We constructed a microscope-based instrument capable of simultaneous, spatially coincident optical trapping and single-molecule fluorescence. The capabilities of this apparatus were demonstrated by studying the force-induced strand separation of a dye-labeled, 15-base-pair region of double-stranded DNA (dsDNA), with force applied either parallel ('unzipping' mode) or perpendicular ('shearing' mode) to the long axis of the region. Mechanical transitions corresponding to DNA hybrid rupture occurred simultaneously with discontinuous changes in the fluorescence emission. The rupture force was strongly dependent on the direction of applied force, indicating the existence of distinct unbinding pathways for the two force-loading modes. From the rupture force histograms, we determined the distance to the thermodynamic transition state and the thermal off rates in the absence of load for both processes.

CRAC channels generate Ca 2؉ signals critical for the activation of immune cells and exhibit an intriguing pore profile distinguished by extremely high Ca 2؉ selectivity, low Cs ؉ permeability, and small unitary conductance. To identify the ion conduction pathway and gain insight into the structural bases of these permeation characteristics, we introduced cysteine residues in the CRAC channel pore subunit, Orai1, and probed their accessibility to various thiolreactive reagents. Our results indicate that the architecture of the ion conduction pathway is characterized by a flexible outer vestibule formed by the TM1-TM2 loop, which leads to a narrow pore flanked by residues of a helical TM1 segment. Residues in TM3, and specifically, E190, a residue considered important for ion selectivity, are not close to the pore. Moreover, the outer vestibule does not significantly contribute to ion selectivity, implying that Ca 2؉ selectivity is conferred mainly by E106. The ion conduction pathway is sufficiently narrow along much of its length to permit stable coordination of Cd 2؉ by several TM1 residues, which likely explains the slow flux of ions within the restrained geometry of the pore. These results provide a structural framework to understand the unique permeation properties of CRAC channels.Orai1 ͉ STIM1 ͉ store-operated channels C a 2ϩ release-activated Ca 2ϩ (CRAC) channels are the principal route of Ca 2ϩ entry in immune cells and orchestrate functions such as gene expression, motility, and the release of inflammatory mediators (1). Mutations in CRAC channels give rise to devastating immunodeficiencies and abnormalities in muscle, skin, and teeth, highlighting their importance for various organ systems (1). The recent discoveries of STIM1 (the ER Ca 2ϩ sensor), and Orai1 (the CRAC channel pore subunit) have provided major breakthroughs to illuminate the molecular basis of CRAC channel function (2). However, while the identification of these proteins has produced rapid progress in our understanding of the cellular events underlying channel activation, the molecular mechanisms of ion selectivity and permeation remain unclear.CRAC channels are distinguished by an extraordinarily high selectivity for Ca 2ϩ over monovalent ions (P Ca /P Na Ͼ 1,000), a very low unitary conductance (Ͻ1 pS), and low permeability to Cs ϩ and larger monovalent cations (3). The structural underpinnings of these characteristics have been the focus of much debate but are largely unknown. As with most ion channels, the pore properties of CRAC channels are likely shaped by the arrangement and chemistry of pore-lining residues. Thus, to understand the basis of the unique permeation properties of CRAC channels, the residues lining the ion transport pathway need to be elucidated.Orai1 bears little sequence homology to other ion channel proteins, and consequently, there are few clues regarding the contribution of the different parts of the molecule for pore formation. Electrophysiological studies indicate that the exquisite Ca 2ϩ selectivity of CRAC...

