We describe algorithms for solving the Lamm equations for the reaction-diffusion-sedimentation process in analytical ultracentrifugation, and examine the potential and limitations for fitting experimental data. The theoretical limiting case of a small, uniformly distributed ligand rapidly reacting with a larger protein in a "constant bath" of the ligand is recapitulated, which predicts the reaction boundary to sediment with a single sedimentation and diffusion coefficient. As a consequence, it is possible to express the sedimentation profiles of reacting systems as c(s) distribution of noninteracting Lamm equation solutions, deconvoluting the effects of diffusion. For rapid reactions, the results are quantitatively consistent with the "constant bath" approximation, showing c(s) peaks at concentration-dependent positions. For slower reactions, the deconvolution of diffusion is still partially successful, with c(s) resolving peaks that reflect the populations of sedimenting species. The transition between c(s) peaks describing reaction boundaries of moderately strong interactions (K(D) approximately 10(-6) M) or resolving sedimenting species was found to occur in a narrow range of dissociation rate constant between 10(-3) and 10(-4) s(-1). The integration of the c(s) peaks can lead to isotherms of species populations or s-value of the reaction boundary, respectively, which can be used for the determination of the equilibrium binding constant.

The cell wall of Mycobacterium tuberculosis is composed of unique lipids that are important for pathogenesis. Indeed, the first-ever genetic screen in M. tuberculosis identified genes involved in the biosynthesis and transport of the cell wall lipid PDIM (phthiocerol dimycocerosates) as crucial for the survival of M. tuberculosis in mice. Here we show evidence for a novel molecular mechanism of the PDIM-mediated virulence in M. tuberculosis. We characterized the DNA interaction and the regulon of Rv3167c, a transcriptional repressor that is involved in virulence regulation of M. tuberculosis, and discovered that it controls the PDIM operon. A loss-of-function genetic approach showed that PDIM levels directly correlate with the capacity of M. tuberculosis to escape the phagosome and induce host cell necrosis and macroautophagy. In conclusion, our study attributes a novel role of the cell wall lipid PDIM in intracellular host cell modulation, which is important for host cell exit and dissemination of M. tuberculosis.

Variable lymphocyte receptors (VLRs) are leucine-rich repeat (LRR) proteins that mediate adaptive immunity in jawless vertebrates. VLRs are fundamentally different from the antibodies of jawed vertebrates, which consist of immunoglobulin (Ig) domains. We determined the structure of an anti-hen egg white lysozyme (HEL) VLRB, isolated by yeast display, bound to HEL. The VLR, whose affinity resembles that of IgM antibodies, uses nearly all its concave surface to bind the protein, in addition to a loop that penetrates into the enzyme active site. The VLR-HEL structure, combined with sequence analysis, revealed an almost perfect match between ligand-contacting positions and positions with highest sequence diversity. Thus, we have defined the generalized antigen-binding site of VLRs. We further demonstrated that VLRs can be affinity-matured to affinities as high as those of IgG antibodies, making VLRs potential alternatives to antibodies for biotechnology applications.

Protein interactions can promote the reversible assembly of multiprotein complexes, which have been identified as critical elements in many regulatory processes in cells. The biophysical characterization of assembly products, their number and stoichiometry, and the dynamics of their interactions in solution can be very difficult. A classical first-principle approach for the study of purified proteins and their interactions is sedimentation velocity analytical ultracentrifugation. This approach allows one to distinguish different protein complexes based on their migration in the centrifugal field without isolating reversibly formed complexes from the individual components. An important existing limitation for systems with multiple components and assembly products is the identification of the species associated with the observed sedimentation rates. We developed a computational approach for integrating multiple optical signals into the sedimentation coefficient distribution analysis of components, which combines the size-dependent hydrodynamic separation with discrimination of the extinction properties of the sedimenting species. This approach allows one to deduce the stoichiometry and to assign the identity of the assembly products without prior assumptions of the number of species and the nature of their interaction. Although chromophoric labels may be used to enhance the spectral resolution, we demonstrate the ability to work label-free for three-component protein mixtures. We observed that the spectral discrimination can synergistically enhance the hydrodynamic resolution. This method can take advantage of differences in the absorbance spectra of interacting solution components, for example, for the study of protein-protein, protein-nucleic acid or protein-small molecule interactions, and can determine the size, hydrodynamic shape, and stoichiometry of multiple complexes in solution.protein interactions ͉ size distribution

adaptive immunity ͉ agnatha ͉ somatic rearrangement

2B4 belongs to the CD2 family of molecules and is expressed on all NK, γδ, and memory CD8+ (αβ) T cells. The murine NK receptor 2B4 exhibits both inhibitory and activating functions, whereas human 2B4 has been reported to be an activating molecule. How murine 2B4 can act both as an activating and inhibitory receptor and what distinguishes its function from human 2B4 have remained largely unknown. We use here a model system that allows the study of human and murine 2B4 under identical and controlled conditions. These studies reveal that both human and mouse 2B4 can activate or inhibit NK cells. We show here that the level of 2B4 expression and the degree of 2B4 cross-linking play a significant role in the regulation of signaling lymphocyte activation molecule-associated protein-mediated activation by 2B4. A high level of 2B4 expression, heavy cross-linking, and relative paucity of signaling lymphocyte activation molecule-associated protein promote inhibitory function. Our studies demonstrate how a single receptor can have opposing function depending on the degree of receptor expression, extent of its ligation, and the relative abundance of certain adaptor molecules. Because the levels of 2B4 and CD48 are dynamically regulated, these findings have implications for the regulation of NK cell function.

This study evaluated the cellular immune response against Brucella species cytoplasmic protein (CP) in peripheral blood mononuclear cells (PBMC) of 25 patients with brucellosis. In vitro proliferation and cytokine gene expression and production were investigated. PBMC from 14 patients proliferated in response to CP (responder patients [RPs]) and cells from 11 patients did not (nonresponder patients [NRPs]). CP-specific interleukin (IL)-2 and interferon-gamma were significantly induced in PBMC from RPs, compared with cells from NRPs. No significant differences were found in the production of IL-10 between the 2 groups. CP did not induce IL-4 production. A close relationship was observed between the clinical status of the patients and the T cell response against CP. Patient with acute infections responded to CP and induced production of T helper 1 (Th1) cytokines, whereas chronically infected patients did not. Diminished production of Th1 cytokines may contribute to T cell unresponsiveness in chronic human brucellosis.

No abstract