ASU Scholarship Showcase

We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

Human protein diversity arises as a result of alternative splicing, single nucleotide polymorphisms (SNPs) and posttranslational modifications. Because of these processes, each protein can exists as multiple variants in vivo. Tailored strategies are needed to study these protein variants and understand their role in health and disease. In this work we utilized quantitative mass spectrometric immunoassays to determine the protein variants concentration of beta-2-microglobulin, cystatin C, retinol binding protein, and transthyretin, in a population of 500 healthy individuals. Additionally, we determined the longitudinal concentration changes for the protein variants from four individuals over a 6 month period. Along with the native forms of the four proteins, 13 posttranslationally modified variants and 7 SNP-derived variants were detected and their concentration determined. Correlations of the variants concentration with geographical origin, gender, and age of the individuals were also examined. This work represents an important step toward building a catalog of protein variants concentrations and examining their longitudinal changes.

Introduction: Urbanization can considerably impact animal ecology, evolution, and behavior. Among the new conditions that animals experience in cities is anthropogenic noise, which can limit the sound space available for animals to communicate using acoustic signals. Some urban bird species increase their song frequencies so that they can be heard above low-frequency background city noise. However, the ability to make such song modifications may be constrained by several morphological factors, including bill gape, size, and shape, thereby limiting the degree to which certain species can vocally adapt to urban settings. We examined the relationship between song characteristics and bill morphology in a species (the house finch, Haemorhous mexicanus) where both vocal performance and bill size are known to differ between city and rural animals.

Results: We found that bills were longer and narrower in more disturbed, urban areas. We observed an increase in minimum song frequency of urban birds, and we also found that the upper frequency limit of songs decreased in direct relation to bill morphology.

Conclusions: These findings are consistent with the hypothesis that birds with longer beaks and therefore longer vocal tracts sing songs with lower maximum frequencies because longer tubes have lower-frequency resonances. Thus, for the first time, we reveal dual constraints (one biotic, one abiotic) on the song frequency range of urban animals. Urban foraging pressures may additionally interact with the acoustic environment to shape bill traits and vocal performance.

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth’s oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S₀ to S₄, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S₁ state and after double laser excitation (putative S₃ state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn₄CaO₅ core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the ‘dangler’ Mn) and the Mn₃CaO_x cubane in the S₂ to S₃ transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.

It has been suggested that the extended intensity profiles surrounding Bragg reflections that arise when a series of finite crystals of varying size and shape are illuminated by the intense, coherent illumination of an x-ray free-electron laser may enable the crystal’s unit-cell electron density to be obtained ab initio via well-established iterative phasing algorithms. Such a technique could have a significant impact on the field of biological structure determination since it avoids the need for a priori information from similar known structures, multiple measurements near resonant atomic absorption energies, isomorphic derivative crystals, or atomic-resolution data. Here, we demonstrate this phasing technique on diffraction patterns recorded from artificial two-dimensional microcrystals using the seeded soft x-ray free-electron laser FERMI. We show that the technique is effective when the illuminating wavefront has nonuniform phase and amplitude, and when the diffraction intensities cannot be measured uniformly throughout reciprocal space because of a limited signal-to-noise ratio.

Vertebrates cannot synthesize carotenoid pigments de novo, so to produce carotenoid-based coloration they must ingest carotenoids. Most songbirds that deposit red carotenoids in feathers, bills, eyes, or skin ingest only yellow or orange dietary pigments, which they oxidize to red pigments via a ketolation reaction. It has been hypothesized that carotenoid ketolation occurs in the liver of vertebrates, but this hypothesis remains to be confirmed. To better understand the role of hepatocytes in the production of ketolated carotenoids in songbirds, we measured the carotenoid content of subcellular components of hepatocytes from wild male house finches (Haemorhous mexicanus) that were molting red, ketocarotenoid-containing feathers (e.g., 3-hydroxy-echinenone). We homogenized freshly collected livers of house finches and isolated subcellular fractions, including mitochondria. We found the highest concentration of ketocarotenoids in the mitochondrial fraction. These observations are consistent with the hypothesis that carotenoid pigments are oxidized on or within hepatic mitochondria, esterified, and then transported to the Golgi apparatus for secretory processing.

Proteins can exist as multiple proteoforms in vivo, as a result of alternative splicing and single-nucleotide polymorphisms (SNPs), as well as posttranslational processing. To address their clinical significance in a context of diagnostic information, proteoforms require a more in-depth analysis. Mass spectrometric immunoassays (MSIA) have been devised for studying structural diversity in human proteins. MSIA enables protein profiling in a simple and high-throughput manner, by combining the selectivity of targeted immunoassays, with the specificity of mass spectrometric detection. MSIA has been used for qualitative and quantitative analysis of single and multiple proteoforms, distinguishing between normal fluctuations and changes related to clinical conditions. This mini review offers an overview of the development and application of mass spectrometric immunoassays for clinical and population proteomics studies. Provided are examples of some recent developments, and also discussed are the trends and challenges in mass spectrometry-based immunoassays for the next-phase of clinical applications.

