need not be the case. Pollination by discriminating visitors may still lead to fruit set, with the most accurate mimics expected to have the highest fitness. Batesian floral mimicry may thus be best indicated when mimic fitness is positively correlated with its resemblance to model flowers.

Do crab spiders perceive Batesian mimicry in hoverflies?

Many putative Batesian mimics only approximately resemble their supposed models, and such “imperfect” mimics are readily distinguished from defended species by humans and other vertebrates. One explanation for the existence of imperfect mimics is that the most important predators of many mimics have very different sensory and cognitive abilities from those of a typical vertebrate. In such circumstances, selection for more accurate mimicry, as perceived by humans, may be reduced. Little is known, however, about how invertebrate predators perceive and respond to mimicry in insect prey. Here, we investigate the foraging behaviour of the crab spider Synema globosum, an important predator of flower-visiting insects at our field site which frequently encounters both Batesian mimics (hoverflies - Diptera: Syrphidae) and their models (bees and wasps - Hymenoptera). In the field, we found that spiders can distinguish among dipteran and hymenopteran prey taxa, frequently attacking some models and mimics, but avoiding others. Laboratory experiments suggest that some apparently accurate mimic taxa are more likely to be avoided when spiders have prior experience of an aversive wasp model. Avoidance by spiders of black and yellow striped artificial prey suggests visual cues play a role in prey selection, but there was no evidence that olfactory cues are used to identify dangerous or noxious species. Overall, our results provide some support for the hypothesis that invertebrate predator behaviour can generate selection on visual signals in putative Batesian mimics.

Is the Evolution of Inaccurate Mimicry a Result of Selection by a Suite of Predators? A Case Study Using Myrmecomorphic Spiders

Several hypotheses have been put forward to explain the evolution of inaccurate mimicry. Here we investigated the novel hypothesis that inaccurate mimicry (in color and shape) is maintained by opposing selective pressures from a suite of different predators: model-aversive visually oriented predators and model- and mimic-specialized predators indifferent to mimetic cues. We hypothesize that spiders resembling ants in color and shape escape predators that typically avoid ants but fall prey to ant-eating predators. We tested whether inaccurate myrmecomorphic spiders are perceived as their models by two types of predators and whether they can escape from these predators. We found that model-specialized (ant-eating) predators captured mimics significantly less frequently than their ant models, because mimics changed their behavior by fleeing predatory attacks. The fastest escape was found in less accurate mimics, indicating a negative association between visual resemblance and effectiveness of defenses. In trials with spider-eating predators, mimics were not captured more frequently than their models. The quality of defensive mechanisms appears to result from opposing selection forces exerted by the predator complex: mimics are more accurate (in color and shape) in microhabitats dominated by model-aversive predators and less accurate in microhabitats with model- and mimic-specialized predators.

Life-history constraints in inaccurate Batesian myrmecomorphic spiders (Araneae: Corinnidae, Gnaphosidae)

Accurate Batesian mimicry is known to impose constraints on some traits of the mimic, such as foraging or reproductive behaviour. It is not known whether life-history traits of inaccurate Batesian mimics are constrained as well. We studied selected life- history traits of three spider species, Liophrurillus flavitarsis, Phrurolithus festivus (both Corinnidae), and Micaria sociabilis (Gna- phosidae), that are inaccurate mimics of ants. Namely, we were interested in how myrmecomorphy (ant-like resemblance) constrains their circadian activity, trophic niche and reproductive behaviour. The spiders were found to have diurnal activity like their models, whereas their close relatives have nocturnal activity. The three mimics do not catch ants, nor do they use food resources of ants, but catch various tiny invertebrates that occur in the vicinity of their models. Their trophic niche seems to be constrained by occurrence among ants. Absence of courtship and long lasting copulation, in a position that does not provide protective resemblance, do not seem to be constrained by mimicry in the three species. Comparative analysis of fecundity in mimetic and non-mimetic spiders showed that clutch size is also not constrained. Unlike in accurate mimics, life-history traits of inaccurate myrmecomorphs appear not to be constrained.

