Photoacoustic Calorimetry Studies of O2-Sensing FixL and (R200, I209) Variants from Sinorhizobium meliloti Reveal Conformational Changes Coupled to Ligand Photodissociation from the Heme-PAS Domain.

FixL is an oxygen-sensing heme-PAS protein that regulates nitrogen fixation in the root nodules of plants. In this paper, we present the first photothermal studies of the full-length wild-type FixL protein from Sinorhizobium meliloti and the first thermodynamic profile of a full-length heme-PAS protein. Photoacoustic calorimetry studies reveal a quadriphasic relaxation for SmFixL*WT and the five variant proteins (SmFixL*R200H, SmFixL*R200Q, SmFixL*R200E, SmFixL*R200A, and SmFixL*I209M) with four intermediates from <20 ns to ∼1.5 µs associated with the photodissociation of CO from the heme. The altered thermodynamic profiles of the full-length SmFixL* variant proteins confirm that the conserved heme domain residues R200 and I209 are important for signal transduction. In contrast, the truncated heme domain, SmFixLH128-264, shows only a single, fast monophasic relaxation at <50 ns associated with the fast disruption of a salt bridge and release of CO to the solvent, suggesting that the full-length protein is necessary to observe the conformational changes that propagate the signal from the heme domain to the kinase domain.

Molecular Mechanism of Protein Kinase Recognition and Sorting by the Hsp90 Kinome-Specific Cochaperone Cdc37.

Despite the essential functions of Hsp90, little is known about the mechanism that controls substrate entry into its chaperone cycle. We show that the role of Cdc37 cochaperone reaches beyond that of an adaptor protein and find that it participates in the selective recruitment of only client kinases. Cdc37 recognizes kinase specificity determinants in both clients and nonclients and acts as a general kinase scanning factor. Kinase sorting within the client-to-nonclient continuum relies on the ability of Cdc37 to challenge the conformational stability of clients by locally unfolding them. This metastable conformational state has high affinity for Cdc37 and forms stable complexes through a multidomain cochaperone interface. The interaction with nonclients is not accompanied by conformational changes of the substrate and results in substrate dissociation. Collectively, Cdc37 performs a quality control of protein kinases, where induced conformational instability acts as a "flag" for Hsp90 dependence and stable cochaperone association.

Confinement-guided photophysics in MOFs, COFs, and cages.

In this review, the dependence of the photophysical response of chromophores in the confined environments associated with crystalline scaffolds, such as metal-organic frameworks (MOFs), covalent-organic frameworks (COFs), and molecular cages, has been carefully evaluated. Tunability of the framework aperture, cavity microenvironment, and scaffold topology significantly affects emission profiles, quantum yields, or fluorescence lifetimes of confined chromophores. In addition to the role of the host and its effect on the guest, the methods for integration of a chromophore (e.g., as a framework backbone, capping linker, ligand side group, or guest) are discussed. The overall potential of chromophore-integrated frameworks for a wide-range of applications, including artificial biomimetic systems, white-light emitting diodes, photoresponsive devices, and fluorescent sensors with unparalleled spatial resolution are highlighted throughout the review.

Spatiotemporal variation in the fecal microbiota of mule deer is associated with proximate and future measures of host health.

BACKGROUND: Mule deer rely on fat and protein stored prior to the winter season as an energy source during the winter months when other food sources are sparse. Since associated microorganisms ('microbiota') play a significant role in nutrient metabolism of their hosts, we predicted that variation in the microbiota might be associated with nutrient storage and overwintering in mule deer populations. To test this hypothesis we performed a 16S rRNA marker gene survey of fecal samples from two deer populations in the western United States before and after onset of winter. RESULTS: PERMANOVA analysis revealed the deer microbiota varied interactively with geography and season. Further, using metadata collected at the time of sampling, we were able to identify different fecal bacterial taxa that could potentially act as bioindicators of mule deer health outcomes. First, we identified the abundance of Collinsella (family: Coriobacteriaceae) reads as a possible predictor of poor overwintering outcomes for deer herds in multiple locations. Second, we showed that reads assigned to the Bacteroides and Mollicutes Order RF39 were both positively correlated with deer protein levels, leading to the idea that these sequences might be useful in predicting mule deer protein storage. CONCLUSIONS: These analyses confirm that variation in the microbiota is associated with season-dependent health outcomes in mule deer, which may have useful implications for herd management strategies.

