Entropic Control of an Excited Folded-Like Conformation in a Disordered Protein Ensemble

Many intrinsically disordered proteins switch between unfolded and folded-like forms in the presence of their binding partner. The possibility of a pre-equilibrium between the two macrostates is challenging to discern given the complex conformational landscape. Here, we show that CytR, a disordered DNA-binding domain, samples a folded-like excited state in its native ensemble through equilibrium multi-probe spectroscopy, kinetics and an Ising-like statistical mechanical model. The population of the excited state increases upon stabilization of the native ensemble with an osmolyte, while decreasing with increasing temperatures. A conserved proline residue, the mutation of which weakens the binding affinity to the target promoter, is found to uniquely control the population of the minor excited state. Semi-quantitative statistical mechanical modeling reveals that the conformational diffusion coefficient of disordered CytR is an order of magnitude slower than the estimates from folded domains. The osmolyte and proline mutation smoothen and roughen up the landscape, respectively, apart from modulation of populations. Our work uncovers general strategies to probe for excited structured states in disordered ensembles, and to measure and modulate the roughness of the disordered landscapes, inter-conversion rates of species and their populations.     

Crystallization and preliminary X-ray analysis of the cytoplasmic domain of human erythrocyte band 3

A cytoplasmic domain of the human erythrocyte membrane protein band 3 (M_r = 42,500), residues 1–379, expressed in and purified from E. coli, has been crystallized by the method of vapor diffusion in sitting drops with subsequent streak-seeding at room temperature. Initial crystals were grown from solutions containing 65–68% saturated ammonium sulfate at pH 4.9 and 2 mg/ml protein. Subsequent streak-seeding into solutions of 50–53% ammonium sulfate at pH 4.9 and 7 mg/ml protein produced single crystals suitable fur X-ray analysis, which contained pure protein as revealed by gel electrophoresis. The crystals belong to the monoclinic space group C2 with cell dimensions of a = 178.8 Å, b = 90.5 Å, c = 122.1 Å, and β = 131.3° and diffract at least to 2.7 Å resolution (at 100 K). A self-rotation function shows the presence of approximate 222 local symmetry. © 1995 Wiley-Liss, Inc.     

Fine structure of the respiratory organs of the Climbing perch, Anabas testudineus (Pisces: Anabantidae)

An electron microscopic study has been made of the three respiratory organs of climbing perch. The gill structure is similar to that of the other telcosts but the thickness of the water/blood barrier is much greater, being as great as 20 μm in some specimens. The increased thickness is due to a multilayered epithelium which is thinner (3.5–7 μm) over the marginal channel of the secondary lamellae. The other two main layers, basement membrane and pillar cell flange, are relatively thin (about 1 μm).
The pillar cells have a typical structure, but in certain regions they are contiguous with one another and line well-defined blood channels. Some of the columns of basement membrane material in such regions may be common to adjacent pillar cells.
The air-breathing organs are (a) the lining of the suprabranchial chambers , and (b) the labyrinthine plates attached to the dorsal region of branchial arches. Electron microscopy showed that their structure is well adapted for gas exchange, the air/blood barriers being only 0.12–0.3 μm, comprising an epithelial layer, basement membrane, and thin capillary endothelium. The many parallel blood channels of the respiratory islets of both organs are separated by pillar-like structures which differ from the pillar cells of the secondary lamellae. Thus the hypothesis that the air-breathing organs represent modified gills is not supported by this study.
The fine structure of the non-respiratory region of the air-breathing organs is similar to that of the skin, and includes chemoreceptor-like cells. Evidence concerning the possible homology of pillar cells with plain muscle cells is discussed.     

Gill microcirculation of the air-breathing climbing perch, Anabas testudineus (Bloch):relationships with the accessory respiratory organs and systemic circulation

The general macrocirculation and branchial microcirculation of the air-breathing climbing perch, Anabas testudineus, was examined by light and scanning electron microscopy of vascular corrosion replicas. The ventral aorta arises from the heart as a short vessel that immediately bifurcates into a dorsal and a ventral branch. The ventral branch distributes blood to gill arches 1 and 2, the dorsal branch to arches 3 and 4. The vascular organization of arches 1 and 2 is similar to that described for aquatic breathing teleosts. The respiratory lamellae are well developed but lack a continuous inner marginal channel. The filaments contain an extensive nutritive and interlamellar network; the latter traverses the filament between, but in register with, the inner lamellar margins. Numerous small, tortuous vessels arise from the efferent filamental and branchial arteries and anastomose with each other to form the nutrient supply for the filament, adductor muscles, and arch supportive tissues. The efferent branchial arteries of arches 1 and 2 supply the accessory air-breathing organs. Arches 3 and 4 are modified to serve primarily as large-bore shunts between the dorsal branch of the ventral aorta and the dorsal aorta. In many filaments from arches 3 and 4, the respiratory lamellae are condensed and have only 1-3 large channels. In some instances in arch 4, shunt vessels arise from the afferent branchial artery and connect directly with the efferent filamental artery. The filamental nutrient and interlamellar systems are poorly developed or absent. The respiratory and systemic pathways in Anabas are arranged in parallel. Blood flows from the ventral branch of the ventral aorta, through gill arches 1 and 2, into the accessory respiratory organs, and then returns to the heart. Blood, after entering the dorsal branch of the ventral aorta, passes through gill arches 3 and 4 and proceeds to the systemic circulation. This arrangement optimizes oxygen delivery to the tissues and minimizes intravascular pressure in the branchial and air-breathing organs. The efficiency of this system is limited by the mixing of respiratory and systemic venous blood at the heart.     