Anilinic N‐Oxides Support Cytochrome P450‐Mediated N‐Dealkylation through Hydrogen‐Atom Transfer

The mechanism of N-dealkylation mediated by cytochrome P450 (P450) has long been studied and argued as either a single electron transfer (SET) or a hydrogen atom transfer (HAT) from the amine to the oxidant of the P450, the reputed iron-oxene. In our study, tertiary anilinic N-oxides were used as oxygen surrogates to directly generate a P450-mediated oxidant that is capable of N-dealkylating the dimethylaniline derived from oxygen donation. These surrogates were employed to probe the generated reactive oxygen species and the subsequent mechanism of N-dealkylation to distinguish between the HAT and SET mechanisms. In addition to the expected N-demethylation of the product aniline, 2,3,4,5,6-pentafluoro-N,N-dimethylaniline N-oxide (PFDMAO) was found to be capable of N-dealkylating both N,N-dimethylaniline (DMA) and N-cyclopropyl-N-methylaniline (CPMA). Rate comparisons of the N-demethylation of DMA supported by PFDMAO show a 27-fold faster rate than when supported by N,N-dimethylaniline N-oxide (DMAO). Whereas intermolecular kinetic isotope effects were masked, intramolecular measurements showed values reflective of those seen previously in DMAO- and the native NADPH/O(2)-supported systems (2.33 and 2.8 for the N-demethylation of PFDMA and DMA from the PFDMAO system, respectively). PFDMAO-supported N-dealkylation of CPMA led to the ring-intact product N-cyclopropylaniline (CPA), similar to that seen with the native system. The formation of CPA argues against a SET mechanism in favor of a P450-like HAT mechanism. We suggest that the similarity of KIEs, in addition to the formation of the ring-intact CPA, argues for a similar mechanism of Compound I (Cpd I) formation followed by HAT for N-dealkylation by the native and N-oxide-supported systems and demonstrate the ability of the N-oxide-generated oxidant to act as an accurate mimic of the native P450 oxidant.

Biomimicry of Surfactant Protein C

Since the widespread use of exogenous lung surfactant to treat neonatal respiratory distress syndrome, premature infant survival and respiratory morbidity have dramatically improved. Despite the effectiveness of the animal-derived surfactant preparations, there still remain some concerns and difficulties associated with their use. This has prompted investigation into the creation of synthetic surfactant preparations. However, to date, no clinically used synthetic formulation is as effective as the natural material. This is largely because the previous synthetic formulations lacked analogues of the hydrophobic proteins of the lung surfactant system, SP-B and SP-C, which are critical functional constituents. As a result, recent investigation has turned toward the development of a new generation of synthetic, biomimetic surfactants that contain synthetic phospholipids along with a mimic of the hydrophobic protein portion of lung surfactant. In this Account, we detail our efforts in creating accurate mimics of SP-C for use in a synthetic surfactant replacement therapy. Despite SP-C's seemingly simple structure, the predominantly helical protein is extraordinarily challenging to work with given its extreme hydrophobicity and structural instability, which greatly complicates the creation of an effective SP-C analogue. Drawing inspiration from Nature, two promising biomimetic approaches have led to the creation of rationally designed biopolymers that recapitulate many of SP-C's molecular features. The first approach utilizes detailed SP-C structure-activity relationships and amino acid folding propensities to create a peptide-based analogue, SP-C33. In SP-C33, the problematic and metastable polyvaline helix is replaced with a structurally stable polyleucine helix and includes a well-placed positive charge to prevent aggregation. SP-C33 is structurally stable and eliminates the association propensity of the native protein. The second approach follows the same design considerations but makes use of a non-natural, poly-N-substituted glycine or "peptoid" scaffold to circumvent the difficulties associated with SP-C. By incorporating unique biomimetic side chains in a non-natural backbone, the peptoid mimic captures both SP-C's hydrophobic patterning and its helical secondary structure. Despite the differences in structure, both SP-C33 and the SP-C peptoid mimic capture many requisite features of SP-C. In a surfactant environment, these analogues also replicate many of the key surface activities necessary for a functional biomimetic surfactant therapy while overcoming the difficulties associated with the natural protein. With improved stability, greater production potential, and elimination of possible pathogenic contamination, these biomimetic surfactant formulations offer not only the potential to improve the treatment of respiratory distress syndrome but also the opportunity to treat other respiratory-related disorders.

Effects of Hydrophobic Helix Length and Side Chain Chemistry on Biomimicry in Peptoid Analogues of SP-C

The hydrophobic proteins of lung surfactant (LS), SP-B and SP-C, are critical constituents of an effective surfactant replacement therapy for the treatment of respiratory distress syndrome. Because of concerns and difficulties associated with animal-derived surfactants, recent investigations have focused on the creation of synthetic analogues of the LS proteins. However, creating an accurate mimic of SP-C that retains its biophysical surface activity is extraordinarily challenging given the lipopeptide's extreme hydrophobicity and propensity to misfold and aggregate. One successful approach that overcomes these difficulties is the use of poly-N-substituted glycines, or peptoids, to mimic SP-C. To develop a non-natural, bioactive mimic of SP-C and to investigate the effects of side chain chemistry and length of the helical hydrophobic region, we synthesized, purified, and performed in vitro testing of two classes of peptoid SP-C mimics: those having a rigid alpha-chiral aromatic helix and those having a biomimetic alpha-chiral aliphatic helix. The length of the two classes of mimics was also systematically altered. Circular dichroism spectroscopy gave evidence that all of the peptoid-based mimics studied here emulated SP-C's secondary structure, forming stable helical structures in solution. Langmuir-Wilhelmy surface balance, fluorescence microscopy, and pulsating bubble surfactometry experiments provide evidence that the aromatic-based SP-C peptoid mimics, in conjunction with a synthetic lipid mixture, have superior surface activity and biomimetic film morphology in comparison to the aliphatic-based mimics and that there is an increase in surface activity corresponding to increasing helical length.