Modulation of Osmium(II) Tris(2,2'-bipyridine) Photophysics through Encapsulation within Zinc(II) Trimesic Acid Metal Organic Frameworks.

The Os(II) tris(2,2'-bipyridine) (OsBpy) complex exhibits optical properties that are particularly attractive for light harvesting systems due to the broad absorption spectrum extending throughout the solar spectrum. However, the relatively short lifetime of the triplet metal to ligand charge transfer state (3MLCT) relative to the related Ru(II)tris(2,2'-bipyridine) (RuBpy) has limited applications. Here, the encapsulation of OsBpy within two distinct Zn(II)-trimesic acid MOFs, HKUST-1(Zn) and USF-2 is demonstrated in an effort to extend the 3MLCT lifetime. Encapsulation results in a hypsochromatic shift of the steady-state emission band in both frameworks resulting from a destabilization of the 3MLCT. The encapsulated OsBpy also exhibits extended emission lifetimes in both HKUST-1(Zn) (104 ns in MOF vs 50 ns in methanol) and USF-2 (81 ns in MOF vs 50 ns in methanol) arising from changes in the nonradiative decay constants in both systems. The data are also consistent with vibronic perturbations involved in mixing between higher energy 3MLCT* and ligand field states.

Seasonal movements and habitat use of African buffalo in Ruaha National Park, Tanzania.

BACKGROUND: Assessing wildlife movements and habitat use is important for species conservation and management and can be informative for understanding population dynamics. The African buffalo (Syncerus caffer) population of Ruaha National Park, Tanzania has been declining, and little was known about the movement, habitat selection, and space use of the population, which is important for understanding possible reasons behind the decline. A total of 12 African buffalo cows from four different herds were collared with satellite transmitters. Movements were assessed over 2 years from 11 animals. RESULTS: The space use of the individual collared buffaloes as an approximation of the 95% home range size estimated using Brownian bridge models, ranged from 73 to 601 km2. The estimated home ranges were larger in the wet season than in the dry season. With the exception of one buffalo all collared animals completed a wet season migration of varying distances. A consistent pattern of seasonal movement was observed with one herd, whereas the other herds did not behave the same way in the two wet seasons that they were tracked. Herd splitting and herd switching occurred on multiple occasions. Buffaloes strongly associated with habitats near the Great Ruaha River in the dry season and had little association to permanent water sources in the wet season. Daily movements averaged 4.6 km (standard deviation, SD = 2.6 km), with the longest distances traveled during November (mean 6.9 km, SD = 3.6 km) at the end of the dry season and beginning of the wet season. The shortest daily distances traveled occurred in the wet season in April-June (mean 3.6 km, SD = 1.6-1.8 km). CONCLUSION: The Great Ruaha River has experienced significant drying in the last decades due to water diversions upstream, which likely has reduced the suitable range for buffaloes. The loss of dry season habitat due to water scarcity has likely contributed to the population decline of the Ruaha buffaloes.

Spatial processes decouple management from objectives in a heterogeneous landscape: predator control as a case study.

Predator control is often implemented with the intent of disrupting top-down regulation in sensitive prey populations. However, ambiguity surrounding the efficacy of predator management, as well as the strength of top-down effects of predators in general, is often exacerbated by the spatially implicit analytical approaches used in assessing data with explicit spatial structure. Here, we highlight the importance of considering spatial context in the case of a predator control study in south-central Utah. We assessed the spatial match between aerial removal risk in coyotes (Canis latrans) and mule deer (Odocoileus hemionus) resource selection during parturition using a spatially explicit, multi-level Bayesian model. With our model, we were able to evaluate spatial congruence between management action (i.e., coyote removal) and objective (i.e., parturient deer site selection) at two distinct scales: the level of the management unit and the individual coyote removal. In the case of the former, our results indicated substantial spatial heterogeneity in expected congruence between removal risk and parturient deer site selection across large areas, and is a reflection of logistical constraints acting on the management strategy and differences in space use between the two species. At the level of the individual removal, we demonstrated that the potential management benefits of a removed coyote were highly variable across all individuals removed and in many cases, spatially distinct from parturient deer resource selection. Our methods and results provide a means of evaluating where we might anticipate an impact of predator control, while emphasizing the need to weight individual removals based on spatial proximity to management objectives in any assessment of large-scale predator control. Although we highlight the importance of spatial context in assessments of predator control strategy, we believe our methods are readily generalizable in any management or large-scale experimental framework where spatial context is likely an important driver of outcomes.

