A high-throughput method for quantifying Drosophila fecundity.

Measurements of Drosophila fecundity are used in a wide variety of studies, such as investigations of stem cell biology, nutrition, behavior, and toxicology. In addition, because fecundity assays are performed on live flies, they are suitable for longitudinal studies such as investigations of aging or prolonged chemical exposure. However, standard Drosophila fecundity assays have been difficult to perform in a high-throughput manner because experimental factors such as the physiological state of the flies and environmental cues must be carefully controlled to achieve consistent results. In addition, exposing flies to a large number of different experimental conditions (such as chemical additives in the diet) and manually counting the number of eggs laid to determine the impact on fecundity is time-consuming. We have overcome these challenges by combining a new multiwell fly culture strategy with a novel 3D-printed fly transfer device to rapidly and accurately transfer flies from one plate to another; the RoboCam, a low-cost, custom built robotic camera to capture images of the wells automatically; and an image segmentation pipeline to automatically identify and quantify eggs. We show that this method is compatible with robust and consistent egg laying throughout the assay period; and demonstrate that the automated pipeline for quantifying fecundity is very accurate (r2 = 0.98 for the correlation between the automated egg counts and the ground truth) In addition, we show that this method can be used to efficiently detect the effects on fecundity induced by dietary exposure to chemicals. Taken together, this strategy substantially increases the efficiency and reproducibility of high throughput egg laying assays that require exposing flies to multiple different media conditions.

Maize-Produced Ag2 as a Subunit Vaccine for Valley Fever.

Coccidioides is the causative agent of San Joaquin Valley fever, a fungal disease prevalent in the semiarid regions of the Americas. Efforts to develop a fungal vaccine over the last 2 decades were unsuccessful. A candidate antigen, Antigen 2 (Ag2), is notoriously difficult to express in Escherichia coli, and this study sought to accumulate the antigen at high levels in maize. Transformed maize lines accumulated recombinant Ag2 at levels >1 g/kg. Mice immunized with this antigen and challenged with live Coccidioides arthroconidia showed a reduction in the fungal load when Ag2 derived from either E. coli or maize was loaded into glucan chitin particles. A fusion of Ag2 to dendritic cell carrier peptide (DCpep) induced a T-helper type 17 response in the spleen when orally delivered, indicative of a protective immune response. The maize production platform and the glucan chitin particle adjuvant system show promise for development of a Coccidioides vaccine, but further testing is needed to fully assess the optimal method of administration.

Microviscosity in E. coli Cells from Time-Resolved Linear Dichroism Measurements.

A protein's folding or function depends on its mobility through the viscous environment that is defined by the presence of macromolecules throughout the cell. The relevant parameter for this mobility is microviscosity-the viscosity on a time and distance scale that is important for protein folding/function movements. A quasi-null, ultrasensitive time-resolved linear dichroism (TRLD) spectroscopy is proving to be a useful tool for measurements of viscosity on this scale, with previous in vitro studies reporting on the microviscosities of crowded environments mimicked by high concentrations of different macromolecules. This study reports the microviscosity experienced by myoglobin in the E. coli cell's heterogeneous cytoplasm by using TRLD to measure rotational diffusion times. The results show that photolyzed deoxyMb ensembles randomize through environment-dependent rotational diffusion with a lifetime of 34 ± 6 ns. This value corresponds to a microviscosity of 2.82 ± 0.42 cP, which is consistent with previous reports of cytoplasmic viscosity in E. coli. The results of these TRLD studies in E. coli (1) provide a measurement of myoglobin mobility in the cytoplasm, (2) taken together with in vitro TRLD studies yield new insights into the nature of the cytoplasmic environment in cells, and (3) demonstrate the feasibility of TRLD as a probe of intracellular viscosity.

Role of Heme Pocket Water in Allosteric Regulation of Ligand Reactivity in Human Hemoglobin.

