Dissection of Nodule Development by Supplementation of \u3cem\u3eRhizobium leguminosarum\u3c/em\u3e biovar \u3cem\u3ephaseoli\u3c/em\u3e Purine Auxotrophs with 4-Aminoimidazole-5-Carboxamide Riboside

Purine auxotrophs of Rhizobium leguminosarum biovar phaseoli CFN42 elicit uninfected pseudonodules on bean (Phaseolus vulgaris L.). Addition of 4-aminoimidazole-5-carboxamide (AICA) riboside to the root medium during incubation of the plant with these mutants leads to enhanced nodule development, although nitrogenase activity is not detected. Nodules elicited in this manner had infection threads and anatomical features characteristic of normal nodules, such as peripheral vasculature rather than the central vasculature of the pseudonodules that were elicited without AICA riboside supplementation. Although 105 to 106 bacteria could be recovered from these nodules after full development, bacteria were not observed in the interior nodule cells. Instead, large cells with extensive internal membranes were present. Approximately 5% of the normal amount of leghemoglobin and 10% of the normal amount of uricase were detected in these nodules. To promote the development of true nodules rather than pseudonodules, AICA riboside was required no later than the second day through no more than the sixth day following inoculation. After this period, removal of AICA riboside from the root medium did not prevent the formation of true nodules. This observation suggests that there is a critical stage of infection, reached before nodule emergence, at which development becomes committed to forming a true nodule rather than a pseudonodule

Infection of Soybean and Pea Nodules by \u3cem\u3eRhizobium\u3c/em\u3e spp. Purine Auxotrophs in the Presence of 5-aminoimidazole-4-Carboxamide Riboside

Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process. Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-amino-imidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR. The addition of purine to the root environment does not have this effect. In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium leguminosarum 128C56 (bv. viciae) were examined. Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR. R. fredii HH303 and R. leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source. All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts. On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside. On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection. Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria. The ability to AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth

Constructing a folding model for protein S6 guided by native fluctuations deduced from NMR structures

The diversity in a set of protein nuclear magnetic resonance (NMR) structures provides an estimate of native state fluctuations that can be used to refine and enrich structure-based protein models (SBMs). Dynamics are an essential part of a protein’s functional native state. The dynamics in the native state are controlled by the same funneled energy landscape that guides the entire folding process. SBMs apply the principle of minimal frustration, drawn from energy landscape theory, to construct a funneled folding landscape for a given protein using only information from the native structure. On an energy landscape smoothed by evolution towards minimal frustration, geometrical constraints, imposed by the native structure, control the folding mechanism and shape the native dynamics revealed by the model. Native-state fluctuations can alternatively be estimated directly from the diversity in the set of NMRstructures for a protein. Based on this information, we identify a highly flexible loop in the ribosomal protein S6 and modify the contact map in a SBM to accommodate the inferred dynamics. By taking into account the probable native state dynamics, the experimental transition state is recovered in the model, and the correct order of folding events is restored. Our study highlights how the shared energy landscape connects folding and function by showing that a better description of the native basin improves the prediction of the folding mechanism

Capturing Transition Paths and Transition States for Conformational Rearrangements in the Ribosome

AbstractTo reveal the molecular determinants of biological function, one seeks to characterize the interactions that are formed in conformational and chemical transition states. In other words, what interactions govern the molecule’s energy landscape? To accomplish this, it is necessary to determine which degrees of freedom can unambiguously identify each transition state. Here, we perform simulations of large-scale aminoacyl-transfer RNA (aa-tRNA) rearrangements during accommodation on the ribosome and project the dynamics along experimentally accessible atomic distances. From this analysis, we obtain evidence for which coordinates capture the correct number of barrier-crossing events and accurately indicate when the aa-tRNA is on a transition path. Although a commonly used coordinate in single-molecule experiments performs poorly, this study implicates alternative coordinates along which rearrangements are accurately described as diffusive movements across a one-dimensional free-energy profile. From this, we provide the theoretical foundation required for single-molecule techniques to uncover the energy landscape governing aa-tRNA selection by the ribosome

Quantum entanglement between electronic and vibrational degrees of freedom in molecules

