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Stranded [Alpha 2.1]

The crystal structure of Serratia endonuclease has been solved to 2.1 Å by multiple isomorphous replacement. This magnesium-dependent enzyme is equally active against single- and double-stranded DNA, as well as RNA, without any apparent base preference. The Serratia endonuclease fold is distinct from that of other nucleases that have been solved by X-ray diffraction. The refined structure consists of a central layer containing six antiparallel β-strands which is flanked on one side by a helical domain and on the opposite side by one dominant helix and a very long coiled loop. Electrostatic calculations reveal a strongly polarized molecular surface and suggest that a cleft between this long helix and loop, near His 89, may contain the active site of the enzyme.

Stranded [Alpha 2.1]

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Parkinson's disease is the second most common brain disorder of the elderly. It is thought to be caused by environmental and genetic factors. However, little is known about the genes and processes involved. Pathologically, Parkinson's disease is recognized by inclusions in the brain that contain a disease-specific protein: alpha-synuclein. We created a small animal model (C. elegans) in which we could follow the formation of alpha-synuclein inclusions in living and aging animals. With a genome-wide RNAi screen we identified 80 genes whose expression influences inclusion formation. These genes include evolutionarily conserved regulators of longevity, suggesting a link between inclusion formation and the molecular mechanism of aging. Our results offer a refined understanding of how Parkinson's disease arises during aging and we identify processes and genes that may underlie an increased susceptibility for the disease, which is important for improving diagnostics and developing strategies for therapeutic intervention.

The selection process began with a single-stranded DNA (ssDNA) library consisting of 1015 different molecules designed by our laboratory as previously described (Figure 1) [22]. In brief, the library, termed RMW.N34, consists of two 23-base constant regions for polymerase chain reaction (PCR) amplification flanking a 34-base random region (commercially synthesized by Eurofins MWG Operon; Huntsville, AL, USA). A total of 12 rounds of SELEX were performed (Table 1) to identify ssDNA molecules that bound specifically to toxin B and not to negative targets (Figure 2).

In Caenorhabditis elegans, injection of double-stranded RNA (dsRNA) results in the specific inactivation of genes containing homologous sequences, a technique termed RNA-mediated interference (RNAi). It has previously been shown that RNAi can also be achieved by feeding worms Escherichia coli expressing dsRNA corresponding to a specific gene; this mode of dsRNA introduction is conventionally considered to be less efficient than direct injection, however, and has therefore seen limited use, even though it is considerably less labor-intensive.

RNA-mediated interference (RNAi) is the phenomenon first described in the nematode Caenorhabditis elegans in which introduction of double-stranded RNA (dsRNA) results in potent and specific inactivation of the corresponding gene through the degradation of endogenous mRNA [1,2]. This technique rapidly produces gene-specific loss-of-function or hypomorphic phenotypes, and potent interference is also observed in the progeny of the affected animal. Thus, because RNAi results in a robust, specific and durable interference effect, and also because RNAi is the simplest and most efficient method for inactivating genes in C. elegans, it has been rapidly embraced as a reverse-genetics tool for determining the functions of specific genes.

Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination and repair in Bacteria, Archaea and Eukarya. In recent years, there has been an increasing interest in SSBs, since they find numerous applications in diverse molecular biology and analytical methods.

The sequence analysis of the D. psychrophila (GenBank accession No. NC_006138;[16]), F. psychrophilum (GenBank accession No. NC_009613;[17]), P. arcticus (GenBank accession No. NC_007204;[18]), P. cryohalolentis (GenBank accession No. NC_007969; Gene Bank Project: PRJNA58373), P. ingrahamii (GenBank accession No. NC_008709;[19]), P. profundum (GenBank accession No. NC_006370;[20]) and P. torquis (GenBank accession No. NC_018721;[15]) genomes indicated the presence of a single ssb gene. In the case of F. psychrophilum, P. ingrahamii and P. torquis, there were additional genes possessing sequences similar to the ssDNA binding domain. The product of the additional gene from F. psychrophilum was a protein of unknown function, while that from P. ingrahamii was the PriB. In P. torquis, it was a short (102 aa), single-stranded DNA binding protein without a characteristic sequence of last amino acid residues, in view of which, we omitted that protein from our research. On the basis of the ssb gene organization and the number of ssb genes paralogs, bacteria have been classified in four different groups[21]. P. arcticus, P. cryohalolentis and P. profundum are classified as group III, which contains bacteria with ssb gene organization uvrA-ssb, whereas D. psychrophila, F. psychrophilum, P. ingrahamii, and P. torquis are classified as group IV, which contains bacteria with ssb placed neither between rpsF and rpsR nor divergently located to uvrA.

