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Biomedical subjects

Sharon Grayer Wolf

Publications and source records attributed to Sharon Grayer Wolf.

6 recordsLinked to original sources
PubMed2006

Plant transformation by Agrobacterium tumefaciens: modulation of single-stranded DNA-VirE2 complex assembly by VirE1.

Agrobacterium tumefaciens infects plant cells by the transfer of DNA. A key factor in this process is the bacterial virulence protein VirE2, which associates stoichiometrically with the transported single-stranded (ss) DNA molecule (T-strand). As observed in vitro by transmission electron microscopy, VirE2-ssDNA readily forms an extended helical complex with a structure well suited to the tasks of DNA protection and nuclear import. Here we have elucidated the role of the specific molecular chaperone VirE1 in regulating VireE2-VirE2 and VirE2-ssDNA interactions. VirE2 alone formed functional filamentous aggregates capable of ssDNA binding. In contrast, co-expression with VirE1 yielded monodisperse VirE1-VirE2 complexes. Cooperative binding of VirE2 to ssDNA released VirE1, resulting in a controlled formation mechanism for the helical complex that is further promoted by macromolecular crowding. Based on this in vitro evidence, we suggest that the constrained volume of the VirB channel provides a natural site for the exchange of VirE2 binding from VirE1 to the T-strand.

Agrobacterium tumefaciens↗
PubMed2006

Aspen SP1, an exceptional thermal, protease and detergent-resistant self-assembled nano-particle.

Stable protein 1 (SP1) is a homo-oligomeric protein isolated from aspen (Populus tremula aspen) plants which forms a ring-shape dodecameric particle with a central cavity. The oligomeric form of SP1 is an exceptionally stable structure that is resistant to proteases (e.g., trypsin, V8, and proteinase K), high temperatures, organic solvents, and high levels of ionic detergent. Analytical ultra-centrifugation, chemical cross-linking, matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF-MS), and transmission electron microscopy were used to further characterize the SP1 dodecamer. Introduction of a single cysteine at the N-terminus of SP1 enabled the formation of disulfide bridges within the SP1 dodecamer, concurrent with increased melting point. A six-histidine tag was introduced at the N-terminus of SP1 to generate 6HSP1, and the DeltaNSP1 mutant was generated by a deletion of amino acids 2-6 at the N-terminus. Both 6HSP1 and DeltaNSP1 maintained their ability to assemble a stable dodecamer. Remarkably, these SP1 homo-dodecamers were able to re-assemble into stable hetero-dodecamers following co-electro-elution from SDS-PAGE. The exceptional stability of the SP1-nano ring and its ability to self-assemble hetero-complexes paves the way to further research in utilizing this unique protein in nano-biotechnology.

Crystallization↗
PubMed2006

Common and divergent roles for members of the mouse DCX superfamily.

The doublecortin-like (DCX) domains serve as protein-interaction platforms. DCX tandem domains appear in the product of the X-linked doublecortin (DCX) gene, in retinitis pigmentosa-1 (RP1), as well as in other gene products. Mutations in the human DCX gene are associated with abnormal neuronal migration, epilepsy, and mental retardation; mutations in RP1 are associated with a form of inherited blindness, while DCDC2 has been associated with dyslectic reading disabilities. Motivated by the possible importance of this gene family, a thorough analysis to detect all family members in the mouse was conducted. The DCX-repeat gene superfamily is composed of eleven paralogs, and we cloned the DCX domains from nine different genes. Our study questioned which functions attributed to the DCX domain, are conserved among the different members. Our results suggest that the proteins with the DCX-domain have conserved and unique roles in microtubule regulation and signal transduction. All the tested proteins stimulated microtubule assembly in vitro. Proteins with tandem repeats stabilized the microtubule cytoskeleton in transfected cells, while those with single repeats localized to actin-rich subcellular structures, or the nucleus. All tested proteins interacted with components of the JNK/MAP-kinase pathway, while only a subset interacted with Neurabin 2, and a nonoverlapping group demonstrated actin association. The sub-specialization of some members due to confined intracellular localization, and protein interactions may explain the success of this superfamily.

Amino Acid Sequence↗
PubMed2004

Three-dimensional reconstruction of Agrobacterium VirE2 protein with single-stranded DNA.

Agrobacterium tumefaciens infects plant cells by a unique mechanism involving an interkingdom genetic transfer. A single-stranded DNA substrate is transported across the two cell walls along with the bacterial virulence proteins VirD2 and VirE2. A single VirD2 molecule covalently binds to the 5'-end of the single-stranded DNA, while the VirE2 protein binds stoichiometrically along the length of the DNA, without sequence specificity. An earlier transmission/scanning transmission electron microscopy study indicated a solenoidal ("telephone coil") organization of the VirE2-DNA complex. Here we report a three-dimensional reconstruction of this complex using electron microscopy and single-particle image-processing methods. We find a hollow helical structure of 15.7-nm outer diameter, with a helical rise of 51.5 nm and 4.25 VirE2 proteins/turn. The inner face of the protein units contains a continuous wall and an inward protruding shelf. These structures appear to accommodate the DNA binding. Such a quaternary arrangement naturally sequesters the DNA from cytoplasmic nucleases and suggests a mechanism for its nuclear import by decoration with host cell factors. Coexisting with the helices, we also found VirE2 tetrameric ring structures. A two-dimensional average of the latter confirms the major features of the three-dimensional reconstruction.

Active Transport, Cell Nucleus↗
PubMed2004

Structure of the DNA-SspC complex: implications for DNA packaging, protection, and repair in bacterial spores.

Bacterial spores have long been recognized as the sturdiest known life forms on earth, revealing extraordinary resistance to a broad range of environmental assaults. A family of highly conserved spore-specific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP), plays a major role in mediating spore resistance. The mechanism by which these proteins exert their protective activity remains poorly understood, in part due to the lack of structural data on the DNA-SASP complex. By using cryoelectron microscopy, we have determined the structure of the helical complex formed between DNA and SspC, a characteristic member of the alpha/beta-type SASP family. The protein is found to fully coat the DNA, forming distinct protruding domains, and to modify DNA structure such that it adopts a 3.2-nm pitch. The protruding SspC motifs allow for interdigitation of adjacent DNA-SspC filaments into a tightly packed assembly of nucleoprotein helices. By effectively sequestering DNA molecules, this dense assembly of filaments is proposed to enhance and complement DNA protection obtained by DNA saturation with the alpha/beta-type SASP.

Bacillus subtilis↗