We consider the quantum entanglement of the electronic and vibrational degrees of freedom in molecules with a tendency towards double welled potentials using model coupled harmonic diabatic potential-energy surfaces. The von Neumann entropy of the reduced density matrix is used to quantify the electron-vibration entanglement for the lowest two vibronic wavefunctions in such a bipartite system. Significant entanglement is found only in the region in which the ground vibronic state contains a density profile that is bimodal (i.e., contains two separate local minima). However, in this region two distinct types of entanglement are found: (1) entanglement that arises purely from the degeneracy of energy levels in the two potential wells and which is destroyed by slight asymmetry, and (2) entanglement that involves strongly interacting states in each well that is relatively insensitive to asymmetry. These two distinct regions are termed fragile degeneracy-induced entanglement and persistent entanglement, respectively. Six classic molecular systems describable by two diabatic states are considered: ammonia, benzene, semibullvalene, pyridine excited triplet states, the Creutz-Taube ion, and the radical cation of the "special pair" of chlorophylls involved in photosynthesis. These chemically diverse systems are all treated using the same general formalism and the nature of the entanglement that they embody is elucidated

α1-Antitrypsin deficiency.

α1-Antitrypsin deficiency (A1ATD) is an inherited disorder caused by mutations in SERPINA1, leading to liver and lung disease. It is not a rare disorder but frequently goes underdiagnosed or misdiagnosed as asthma, chronic obstructive pulmonary disease (COPD) or cryptogenic liver disease. The most frequent disease-associated mutations include the S allele and the Z allele of SERPINA1, which lead to the accumulation of misfolded α1-antitrypsin in hepatocytes, endoplasmic reticulum stress, low circulating levels of α1-antitrypsin and liver disease. Currently, there is no cure for severe liver disease and the only management option is liver transplantation when liver failure is life-threatening. A1ATD-associated lung disease predominately occurs in adults and is caused principally by inadequate protease inhibition. Treatment of A1ATD-associated lung disease includes standard therapies that are also used for the treatment of COPD, in addition to the use of augmentation therapy (that is, infusions of human plasma-derived, purified α1-antitrypsin). New therapies that target the misfolded α1-antitrypsin or attempt to correct the underlying genetic mutation are currently under development

Structural insights into crista junction formation by the Mic60-Mic19 complex

Mitochondrial cristae membranes are the oxidative phosphorylation sites in cells. Crista junctions (CJs) form the highly curved neck regions of cristae and are thought to function as selective entry gates into the cristae space. Little is known about how CJs are generated and maintained. We show that the central coiled-coil (CC) domain of the mitochondrial contact site and cristae organizing system subunit Mic60 forms an elongated, bow tie–shaped tetrameric assembly. Mic19 promotes Mic60 tetramerization via a conserved interface between the Mic60 mitofilin and Mic19 CHCH (CC-helix-CC-helix) domains. Dimerization of mitofilin domains exposes a crescent-shaped membrane-binding site with convex curvature tailored to interact with the curved CJ neck. Our study suggests that the Mic60-Mic19 subcomplex traverses CJs as a molecular strut, thereby controlling CJ architecture and function

SMOG@ctbp: simplified deployment of structure-based models in GROMACS

Molecular dynamics simulations with coarse-grained and/or simplified Hamiltonians are an effective means of capturing the functionally important long-time and large-length scale motions of proteins and RNAs. Structure-based Hamiltonians, simplified models developed from the energy landscape theory of protein folding, have become a standard tool for investigating biomolecular dynamics. SMOG@ctbp is an effort to simplify the use of structure-based models. The purpose of the web server is two fold. First, the web tool simplifies the process of implementing a well-characterized structure-based model on a state-of-the-art, open source, molecular dynamics package, GROMACS. Second, the tutorial-like format helps speed the learning curve of those unfamiliar with molecular dynamics. A web tool user is able to upload any multi-chain biomolecular system consisting of standard RNA, DNA and amino acids in PDB format and receive as output all files necessary to implement the model in GROMACS. Both Cα and all-atom versions of the model are available. SMOG@ctbp resides at http://smog.ucsd.edu