Single-molecule imaging of l-type Ca(2+) channels in live cells

L-type Ca(2+) channels are an important means by which a cell regulates the Ca(2+) influx into the cytosol on electrical stimulation. Their structure and dynamics in the plasma membrane, including their molecular mobility and aggregation, is of key interest for the in-depth understanding of their function. Construction of a fluorescent variant by fusion of the yellow-fluorescent protein to the ion channel and expression in a human cell line allowed us to address its dynamic embedding in the membrane at the level of individual channels in vivo. We report on the observation of individual fluorescence-labeled human cardiac L-type Ca(2+) channels using wide-field fluorescence microscopy in living cells. Our fluorescence and electrophysiological data indicate that L-type Ca(2+) channels tend to form larger aggregates which are mobile in the plasma membrane.     

End-point targeted molecular dynamics: large-scale conformational changes in potassium channels

Large-scale conformational changes in proteins that happen often on biological time scales may be relatively rare events on the molecular dynamics time scale. We have implemented an approach to targeted molecular dynamics called end-point targeted molecular dynamics that transforms proteins between two specified conformational states through the use of nonharmonic “soft” restraints. A key feature of the method is that the protein is free to discover its own conformational pathway through the plethora of possible intermediate states. The method is applied to the Shaker K_v1.2 potassium channel in implicit solvent. The rate of cycling between the open and closed states was varied to explore how slow the cycling rate needed to be to ensure that microscopic reversibility along the transition pathways was well approximated. Results specific to the K⁺ channel include: 1), a variation in backbone torsion angles of residues near the Pro-Val-Pro motif in the inner helix during both opening and closing; 2), the identification of possible occlusion sites in the closed channel located among Pro-Val-Pro residues and downstream; 3), a difference in the opening and closing pathways of the channel; and 4), evidence of a transient intermediate structural substate. The results also show that likely intermediate conformations during the opening-closing process can be generated in computationally tractable simulation times.     

A fluorescence temperature-jump study on Ca2(+)-induced conformational changes in calmodulin

Calmodulin has been shown to alter its conformation so as to interact with a number of target proteins upon Ca2+ binding. A Ca2(+)-binding study of calmodulin was performed by monitoring the fluorescence of intrinsic tyrosine residues and the probe 1-anilinonaphthalene-8-sulfonate (ANS). ANS fluorescence was shown to reflect Ca2+ binding to both high- and low-affinity sites. On the one hand, tyrosine fluorescence was sensitive only to the high-affinity Ca2+ binding. Temperature-jump investigation of the ternary complex of Ca2(+)-calmodulin-ANS in combination with monitoring of ANS fluorescence demonstrated the kinetic characteristics of the conformational change. The relaxation process was attributed to Ca2(+)-induced conformational change and the rate constants of this process were evaluated. On the basis of the rate constants of the conformational change, a rapid response of calmodulin in Ca2+ signaling is suggested.     

In vitro fluorescence assay to study the folding of Kv ion channels

A fluorescence assay to check the folding of potassium Kv channels expressed in vitro has been developed. For this aim, the fluorescently labeled channel blocker, recombinant agitoxin of yellow scorpion was employed. The level of expression of various Kv channels in vitro has been tested. It has been demonstrated that Kv2 channels form clusters on the cell surface, which are not associated with actin filaments. On the other hand, Kv10 channels form larger clusters, which are associated with actin, indicating the principal differences in the organization of cytoplasmic domains of Kv2 and Kv10 channels.     

A spin label study of conformational changes in cytochrome c

Spin-labeled pig heart cytochromes c singly modified at Met-65, Tyr-74 and at one of the lysine residues, Lys-72 or Lys-73, were investigated by the ESR method under conditions of different ligand and redox states of the heme and at various pH values. Replacement of Met-80 by the external ligand, cyanide, was shown to produce a sharp increase in the mobility of all the three bound labels while reduction of the spin-labeled ferricytochromes c did not cause any marked changes in their ESR spectra. In the pH range 6-13, two conformational transitions in ferricytochrome c were observed which preceded its alkaline denaturation: the first with pK 9.3 registered by the spin label at the Met-65 position, and the second with pK 11.1 registered by the labels bound to Tyr-74 and Lys-72(73). The conformational changes in the 'left-hand part' of ferricytochrome c are most probably induced in both cases by the exchange of internal protein ligands at the sixth coordination site of the heme.     

