CHARMM Force-Fields with Modified Polyphosphate Parameters Allow Stable Simulation of the ATP-Bound Structure of Ca²⁺-ATPase

Adenosine triphosphate (ATP) is an indispensable energy source in cells. In a wide variety of biological phenomena like glycolysis, muscle contraction/relaxation, and active ion transport, chemical energy released from ATP hydrolysis is converted to mechanical forces to bring about large-scale conformational changes in proteins. Investigation of structure–function relationships in these proteins by molecular dynamics (MD) simulations requires modeling of ATP in solution and ATP bound to proteins with accurate force-field parameters. In this study, we derived new force-field parameters for the triphosphate moiety of ATP based on the high-precision quantum calculations of methyl triphosphate. We tested our new parameters on membrane-embedded sarcoplasmic reticulum Ca²⁺-ATPase and four soluble proteins. The ATP-bound structure of Ca²⁺-ATPase remains stable during MD simulations, contrary to the outcome in shorter simulations using original parameters. Similar results were obtained with the four ATP-bound soluble proteins. The new force-field parameters were also tested by investigating the range of conformations sampled during replica-exchange MD simulations of ATP in explicit water. Modified parameters allowed a much wider range of conformational sampling compared with the bias toward extended forms with original parameters. A diverse range of structures agrees with the broad distribution of ATP conformations in proteins deposited in the Protein Data Bank. These simulations suggest that the modified parameters will be useful in studies of ATP in solution and of the many ATP-utilizing proteins

CHARMM Force-Fields with Modified Polyphosphate Parameters Allow Stable Simulation of the ATP-Bound Structure of Ca²⁺-ATPase

Adenosine triphosphate (ATP) is an indispensable energy source in cells. In a wide variety of biological phenomena like glycolysis, muscle contraction/relaxation, and active ion transport, chemical energy released from ATP hydrolysis is converted to mechanical forces to bring about large-scale conformational changes in proteins. Investigation of structure–function relationships in these proteins by molecular dynamics (MD) simulations requires modeling of ATP in solution and ATP bound to proteins with accurate force-field parameters. In this study, we derived new force-field parameters for the triphosphate moiety of ATP based on the high-precision quantum calculations of methyl triphosphate. We tested our new parameters on membrane-embedded sarcoplasmic reticulum Ca²⁺-ATPase and four soluble proteins. The ATP-bound structure of Ca²⁺-ATPase remains stable during MD simulations, contrary to the outcome in shorter simulations using original parameters. Similar results were obtained with the four ATP-bound soluble proteins. The new force-field parameters were also tested by investigating the range of conformations sampled during replica-exchange MD simulations of ATP in explicit water. Modified parameters allowed a much wider range of conformational sampling compared with the bias toward extended forms with original parameters. A diverse range of structures agrees with the broad distribution of ATP conformations in proteins deposited in the Protein Data Bank. These simulations suggest that the modified parameters will be useful in studies of ATP in solution and of the many ATP-utilizing proteins

Energetics of the Presequence-Binding Poses in Mitochondrial Protein Import Through Tom20

Tom20 is located at the outer membrane of mitochondria and functions as a receptor for the N-terminal presequence of mitochondrial-precursor proteins. Recently, three atomic structures of the Tom20-presequence complex were determined using X-ray crystallography and classified into A-, M-, and Y-poses in terms of their presequence-binding modes. Combined with biochemical and NMR data, a dynamic-equilibrium model between the three poses has been proposed. To investigate this mechanism in further detail, we performed all-atom molecular dynamics (MD) simulations and replica-exchange MD (REMD) simulations of the Tom20-presequence complex in explicit water. In the REMD simulations, one major distribution and another minor one were observed in the converged free-energy landscape at 300 K. In the major distribution, structures similar to A- and M-poses exist, whereas those similar to Y-pose are located in the minor one, suggesting that A-pose in solution is more stable than Y-pose. A <i>k</i>-means clustering algorithm revealed a new pose not yet obtained by X-ray crystallography. This structure has double salt bridges between Arg14′ in the presequence and Glu78 or Glu79 in Tom20 and can explain the binding affinity of the complex in previous pull-down assay experiments. Structural clustering and analyses of contacts between Tom20 and the presequence suggest smooth conformational changes from Y- to A-poses through low activation barriers. M-pose lies between Y- and A-poses as a metastable state. The REMD simulations thus provide insights into the energetics of the multiple-binding forms and help to detail the progressive conformational states in the dynamic-equilibrium model based on the experimental data

Simultaneous Formation and Spatial Patterning of ZnO on ITO Surfaces by Local Laser-Induced Generation of Microbubbles in Aqueous Solutions of [Zn(NH₃)₄]²⁺

We demonstrate the simultaneous formation and spatial patterning of ZnO nanocrystals on an indium–tin oxide (ITO) surface upon local heating using a laser (1064 nm) and subsequent formation of microbubbles. Laser irradiation of an ITO surface in aqueous [Zn­(NH₃)₄]²⁺ solution (1.0 × 10^–2 M at pH 12.0) under an optical microscope produced ZnO nanocrystals, the presence of which was confirmed by X-ray diffraction analysis and Raman microspectroscopy. Scanning the focused laser beam over the ITO surface generated a spatial ZnO pattern (height: ∼60 nm, width: ∼1 μm) in the absence of a template or mask. The Marangoni convection generated in the vicinity of the microbubbles resulted in a rapid concentration/accumulation of [Zn­(NH₃)₄]²⁺ around the microbubbles, which led to the formation of ZnO at the solid–bubble–solution three-phase contact line around the bubbles and thus afforded ZnO nanocrystals on the ITO surface upon local heating with a laser

Formation of M‑Like Intermediates in Proteorhodopsin in Alkali Solutions (pH ≥ ∼8.5) Where the Proton Release Occurs First in Contrast to the Sequence at Lower pH

Proteorhodopsin (PR) is an outward light-driven proton pump observed in marine eubacteria. Despite many structural and functional similarities to bacteriorhodopsin (BR) in archaea, which also acts as an outward proton pump, the mechanism of the photoinduced proton release and uptake is different between two H⁺-pumps. In this study, we investigated the pH dependence of the photocycle and proton transfer in PR reconstituted with the phospholipid membrane under alkaline conditions. Under these conditions, as the medium pH increased, a blue-shifted photoproduct (defined as M_a), which is different from M, with a p<i>K</i>_a of ca. 9.2 was produced. The sequence of the photoinduced proton uptake and release during the photocycle was inverted with the increase in pH. A p<i>K</i>_a value of ca. 9.5 was estimated for this inversion and was in good agreement with the p<i>K</i>_a value of the formation of M_a (∼9.2). In addition, we measured the photoelectric current generated by PRs attached to a thin polymer film at varying pH. Interestingly, increases in the medium pH evoked bidirectional photocurrents, which may imply a possible reversal of the direction of the proton movement at alkaline pH. On the basis of these findings, a putative photocycle and proton transfer scheme in PR under alkaline pH conditions was proposed