Temporal patterns of ungulate herbivory and phenology of aspen regeneration and defense.

Ungulate herbivory can create strong top-down effects on forest recruitment, especially after fire. Defense strategies of tree species against ungulate herbivory include escape through vertical growth and resistance through the production of defense compounds. Using a four-way fence design and camera traps we characterized the differential impacts of ungulate herbivores (deer, elk, cattle) on aspen forest recruitment and plant defense responses and how they vary depending on the timing of herbivory. Aspen height growth was greatest between June and August and ungulate use of aspen was highest in July and August. Three years after fire, height of aspen differed among fence treatments with full ungulate exclusion > deer-only plots > native ungulate plots > fenceless plots: 108 ± 4 cm, 94 ± 4 cm, 89 ± 4 cm, and 65 ± 4 cm, respectively. Fenceless plots had the highest rates of removal of apical meristems by the end of 2014 and 2015 (61% and 53%, respectively). Native ungulate plots, and deer-only plots both had similar removal of apical meristems in 2014 (37% and 39%, respectively). The highest phenolic glycoside concentrations were associated with an 80% reduction in meristem removal and four-fold greater aspen height by the end of summer. Low nitrogen was associated with an 86% reduction in apical meristem removal and threefold greater aspen height. In conclusion, our study suggests that high ungulate abundance can have detrimental impacts on forest recruitment and that high aspen defense chemistry and lower leaf N deters ungulate herbivory, especially in the late summer.

Time resolved calorimetry of photo-induced folding in horse heart cytochrome c at high pH.

Here the molar volume and enthalpy changes associated with the early events in the folding of ferrocytochrome c (Cc) at high pH have been examined using time resolved photoacoustic calorimetry (PAC). The data reveal an overall volume change of 1.3 ± 0.3 mL mol-1 and an enthalpy change of 13 ± 7 kcal mol -1 occurring subsequent to photodissociation of the unfolded CO bound Cc species in <∼20 ns. Two additional kinetic phases are observed that are associated with non-native His binding (ΔH and ΔV of 2 ± 4 kcal mol-1 and -0.5 mL mol-1, τ âˆ¼ 2.5 µs ) and Met binding (ΔH and ΔV -0.4 ± 2 kcal mol-1 and -0.1 ± 0.1 mL mol-1, τ∼ 660 ns). Considering only protein conformational changes (excluding volume and enthalpies associated with heme ligation events) the initial conformational event exhibits a ΔH and ΔV of 6 ± 3 kcal mol-1 and -3±0.1 mL mol-1, respectively, that are attributed to a small contraction of the unfolded protein. The corresponding enthalpy associated with both native and non-native ligand binding are found to be -5±4 kcal mol-1 (Fe-Met) and +20 ± 4 kcal mol-1 (Fe-His) with the change in volume for both phases being essential negligible. This would indicate that non-native ligand binding likely occurs from an already collapsed conformation.

Synthesis, characterization, and cellular localization of a fluorescent probe of the antimalarial 8-aminoquinoline primaquine.

Primaquine (PQ) is the only commercially available drug that clears dormant liver stages of malaria and blocks transmission to mosquito vectors. Although an old drug, much remains to be known about the mechanism(s) of action. Herein we develop a fluorescent tagged PQ to discover cellular localization in the human malaria parasite, Plasmodium falciparum. Successful synthesis and characterization of a primaquine-coumarin fluorescent probe (PQCP) demonstrated potency equivalent to the parent drug and the probe was not cytotoxic to HepG2 carcinoma cells. Cellular localization was found primarily in the cytosol of the asexual erythrocytic and gametocyte stages of parasite development.

Photoacoustic calorimetry studies of CO photo-dissociation from chloramine-T modified horse heart cytochrome-c.