Water molecules can enter the heme pockets of unliganded myoglobins and hemoglobins, hydrogen bond with the distal histidine, and introduce steric barriers to ligand binding. The spectrokinetics of photodissociated CO complexes of human hemoglobin and its isolated α and ß chains were analyzed for the effect of heme hydration on ligand rebinding. A strong coupling was observed between heme hydration and quaternary state. This coupling may contribute significantly to the 20-60-fold difference between the R- and T-state bimolecular CO binding rate constants and thus to the modulation of ligand reactivity that is the hallmark of hemoglobin allostery. Heme hydration proceeded over the course of several kinetic phases in the tetramer, including the R to T quaternary transition. An initial 150 ns hydration phase increased the R-state distal pocket water occupancy, nw(R), to a level similar to that of the isolated α (∼60%) and ß (∼10%) chains, resulting in a modest barrier to ligand binding. A subsequent phase, concurrent with the first step of the R → T transition, further increased the level of heme hydration, increasing the barrier. The final phase, concurrent with the final step of the allosteric transition, brought the water occupancy of the T-state tetramer, nw(T), even higher and close to full occupancy in both the α and ß subunits (∼90%). This hydration level could present an even larger barrier to ligand binding and contribute significantly to the lower iron reactivity of the T state toward CO.

root uv-b sensitive mutants are suppressed by specific mutations in ASPARTATE AMINOTRANSFERASE2 and by exogenous vitamin B6.

Vitamin B6 (vitB6) serves as an essential cofactor for more than 140 enzymes. Pyridoxal 5'-phosphate (PLP), active cofactor form of vitB6, can be photolytically destroyed by trace amounts of ultraviolet-B (UV-B). How sun-exposed organisms cope with PLP photosensitivity and modulate vitB6 homeostasis is currently unknown. We previously reported on two Arabidopsis mutants, rus1 and rus2, that are hypersensitive to trace amounts of UV-B light. We performed mutagenesis screens for second-site suppressors of the rus mutant phenotype and identified mutations in the ASPARTATE AMINOTRANSFERASE2 (ASP2) gene. ASP2 encodes for cytosolic aspartate aminotransferase (AAT), a PLP-dependent enzyme that plays a key role in carbon and nitrogen metabolism. Genetic analyses have shown that specific amino acid substitutions in ASP2 override the phenotypes of rus1 and rus2 single mutants as well as rus1 rus2 double mutant. These substitutions, all shown to reside at specific positions in the PLP-binding pocket, resulted in no PLP binding. Additional asp2 mutants that abolish AAT enzymatic activity, but which alter amino acids outside of the PLP-binding pocket, fail to suppress the rus phenotype. Furthermore, exogenously adding vitB6 in growth media can rescue both rus1 and rus2. Our data suggest that AAT plays a role in vitB6 homeostasis in Arabidopsis.

Kinetic spectroscopy of heme hydration and ligand binding in myoglobin and isolated hemoglobin chains: an optical window into heme pocket water dynamics.

The entry of a water molecule into the distal heme pocket of pentacoordinate heme proteins such as myoglobin and the alpha,beta chains of hemoglobin can be detected by time-resolved spectroscopy in the heme visible bands after photolysis of the CO complex. Reviewing the evidence from spectrokinetic studies of Mb variants, we find that this optical method measures the occupancy of non(heme)coordinated water in the distal pocket, n(w), with high fidelity. This evidence further suggests that perturbation of the kinetic barrier presented by distal pocket water is often the dominant mechanism by which active site mutations affect the bimolecular rate constant for CO binding. Water entry into the heme pockets of isolated hemoglobin subunits was detected by optical methods. Internal hydration is higher in the native alpha chains than in the beta chains, in agreement with previous crystallographic results for the subunits within Hb tetramers. The kinetic parameters obtained from modeling of the water entry and ligand rebinding in Mb mutants and native Hb chains are consistent with an inverse dependence of the bimolecular association rate constant on the water occupancy factor. This correlation suggests that water and ligand mutually exclude one another from the distal pockets of both types of hemoglobin chains and myoglobin.

Optical detection of disordered water within a protein cavity.