In chemical cross-linking experiments using glutaraldehyde, the Dps SSB, Fps SSB and Pto SSB complexes were found at a position corresponding to a molecular mass of approximately 80 kDa, the Ppr SSB complexes were found at a position corresponding to a molecular mass of about 100 kDa, the Par SSB and Pcr SSB complexes were found at a position corresponding to a molecular mass of around 116 kDa, and the Pin SSB complexes were found at a position corresponding to a molecular mass of approximately 140 kDa (Figure 2A). We observed that the psychrophilic SSB proteins in question have anomalous mobility in SDS-PAGE gels than would be expected on the basis of their predicted molecular masses. This phenomenon has also been observed in SSBs from Shewanella strains[27] and could be a characteristic feature of psychrophilic single-stranded DNA-binding proteins. The SSBs from D. psychrophila, F. psychrophilum and P. torquis were found at a position corresponding to a molecular mass of around 20 kDa (Figure 2A), while their calculated molecular masses are 15.6, 15.9 and 17.1 kDa, respectively. The Ppr SSB was found at a position corresponding to a molecular mass of approximately 25 kDa, while its calculated molecular mass is 20.4 kDa (Figure 2A). The Par SSB, Pcr SSB and Pin SSB were found at positions corresponding to molecular masses of around 25, 27 and 32 kDa, although their predicted molecular masses are 22.8, 23.3 and 25.1 kDa, accordingly (Figure 2A). Taken together, these results confirmed our prediction that the Dps SSB, Fps SSB, Par SSB, Pcr SSB, Pin SSB, Ppr SSB and Pto SSB exist as homotetramers in solution.

An analytical gel filtration chromatography analysis of the purified psychrophilic SSBs revealed a single peak for each protein. As calculated using a regression curve equation, there was a peak with a molecular mass of 59 kDa for the Dps SSB, 69.5 kDa for the Fps SSB, 94.4 kDa for the Par SSB, 96.1 kDa for the Pcr SSB, 102.8 kDa for the Pin SSB, 85.4 kDa for the Ppr SSB, and 72.3 kDa for the Pto SSB, (Figure 2C). The native molecular mass of each peak represents 3.8 for the Dps SSB mass monomer, 4.4 for the Fps SSB mass monomer, 4.1 for the Par SSB, Pcr SSB and Pin SSB mass monomers, and 4.2 for the Ppr SSB and Pto SSB mass monomers, respectively. Psychrophilic single-stranded DNA binding proteins therefore exist in solution as homotetramers.

Proteins are one of the most abundant organic molecules in living systems and have the most diverse range of functions of all macromolecules. Proteins may be structural, regulatory, contractile, or protective; they may serve in transport, storage, or membranes; or they may be toxins or enzymes. Each cell in a living system may contain thousands of different proteins, each with a unique function. Their structures, like their functions, vary greatly. They are all, however, polymers of alpha amino acids, arranged in a linear sequence and connected together by covalent bonds.

The major building block of proteins are called alpha (α) amino acids. As their name implies they contain a carboxylic acid functional group and an amine functional group. The alpha designation is used to indicate that these two functional groups are separated from one another by one carbon group. In addition to the amine and the carboxylic acid, the alpha carbon is also attached to a hydrogen and one additional group that can vary in size and length. In the diagram below, this group is designated as an R-group. Within living organisms there are 20 amino acids used as protein building blocks. They differ from one another only at the R-group position. The basic structure of an amino acid is shown below:

There are a total of 20 alpha amino acids that are commonly incorporated into protein structures (Figure 2.x). The different R-groups have different characteristics based on the nature of atoms incorporated into the functional groups. There are R-groups that predominantly contain carbon and hydrogen and are very nonpolar or hydrophobic. Others contain polar uncharged functional groups such as alcohols, amides, and thiols. A few amino acids are basic (containing amine functional groups) or acidic (containing carboxylic acid functional groups). These amino acids are capable of forming full charges and can have ionic interactions. Each amino acid can be abbreviated using a three letter and a one letter code. 041b061a72


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