Hydrostatic pressure-induced conformational changes in phosphatidylcholine headgroups: a 2H NMR study.

The effects of pressure and temperature on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine headgroup conformations were examined using deuterium nuclear magnetic resonance. Isothermal compression was found to produce a decrease in the choline alpha deuteron quadrupole splitting and increases in the choline beta and gamma deuteron quadrupole splittings. A similar counterdirectional change, seen in the presence of positive surface charge, has been attributed to tilting of the headgroup away from the bilayer surface in response to the torque exerted on the phosphocholine dipole by positive surface charges. The direction of the change in headgroup deuteron quadrupole splitting is consistent with the pressure-induced reduction in area per lipid in the liquid crystalline phase, which can be inferred from the ordering of phospholipid acyl chains under comparable conditions. The temperature dependences of the headgroup deuteron quadrupole splittings were also examined. It was found that at elevated pressure, the alpha splitting was insensitive to temperature, whereas the beta and gamma splittings decreased. The response of the beta deuteron splitting to temperature was found to be weaker at elevated pressure than at ambient pressure.     

Redox-induced conformational changes in plastocyanin: an infrared study.

The conformational changes associated with the redox transition of plastocyanin (PC) were investigated by absorption and reaction-induced infrared spectroscopy. In addition to spectral features readily ascribed to beta and turn protein secondary structures, the amide I band shows a major component band at 1647 cm(-1) in both redox states of the protein. The sensitivity of this component to deuteration and increasing temperature suggests that PC adopts an unusual secondary structure in solution, which differs from those described for other type I copper proteins, such as azurin and halocyanin. The conformations of oxidized and reduced PC are different, as evidenced (1) by analysis of their amide I band contour and the electrochemically induced oxidized-minus-reduced difference spectrum and (2) by their different thermal stability. The redox-induced difference spectrum exhibits a number of difference bands within the conformationally sensitive amide I band that could be assigned to peptide C=O modes, in light of their small shift upon deuteration, and to signals attributable to side chain vibrational modes of Tyr residues. Lowering the pH to 4.8 induces destabilization of both redox states of the protein, more pronounced for reduced PC, without significantly affecting their secondary structure. Besides the conformational differences obtained at neutral pH, the oxidized-minus-reduced difference spectrum shows two broad and strong negative bands at 1405 and 1571 cm(-1), assigned to COO(-) vibrations, and a broad positive band at 1710 cm(-1), attributed to the C=O vibration of a COOH group(s). These bands are indicative of a protonation of (an) Asp or Glu side chain(s) upon plastocyanin oxidation at acidic pH.     

Applying two-photon excitation fluorescence lifetime imaging microscopy to study photosynthesis in plant leaves

This study investigates to which extent two-photon excitation (TPE) fluorescence lifetime imaging microscopy can be applied to study picosecond fluorescence kinetics of individual chloroplasts in leaves. Using femtosecond 860 nm excitation pulses, fluorescence lifetimes can be measured in leaves of Arabidopsis thaliana and Alocasia wentii under excitation-annihilation free conditions, both for the F ₀- and the F _m-state. The corresponding average lifetimes are ~250 ps and ~1.5 ns, respectively, similar to those of isolated chloroplasts. These values appear to be the same for chloroplasts in the top, middle, and bottom layer of the leaves. With the spatial resolution of ~500 nm in the focal (xy) plane and 2 μm in the z direction, it appears to be impossible to fully resolve the grana stacks and stroma lamellae, but variations in the fluorescence lifetimes, and thus of the composition on a pixel-to-pixel base can be observed.     

