Pathogenic Gram-negative bacteria use a type three secretion system (TTSS) to deliver virulence factors into host cells. Although the order in which proteins incorporate into the growing TTSS is well described, the underlying assembly mechanisms are still unclear. Here we show that the TTSS needle protomer refolds spontaneously to extend the needle from the distal end. We developed a functional mutant of the needle protomer from Shigella flexneri and Salmonella typhimurium to study its assembly in vitro. We show that the protomer partially refolds from -helix into -strand conformation to form the TTSS needle. Reconstitution experiments show that needle growth does not require ATP. Thus, like the structurally related flagellar systems, the needle elongates by subunit polymerization at the distal end but requires protomer refolding. Our studies provide a starting point to understand the molecular assembly mechanisms and the structure of the TTSS at atomic level.

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Bis(2,4,6-tri-tert-butylphenyl)germylene (1), which had been reported to undergo an insertion of the germanium atom into a C−H bond of an o-tert-butyl group at room temperature, was found to be quite stable. Such a C−H insertion does occur in solution, forming germaindane 2, but only in the presence of a Lewis acid such as the starting material GeCl2. 1 can be stored unchanged at −30 °C for months; at 20 °C it decomposes within several weeks under release of 1,3,5-tri-tert-butylbenzene. Compound 1 was characterized by X-ray diffraction methods. The X-ray structure of 1 shows that the two aryl ligands have very different geometries:  one aryl group is greatly distorted to give a “boat” form and the Ge atom is remote from any of the ring planes, while the second aryl group is only slightly deformed and the Ge atom lies in the ring plane. UV/vis spectra exhibit an intense absorption at 430 nm in solution and at 405 nm in the solid state, which represents an enormous hypsochromic shift compared to other germylenes with bulky aryl ligands. Oxidation of 1 with trimethylamine N-oxide leads to a germanone, which rearranges very rapidly by a C−H insertion process yielding the germaindanol 3.

The stability against gamma radiation of MeTODGA (methyl tetraoctyldiglycolamide) and Me2TODGA (dimethyl tetraoctyldiglycolamide), derivatives from the well-known extractant TODGA (N,N,N',N'-tetraoctyldiglycolamide), were studied and compared. Solutions of MeTODGA and Me2TODGA in alkane diluents were subjected to (60)Co γ-irradiation in the presence and absence of nitric acid and analyzed using LC-MS to determine their rates of radiolytic concentration decrease, as well as to identify radiolysis products. The results of product identification from three different laboratories are compared and found to be in good agreement. The diglycolamide (DGA) concentrations decreased exponentially with increasing absorbed dose. The MeTODGA degradation rate constants (dose constants) were uninfluenced by the presence of nitric acid, but the acid increased the rate of degradation for Me2TODGA. The degradation products formed by irradiation are also initially produced in greater amounts in acid-contacted solution, but products may also be degraded by continued radiolysis. The identified radiolysis products suggest that the weakest bonds are those in the diglycolamide center of these molecules.

Ler, a member of the H-NS protein family, is the master regulator of the LEE pathogenicity island in virulent Escherichia coli strains. Here, we determined the structure of a complex between the DNA-binding domain of Ler (CT-Ler) and a 15-mer DNA duplex. CT-Ler recognizes a preexisting structural pattern in the DNA minor groove formed by two consecutive regions which are narrower and wider, respectively, compared with standard B-DNA. The compressed region, associated with an AT-tract, is sensed by the side chain of Arg90, whose mutation abolishes the capacity of Ler to bind DNA. The expanded groove allows the approach of the loop in which Arg90 is located. This is the first report of an experimental structure of a DNA complex that includes a protein belonging to the H-NS family. The indirect readout mechanism not only explains the capacity of H-NS and other H-NS family members to modulate the expression of a large number of genes but also the origin of the specificity displayed by Ler. Our results point to a general mechanism by which horizontally acquired genes may be specifically recognized by members of the H-NS family.

The HIV‐1 encoded virus protein U (VpU) is required for efficient viral release from human host cells and for induction of CD4 degradation in the endoplasmic reticulum. The cytoplasmic domain of the membrane protein VpU (VpUcyt) is essential for the latter activity. The structure and dynamics of VpUcyt were characterized in the presence of membrane simulating dodecylphosphatidylcholine (DPC) micelles by high‐resolution liquid state NMR. VpUcyt is unstructured in aqueous buffer. The addition of DPC micelles induces a well‐defined membrane proximal α‐helix (residues I39–E48) and an additional helical segment (residues L64–R70). A tight loop (L73–V78) is observed close to the C‐terminus, whereas the interhelical linker (R49–E63) remains highly flexible. A 3D structure of VpUcyt in the presence of DPC micelles was calculated from a large set of proton–proton distance constraints. The topology of micelle‐associated VpUcyt was derived from paramagnetic relaxation enhancement of protein nuclear spins after the introduction of paramagnetic probes into the interior of the micelle or the aqueous buffer. Qualitative analysis of secondary chemical shift and paramagnetic relaxation enhancement data in conjunction with dynamic information from heteronuclear NOEs and structural insight from homonuclear NOE‐based distance constraints indicated that micelle‐associated VpUcyt retains a substantial degree of structural flexibility.

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The 2,4-di-tert-butyl-6-(N,N-dimethylaminomethyl)phenyl (Mamx) ligand allows the synthesis of the germylenes MamxGeCl (1), Mamx2Ge (2), Mamx(Tip)Ge (3; Tip = 2,4,6-iPr3C6H2), MamxGeN3 (4), MamxGeN(SiMe3)2 (5), and MamxGe[Fe(CO)2Cp*] (6), which are all stabilized by coordination of the amino side chain. Reactions of 2 and 3 with MeI yield the ionic compounds Mamx(Tip)(Me)Ge+I- (7) and Mamx2(Me)Ge+I- (8), respectively; the X-ray crystal structure of 7 shows a trigonal-planar coordination at the Ge center and a nearly perpendicular Ge−N bond.