A mammalian circadian clock model incorporating daytime expression elements.

Models of the mammalian clock have traditionally been based around two feedback loops-the self-repression of Per/Cry by interfering with activation by BMAL/CLOCK, and the repression of Bmal/Clock by the REV-ERB proteins. Recent experimental evidence suggests that the D-box, a transcription factor binding site associated with daytime expression, plays a larger role in clock function than has previously been understood. We present a simplified clock model that highlights the role of the D-box and illustrate an approach for finding maximum-entropy ensembles of model parameters, given experimentally imposed constraints. Parameter variability can be mitigated using prior probability distributions derived from genome-wide studies of cellular kinetics. Our model reproduces predictions concerning the dual regulation of Cry1 by the D-box and Rev-ErbA/ROR response element (RRE) promoter elements and allows for ensemble-based predictions of phase response curves (PRCs). Nonphotic signals such as Neuropeptide Y (NPY) may act by promoting Cry1 expression, whereas photic signals likely act by stimulating expression from the E/E' box. Ensemble generation with parameter probability restraints reveals more about a model's behavior than a single optimal parameter set.

Site-directed coordination chemistry with P22 virus-like particles.

Protein cage nanoparticles (PCNs) are attractive platforms for developing functional nanomaterials using biomimetic approaches for functionalization and cargo encapsulation. Many strategies have been employed to direct the loading of molecular cargos inside a wide range of PCN architectures. Here we demonstrate the exploitation of a metal-ligand coordination bond with respect to the direct packing of guest molecules on the interior interface of a virus-like PCN derived from Salmonella typhimurium bacteriophage P22. The incorporation of these guest species was assessed using mass spectrometry, multiangle laser light scattering, and analytical ultracentrifugation. In addition to small-molecule encapsulation, this approach was also effective for the directed synthesis of a large macromolecular coordination polymer packed inside of the P22 capsid and initiated on the interior surface. A wide range of metals and ligands with different thermodynamic affinities and kinetic stabilities are potentially available for this approach, highlighting the potential for metal-ligand coordination chemistry to direct the site-specific incorporation of cargo molecules for a variety of applications.

Efficient light harvesting in a dark, hot, acidic environment: the structure and function of PSI-LHCI from Galdieria sulphuraria.

Photosystem I-light harvesting complex I (PSI-LHCI) was isolated from the thermoacidophilic red alga Galdieria sulphuraria, and its structure, composition, and light-harvesting function were characterized by electron microscopy, mass spectrometry, and ultrafast optical spectroscopy. The results show that Galdieria PSI is a monomer with core features similar to those of PSI from green algae, but with significant differences in shape and size. A comparison with the crystal structure of higher plant (pea) PSI-LHCI indicates that Galdieria PSI binds seven to nine light-harvesting proteins. Results from ultrafast optical spectroscopy show that the functional coupling of the LHCI proteins to the PSI core is tighter than in other eukaryotic PSI-LHCI systems reported thus far. This tight coupling helps Galdieria perform efficient light harvesting under the low-light conditions present in its natural endolithic habitat.

A novel photosynthetic strategy for adaptation to low-iron aquatic environments.

Iron (Fe) availability is a major limiting factor for primary production in aquatic environments. Cyanobacteria respond to Fe deficiency by derepressing the isiAB operon, which encodes the antenna protein IsiA and flavodoxin. At nanomolar Fe concentrations, a PSI-IsiA supercomplex forms, comprising a PSI trimer encircled by two complete IsiA rings. This PSI-IsiA supercomplex is the largest photosynthetic membrane protein complex yet isolated. This study presents a detailed characterization of this complex using transmission electron microscopy and ultrafast fluorescence spectroscopy. Excitation trapping and electron transfer are highly efficient, allowing cyanobacteria to avoid oxidative stress. This mechanism may be a major factor used by cyanobacteria to successfully adapt to modern low-Fe environments.

Ion accumulation in a protein nanocage: finding noisy temporal sequences using a genetic algorithm.

Many pathogenic bacteria are able to survive attack by the host's immune system because of antioxidant systems that mitigate the effects of reactive oxygen species. Dps is a hollow 12-subunit protein nanocage that prevents oxidative damage by oxidizing and sequestering intracellular Fe(2+); the resulting Fe(3+) forms an iron oxyhydroxide nanoparticle in the cage interior. Charged sites on the protein nanocage create an electrostatic gradient that guides ions through well-defined pores that connect the cage interior with the surrounding solution and toward nucleation sites on the cage interior. In this study, we use all-atom molecular dynamics to simulate the motion of simple cations into the dodecameric cage formed by the Dps protein from Listeria monocytogenes. Ion trajectories are analyzed by using a novel, to our knowledge, genetic algorithm to determine the temporal sequence of ion-protein interactions. Ions enter Dps through well-defined pores at the ferritinlike C(3) axes, with negatively-charged residues on the outside of the cage forming a fairly well-defined entrance pathway. This method of trajectory analysis may be broadly applicable in situations where the spatial localization of ions or other small molecules is electrostatically driven by a biomolecule.

From metal binding to nanoparticle formation: monitoring biomimetic iron oxide synthesis within protein cages using mass spectrometry.

Mass measurements of metal-mineralized protein cages allowed quantitative examination of the effects of metal-ion concentration on the final nanoparticle size. Modeling using a kinetic master equation suggests that particle growth involves both a binding phase and a growth phase (see picture; I: relative abundance; LiDps: a DNA binding protein; (n)Fe: number of Fe atoms).

Fitting low-resolution cryo-EM maps of proteins using constrained geometric simulations.