Single-molecule measurements of the activities of a variety of enzymes show that rates of catalysis may vary markedly between different molecules in putatively homogenous enzyme preparations. We measured the rate at which purified Escherichia coli RNA polymerase moves along a ϳ2650-bp DNA during transcript elongation in vitro at 0.5 mM nucleoside triphosphates. Individual molecules of a specifically biotinated RNA polymerase derivative were tagged with 199-nm diameter avidin-coated polystyrene beads; enzyme movement along a surface-linked DNA molecule was monitored by observing changes in bead Brownian motion by light microscopy. The DNA was derived from a naturally occurring transcription unit and was selected for the absence of regulatory sequences that induce lengthy pausing or termination of transcription. With rare exceptions, individual enzyme molecules moved at a constant velocity throughout the transcription reaction; the distribution of velocities across a population of 140 molecules was unimodal and was well fit by a Gaussian. However, the width of the Gaussian, ‫؍‬ 6.7 bp/s, was considerably larger than the precision of the velocity measurement (1 bp/s). The observations show that different transcription complexes have differences in catalytic rate (and thus differences in structure) that persist for thousands of catalytic turnovers. These differences may provide a parsimonious explanation for the complex transcription kinetics observed in bulk solution.Different individual molecules within a purified enzyme or ribozyme preparation can display greatly differing rate constants for catalytic turnover (1-3). Some of this variability may be due to the presence of enzyme molecules with differing covalent structures; these may arise from the presence of two or more related genes, from alternative splicing of a single pre-mRNA, or from post-translational protein modifications (4). However, some heterogeneity may also arise from the adoption by enzyme molecules with identical covalent structures of different conformational states that persist through multiple catalytic turnovers (5, 6). It has been proposed that such conformational heterogeneity may be a heretofore unappreciated feature of macromolecular catalysts in general (2).Bacterial RNA polymerases (RNAPs), 1 and their structurally and mechanistically similar homologs, eukaryotic RNAP II enzymes, are large, multisubunit proteins that synthesize all cellular mRNAs (7,8). These enzymes are processive; a given RNA molecule is synthesized in its entirety by the same RNAP molecule. In bacterial RNAPs, most RNA synthesis takes place in a stable transcription elongation complex (TEC) containing the core RNAP subunits, the template DNA, and the nascent RNA transcript. The TEC structure has been extensively characterized (9 -12). A variety of proteins and DNA sequence elements in both prokaryotes and eukaryotes regulate gene expression by altering the extent to which active TECs terminate transcription upstream of or within genes (13-15). The efficiency of termi...

CRAC channels generate Ca 2؉ signals critical for the activation of immune cells and exhibit an intriguing pore profile distinguished by extremely high Ca 2؉ selectivity, low Cs ؉ permeability, and small unitary conductance. To identify the ion conduction pathway and gain insight into the structural bases of these permeation characteristics, we introduced cysteine residues in the CRAC channel pore subunit, Orai1, and probed their accessibility to various thiolreactive reagents. Our results indicate that the architecture of the ion conduction pathway is characterized by a flexible outer vestibule formed by the TM1-TM2 loop, which leads to a narrow pore flanked by residues of a helical TM1 segment. Residues in TM3, and specifically, E190, a residue considered important for ion selectivity, are not close to the pore. Moreover, the outer vestibule does not significantly contribute to ion selectivity, implying that Ca 2؉ selectivity is conferred mainly by E106. The ion conduction pathway is sufficiently narrow along much of its length to permit stable coordination of Cd 2؉ by several TM1 residues, which likely explains the slow flux of ions within the restrained geometry of the pore. These results provide a structural framework to understand the unique permeation properties of CRAC channels. Orai1 ͉ STIM1 ͉ store-operated channels C a 2ϩ release-activated Ca 2ϩ (CRAC) channels are the principal Effects of Cysteine Mutations. Barring a few exceptions (H113C, E106C, and M101C), Orai1 Cys substitutions were well tolerated and produced currents Ͼ5 pA/pF in amplitude and with permeation properties characteristic of I CRAC (Table S1). Currents in the E106C and H113C mutants (Ͻ0.5 pA/pF) were indistinguishable from leak and were not further tested. Currents arising from M101C averaged only 1 pA/pF, but despite their small size, exhibited properties such as inward rectification and positive reversal potentials characteristic of I CRAC. Inactivity of E106C channels is consistent with previous reports showing that Ala and Gln substitutions of this critical residue abrogate channel activity (7-10). The

ClC chloride channels, which are ubiquitously expressed in mammals, have a unique double-barreled structure, in which each monomer forms its own pore. Identification of pore-lining elements is important for understanding the conduction properties and unusual gating mechanisms of these channels. Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels. In this study we used cysteine-scanning mutagenesis and modification (SCAM) to screen >50 residues in the intracellular vestibule of ClC-0. We identified 14 positions sensitive to the negatively charged thiol-modifying reagents sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) or sodium 4-acetamido-4'-maleimidylstilbene-2'2-disulfonic acid (AMS) and show that 11 of these alter pore properties when modified. In addition, two MTSES-sensitive residues, on different helices and in close proximity in the prokaryotic structures, can form a disulfide bond in ClC-0. When mapped onto prokaryotic structures, MTSES/AMS-sensitive residues cluster around bound chloride ions, and the correlation is even stronger in the ClC-0 homology model developed by Corry et al. (2004). These results support the hypothesis that both secondary and tertiary structures in the intracellular vestibule are conserved among ClC family members, even in regions of very low sequence similarity.