A simulation model of <Emphasis Type="Italic">Escherichia coli</Emphasis> osmoregulatory switch using E-CELL system

Background  

Bacterial signal transduction mechanism referred to as a "two component regulatory systems" contributes to the overall adaptability of the bacteria by regulating the gene expression. Osmoregulation is one of the well-studied two component regulatory systems comprising of the sensor, EnvZ and the cognate response regulator, OmpR, which together control the expression of OmpC and OmpF porins in response to the osmolyte concentration.     

A steady state analysis indicates that negative feedback regulation of PTP1B by Akt elicits bistability in insulin-stimulated GLUT4 translocation

Background  

The phenomenon of switch-like response to graded input signal is the theme involved in various signaling pathways in living systems. Positive feedback loops or double negative feedback loops embedded with nonlinearity exhibit these switch-like bistable responses. Such feedback regulations exist in insulin signaling pathway as well.     

Development of the paravascular bed of the vessels of the human heart during the prenatal period of ontogenesis

The paravascular bed of the cardiac vessels has been studied in 128 human fetuses at the age of 3-9 lunar months. Anatomical and histological techniques have been used, morphometrical analysis has been carried out. The paravascular bed of the cardiac wall vessels begins to form from the vascular epicardial network and from the paraneural vessels in 5-month-old fetuses. The paravasal longitudinal tracts are the first to form (the venous ones preceed the arterial). During the seventh month the nutritive vessels and the intramural networks of the main cardiac arteries and veins develop. The formation of the paraarterial bed is connected with the vascular diameter and with thickness of the arterial walls. Certain regularities in development of the venous paravascular bed are defined. By the beginning of the 8th month there are all main components of the paravascular bed of the cardiac vessels.     

Backbone dynamics of (1–71)- and (1–36)bacterioopsin studied by two-dimensional 1H-15N NMR spectroscopy

Summary The backbone dynamics of uniformly ¹⁵N-labelled fragments (residues 1–71 and 1–36) of bacterioopsin, solubilized in two media (methanol-chloroform (1:1), 0.1 M ²HCO₂NH₄, or SDS micelles) have been investigated using 2D proton-detected heteronuclear ¹H-¹⁵N NMR spectroscopy at two spectrometer frequencies, 600 and 400 MHz. Contributions of the conformational exchange to the transverse relaxation rates of individual nitrogens were elucidated using a set of different rates of the CPMG spin-lock pulse train and were essentially suppressed by the high-frequency CPMG spin-lock. We found that most of the backbone amide groups of (1–71)bacterioopsin in SDS micelles are involved in the conformational exchange process over a rate range of 10³ to 10⁴ s^-1. This conformational exchange is supposed to be due to an interaction between two -helixes of (1–71)bacterioopsin, since the hydrolysis of the peptide bond in the loop region results in the disappearance of exchange line broadening. ¹⁵N relaxation rates and ¹H-¹⁵N NOE values were interpreted using the model-free approach of Lipari and Szabo [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546–4559]. In addition to overall rotation of the molecule, the backbone N-H vectors of the peptides are involved in two types of internal motions: fast, on a time scale <20 ps, and intermediate, on a time scale close to 1 ns. The intermediate dynamics in the -helical stretches was mostly attributed to bending motions. A decrease in the order parameter of intermediate motions was also observed for residues next to Pro⁵⁰, indicating an anisotropy of the overall rotational diffusion of the molecule. Distinctly mobile regions are identified by a large decrease in the order parameter of intermediate motions and correspond to the N- and C-termini, and to a loop connecting the -helixes of (1–71)bacterioopsin. The internal dynamics of the -helixes on the millisecond and nanosecond time scales should be taken into account in the development of a model of the functioning bacteriorhodopsin.Abbreviations BO bacterioopsin - 2D two-dimensional - CPMG Carr-Purcell-Meiboom-Gill (Carr and Purcell, 1954) - SDS sodium dodecyl(²H₂₅) sulfate - R(S_x), R(S_z) ¹⁵N transverse and longitudinal relaxation rates, respectively     

Quantification of the glycogen cascade system: the ultrasensitive responses of liver glycogen synthase and muscle phosphorylase are due to distinctive regulatory designs

Background

Signaling pathways include intricate networks of reversible covalent modification cycles. Such multicyclic enzyme cascades amplify the input stimulus, cause integration of multiple signals and exhibit sensitive output responses. Regulation of glycogen synthase and phosphorylase by reversible covalent modification cycles exemplifies signal transduction by enzyme cascades. Although this system for regulating glycogen synthesis and breakdown appears similar in all tissues, subtle differences have been identified. For example, phosphatase-1, a dephosphorylating enzyme of the system, is regulated quite differently in muscle and liver. Do these small differences in regulatory architecture affect the overall performance of the glycogen cascade in a specific tissue? We address this question by analyzing the regulatory structure of the glycogen cascade system in liver and muscle cells at steady state.