Using fluorescence correlation spectroscopy to study conformational changes in denatured proteins

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool that allows dynamics and hydrodynamics of biomolecules to be studied under a broad range of experimental conditions. One application of FCS of current interest is the determination of the size of protein molecules in the various states they sample along their folding reaction coordinate, which can be accessed through the measurement of diffusion coefficients. It has been pointed out that the analysis of FCS curves is prone to artifacts that may lead to erroneous size determination. To set the stage for FCS studies of unfolded proteins, we first show that the diffusion coefficients of small molecules as well as proteins can be determined accurately even in the presence of high concentrations of co-solutes that change the solution refractive index significantly. Indeed, it is found that the Stokes-Einstein relation between the measured diffusion coefficient and solution viscosity holds even in highly concentrated glycerol or guanidinium hydrochloride (GuHCl) solutions. These measurements form the basis for an investigation of the structure of the denatured state of two proteins, the small protein L and the larger, three-domain protein adenylate kinase (AK). FCS is found useful for probing expansion in the denatured state beyond the unfolding transition. It is shown that the denatured state of protein L expands as the denaturant concentration increases, in a process akin to the transition from a globule to a coil in polymers. This process continues at least up to 5 M GuHCl. On the other hand, the denatured state of AK does not seem to expand much beyond 2 M GuHCl, a result that is in qualitative accord with single-molecule fluorescence histograms. Because both the unfolding transition and the coil-globule transition of AK occur at a much lower denaturant concentration than those of protein L, a possible correlation between the two phenomena is suggested.     

Iterative cluster-NMA: A tool for generating conformational transitions in proteins

Computational models provide insight into the structure-function relationship in proteins. These approaches, especially those based on normal mode analysis, can identify the accessible motion space around a given equilibrium structure. The large magnitude, collective motions identified by these methods are often well aligned with the general direction of the expected conformational transitions. However, these motions cannot realistically be extrapolated beyond the local neighborhood of the starting conformation. In this article, the iterative cluster-NMA (icNMA) method is presented for traversing the energy landscape from a starting conformation to a desired goal conformation. This is accomplished by allowing the evolving geometry of the intermediate structures to define the local accessible motion space, and thus produce an appropriate displacement. Following the derivation of the icNMA method, a set of sample simulations are performed to probe the robustness of the model. A detailed analysis of beta1,4-galactosyltransferase-T1 is also given, to highlight many of the capabilities of icNMA. Remarkably, during the transition, a helix is seen to be extended by an additional turn, emphasizing a new unknown role for secondary structures to absorb slack during transitions. The transition pathway for adenylate kinase, which has been frequently studied in the literature, is also discussed.     

Cofilin induced conformational changes in F-actin expose subdomain 2 to proteolysis

Cofilin/ADF affects strongly the structure of actin filaments and especially the intermolecular contacts of the DNase I binding loop (D-loop) in subdomain 2. In G-actin, the D-loop is cleaved by subtilisin between Met47 and Gly48, while in F-actin this cleavage is inhibited. Here, we report that yeast cofilin, which is resistant to both subtilisin and trypsin, accelerates greatly the rate of subtilisin cleavage of this loop in F-actin at pH 6.8 and at pH 8.0. Similarly, cofilin accelerates strongly the tryptic cleavage in F-actin of loop 60-69 in subdomain 2, at Arg62 and Lys68. The acceleration of the loops' proteolysis cannot be attributed to an increased treadmilling of F-actin for the following reasons: (i) the rate of subtilisin cleavage is independent of pH between pH 6.8 and 8.0, unlike F-actin depolymerization, which is pH-dependent; (ii) at high concentrations of protease the cleavage rate of F-actin in the presence of cofilin is faster than the rate of monomer dissociation from the pointed end of TRC-labeled F-actin, which limits the rate of treadmilling; and (iii) cofilin also accelerates the rate of subtilisin cleavage of F-actin in which the treadmilling is blocked by interprotomer cross-linking of the D-loop to the C terminus on an adjacent protomer. This suggests a substantial flexibility of the D-loop in the cross-linked F-actin. The increased cleavage rates of the D-loop and loop 60-69 reveal extensive exposure of subdomain 2 in F-actin to proteolytic enzymes by cofilin.     

