Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways. Here, we have studied the structure and dynamics of the small enzyme ribonuclease HI (RNase H) in the presence of the chemical denaturant guanidinium chloride (GdmCl) using single-molecule fluorescence microscopy, with a particular focus on the characterization of the unfoldedstate ensemble. A dye pair was specifically attached to the enzyme to measure structural changes through Fö rster resonance energy transfer (FRET). Enzyme immobilization on star-polymer surfaces that were specially developed for negligible interaction with folded and unfolded proteins enabled us to monitor conformational changes of individual proteins for several hundred seconds. FRET efficiency histograms were calculated from confocal scan images. They showed an expansion of the unfolded proteins with increasing GdmCl concentration. Cross-correlation analysis of donor and acceptor fluorescence intensity time traces from single molecules revealed reconfiguration of the polypeptide chain on a timescale of Ϸ20 s at 1.7 M GdmCl. Slow conformational dynamics gave rise to characteristic, stepwise FRET efficiency changes. Transitions between folded and unfolded enzyme molecules occurred on the 100-s timescale, in excellent agreement with bulk denaturation experiments. Transitions between unfolded conformations were more frequent, with characteristic times of Ϸ2 s. These data were analyzed to obtain information on the free energy landscape of RNase H in the presence of chemical denaturants.fluorescence resonance energy transfer ͉ guanidinium chloride ͉ protein folding ͉ RNase H ͉ single-molecule spectroscopy P roteins are complex systems that can exist in a huge number of different conformations. Even in the properly folded, native state, the polypeptide chain adopts many slightly different conformations that can be depicted as local minima in a rugged energy landscape, and relaxations and fluctuations in the landscape are crucially involved in functional processes (1, 2). Conformational heterogeneity is even more of a concern in studies of protein-folding reactions because of the vast number of possible arrangements of the unfolded polypeptide chain and the many complex pathways leading from the ensemble of unfolded to the native conformations in an overall funnel-shaped energy landscape (3-7).Because of Anfinsen's key observation that the native fold is already encoded in the sequence of amino acids (8), the proteinfolding problem has attracted enormous attention, and the field has progressed in a healthy interplay between theory and experiment. A wide variety of biophysical approaches were developed to follow the dynamics of the polypeptide en route to the native state. Valuable insights have been achieved by timeresolved spectroscopic experiments on bu...

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Granule-mediated cytotoxicity is the main effector mechanism of cytotoxic CD8+ T cells. We report that CD8+ T cells from acid sphingomyelinase (ASMase)-deficient (ASMase-KO) mice are defective in exocytosis of cytolytic effector molecules; this defect resulted in attenuated cytotoxic activity of ASMase-KO CD8+ T cells and delayed elimination of lymphocytic choriomeningitis virus from ASMase-KO mice. Cytolytic granules of ASMase-KO and wild-type CD8+ T cells were equally loaded with granzymes and perforin, and correctly directed to the immunological synapse. In wild-type CD8+ T cells, secretory granules underwent shrinkage by 82% after fusion with the plasma membrane. In ASMase-KO CD8+ T cells, the contraction of secretory granules was markedly impaired. Thus, ASMase is required for contraction of secretory granules and expulsion of cytotoxic effector molecules.

The eight members of the calcium channel γ subunit family are integral membrane proteins that regulate the expression and behaviour of voltage and ligand gated ion channels. While a subgroup consisting of γ 2 , γ 3 , γ 4 and γ 8 (the TARPs) modulate AMPA receptor localization and function, the γ 1 and γ 6 subunits conform to the original description of these proteins as regulators of voltage gated calcium channels. We have previously shown that the γ 6 subunit is highly expressed in atrial myocytes and that it is capable of acting as a negative modulator of low voltage activated calcium current. In this study we extend our understanding of γ 6 subunit modulation of low voltage activated calcium current. Using engineered chimeric constructs, we demonstrate that the first transmembrane domain (TM1) of γ 6 is necessary for its inhibitory effect on Cav3.1 current. Mutational analysis is then used to identify a unique GxxxA motif within TM1 that is required for the function of the subunit strongly suggesting the involvement of helix-helix interactions in its effects. Results from co-immunoprecipitation experiments confirm a physical association of γ 6 with the Cav3.1 channel in both HEK cells and atrial myocytes. Single channel analysis reveals that binding of γ 6 reduces channel availability for activation. Taken together, the results of this study provide both a molecular and a mechanistic framework for understanding the unique ability of the γ 6 calcium channel subunit to modulate low voltage activated (Cav3.1) calcium current density. Calcium channel γ subunits comprise a family of eight proteins that share a common topology consisting of four transmembrane domains with intracellular N-and C-terminal ends. The first member of this protein family to be described, γ 1 , was isolated as a subunit of the high-voltage activated (HVA), Cav1.1 calcium channel found in skeletal muscle (Jay et al. 1990). Unlike other calcium channel accessory subunits (β, α 2 δ) which enhance calcium current, γ 1 was shown to accelerate L-type calcium current activation and inactivation in heterologous systems when coexpressed with the Cav1.2 (also an HVA) α 1 subunit (Singer et al. 1991;Eberst et al. 1997). Skeletal muscle isolated from knockout mice lacking the γ 1 gene have increased HVA calcium current density confirming a physiological role of γ 1 as a negative regulator of HVA, L-type calcium current density in developing skeletal myocytes (Freise et al. 2000;Held et al. 2002).Phylogenetic and sequence homology analysis indicates that the recently described γ 6 protein is the closest homologue of γ 1 within the γ subunit family (Burgess et al. 2001;Chu et al. 2001). Both γ 1 and γ 6 have short C-terminal regions that lack the consensus PDZ1-binding motif that is a notable characteristic of the four γ subunits (γ 2 , γ 3 , γ 4 and γ 8 ) known collectively as the TARP proteins that regulate AMPA receptor trafficking and function (Tomita et al. 2003;Osten & Stern-Bach, 2006). The γ 1 and γ 6 subunits also share similarities in their ti...