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.
Enzymes and motor proteins are dynamic macromolecules that coexist in a number of conformations of similar energies. Protein function is usually accompanied by a change in structure and flexibility, often induced upon binding to ligands. However, while measuring protein flexibility changes between active and resting states is of therapeutic significance, it remains a challenge. Recently, our group has demonstrated that breadth of signal amplitudes in measured electrical signatures as an ensemble of individual protein molecules is driven through solid-state nanopores and correlates with protein conformational dynamics. Here, we extend our study to resolve subtle flexibility variation in dihydrofolate reductase mutants from unlabeled single molecules in solution. We first demonstrate using a canonical protein system, adenylate kinase, that both size and flexibility changes can be observed upon binding to a substrate that locks the protein in a closed conformation. Next, we investigate the influence of voltage bias and pore geometry on the measured electrical pulse statistics during protein transport. Finally, using the optimal experimental conditions, we systematically study a series of wild-type and mutant dihydrofolate reductase proteins, finding a good correlation between nanopore-measured protein conformational dynamics and equilibrium bulk fluorescence probe measurements. Our results unequivocally demonstrate that nanopore-based measurements reliably probe conformational diversity in native protein ensembles.
Growing evidence points toward a very dynamic role for metals in biology. This suggests that physiological circumstance may mandate metal ion redistribution among ligands. This work addresses a critical need for technology that detects, identifies, and measures the metal-containing components of complex biological matrixes. We describe a direct, user-friendly approach for identifying and quantifying metal-protein adducts in complex samples using native- or SDS-PAGE, blotting, and rapid synchrotron X-ray fluorescence mapping with micro-XANES (X-ray absorption near-edge structure) of entire blots. The identification and quantification of each metal bound to a protein spot has been demonstrated, and the technique has been applied in two exemplary cases. In the first, the speciation of the in vitro binding of exogenous chromium to blood serum proteins was influenced markedly by both the oxidation state of chromium exposed to the serum proteins and the treatment conditions, which is of relevance to the biochemistry of Cr dietary supplements. In the second case, in vivo changes in endogenous metal speciation were examined to probe the influence of oxygen depletion on iron speciation in Shewanella oneidensis.
The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares ϳ25% sequence identity with gp160 from the human immunodeficiency virus, type I, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g. CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.
A strategy to overexpress T cell receptors (TCRs) in Lec3.2.8.1 cells has been developed using the "Velcro" leucine zipper sequence to facilitate ␣- pairing. Upon secretion in culture media, the VSV-8-specific/H2-K b -restricted N15 TCR could be readily immunopurified using the anti-leucine zipper monoclonal antibody 2H11, with a yield of 5-10 mg/liter. Mass , with the former diffracting to 2.8-Å resolution. These findings show that neither intact glycans nor the conserved and partially exposed Cys-183 is required for protein stability. Furthermore, our results suggest that the H57 Fab fragment aids in the crystallization of TCRs by altering their molecular surface and/or stabilizing inherent conformational mobility.The TCR 1 complex consists of multiple transmembrane polypeptide chains on the surface of T lymphocytes (1-3). The disulfide-linked ␣ heterodimer (Ti) is the clonally unique component that possesses a recognition site for antigen in the context of a major histocompatibility complex protein (4, 5). Sequence analysis of ␣ and  subunits strongly supports that their ectodomains form a recognition unit reminiscent of an immunoglobulin Fab fragment (6 -8). This notion has been confirmed in crystallographic studies of TCR subunit fragments (9, 10). On the other hand, the invariant CD3 components (␥, ␦, ⑀, , and ) possess lengthy cytoplasmic tails containing immune cell tyrosine-based activation motifs and are involved in signal transduction (11, 12). To date, most of the attributes of TCR recognition have been studied largely indirectly because of the intimate membrane association of this complex.To understand the process by which T cells recognize pathogens in explicit molecular terms, recent efforts have begun to focus on the structural nature of the TCR. However, many efforts to express soluble TCR ␣ heterodimers in both prokaryotic and eukaryotic systems have been hampered by inefficient pairing of ␣ and  subunits in the absence of their respective transmembrane regions and associated CD3 components (reviewed in Refs. 13-21). We have recently developed a methodology to overcome this obstacle by adding 30-amino acid-long segments to the carboxyl termini of ␣ and  extracellular domains via a thrombin-cleavable flexible linker (22). These peptide segments (Base-p1 for ␣ and Acid-p1 for ) were previously shown to selectively associate to form a stable heterodimeric coiled coil termed the leucine zipper (23). Homodimeric structures are not favored due to the electrostatic repulsion among amino acid side chains. Furthermore, the yield of these engineered proteins was 5-10-fold greater than that of the TCR expressed in the absence of the synthetic leucine zipper (22). Through the use of a panel of mAbs directed at native ␣ and  epitopes within constant and variable regions, it was further shown that the fusion heterodimer was native.Efforts to obtain TCR crystals from such material, however, were unsuccessful, perhaps owing to the glycosylation heterogeneity inherent in the baculovirus system and/or ...
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