Background: The coevolution of male traits and female mate preferences has led to the elaboration and diversification of sexually selected traits; however the mechanisms that mediate trait-preference coevolution are largely unknown. Carotenoid acquisition and accumulation are key determinants of the expression of male sexually selected carotenoid-based coloration and a primary mechanism maintaining the honest information content of these signals. Carotenoids also influence female health and reproduction in ways that may alter the costs and benefits of mate choice behaviors and thus provide a potential biochemical link between the expression of male traits and female preferences. To test this hypothesis, we manipulated the dietary carotenoid levels of captive female house finches (Carpodacus mexicanus) and assessed their mate choice behavior in response to color-manipulated male finches.

Results: Females preferred to associate with red males, but carotenoid supplementation did not influence the direction or strength of this preference. Females receiving a low-carotenoid diet were less responsive to males in general, and discrimination among the colorful males was positively linked to female plasma carotenoid levels at the beginning of the study when the diet of all birds was carotenoid-limited.

Conclusions: Although female preference for red males was not influenced by carotenoid intake, changes in mating responsiveness and discrimination linked to female carotenoid status may alter how this preference is translated into choice. The reddest males, with the most carotenoid rich plumage, tend to pair early in the breeding season. If carotenoid-related variations in female choice behavior shift the timing of pairing, then they have the potential to promote assortative mating by carotenoid status and drive the evolution of carotenoid-based male plumage coloration.

Introduction: Apolipoprotein C-III (apoC-III) regulates triglyceride (TG) metabolism. In plasma, apoC-III exists in non-sialylated (apoC-III_0a without glycosylation and apoC-III[subscript 0b] with glycosylation), monosialylated (apoC-III₁) or disialylated (apoC-III₂) proteoforms. Our aim was to clarify the relationship between apoC-III sialylation proteoforms with fasting plasma TG concentrations.

Methods: In 204 non-diabetic adolescent participants, the relative abundance of apoC-III plasma proteoforms was measured using mass spectrometric immunoassay.

Results: Compared with the healthy weight subgroup (n = 16), the ratios of apoC-III_0a, apoC-III_0b, and apoC-III₁ to apoC-III₂ were significantly greater in overweight (n = 33) and obese participants (n = 155). These ratios were positively correlated with BMI z-scores and negatively correlated with measures of insulin sensitivity (S[subscript i]). The relationship of apoC-III₁ / apoC-III₂ with S_i persisted after adjusting for BMI (p = 0.02). Fasting TG was correlated with the ratio of apoC-III_0a / apoC-III₂ (r = 0.47, p<0.001), apoC-III_0b / apoC-III₂ (r = 0.41, p<0.001), apoC-III₁ / apoC-III₂ (r = 0.43, p<0.001). By examining apoC-III concentrations, the association of apoC-III proteoforms with TG was driven by apoC-III_0a (r = 0.57, p<0.001), apoC-III_0b (r = 0.56. p<0.001) and apoC-III₁ (r = 0.67, p<0.001), but not apoC-III₂ (r = 0.006, p = 0.9) concentrations, indicating that apoC-III relationship with plasma TG differed in apoC-III₂ compared with the other proteoforms.

Conclusion: We conclude that apoC-III_0a, apoC-III_0b, and apoC-III₁, but not apoC-III₂ appear to be under metabolic control and associate with fasting plasma TG. Measurement of apoC-III proteoforms can offer insights into the biology of TG metabolism in obesity.

Background: Diet-derived carotenoid pigments are concentrated in the retinas of birds and serve a variety of functions, including photoprotection. In domesticated bird species (e.g., chickens and quail), retinal carotenoid pigmentation has been shown to respond to large manipulations in light exposure and provide protection against photodamage. However, it is not known if or how wild birds respond to ecologically relevant variation in sun exposure.

Methods: We manipulated the duration of natural sunlight exposure and dietary carotenoid levels in wild-caught captive House Finches (Haemorhous mexicanus), then measured carotenoid accumulation and oxidative stress in the retina.

Results: We found no significant effects of sun exposure on retinal levels of carotenoids or lipid peroxidation, in replicate experiments, in winter (Jan–Mar) and spring/summer (May–June). Dietary carotenoid supplementation in the spring/summer experiment led to significantly higher retinal carotenoid levels, but did not affect lipid peroxidation. Carotenoid levels differed significantly between the winter and spring/summer experiments, with higher retinal and lower plasma carotenoid levels in birds from the later experiment.

Conclusion: Our results suggest that variation in the duration of exposure to direct sunlight have limited influence on intraspecific variation in retinal carotenoid accumulation, but that accumulation may track other seasonal–environmental cues and physiological processes.