Internal water molecules are important to protein structure and function, but positional disorder and low occupancies can obscure their detection by X-ray crystallography. Here, we show that water can be detected within the distal cavities of myoglobin mutants by subtle changes in the absorbance spectrum of pentacoordinate heme, even when the presence of solvent is not readily observed in the corresponding crystal structures. A well-defined, noncoordinated water molecule hydrogen bonded to the distal histidine (His64) is seen within the distal heme pocket in the crystal structure of wild type (wt) deoxymyoglobin. Displacement of this water decreases the rate of ligand entry into wt Mb, and we have shown previously that the entry of this water is readily detected optically after laser photolysis of MbCO complexes. However, for L29F and V68L Mb no discrete positions for solvent molecules are seen in the electron density maps of the crystal structures even though His64 is still present and slow rates of ligand binding indicative of internal water are observed. In contrast, time-resolved perturbations of the visible absorption bands of L29F and V68L deoxyMb generated after laser photolysis detect the entry and significant occupancy of water within the distal pockets of these variants. Thus, the spectral perturbation of pentacoordinate heme offers a potentially robust system for measuring nonspecific hydration of the active sites of heme proteins.

The pH dependence of heme pocket hydration and ligand rebinding kinetics in photodissociated carbonmonoxymyoglobin.

We monitored the occupancy of a functionally important non-coordinated water molecule in the distal heme pocket of sperm whale myoglobin over the pH range 4.3-9.4. Water occupancy was assessed by using time-resolved spectroscopy to detect the perturbation of the heme visible band absorption spectrum caused by water entry after CO photodissociation ( Goldbeck, R. A., Bhaskaran, S., Ortega, C., Mendoza, J. L., Olson, J. S., Soman, J., Kliger, D. S., and Esquerra, R. M. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1254-1259 ). We found that the water occupancy observed during the time interval between ligand photolysis and diffusive recombination decreased by nearly 20% as the pH was lowered below 6. This decrease accounted for most of the concomitant increase in the observed CO bimolecular recombination rate constant, as the lower water occupancy presented a smaller kinetic barrier to CO entry into the pocket at lower pH. These results were consistent with a model in which the distal histidine, which stabilizes the water molecule within the distal pocket by accepting a hydrogen bond, tends to swing out of the pocket upon protonation and destabilize the water occupancy at low pH. Extrapolation of this model to lower pH suggests that the additional increase in ligand association rate constant observed previously in stopped-flow studies at pH 3 may also be due in part to reduced distal water occupancy concomitant with further His64 protonation and coupled protein conformational change.

Water and ligand entry in myoglobin: assessing the speed and extent of heme pocket hydration after CO photodissociation.

A previously undescribed spectrokinetic assay for the entry of water into the distal heme pocket of wild-type and mutant myoglobins is presented. Nanosecond photolysis difference spectra were measured in the visible bands of sperm whale myoglobin as a function of distal pocket mutation and temperature. A small blue shift in the 560-nm deoxy absorption peak marked water entry several hundred nanoseconds after CO photodissociation. The observed rate suggests that water entry is rate-limited by the escape of internal dissociated CO. The heme pocket hydration and geminate recombination yields were found to be the primary factors controlling the overall bimolecular association rate constants for CO binding to the mutants studied. The kinetic analysis provides estimates of 84%, 60%, 40%, 0%, and 99% for the steady-state hydrations of wild-type, H64Q, H64A, H64L, and V68F deoxymyoglobin, respectively. The second-order rate constants for CO and H(2)O entry into the empty distal pocket of myoglobin are markedly different, 8 x 10(7) and 2 x 10(5) M(-1).s(-1), respectively, suggesting that hydrophobic partitioning of the apolar gas from the aqueous phase into the relatively apolar protein interior lowers the free energy barrier for CO entry.

The effect of non-enzymatic glycation on the unfolding of human serum albumin.

We monitored the unfolding of human serum albumin (HSA) and glycated human serum albumin (gHSA) subjected to guanidine hydrochloride (GndHCl) by using fluorescence and circular dichroism (CD) spectroscopy. A two-state model with sloping baselines best described the Trp-214 fluorescence unfolding measurements, while a three-state model best described the far-UV CD unfolding data. Glycation of HSA increased the [D](50%) point by approximately 0.20M. This corresponded to an increase in the free energy of unfolding of gHSA relative to HSA of 2.6kJ/mol. The intrinsic fluorescence of Trp-214 in gHSA is 0.72 of that of HSA and the far-UV CD spectrum of gHSA is nearly identical to that of HSA. These results showed that glycation altered the local structure around Trp-214 while not significantly impacting the secondary structure, and this alteration translated into an overall change in the stability of gHSA compared to HSA.

