Yutaka Ueno

The voltage-sensitive sodium channel is a bell-shaped molecule with several cavities

Voltage-sensitive membrane channels, the sodium channel, the potassium channel and the calcium channel operate together to amplify, transmit and generate electric pulses in higher forms of life. Sodium and calcium channels are involved in cell excitation, neuronal transmission, muscle contraction and many functions that relate directly to human diseases. Sodium channels—glycosylated proteins with a relative molecular mass of about 300,000 (ref. 5)—are responsible for signal transduction and amplification, and are chief targets of anaesthetic drugs and neurotoxins. Here we present the three-dimensional structure of the voltage-sensitive sodium channel from the eel Electrophorus electricus. The 19 Å structure was determined by helium-cooled cryo-electron microscopy and single-particle image analysis of the solubilized sodium channel. The channel has a bell-shaped outer surface of 135 Å in height and 100 Å in side length at the square-shaped bottom, and a spherical top with a diameter of 65 Å. Several inner cavities are connected to four small holes and eight orifices close to the extracellular and cytoplasmic membrane surfaces. Homologous voltage-sensitive calcium and tetrameric potassium channels, which regulate secretory processes and the membrane potential, may possess a related structure.

Fragment molecular orbital method: application to molecular dynamics simulation, 'ab initio FMO-MD'

Abstract A quantum molecular simulation method applicable to biological molecules is proposed. Ab initio fragment molecular orbital method-based molecular dynamics (FMO-MD) combines molecular dynamics simulation with the ab initio fragment molecular orbital method. Here, FMO computes the force acting on each atom’s nucleus while MD computes the nuclei’s time-dependent evolutions. FMO-MD successfully simulated a small polypeptide, demonstrating the method’s applicability to biological molecules.

Molecular dynamics simulations revealed Ca2+-dependent conformational change of Calmodulin

Molecular dynamics simulations were performed to simulate Ca2+‐dependent conformational change of calmodulin (CaM). Simulations of the fully Ca2+‐bound form of CaM (Holo‐CaM) and the Ca2+‐free form (Apo‐CaM) were performed in solution for 4 ns starting from the X‐ray crystal structure of Holo‐CaM. A striking difference was observed between the trajectories of Holo‐CaM and Apo‐CaM: the central helix remained straight in the former but became largely bent in the latter. Also, the flexibility of Apo‐CaM was higher than that of Holo‐CaM. The results indicated that the bound Ca2+ ions harden the structure of CaM.

A Three-Dimensional FRET Analysis to Construct an Atomic Model of the Actin–Tropomyosin Complex on a Reconstituted Thin Filament

Fluorescence resonance energy transfer (FRET) was used to construct an atomic model of the actin-tropomyosin (Tm) complex on a reconstituted thin filament. We generated five single-cysteine mutants in the 146-174 region of rabbit skeletal muscle α-Tm. An energy donor probe was attached to a single-cysteine Tm residue, while an energy acceptor probe was located in actin Gln41, actin Cys374, or the actin nucleotide binding site. From these donor-acceptor pairs, FRET efficiencies were determined with and without Ca(2+). Using the atomic coordinates for F-actin and Tm, we searched all possible arrangements for Tm segment 146-174 on F-actin to calculate the FRET efficiency for each donor-acceptor pair in each arrangement. By minimizing the squared sum of deviations for the calculated FRET efficiencies from the observed FRET efficiencies, we determined the location of the Tm segment on the F-actin filament. Furthermore, we generated a set of five single-cysteine mutants in each of the four Tm regions 41-69, 83-111, 216-244, and 252-279. Using the same procedures, we determined each segments location on the F-actin filament. In the best-fit model, Tm runs along actin residues 217-236, which were reported to compose the Tm binding site. Electrostatic, hydrogen-bonding, and hydrophobic interactions are involved in actin and Tm binding. The C-terminal region of Tm was observed to contact actin more closely than did the N-terminal region. Tm contacts more residues on actin without Ca(2+) than with it. Ca(2+)-induced changes on the actin-Tm contact surface strongly affect the F-actin structure, which is important for muscle regulation.

Structural Alterations of Thin Actin Filaments in Muscle Contraction by Synchrotron X-ray Fiber Diffraction

Strong evidence has been accumulated that the conformational changes of the thin actin filaments are occurring and playing an important role in the entire process of muscle contraction. The conformational changes and the mechanical properties of the thin actin filaments we have found by X-ray fiber diffraction on skeletal muscle contraction are explored. Recent studies on the conformational changes of regulatory proteins bound to actin filaments upon activation and in the force generation process are also described. Finally, the roles of structural alterations and dynamics of the actin filaments are discussed in conjunction with the regulation mechanism and the force generation mechanism.

