sugar transport ͉ phosphorylation ͉ x-ray crystallography T he phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) (1) catalyzes the synchronized uptake and phosphorylation of a number of carbohydrates in eubacteria (group translocation) (2, 3). With some variations, the PTS comprises three proteins. In the cytoplasm, PEP phosphorylates enzyme I (EI), which then transfers the phosphoryl group to the histidine phosphocarrier protein, HPr. From HPr, the phosphoryl group is transferred to various sugar-specific membrane associated transporters [enzyme II (EII)], each comprising two cytoplasmic domains, EIIA and EIIB, and an integral membrane domain EIIC. Within EII, EIIA accepts the phosphoryl group from HPr and donates it to EIIB, whereupon EIIC mediates sugar translocation. In addition to controlling sugar translocation, the phosphorylation state of PTS proteins is associated with regulation of metabolic pathways and signaling in bacterial cells (4-8).The Ϸ64-kDa EI is a homodimer, which is more tightly associated at the phosphorylated state than the unphosphorylated state (9-14). The phosphorylation by PEP requires Mg 2ϩ and targets the N atom of His-189 (numbering scheme of EI from Escherichia coli) (15). The dimer association rate constant is two to three orders of magnitude slower than typical rates measured for other dimeric proteins, suggesting that oligomerization is accompanied by major conformational rearrangements (13,16,17). The monomer-dimer equilibrium has been studied in vitro by various methods (18-21), and it has been proposed that the transition plays a regulatory role in the PEP:sugar phosphotransferase system. Yet, transient kinetic studies indicated that the EI dimer phosphorylates HPr without dissociating into monomers (17).Proteolytic cleavage of EI produces two domains (22, 23). The EI N-terminal domain (EIN, residues 1-230) contains the residue that transfers the phosphoryl group, 24) and the HPr-binding domain, whereas the EI C-terminal domain (EIC, residues 261-575) binds PEP in the presence of Mg 2ϩ (the PEP-binding domain) (22,25) and mediates dimerization (26,27). Site-directed mutagenesis showed that Cys-502, located on EIC, is essential for phosphorylation of His-189 by PEP (28). The structure of EIN from E. coli has been determined by x-ray crystallography (29) and NMR spectroscopy (30).

Each T cell receptor (TCR) recognizes a peptide antigen bound to a major histocompatibility complex (MHC)molecule via a clonotypic αβ heterodimeric structure (Ti) non-covalently associated with the monomorphic CD3 signaling components. A crystal structure of an αβ TCR-anti-TCR Fab complex shows an Fab fragment derived from the H57 monoclonal antibody (mAb), interacting with the elongated FG loop of the Cβ domain, situated beneath the Vβ domain. This loop, along with the partially exposed ABED β sheet of Cβ, and glycans attached to both Cβ and Cα domains, forms a cavity of sufficient size to accommodate a single non-glycosylated Ig domain such as the CD3ε ectodomain. That this asymmetrically localized site is embedded within the rigid constant domain module has implications for the mechanism of signal transduction in both TCR and pre-TCR complexes. Furthermore, quaternary structures of TCRs vary significantly even when they bind the same MHC molecule, as manifested by a unique twisting of the V module relative to the C module.

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Measurements of mechanical properties of biological cells are of great importance because changes in these properties can be strongly associated with the progression of cell differentiation and cell diseases. Although state of the art methods, such as atomic force microscopy, optical tweezers and micropipette aspiration, have been widely used to measure the mechanical properties of biological cells, all these methods involve direct contact with the cell and the measurements could be affected by the contact or any local deformation. In addition, all these methods typically deduced the Young's modulus of the cells based on their measurements. Herein, we report a new method for fast and direct measurement of the compressibility or bulk modulus of various cell lines on a microchip. In this method, the whole cell is exposed to acoustic radiation force without any direct contact. The method exploits the formation of an acoustic standing wave within a straight microchannel. When the polystyrene beads and cells are introduced into the channel, the acoustic radiation force moves them to the acoustic pressure node and the movement speed is dependent on the compressibility. By fitting the experimental and theoretical trajectories of the beads and the cells, the compressibility of the cells can be obtained. We find that the compressibility of various cancer cells (MCF-7: 4.22 ± 0.19 × 10(-10) Pa(-1), HEPG2: 4.28 ± 0.12 × 10(-10) Pa(-1), HT-29: 4.04 ± 0.16 × 10(-10) Pa(-1)) is higher than that of normal breast cells (3.77 ± 0.09 × 10(-10) Pa(-1)) and fibroblast cells (3.78 ± 0.17 × 10(-10) Pa(-1)). This work demonstrates a novel acoustic-based method for on-chip measurements of cell compressibility, complementing existing methods for measuring the mechanical properties of biological cells.