The molecular code for hemoglobin allostery revealed by linking the thermodynamics and kinetics of quaternary structural change. 1. Microstate linear free energy relations.

A novel model linking the thermodynamics and kinetics of hemoglobin's allosteric (R --> T) and ligand binding reactions is applied to photolysis data for human HbCO. To describe hemoglobin's kinetics at the microscopic level of structural transitions and ligand-binding events for individual [ij]-ligation microstates ((ij)R --> (ij)T, (ij)R + CO --> ((i)(+1))(k)R, and (ij)T + CO --> ((i)(+1))(k)T), the model calculates activation energies, (ij)DeltaG(++), from previously measured cooperative free energies of the equilibrium microstates (Huang, Y., and Ackers, G. K. (1996) Biochemistry 35, 704-718) by using linear free energy relations ((ij)DeltaG(++) - (01)DeltaG(++) = alpha[(ij)DeltaG - (01)DeltaG], where the parameter alpha, describing the variation of activation energy with reaction energy perturbation, can depend on the natures of both the reaction and the perturbation). The alpha value measured here for the allosteric dynamics, 0.21 +/- 0.03, corresponds closely to values observed previously, strongly suggesting that the thermodynamic microstate energies directly underlie the allosteric kinetics (as opposed to the alpha((ij)DeltaG(RT)) serving merely as arbitrary fitting parameters). Besides systematizing the study of hemoglobin kinetics, the utility of the microstate linear free energy model lies in the ability to test microscopic aspects of allosteric dynamics such as the "symmetry rule" for quaternary change deduced previously from thermodynamic evidence (Ackers, G. K., et al. (1992) Science 255, 54-63). Reflecting a remarkably detailed correspondence between thermodynamics and kinetics, we find that a kinetic model that includes the large free energy splitting between doubly ligated T microstates implied by the symmetry rule fits the data significantly better than one that does not.

The molecular code for hemoglobin allostery revealed by linking the thermodynamics and kinetics of quaternary structural change. 2. Cooperative free energies of (alphaFeCObetaFe)2 and (alphaFebetaFeCO)2 T-state tetramers.

Ligand photodissociation experiments are used to measure the prephotolysis equilibria between doubly liganded R and T quaternary conformers of the symmetric Fe-Co HbCO hybrids, (alpha(FeCO)beta(Co))(2) and (alpha(Co)beta(FeCO))(2). The free energies obtained from these data are used to calculate the cooperative free energies of the (alpha(FeCO)beta(Fe))(2) and (alpha(Fe)beta(FeCO))(2) intermediate CO-ligation states of normal hemoglobin in the T conformation, quantities important to the evaluation of current models of cooperativity. The symmetry rule model, incorporating sequential cooperativity of T-state ligand binding within an alphabeta dimer in addition to the traditional two-state cooperativity of the tetramer, predicts a larger free energy penalty for disturbing both dimers in a doubly liganded T tetramer than would be expected in the two-state model as currently formulated. (Cooperative energy penalties are simply proportional to the number of tetramer-bound ligands in the traditional two-state model.) The value found here for the energies of doubly liganded T microstates in which both dimers are perturbed, 7.9 +/- 0.3 kcal/mol, is consistent with the symmetry rule model but significantly higher than that expected (5-6 kcal/mol) in the two-state model of cooperativity.

Hydrogen bonding to Trp beta37 is the first step in a compound pathway for hemoglobin allostery.

Human hemoglobin is widely thought to change from the R to the T quaternary structure in a single rate process requiring tens of microseconds. Here we present kinetic evidence that the R --> T allosteric pathway in hemoglobin requires more than one step. We use magnetic circular dichroism (MCD) spectroscopy of the aromatic amino acid bands to show that formation of a tryptophan-aspartate hydrogen bond in the hinge region of the dimer-dimer interface is part of an obligatory R --> T step proceeding more than a factor of 10 faster than the kinetic step previously identified in heme-band absorption studies.