Treatment of horse heart Cytochrome-c (Cc) with N-chloro-4-toluosulfonamide (Chloramine-t, CT) results in the oxidation of methionine (Met) residues to the corresponding sulfoxide including the distal heme ligand, Met80. The resulting Fe-sulfoxide coordination is sufficiently labile in the ferrous form to be displaced by gaseous ligands, including CO. Photolysis of the CO-CT-Cc complex provides an opportunity to examine ligand binding dynamics that are associated with a relatively rigid distal heme pocket. In this work, photoacoustic calorimetry (PAC) was utilized to obtain the kinetics as well as enthalpy and molar volume changes subsequent to CO photo-dissociation from CO-CT-Cc. Previous photolysis studies of CO-CT-Cc have led to a proposed model for ligand recombination in which the Met80-sulfoxide and CO recombine with the heme on relatively slow timescales (50 µs and ∼500 µs, respectively). The PAC data presented here reveals two additional kinetic phases with lifetimes of <20 ns and 534 ± 75 ns. The fast phase (<20 ns) is associated with an ΔH of 44 ± 5 kcal mol-1 and ΔV of -0.5 ± 0.5 mL mol-1, whereas the slower phase (534 ns) is associated with a small ΔH of 2 ± 3 kcal mol-1 and ΔV of 1 ± 0.5 mL mol-1.

Sulfono-γ-AApeptides as a new class of nonnatural helical foldamer.

Foldamers offer an attractive opportunity for the design of novel molecules that mimic the structures and functions of proteins and enzymes including biocatalysis and biomolecular recognition. Herein we report a new class of nonnatural helical sulfono-γ-AApeptide foldamers of varying lengths. The crystal structure of the sulfono-γ-AApeptide monomer S6 illustrates the intrinsic folding propensity of sulfono-γ-AApeptides, which likely originates from the bulkiness of tertiary sulfonamide moiety. The two-dimensional solution NMR spectroscopy data for the longest sequence S1 demonstrates a 10/16 right-handed helical structure. Optical analysis using circular dichroism further supports well- defined helical conformation of sulfono-γ-AApeptides in solution containing as few as five building blocks. Future development of sulfono-γ-AApeptides may lead to new foldamers with discrete functions, enabling expanded application in chemical biology and biomedical sciences.

Hybrid-state emission in a polythienylenevinylene derivative with an electron deficient moiety.

The photoluminescence (PL) of a novel imide-substituted poly(3-thienylenevinylene) derivative (imidePTV) was studied in film and solution. PL quantum efficiency was measured to be more than two orders of magnitude larger than its nonluminescent counterpart, namely, alkyl-substituted PTV and was interpreted as evidence for a near degeneracy of optically allowed 1(1)Bu and optically forbidden 2(1)Ag excitonic states. As a result, coexistence of 2(1)Ag and 1(1)Bu emissions was observed, and the predominance was found to be sensitive to temperature and morphological environment. PL of solutions in solvents of higher polarity and polarizability and from low-temperature films was dominated by the transition from the dipole allowed 1(1)Bu state. On the other hand, the PL spectra of films at high temperature and solutions in solvents of low polarity and polarizability were primarily from the 2(1)Ag state that obtains a finite transmission moment from an asymmetric perturbation mixing with the 1(1)Bu.

Ruthenium(II) tris(2,2'-bipyridine)-templated zinc(II) 1,3,5-tris(4-carboxyphenyl)benzene metal organic frameworks: structural characterization and photophysical properties.

The ability to confine photoactive catalysts within metal organic framework (MOF) materials affords the opportunity to expand the functional diversity of these materials into solar-based applications. Here, two new Ru(II) tris(2,2'-bipyridine) (RuBpy)-based photoactive materials derived from reactions between Zn(II) ions and 1,3,5-tris(4-carboxyphenyl)benzene and templated by the presence of RuBpy (RWLC-1 and RWLC-2) are described with regard to structure and RuBpy photophysics. RuBpy cations have been successfully encapsulated within the cavities (RWLC-1) and channels (RWLC-2) of the new negatively charged frameworks, both of which are synthesized simultaneously in a single reaction vial. Single-crystal X-ray diffraction studies allowed for determination of the RuBpy position within crystal voids. RuBpy encapsulated in each of the two new MOFs exhibits biphasic triplet metal to ligand charge transfer ((3)MLCT) emission decay lifetimes (τRWLC-1-fast = 237 ns, τRWLC-1-slow = 1.60 µs, τRWLC-2-fast = 171 ns, and τRWLC-2-slow = 797 ns at 25 °C) consistent with two populations of RuBpy complexes, one being encapsulated in highly space-restricted cavities giving rise to a longer (3)MLCT lifetime, while the second is encapsulation within a larger nonperiodic pore or defect with a coencapsulated quencher giving rise to short emission lifetimes. Taken together, these results represent examples of the templating ability of RuBpy to produce novel materials with distinct photophysical environments of the encapsulated guests.

