Completion of the Vimentin Rod Domain Structure Using Experimental Restraints: A New Tool for Exploring Intermediate Filament Assembly and Mutations

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Protein folding by the effects of macromolecular crowding

Unfolded states of ribonuclease A were used to investigate the effects of macromolecular crowding on macromolecular compactness and protein folding. The extent of protein folding and compactness were measured by circular dichroism spectroscopy, fluorescence correlation spectroscopy, and NMR spectroscopy in the presence of polyethylene glycol (PEG) or Ficoll as the crowding agent. The unfolded state of RNase A in a 2.4 M urea solution at pH 3.0 became native in conformation and compactness by the addition of 35% PEG 20000 or Ficoll 70. In addition, the effects of macromolecular crowding on inert macromolecule compactness were investigated by fluorescence correlation spectroscopy using Fluorescence-labeled PEG as a test macromolecule. The size of Fluorescence-labeled PEG decreased remarkably with an increase in the concentration of PEG 20000 or Ficoll 70. These results show that macromolecules are favored compact conformations in the presence of a high concentration of macromolecules and indicate the importance of a crowded environment for the folding and stabilization of globular proteins. Furthermore, the magnitude of the effects on macromolecular crowding by the different sizes of background molecules was investigated. RNase A and Fluorescence-labeled PEG did not become compact, and had folded conformation by the addition of PEG 200. The effect of the chemical potential on the compaction of a test molecule in relation to the relative sizes of the test and background molecules is also discussed.     

A structural-informatics approach for mining beta-sheets: locating sheets in intermediate-resolution density maps

Here, we report a new computational method, called sheetminer, for mining beta-sheets in the density maps at intermediate resolutions of 6 to 10A. The method employs a multi-step ad hoc morphological analysis of density maps to identify the unique characteristics of beta-sheets. It was tested on density maps from 12 protein crystal structures that were artificially blurred to intermediate resolutions. There are a total of 35 independent beta-sheets with a wide distribution of morphology. The method successfully located 34 of them and missed only one. The method was also applied to an experimental 9A electron cryomicroscopic structure and an 8A X-ray density map. In both cases, the sheet-searching results were found to agree very well with known high-resolution crystal structures. Collectively, these results demonstrate clearly the robustness of sheetminer in locating the regions belonging to beta-sheets in the intermediate-resolution density maps. Furthermore, sheetminer is completely complementary to all other existing computational methods, including helixhunter and threading algorithms. Their combined usage has the potential to significantly enhance the computational modeling capacity for a much more complete interpretation of structural data at intermediate resolutions, from which extraction of functional information would be more effective. This is particularly important in the field of structural genomics, in which the fast screening approach may not always yield crystals that diffract to atomic resolution. An exciting future application of sheetminer is as a valuable tool for revealing the structures of amyloid fibrils that are rich in beta-motifs.     

Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana

Plant viruses must enter the host vascular system in order to invade the young growing parts of the plant rapidly. Functional entry sites into the leaf vascular system for rapid systemic infection have not been determined for any plant/virus system. Tobacco mosaic virus (TMV) entry into minor, major and transport veins from non-vascular cells of Nicotiana benthamiana in source tissue and its exit from veins in sink tissue was studied using a modified virus expressing green fluorescent protein (GFP). Using a surgical procedure that isolated specific leaf and stem tissues from complicating vascular tissues, we determined that TMV could enter minor, major or transport veins directly from non-vascular cells to produce a systemic infection. TMV first accumulated in abaxial or external phloem-associated cells in major veins and petioles of the inoculated leaf and stems below the inoculated leaf. It also initially accumulated exclusively in internal or adaxial phloem-associated cells in stems above the inoculated leaf and petioles or major veins of sink leaves. This work shows the functional equivalence of vein classes in source leaves for entry of TMV, and the lack of equivalence of vein classes in sink leaves for exit of TMV. Thus, the specialization of major veins for transport rather than loading of photoassimilates in source tissue does not preclude virus entry. During transport, the virus initially accumulates in specific vascular-associated cells, indicating that virus accumulation in this tissue is highly regulated. These findings have important implications for studies on the identification of symplasmic domains and host macromolecule vascular transport.     

Phage P22 procapsids equilibrate with free coat protein subunits

Assembly of bacteriophage P22 procapsids has long served as a model for assembly of spherical viruses. Historically, assembly of viruses has been viewed as a non-equilibrium process. Recently alternative models have been developed that treat spherical virus assembly as an equilibrium process. Here we have investigated whether P22 procapsid assembly reactions achieve equilibrium or are irreversibly trapped. To assemble a procapsid-like particle in vitro, pure coat protein monomers are mixed with scaffolding protein. We show that free subunits can exchange with assembled structures, indicating that assembly is a reversible, equilibrium process. When empty procapsid shells (procapsids with the scaffolding protein stripped out) were diluted so that the concentration was below the dissociation constant ( approximately 5 microM) for coat protein monomers, free monomers were detected. The released monomers were assembly-competent; when NaCl was added to metastable partial capsids that were aged for an extended period, the released coat subunits were able to rapidly re-distribute from the partial capsids and form whole procapsids. Lastly, radioactive monomeric coat subunits were able to exchange with the subunits from empty procapsid shells. The data presented illustrate that coat protein monomers are able to dissociate from procapsids in an active state, that assembly of procapsids is consistent with reactions at equilibrium and that the reaction follows the law of mass action.     

Hydrogen exchange of disordered proteins in Escherichia coli

A truly disordered protein lacks a stable fold and its backbone amide protons exchange with solvent at rates predicted from studies of unstructured peptides. We have measured the exchange rates of two model disordered proteins, FlgM and α-synuclein, in buffer and in Escherichia coli using the NMR experiment, SOLEXSY. The rates are similar in buffer and cells and are close to the rates predicted from data on small, unstructured peptides. This result indicates that true disorder can persist inside the crowded cellular interior and that weak interactions between proteins and macromolecules in cells do not necessarily affect intrinsic rates of exchange.     

Synthetic symmetrization in the crystallization and structure determination of CelA from Thermotoga maritima

Protein crystallization continues to be a major bottleneck in X‐ray crystallography. Previous studies suggest that symmetric proteins, such as homodimers, might crystallize more readily than monomeric proteins or asymmetric complexes. Proteins that are naturally monomeric can be made homodimeric artificially. Our approach is to create homodimeric proteins by introducing single cysteines into the protein of interest, which are then oxidized to form a disulfide bond between the two monomers. By introducing the single cysteine at different sequence positions, one can produce a variety of synthetically dimerized versions of a protein, with each construct expected to exhibit its own crystallization behavior. In earlier work, we demonstrated the potential utility of the approach using T4 lysozyme as a model system. Here we report the successful application of the method to Thermotoga maritima CelA, a thermophilic endoglucanase enzyme with low sequence identity to proteins with structures previously reported in the Protein Data Bank. This protein had resisted crystallization in its natural monomeric form, despite a broad survey of crystallization conditions. The synthetic dimerization of the CelA mutant D188C yielded well‐diffracting crystals with molecules in a packing arrangement that would not have occurred with native, monomeric CelA. A 2.4 Å crystal structure was determined by single anomalous dispersion using a seleno‐methionine derivatized protein. The results support the notion that synthetic symmetrization can be a useful approach for enlarging the search space for crystallizing monomeric proteins or asymmetric complexes.