Recent experimental advances in producing density maps from cryo-electron microscopy (cryo-EM) have challenged theorists to develop improved techniques to provide structural models that are consistent with the data and that preserve all the local stereochemistry associated with the biomolecule. We develop a new technique that maintains the local geometry and chemistry at each stage of the fitting procedure. A geometric simulation is used to drive the structure from some appropriate starting point (a nearby experimental structure or a modeled structure) toward the experimental density, via a set of small incremental motions. Structural motifs such as alpha-helices can be held rigid during the fitting procedure as the starting structure is brought into alignment with the experimental density. After validating this procedure on simulated data for adenylate kinase and lactoferrin, we show how cryo-EM data for two different GroEL structures can be fit using a starting x-ray crystal structure. We show that by incorporating the correct local stereochemistry in the modeling, structures can be obtained with effective resolution that is significantly higher than might be expected from the nominal cryo-EM resolution.

A design principle for a posttranslational biochemical oscillator.

Multisite phosphorylation plays an important role in biological oscillators such as the circadian clock. Its general role, however, has been elusive. In this theoretical study, we show that a simple substrate with two modification sites acted upon by two opposing enzymes (e.g., a kinase and a phosphatase) can show oscillations in its modification state. An unbiased computational analysis of this oscillator reveals two common characteristics: a unidirectional modification cycle and sequestering of an enzyme by a specific modification state. These two motifs cause a substrate to act as a coupled system in which a unidirectional cycle generates single-molecule oscillators, whereas sequestration synchronizes the population by limiting the available enzyme under conditions in which substrate is in excess. We also demonstrate the conditions under which the oscillation period is temperature compensated, an important feature of the circadian clock. This theoretical model will provide a framework for analyzing and synthesizing posttranslational oscillators.

Alteration of proteins and pigments influence the function of photosystem I under iron deficiency from Chlamydomonas reinhardtii.

BACKGROUND: Iron is an essential micronutrient for all organisms because it is a component of enzyme cofactors that catalyze redox reactions in fundamental metabolic processes. Even though iron is abundant on earth, it is often present in the insoluble ferric [Fe (III)] state, leaving many surface environments Fe-limited. The haploid green alga Chlamydomonas reinhardtii is used as a model organism for studying eukaryotic photosynthesis. This study explores structural and functional changes in PSI-LHCI supercomplexes under Fe deficiency as the eukaryotic photosynthetic apparatus adapts to Fe deficiency. RESULTS: 77K emission spectra and sucrose density gradient data show that PSI and LHCI subunits are affected under iron deficiency conditions. The visible circular dichroism (CD) spectra associated with strongly-coupled chlorophyll dimers increases in intensity. The change in CD signals of pigments originates from the modification of interactions between pigment molecules. Evidence from sucrose gradients and non-denaturing (green) gels indicates that PSI-LHCI levels were reduced after cells were grown for 72 h in Fe-deficient medium. Ultrafast fluorescence spectroscopy suggests that red-shifted pigments in the PSI-LHCI antenna were lost during Fe stress. Further, denaturing gel electrophoresis and immunoblot analysis reveals that levels of the PSI subunits PsaC and PsaD decreased, while PsaE was completely absent after Fe stress. The light harvesting complexes were also susceptible to iron deficiency, with Lhca1 and Lhca9 showing the most dramatic decreases. These changes in the number and composition of PSI-LHCI supercomplexes may be caused by reactive oxygen species, which increase under Fe deficiency conditions. CONCLUSIONS: Fe deficiency induces rapid reduction of the levels of photosynthetic pigments due to a decrease in chlorophyll synthesis. Chlorophyll is important not only as a light-harvesting pigment, but also has a structural role, particularly in the pigment-rich LHCI subunits. The reduced level of chlorophyll molecules inhibits the formation of large PSI-LHCI supercomplexes, further decreasing the photosynthetic efficiency.

A click chemistry based coordination polymer inside small heat shock protein.

A branched iron-phenanthroline based coordination polymer has been constructed in a water based system using a click chemistry approach to link monomeric coordination complexes together within a protein cage nanoarchitecture, which acts both as a template and a sized constrained reaction environment.

Size and crystallinity in protein-templated inorganic nanoparticles.

Protein cages such as ferritins and virus capsids have been used as containers to synthesize a wide variety of protein-templated inorganic nanoparticles. While identification of the inorganic crystal phase has been successful in some cases, very little is known about the detailed nanoscale structure of the inorganic component. We have used pair distribution function analysis of total X-ray scattering to measure the crystalline domain size in nanoparticles of ferrihydrite, γ-Fe2O3, Mn3O4, CoPt, and FePt grown inside 24-meric ferritin cages from H. sapiens and P. furiosus. The material properties of these protein-templated nanoparticles are influenced by processes at a variety of length scales: the chemistry of the material determines the precise arrangement of atoms at very short distances, while the interior volume of the protein cage constrains the maximum nanoparticle size attainable. At intermediate length scales, the size of coherent crystalline domains appears to be constrained by the arrangement of crystal nucleation sites on the interior of the cage. Based on these observations, some potential synthetic strategies for the control of crystalline domain size in protein-templated nanoparticles are suggested.

Docking of photosystem I subunit C using a constrained geometric simulation.

The elucidation of assembly pathways of multi-subunit protein complexes is a problem of great interest in structural biology and biomolecular modeling. In this study, we use a new computer algorithm for the simulation of large-scale motion in proteins to dock the subunit PsaC onto Photosystem I. We find that a complicated docking pathway involving multiple conformational changes can be quickly simulated by actively targeting only a few residues at a time to their target positions. Simulations for two possible docking scenarios are explored, and experimental approaches to distinguish between them are discussed.