Evaluating movement-based methods for estimating the frequency and timing of parturition in mule deer.

BACKGROUND: Information on reproduction of harvested species such as mule deer (Odocoileus hemionus) is vital for conservation and management. Furthermore, parturition in ungulates may be detected using patterns of movement logged by GPS transmitters. Several movement-based methods have been developed to detect parturition in ungulates including the Peterson method, behavioral change point analysis (BCPA), rolling minimum convex polygons (rMCP), individual-based method (IBM), and population-based method (PBM). Our objectives were to (1) test the accuracy and the precision of each previously described method and (2) develop an improved method optimized for mule deer that incorporated aspects of the other methods. METHODS: We determined parturition timing and status for female mule deer fitted with GPS collars and implanted with vaginal implant transmitters (VITs). We used movement patterns before and after parturition to set movement thresholds for each movement-based method. Following model training, we used location and birth date data from an external dataset to test the effectiveness of each movement-based method. Additionally, we developed a novel method for detecting parturition called the analysis of parturition indicators (API). We used two regression analyses to determine the accuracy and precision of estimates generated by each method. RESULTS: The six methods we employed varied in accuracy, with the API, rMCP, and BCPA being most accurate. Precision also varied among methods, with the API, rMCP, and PBM generating the most precise estimates of parturition dates. The API and the rMCP performed similarly and better overall than any of the other existing methods. CONCLUSIONS: We found that movement-based methods could be used to accurately and precisely detect parturition in mule deer. Further, we determined that the API and rMCP methods had the greatest overall success at detecting parturition in mule deer. The relative success of the API and rMCP may be attributed to the fact that both methods use home range size to detect parturition and are validated using known parturition dates of collared deer. We present the API as an efficient method of estimating birth status and timing of parturition of mule deer fitted with GPS transmitters, as well as affirm the effectiveness of a previously developed method, rMCP.

A fine-scale examination of parturition timing in temperate ungulates.

Parturition timing has long been a topic of interest in ungulate research. However, few studies have examined parturition timing at fine scale (e.g., <1 day). Predator activity and environmental conditions can vary considerably with diel timing, which may result in selective pressure for parturition to occur during diel times that maximize the likelihood of neonate survival. We monitored parturition events and early-life survival of elk (Cervus canadensis) and mule deer (Odocoileus hemionus) in Utah, USA to better understand diel timing of parturition in temperate ungulates. Diel timing of parturition was moderately synchronous among conspecifics and influenced by environmental variables on the date of parturition. For elk, parturition events were most common during the morning crepuscular period and generally occurred later (i.e., closer to 12:00) when a relatively large proportion of the moon was illuminated. For mule deer, parturition events were most common during the diurnal period and generally occurred later (i.e., closer to 15:00) on cold, wet dates. Diel timing of parturition did not influence neonate survival, but larger datasets may be required to verify the apparent lack of influence. Although additional work could evaluate alternative variables that might affect parturition timing, our data provide an improved and finer scale understanding of reproductive ecology and phenology in ungulates.

Spectroscopic investigation of the noncovalent association of the nerve agent simulant diisopropyl methylphosphonate (DIMP) with zinc(II) porphyrins.