The natural history of Myrmarachne melanotarsa, a social ant‐mimicking jumping spider

Myrmarachne melanotarsa, an ant‐like jumping spider (Salticidae) from East Africa, is an accurate mimic of Crematogaster sp. and associates unusually closely with its models. M. melanotarsa is remarkable in that it forms dense aggregations and builds large nest complexes (numerous individually‐occupied nests connected to each other by silk). Other salticids (Pseudicius spp., Menemerus spp.) live with M. melanotarsa in the same nest complex. These aggregations, which can exceed 50 conspecific individuals per colony, are considerably larger than those few previously described, and seem to have primarily a protective function. We provide baseline information on the natural history of M. melanotarsa, paying particular attention to predatory behaviour and association with Crematogaster sp., and fit this within current theory on the function of sociality in spiders. Other unusual behaviour of M. melanotarsa includes “mouthing”, in which the spider opens and closes its chelicerae while pressing its mouthparts against nest silk. We investigated the role of prior presence of Crematogaster sp. on nest silk in eliciting this previously unreported behaviour.

Commercially available chemicals that mimic a deposit feeder's (Arenicola marina) digestive solubilization of lipids.

To develop a simple and cost-effective bioavailability test for sediment-bound contaminants, the solubilization strengths of mixtures of four commercially available surfactants and four proteins were compared to that of digestive fluids from a deposit-feeding benthic polychaete Arenicola marina. Initial tests indicated that sodium taurocholate, a vertebrate bile salt, was the most accurate mimic of A. marina gut fluids' solubilization of individual polycyclic aromatic hydrocarbons (PAH). Further testing with nutritional lipids and other hydrophobic contaminants confirmed the similarities of these fluids. Bovine serum albumin (BSA) solubilization of PAH was the most efficient of all the proteins tested. A cocktail of sodium taurocholate and BSA was compared to A. marina's solubilization of 12 PAH from four different contaminated sediments (from Boston, Charleston, Jacksonville, and San Diego harbors). The two solutions released most PAH to similar extents; 40 of 48 PAH-sediment combinations were released at amounts within a factor of 2 in cocktail and gut fluid solutions. Therefore, the cocktail may serve as a surrogate for real gut fluids and allow easier adoption of the in vitro incubation approach to bioavailability testing.

Mutations of Hydrophobic Residues in the N-terminal Domain of Troponin C Affect Calcium Binding and Exchange with the Troponin C-Troponin I96–148 Complex and Muscle Force Production

Interactions between troponin C and troponin I play a critical role in the regulation of skeletal muscle contraction and relaxation. We individually substituted 27 hydrophobic Phe, Ile, Leu, Val, and Met residues in the regulatory domain of the fluorescent troponin C(F29W) with polar Gln to examine the effects of these mutations on: (a) the calcium binding and dynamics of troponin C(F29W) complexed with the regulatory fragment of troponin I (troponin I(96-148)) and (b) the calcium sensitivity of force production. Troponin I(96-148) was an accurate mimic of intact troponin I for measuring the calcium dynamics of the troponin C(F29W)-troponin I complexes. The calcium affinities of the troponin C(F29W)-troponin I(96-148) complexes varied approximately 243-fold, whereas the calcium association and dissociation rates varied approximately 38- and approximately 33-fold, respectively. Interestingly, the effect of the mutations on the calcium sensitivity of force development could be better predicted from the calcium affinities of the troponin C(F29W)-troponin I(96-148) complexes than from that of the isolated troponin C(F29W) mutants. Most of the mutations did not dramatically affect the affinity of calcium-saturated troponin C(F29W) for troponin I(96-148). However, the Phe(26) to Gln and Ile(62) to Gln mutations led to >10-fold lower affinity of calcium-saturated troponin C(F29W) for troponin I(96-148), causing a drastic reduction in force recovery, even though these troponin C(F29W) mutants still bound to the thin filaments. In conclusion, elucidating the determinants of calcium binding and exchange with troponin C in the presence of troponin I provides a deeper understanding of how troponin C controls signal transduction.