Molecular modeling study for conformational changes of Sirtuin 2 due to substrate and inhibitor binding

Sirtuin is a member of NAD(+)-dependent deacetylase family. The structural details of Sirtuin 2 (SIRT2) complex will be very useful to discover the drug which might have beneficial effects on various diseases like cancer, diabetes, etc. Unfortunately, SIRT2 complex structure is not available yet, hence molecular docking was carried out to dock the substrate (NAD(+) and acetylated lysine) and inhibitor (sirtinol) in the NAD(+) binding site. The suitable binding orientation of substrate and inhibitor in the SIRT2 active site was selected and subjected to 5 ns molecular dynamics simulations to adjust the binding orientation of inhibitor and substrate as well as to identify the conformational changes in the active site. The result provides an insight about 3D SIRT2 structural details as well as the importance of F96 in deacetylation function. In addition, our simulations revealed the displacement of F96 upon substrate and inhibitor binding, inducing an extended conformation of loop3 and changing its interactions with the rest of SIRT2. We believe that our study could be helpful to gain a structural insight of SIRT2 and to design the receptor-based inhibitors.     

Molecular modeling study for conformational changes of Sirtuin 2 due to substrate and inhibitor binding

Sirtuin is a member of NAD⁺-dependent deacetylase family. The structural details of Sirtuin 2 (SIRT2) complex will be very useful to discover the drug which might have beneficial effects on various diseases like cancer, diabetes, etc. Unfortunately, SIRT2 complex structure is not available yet, hence molecular docking was carried out to dock the substrate (NAD⁺ and acetylated lysine) and inhibitor (sirtinol) in the NAD⁺ binding site. The suitable binding orientation of substrate and inhibitor in the SIRT2 active site was selected and subjected to 5?ns molecular dynamics simulations to adjust the binding orientation of inhibitor and substrate as well as to identify the conformational changes in the active site. The result provides an insight about 3D SIRT2 structural details as well as the importance of F96 in deacetylation function. In addition, our simulations revealed the displacement of F96 upon substrate and inhibitor binding, inducing an extended conformation of loop3 and changing its interactions with the rest of SIRT2. We believe that our study could be helpful to gain a structural insight of SIRT2 and to design the receptor-based inhibitors.     

A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease

Ligand binding to proteins often is accompanied by conformational transitions. Here, we describe a competition assay based on single molecule Förster resonance energy transfer (smFRET) to investigate the ligand-induced conformational changes of the dengue virus (DENV) NS2B-NS3 protease, which can adopt at least two different conformations. First, a competitive ligand was used to stabilize the closed conformation of the protease. Subsequent addition of the allosteric inhibitor reduced the fraction of the closed conformation and simultaneously increased the fraction of the open conformation, demonstrating that the allosteric inhibitor stabilizes the open conformation. In addition, the proportions of open and closed conformations at different concentrations of the allosteric inhibitor were used to determine its binding affinity to the protease. The K_D value observed is in accordance with the IC₅₀ determined in the fluorometric assay. Our novel approach appears to be a valuable tool to study conformational transitions of other proteases and enzymes.     

Circular dichroic study of conformational changes in ovalbumin

By simulation of the circular dichroic spectra (Greenfield and Fasman (1969)) and using reference spectra of Chen et al. (1974), native ovalbumin was estimated to contain 33% -helix, 5% -structure, and 62% random coil. Ovalbumin resisted conformational changes in solutions of urea and of SDS. However, guanidine induced transition, starting at about 2 M and completing at about 4.5 M. At concentrations exceeding 4.5 M guanidine, ovalbumin existed as 6–7% -helical, 12–13% -structure, and 80–81% random coil. Ovalbumin after denaturation in 6 M guanidine or in 8 M urea (incubated at 4°C for 24 hr) did not recover the native conformation but acquired a new conformation in each case, with a somewhat destabilized helical structure.Abbreviation used CD circular dichroism - SDS sodium dodecyl sulfate