Palmitoylation of the luteinizing hormone/human chorionic gonadotropin receptor regulates receptor interaction with the arrestin-mediated internalization pathway.

The luteinizing hormone/human chorionic gonadotropin receptor (LH/hCGR) undergoes palmitoylation at cysteine residues 621 and 622 located in the carboxyl terminal tail of the receptor. This study examined the biological function of palmitoylation with respect to its effect on receptor internalization. Coexpression of wild-type (WT) or C621/622G mutant receptors with arrestin-2 increased receptor internalization in 293T cells. Furthermore, measurements of rate enhancement upon overexpression of arrestin indicate that the palmitoylation deficient mutant receptor is more prone to utilizing the arrestin mediated internalization pathway than the WT receptor. Coexpression of G-protein-coupled receptor kinase 4 (GRK4) with wild type receptor resulted in an increase in internalization, while coexpression with the mutant receptor did not result in further enhancement of internalization. Additionally, 293T cells expressing mutant receptor were responsive to hCG with respect to production of inositol phosphates. Taken together, these results suggest that the palmitoylation state of the receptor governs internalization by regulating the accessibility of the receptor to the arrestin-mediated internalization pathway.     

Succinic dehydrogenase activity of the respiratory muscles of a fresh-water teleost, Bagarius bagarius (Ham.) (Sisoridae, Pisces).

Functional morphology including the origin, insertion, and innervation of the respiratory muscles in relation to buccal pressure pump and opercular suction pumps in a fresh-water bottom dwelling siluroid fish, Bagarius bagarius have been studied. Histochemical studies were made on the succinic dehydrogenase activity of adductor mandibulae, retractor tentaculi, levator operculi, dilatator operculi, adductor operculi, intermandibularis, interhyoideus, hyohyoideus superior and constrictor branchialis. The intensity of reaction reveals the presence of three types of muscle fibres in some of the respiratory muscles. The muscle containing red muscle fibres are mostly innervated by the branches of the VIIth cranial nerve. The retractor tentaculi consists of superficial white muscle fibres and the interior part is dominated by red muscle fibres. The muscles (adductor operculi, levator operculi, dilatator operculi, interhyoideus, hyohyoideus superior) concerned with the opercular suction pumps are of mixed type and consist of white and red muscle fibres, whereas adductor mandibulae and intermandibularis are made up entirely of white muscle fibres. The adductor muscle bundles of the constrictor branchialis, which are responsible for movement of gill filaments, are dominated by the red muscle fibres. The abductor part, however, is made up entirely of white muscle fibres.     

Variation in brown rat cranial shape shows directional selection over 120 years in New York City

Urbanization exposes species to novel environments and selection pressures that may change morphological traits within a population. We investigated how the shape and size of crania and mandibles changed over time within a population of brown rats (Rattus norvegicus) living in Manhattan, New York, USA, a highly urbanized environment. We measured 3D landmarks on the cranium and mandible of 62 adult individuals sampled in the 1890s and 2010s. Static allometry explained approximately 22% of shape variation in crania and mandible datasets, while time accounted for approximately 14% of variation. We did not observe significant changes in skull size through time or between the sexes. Estimating the P‐matrix revealed that directional selection explained temporal change of the crania but not the mandible. Specifically, rats from the 2010s had longer noses and shorter upper molar tooth rows, traits identified as adaptive to colder environments and higher quality or softer diets, respectively. Our results highlight the continual evolution to selection pressures. We acknowledge that urban selection pressures impacting cranial shape likely began in Europe prior to the introduction of rats to Manhattan. Yet, our study period spanned changes in intensity of artificial lighting, human population density, and human diet, thereby altering various aspects of rat ecology and hence pressures on the skull.     

Isolation, purification and characterization of an antifungal molecule produced by Bacillus licheniformis BC98, and its effect on phytopathogen Magnaporthe grisea

AIMS: Isolation of bacterial antagonist for use in the biological control of phytopathogenic fungi like rice blast fungus, Magnaporthe grisea, and to further purify and characterize the antifungal molecule produced by the antagonist. METHODS AND RESULTS: Bacterial antagonist exhibiting highest antifungal activity against the rice blast fungus M. grisea was isolated from soil and identified as Bacillus licheniformis BC98. Besides M. grisea, the isolate also inhibited the growth of other phytopathogens such as Curvularia lunata and Rhizoctonia bataticola. Biologically active fractions were isolated from the culture filtrate and further fractionated by reverse-phase high-performance liquid chromatography (HPLC) enabling detailed structural characterization of a component of molecular mass 1035 Da. The active peptide was identified as surfactin after 500 MHz (1)H NMR analysis. Microscopic analysis of the effect of the antagonist on M. grisea revealed bulbous hyphae showing patchy and vacuolated cytoplasm when observed under the electron microscope. CONCLUSIONS: The antagonistic lipopeptide secreted by B. licheniformis BC98 and identified as surfactin, induced morphological changes in M. grisea, inhibiting its further growth, and thus exhibiting fungicidal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The antagonist inhibits germination of M. grisea, a potent rice phytopathogen, and therefore appears to be a potential candidate for control of rice blast disease.