Results

The glycogen cascade system in liver and muscle cells was analyzed at steady state and the results were compared with literature data. We found that the cascade system exhibits highly sensitive switch-like responses to changes in cyclic AMP concentration and the outputs are surprisingly different in the two tissues. In muscle, glycogen phosphorylase is more sensitive than glycogen synthase to cyclic AMP, while the opposite is observed in liver. Furthermore, when the liver undergoes a transition from starved to fed-state, the futile cycle of simultaneous glycogen synthesis and degradation switches to reciprocal regulation. Under such a transition, different proportions of active glycogen synthase and phosphorylase can coexist due to the varying inhibition of glycogen-synthase phosphatase by active phosphorylase.

Conclusion

The highly sensitive responses of glycogen synthase in liver and phosphorylase in muscle to primary stimuli can be attributed to distinctive regulatory designs in the glycogen cascade system. The different sensitivities of these two enzymes may exemplify the adaptive strategies employed by liver and muscle cells to meet specific cellular demands.

     

Automatic assignment of protein backbone resonances by direct spectrum inspection in targeted acquisition of NMR data

The necessity to acquire large multidimensional datasets, a basis for assignment of NMR resonances, results in long data acquisition times during which substantial degradation of a protein sample might occur. Here we propose a method applicable for such a protein for automatic assignment of backbone resonances by direct inspection of multidimensional NMR spectra. In order to establish an optimal balance between completeness of resonance assignment and losses of cross-peaks due to dynamic processes/degradation of protein, assignment of backbone resonances is set as a stirring criterion for dynamically controlled targeted nonlinear NMR data acquisition. The result is demonstrated with the 12 kDa ¹³C,¹⁵ N-labeled apo-form of heme chaperone protein CcmE, where hydrolytic cleavage of 29 C-terminal amino acids is detected. For this protein, 90 and 98% of manually assignable resonances are automatically assigned within 10 and 40 h of nonlinear sampling of five 3D NMR spectra, respectively, instead of 600 h needed to complete the full time domain grid. In addition, resonances stemming from degradation products are identified. This study indicates that automatic resonance assignment might serve as a guiding criterion for optimal run-time allocation of NMR resources in applications to proteins prone to degradation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Domain Features of the Peripheral Stalk Subunit H of the Methanogenic A1AO ATP Synthase and the NMR Solution Structure of H1-47

A series of truncated forms of subunit H were generated to establish the domain features of that protein. Circular dichroism analysis demonstrated that H is divided at least into a C-terminal coiled-coil domain within residues 54-104, and an N-terminal domain formed by adjacent α-helices. With a cysteine at the C-terminus of each of the truncated proteins (H_1-47, H_1-54, H_1-59, H_1-61, H_1-67, H_1-69, H_1-71, H_1-78, H_1-80, H_1-91, and H_47-105), the residues involved in formation of the coiled-coil interface were determined. Proteins H_1-54, H_1-61, H_1-69, and H_1-80 showed strong cross-link formation, which was weaker in H_1-47, H_1-59, H_1-71, and H_1-91. A shift in disulfide formation between cysteins at positions 71 and 80 reflected an interruption in the periodicity of hydrophobic residues in the region ₇₁AEKILEETEKE₈₁. To understand how the N-terminal domain of H is formed, we determined for the first time, to our knowledge, the solution NMR structure of H_1-47, which revealed an α-helix between residues 15-42 and a flexible N-terminal stretch. The α-helix includes a kink that would bring the two helices of the C-terminus into the coiled-coil arrangement. H_1-47 revealed a strip of alanines involved in dimerization, which were tested by exchange to single cysteines in subunit H mutants.     

Detailed protein sequence alignment based on Spectral Similarity Score (SSS)

Background  

The chemical property and biological function of a protein is a direct consequence of its primary structure. Several algorithms have been developed which determine alignment and similarity of primary protein sequences. However, character based similarity cannot provide insight into the structural aspects of a protein. We present a method based on spectral similarity to compare subsequences of amino acids that behave similarly but are not aligned well by considering amino acids as mere characters. This approach finds a similarity score between sequences based on any given attribute, like hydrophobicity of amino acids, on the basis of spectral information after partial conversion to the frequency domain.     

Analysis of optimal phenotypic space using elementary modes as applied to Corynebacterium glutamicum

Background  

Quantification of the metabolic network of an organism offers insights into possible ways of developing mutant strain for better productivity of an extracellular metabolite. The first step in this quantification is the enumeration of stoichiometries of all reactions occurring in a metabolic network. The structural details of the network in combination with experimentally observed accumulation rates of external metabolites can yield flux distribution at steady state. One such methodology for quantification is the use of elementary modes, which are minimal set of enzymes connecting external metabolites. Here, we have used a linear objective function subject to elementary modes as constraint to determine the fluxes in the metabolic network of Corynebacterium glutamicum. The feasible phenotypic space was evaluated at various combinations of oxygen and ammonia uptake rates.