85 research outputs found
Unstructured intermediate states in single protein force experiments
Recent single-molecule force measurements on single-domain proteins have
highlighted a three-state folding mechanism where a stabilized intermediate
state (I) is observed on the folding trajectory between the stretched state and
the native state. Here we investigate on-lattice protein-like heteropolymer
models that lead to a three-state mechanism and show that force experiments can
be useful to determine the structure of I. We have mostly found that I is
composed of a core stabilized by a high number of native contacts, plus an
unstructured extended chain. The lifetime of I is shown to be sensitive to
modifications of the protein that spoil the core. We then propose three types
of modifications--point mutations, cuts, and circular permutations--aiming at:
(1) confirming the presence of the core and (2) determining its location,
within one amino acid accuracy, along the polypeptide chain. We also propose
force jump protocols aiming to probe the on/off-pathway nature of I.Comment: 10 page
Structure-function Relationship of N-terminal Deletion Mutants of Cardiac L-type Calcium Channel ß1-subunits
Electrons, Photons, and Force: Quantitative Single-Molecule Measurements from Physics to Biology
Single-molecule measurement techniques have illuminated unprecedented details of chemical behavior, including observations of the motion of a single molecule on a surface, and even the vibration of a single bond within a molecule. Such measurements are critical to our understanding of entities ranging from single atoms to the most complex protein assemblies. We provide an overview of the strikingly diverse classes of measurements that can be used to quantify single-molecule properties, including those of single macromolecules and single molecular assemblies, and discuss the quantitative insights they provide. Examples are drawn from across the single-molecule literature, ranging from ultrahigh vacuum scanning tunneling microscopy studies of adsorbate diffusion on surfaces to fluorescence studies of protein conformational changes in solution
Protein folding dynamics: single-molecule studies of Ribonuclease HI on biocompatible surfaces
Heterogeneity of individual trajectories of a protein adopting its native conformation from the myriad of denatured states can be studied by single-molecule spectroscopy. However, to study timescales longer than a few milliseconds, the protein has to be fixed in space, for example by immobilization on a surface. The delicate structure of proteins requires surface coatings which neither interfere with the energetics of the folding nor introduce additional, surface-induced heterogeneities. In this work, we characterized and compared several surfaces devised for single molecule fluorescence experiments for their biocompatibility. The best surface was the surface prepared from the cross-linked star-shaped poly (ethylene glycol) polymers. We employed this surface to investigate the complex folding landscape of Ribonuclease HI (RNase H). Our single-molecule data revealed that the average radius of gyration of unfolded RNase H increases with increasing GdmCl concentration. We modeled the data as a shift of the equilibrium within a continuum of unfolded conformations and obtained various thermodynamic and structural parameters. The fluorescence time traces recorded from individual molecules showed that unfolded RNase H molecules fluctuate between many conformations on the second timescale. The transitions were spread over all possible initial and final FRET efficiency values, indicating a high diversity in the folding pathways. The existence of long-lived unfolded structures resulted in a significant width of the FRET efficiency distribution of the unfolded state. From the cross-correlation functions of the donor and acceptor emission, we found that the reconfiguration time of unfolded RNase H chains is ~ 20 µs. Applying Kramer’s theory, we evaluated the average height of the barriers between the unfolded substates and the barrier from the unfolded to the folded ensemble. Finally, we presented a two-dimensional scheme of the folding free energy landscape for RNase H
Single-Channel Analysis of the Inhibition of the Calcium Dependent Inactivation by the C-Terminal Modulator Domain of Cav1.3 Channels
Functional Interaction between the N-Termini of Murine L-Type Calcium Channel CaV1.2-and beta-Subunit Splice Variants
Single-Channel Monitoring of Reversible L-Type Ca2+ Channel Ca-v alpha(1)-Ca-v beta Subunit Interaction
Voltage-dependent Ca2+ channels are heteromultimers of Ca-v alpha(1) (pore), Ca-v beta- and Ca-v alpha(2)delta-subunits. The stoichiometry of this complex, and whether it is dynamically regulated in intact cells, remains controversial. Fortunately, Ca-v beta-isoforms affect gating differentially, and we chose two extremes (Ca-v beta(1a) and Ca-v beta(2b)) regarding single-channel open probability to address this question. HEK293 alpha(1C) cells expressing the Ca(v)1.2 subunit were transiently transfected with Ca-v alpha(2)delta 1 alone or with Ca-v beta(1a), Ca-v beta(2b), or (2:1 or 1:1 plasmid ratio) combinations. Both Ca-v beta-subunits increased whole-cell current and shifted the voltage dependence of activation and inactivation to hyperpolarization. Time-dependent inactivation was accelerated by Ca-v beta(1a)-subunits but not by Ca-v beta(2b)-subunits. Mixtures induced intermediate phenotypes. Single channels sometimes switched between periods of low and high open probability. To validate such slow gating behavior, data were segmented in clusters of statistically similar open probability. With Ca-v beta(1a)-subunits alone, channels mostly stayed in clusters (or regimes of alike clusters) of low open probability. Increasing Ca-v beta(2b)-subunits (co-)expressed (1:2, 1:1 ratio or alone) progressively enhanced the frequency and total duration of high open probability clusters and regimes. Our analysis was validated by the inactivation behavior of segmented ensemble averages. Hence, a phenotype consistent with mutually exclusive and dynamically competing binding of different Ca-v beta-subunits is demonstrated in intact cells
Inhibitory effects on L- and N-type calcium channels by a novel Ca-V beta(1) variant identified in a patient with autism spectrum disorder
Voltage-gated calcium channel (VGCC) subunits have been genetically associated with autism spectrum disorders (ASD). The properties of the pore-forming VGCC subunit are modulated by auxiliary beta-subunits, which exist in four isoforms (Ca-V beta(1-4)). Our previous findings suggested that activation of L-type VGCCs is a common feature of Ca-V beta(2) subunit mutations found in ASD patients. In the current study, we functionally characterized a novel Ca-V beta(1b) variant (p.R296C) identified in an ASD patient. We used whole-cell and single-channel patch clamp to study the effect of Ca-V beta(1b_R296C) on the function of L- and N-type VGCCs. Furthermore, we used co-immunoprecipitation followed by Western blot to evaluate the interaction of the Ca-V beta(1b)-subunits with the RGK-protein Gem. Our data obtained at both, whole-cell and single-channel levels, show that compared to a wild-type Ca-V beta(1b), the Ca-V beta(1b_R296C) variant inhibits L- and N-type VGCCs. Interaction with and modulation by the RGK-protein Gem seems to be intact. Our findings indicate functional effects of the Ca-V beta(1b_R296C) variant differing from that attributed to Ca-V beta(2) variants found in ASD patients. Further studies have to detail the effects on different VGCC subtypes and on VGCC expression
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