Measurement of agonist-dependent and -independent signal initiation of alpha(1b)-adrenoceptor mutants by direct analysis of guanine nucleotide exchange on the G protein galpha(11).

Immunoprecipitation of a fusion protein between the alpha(1b)-adrenoceptor and Galpha(11) following a [(35)S]GTPgammaS [guanosine-5'-O-(3-thio)triphosphate] binding assay resulted in incorporation of low levels of nucleotide. The agonist phenylephrine increased incorporation some 30-fold. Agonist-induced binding represented 1.0 mol of [(35)S]GTPgammaS/mol of fusion protein. This was to the G protein linked to the receptor rather than endogenous Galpha(q)/Galpha(11) as a fusion protein containing the alpha(1b)-adrenoceptor and a form of Galpha(11) (G(208)A) unable to exchange guanine nucleotides effectively, bound [(35)S]GTPgammaS very poorly. Fusion proteins between A(293)E, D(142)A, and 3CAM mutants of the alpha(1b)-adrenoceptor and Galpha(11) bound substantially greater levels of [(35)S]GTPgammaS in the absence of agonist than the fusion incorporating the wild-type receptor. Constitutive binding of the nucleotide induced by these mutants was only 20% of the level achieved by phenylephrine. These mutant receptors thus do not provide an accurate mimic of the agonist-occupied state. Phentolamine reduced the binding of [(35)S]GTPgammaS and acted as a partial inverse agonist for each of the constitutively active mutants. [(35)S]GTPgammaS binding to Galpha(11) was elevated by phenylephrine in both wild-type and constitutively active mutant forms of the fusion proteins, but agonist potency and binding affinity were 50 times higher for the fusions containing the mutated receptors. These studies provide the first direct demonstration of the capacity of constitutively active mutants of a receptor to stimulate guanine nucleotide exchange on the alpha subunit of a G(q) family G protein and defines a strategy potentially suitable for any receptor that couples to these G proteins.

Vibrational Structure of Dihydrofolate Bound to R67 Dihydrofolate Reductase

R67 is a Type II dihydrofolate reductase (DHFR) that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate by facilitating the addition of a proton to N5 of DHF and the transfer of a hydride ion from NADPH to C6. Because this enzyme is a plasmid-encoded DHFR from trimethoprim-resistant bacteria, extensive studies on R67 with various methods have been performed to elucidate its reaction mechanism. Here, Raman difference measurements, conducted on the ternary complex of R67.NADP(+).DHF believed to be an accurate mimic of the productive DHFR.NADPH.DHF complex, show that the pK(a) of N5 in the complex is less than 4. This is in clear contrast to the behavior observed in Escherichia coli DHFR, a substantially more efficient enzyme, where the pK(a) of bound DHF at N5 is increased to 6.5 compared with its solution value of 2.6. A comparison of the ternary complexes in R67 and E. coli DHFRs suggests that enzymic raising of the pK(a) at N5 can significantly increase the catalytic efficiency of the hydride transfer step. However, R67 shows that even without such a strategy an effective DHFR can still be designed.

Effects of substitution at serine 40 of tyrosine hydroxylase on catecholamine binding.

Phosphorylation of Ser40 in the regulatory domain of tyrosine hydroxylase activates the enzyme by increasing the rate of dissociation of inhibitory catecholamines [Ramsey, A. J., and Fitzpatrick, P. F. (1998) Biochemistry 37, 8980-8986]. To probe the structural basis for this effect and to ascertain the ability of other amino acids to functionally replace serine and serine phosphate, the effects of replacement of Ser40 with other amino acids were determined. Only minor changes in the Vmax value and the Km values for tyrosine and tetrahydropterin were seen upon replacement of Ser40 with alanine, valine, threonine, aspartate, or glutamate, in line with the minor effects of phosphorylation on steady-state kinetic parameters. More significant effects were seen on the binding of dopamine and dihydroxyphenylalanine. The affinity of the S40T enzyme for either catecholamine was very similar to that of the wild-type enzyme, while the S40E enzyme was similar to the phosphorylated enzyme. The S40D enzyme had an affinity for DOPA comparable to the phosphorylated enzyme but a higher affinity for dopamine than the latter. With both catecholamines, the S40V and S40A enzymes showed intermediate levels of activation. The results suggest that the serine hydroxyl contributes to the stabilization of the catecholamine-inhibited enzyme. In addition, the S40E enzyme will be useful in further studies of the effects of multiple phosphorylation on tyrosine hydroxylase, while the alanine enzyme does not provide an accurate mimic of the unphosphorylated enzyme.