MOSBY: a molecular structure viewer program with portability and extensibility.

A molecular structure viewer program, MOSBY has been developed for studies that use atomic coordinates to understand the structures of protein molecules. The program is designed to be portable with a comprehensive user interface by our high-throughput graphics library. In addition, it cooperates with extension modules customized for individual research topics and analysis. For example, an electron density module loads and displays electron density maps derived in X-ray crystallographic analysis superimposed to an atomic model. A molecular dynamics module reads a trajectory file of the results of molecular dynamics calculations and animates the structure. These plug-in modules are devised to function without modification to the MOSBY program. For variations of analysis and calculations with atomic coordinates, the portability and extensibility illustrated by MOSBY play an important rule in scientific computational tools with active software development.

Three-dimensional reconstruction of single particle electron microscopy: the voltage sensitive sodium channel structure.

Single particle analysis in electron microscopy allows direct observation of the reconstructed three-dimensional structures of protein molecules. This method enables a more comprehensive study of membrane proteins which have been problematic in structural studies using X-ray crystallography. These membrane proteins include the voltage-sensitive ion channel proteins, which play an important rule in neural activities, and have great medical significance. The method described is supported by the development of cryo-electron microscopy and the angular reconstitution method. This review summarizes certain principles governing single particle analysis employing angular reconstitution. This method was applied to our study of the voltage-sensitive sodium channel, and the results are discussed. With improvements in resolutions and statistical analyses, the single particle technique is considered to be advantageous in studies of the structural changes and molecular interactions of protein molecules.

X-ray fiber diffraction modeling of structural changes of the thin filament upon activation of live vertebrate skeletal muscles

In order to clarify the structural changes of the thin filaments related to the regulation mechanism in skeletal muscle contraction, the intensities of thin filament-based reflections in the X-ray fiber diffraction patterns from live frog skeletal muscles at non-filament overlap length were investigated in the relaxed state and upon activation. Modeling the structural changes of the whole thin filament due to Ca2+-activation was systematically performed using the crystallographic data of constituent molecules (actin, tropomyosin and troponin core domain) as starting points in order to determine the structural changes of the regulatory proteins and actin. The results showed that the globular core domain of troponin moved toward the filament axis by ∼6 Å and rotated by ∼16° anticlockwise (viewed from the pointed end) around the filament axis by Ca2+-binding to troponin C, and that tropomyosin together with the tail of troponin T moved azimuthally toward the inner domains of actin by ∼12° and radially by ∼7 Å from the relaxed position possibly to partially open the myosin binding region of actin. The domain structure of the actin molecule in F-actin we obtained for frog muscle thin filament was slightly different from that of the Holmes F-actin model in the relaxed state, and upon activation, all subdomains of actin moved in the direction to closing the nucleotide-binding pocket, making the actin molecule more compact. We suggest that the troponin movements and the structural changes within actin molecule upon activation are also crucial components of the regulation mechanism in addition to the steric blocking movement of tropomyosin.

FOLDING ELASTIC TRANSMEMBRANE HELICES TO FIT IN A LOW-RESOLUTION IMAGE BY ELECTRON MICROSCOPY

Structure prediction of membrane proteins could be constrained and thereby improved by introducing data of the observed molecular shape. We studied a coarse-grained molecular model that relied on residue-based dummy atoms to fold the transmembrane helices of a protein in the observed molecular shape. Based on the inter-residue potential, the α-helices were folded to contact each other in a simulated annealing protocol to search optimized conformation. Fitting the model into a three-dimensional volume was tested for proteins with known structures and resulted in a fairly reasonable arrangement of helices. In addition, the constraint to the packing transmembrane helix with the two-dimensional region was tested and found to work as a very similar folding guide. The obtained models nicely represented α-helices with the desired slight bend. Our structure prediction method for membrane proteins well demonstrated reasonable folding results using a low-resolution structural constraint introduced from recent cell-surface imaging techniques.

Inference of Euler angles for single-particle analysis by means of evolutionary algorithms

Single-particle analysis is one of the methods for structural studies of protein and macromolecules; it requires advanced image analysis of electron micrographics. Reconstructing three-dimensional (3D) structure from microscope images is not an easy analysis because of the low image resolution of images and lack of the directional information of images in 3D structure. To improve the resolution, different projections are aligned, classified, and averaged. Inferring the orientations of these images is so difficult that the task of reconstructing 3D structures depends upon the experience of researchers. But recently, a method to reconstruct 3D structures was automatically devised. In this paper, we propose a new method for determining Euler angles of projections by applying genetic algorithms. We empirically show that the proposed approach has improved the previous one in terms of computational time and acquired precision.