Organophosphonates pose a significant threat as chemical warfare agents, as well as environmental toxins in the form of pesticides. Thus, methodologies to sense and decontaminate these agents are of significant interest. Porphyrins and metalloporphyrins offer an excellent platform to develop chemical threat sensors and photochemical degradation systems. These highly conjugated planar molecules exhibit relatively long-lived singlet and triplet states with high quantum yields and also form self-associated complexes with a wide variety of molecules. A significant aspect of porphyrins is the ability to functionalize the peripheral ring system either directly to the pyrrole rings or to the bridging methine carbons. In this report, steady-state absorption and fluorescence are utilized to probe binding affinities of a series of symmetric and asymmetric zinc(II) metalloporphyrins for the nerve agent simulant diisopropyl methylphosphonate (DIMP) in hexane. The red shifts in the absorption and emission spectra observed for all of the metalloporphyrins probed are discussed in the frame of Gouterman's four orbital model and a common binding motif involving coordination between the metalloporphyrin and DIMP via interaction between the zinc metal center of the porphyrin and phosphoryl oxygen of DIMP (Zn-O═P) is proposed.

Affect is greater than, not equal to, condition: condition and person effects in affective priming paradigms.

Affective primes may impact ensuing behavior through condition and person effects. However, previous research has not experimentally disentangled these two sources of influence in affective priming paradigms. In the current research, we simultaneously examine the influence of condition factors, in terms of prime valence, and person factors, in terms of affect reactivity and personality. In both studies, undergraduate participants (total N = 174) were primed with either positive or negative affective stimuli (words, Study 1; pictures, Study 2) prior to judging the likability of a neutral target (Arabic characters, Study 1; inkblots, Study 2). Although we did observe between-condition differences for positive and negative primes, person-level effects were more consistent predictors of target ratings. Affect reactivity (affect Time 2, controlling Time 1) to the primes predicted evaluative judgments, even in the absence of condition effects. In addition, the personality traits of Neuroticism (Study 1) and behavioral inhibition system sensitivity (Study 2) predicted evaluative judgments of neutral targets following negative affective primes. With effects for condition, affect reactivity, and personality, our results suggest that affective primes influence ensuing behaviors through both informational and affective means. Research using affective priming methodologies should take into account both condition and person-level effects.

Estimating age of mule deer in the field: Can we move beyond broad age categories?

Age of individuals is an intrinsic demographic parameter used in the modeling and management of wildlife. Although analysis of cementum annuli from teeth is currently the most accurate method used to age ungulates, the age of live ungulates in the field can be estimated by examining tooth wear and tooth replacement patterns. However, there may be limitations to aging based on tooth wear as the rate of tooth wear likely varies among individuals due to factors such as age, diet, environment, and sex. Our objective was to determine the reliability of estimating age for mule deer based on tooth wear and tooth replacement patterns. We compared ages estimated by tooth wear (collected at time of capture for a statewide monitoring effort) to ages determined from cementum analysis (from teeth collected after mortalities of radio-tracked animals from the monitoring effort). Accuracy was high; ages estimated from tooth wear were within one year of cementum ages >75% of the time when aged by experienced observers. Bias in accuracy for estimates of age was low but slightly biased toward underestimation (i.e., 0.6 years on average)-especially as cementum age increased. Our results indicate that aging mule deer using patterns in tooth wear can be reliable if observers estimating age have experience using this method.

Time resolved thermodynamics associated with ligand photorelease in heme peroxidases and globins: Open access channels versus gated ligand release.

Heme proteins represent a diverse class of biomolecules responsible for an extremely diverse array of physiological functions including electron transport, monooxygenation, ligand transport and storage, cellular signaling, respiration, etc. An intriguing aspect of these proteins is that such functional diversity is accomplished using a single type of heme macrocycle based upon iron protoporphyrin IX. The functional diversity originates from a delicate balance of inter-molecular interactions within the protein matrix together with well choreographed dynamics that modulate the heme electronic structure as well as ligand entry/exit pathways from the bulk solvent to the active site. Of particular interest are the dynamics and energetics associated with the entry/exit of ligands as this process plays a significant role in regulating the rates of heme protein activity. Time-resolved photoacoustic calorimetry (PAC) has emerged as a powerful tool through which to probe the underlying energetics associated with small molecule dissociation and release to the bulk solvent in heme proteins on time scales from tens of nanoseconds to several microseconds. In this review, the results of PAC studies on various classes of heme proteins are summarized highlighting how different protein structures affect the thermodynamics of ligand migration. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.