Single-molecule imaging of PI(4,5)P and PTEN in vitro reveals a positive feedback mechanism for PTEN membrane binding.

PTEN, a 3-phosphatase of phosphoinositide, regulates asymmetric PI(3,4,5)P signaling for the anterior-posterior polarization and migration of motile cells. PTEN acts through posterior localization on the plasma membrane, but the mechanism for this accumulation is poorly understood. Here we developed an in vitro single-molecule imaging assay with various lipid compositions and use it to demonstrate that the enzymatic product, PI(4,5)P, stabilizes PTEN's membrane-binding.

A gold nano-electrode for single ion channel recordings.

The artificial bilayer single channel recording technique is commonly used to observe the detailed physiological properties of various ion channel proteins. It permits easy control of the solution and membrane lipid composition, and is also compatible with pharmacological screening devices. However, its use is limited due to low measurement efficiency.

A Simple Method for Ion Channel Recordings Using Fine Gold Electrode.

The artificial bilayer single-channel recording technique is commonly used to observe detailed pharmacological properties of various ion channel proteins. It permits easy control of the solution and membrane lipid composition, and is also compatible with pharmacological screening devices. However, its use is limited due to low measurement efficiency.

A single amino acid gates the KcsA channel.

The KcsA channel is a proton-activated potassium channel. We have previously shown that the cytoplasmic domain (CPD) acts as a pH-sensor, and the charged states of certain negatively charged amino acids in the CPD play an important role in regulating the pH-dependent gating. Here, we demonstrate the KcsA channel is constitutively open independent of pH upon mutating E146 to a neutrally charged amino acid.

Single-molecule analysis of the rotation of F₁-ATPase under high hydrostatic pressure.

F1-ATPase is the water-soluble part of ATP synthase and is an ATP-driven rotary molecular motor that rotates the rotary shaft against the surrounding stator ring, hydrolyzing ATP. Although the mechanochemical coupling mechanism of F1-ATPase has been well studied, the molecular details of individual reaction steps remain unclear. In this study, we conducted a single-molecule rotation assay of F1 from thermophilic bacteria under various pressures from 0.

Mechanical modulation of ATP-binding affinity of V1-ATPase.

V(1)-ATPase is a rotary motor protein that rotates the central shaft in a counterclockwise direction hydrolyzing ATP. Although the ATP-binding process is suggested to be the most critical reaction step for torque generation in F(1)-ATPase (the closest relative of V(1)-ATPase evolutionarily), the role of ATP binding for V(1)-ATPase in torque generation has remained unclear. In the present study, we performed single-molecule manipulation experiments on V(1)-ATPase from Thermus thermophilus to investigate how the ATP-binding process is modulated upon rotation of the rotary shaft.

Single-molecule analysis of inhibitory pausing states of V1-ATPase.

V(1)-ATPase, the hydrophilic V-ATPase domain, is a rotary motor fueled by ATP hydrolysis. Here, we found that Thermus thermophilus V(1)-ATPase shows two types of inhibitory pauses interrupting continuous rotation: a short pause (SP, 4.2 s) that occurred frequently during rotation, and a long inhibitory pause (LP, >30 min) that terminated all active rotations.

Principal role of the arginine finger in rotary catalysis of F1-ATPase.

F(1)-ATPase (F(1)) is an ATP-driven rotary motor wherein the γ subunit rotates against the surrounding α(3)β(3) stator ring. The 3 catalytic sites of F(1) reside on the interface of the α and β subunits of the α(3)β(3) ring. While the catalytic residues predominantly reside on the β subunit, the α subunit has 1 catalytically critical arginine, termed the arginine finger, with stereogeometric similarities with the arginine finger of G-protein-activating proteins.

Mechanical modulation of catalytic power on F1-ATPase.

The conformational fluctuation of enzymes has a crucial role in reaction acceleration. However, the contribution to catalysis enhancement of individual substates with conformations far from the average conformation remains unclear. We studied the catalytic power of the rotary molecular motor F(1)-ATPase from thermophilic Bacillus PS3 as it was stalled in transient conformations far from a stable pausing angle.

Rotation and structure of FoF1-ATP synthase.

F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft.

Measurement of the conformational state of F(1)-ATPase by single-molecule rotation.

F(1)-ATPase (F(1)) is the water-soluble portion of ATP synthase and a rotary molecular motor in which the rotary shaft, the gamma subunit, rotates with 120 degrees steps against the alpha(3)beta(3) stator ring upon ATP hydrolysis. While the crystal structures of F(1) exhibit essentially one stable conformational state of F(1), single-molecule rotation studies revealed that there are two stable conformations of F(1) in each 120 degrees step: the ATP-binding dwell state and the catalytic dwell state. This chapter provides the experimental procedure for the determination of which catalytic state the crystal structures of F(1) represent, by the use of a cross-linking technique in the single-molecule rotation assay.

Stiffness of γ subunit of F(1)-ATPase.

F(1)-ATPase is a molecular motor in which the γ subunit rotates inside the α(3)β(3) ring upon adenosine triphosphate (ATP) hydrolysis. Recent works on single-molecule manipulation of F(1)-ATPase have shown that kinetic parameters such as the on-rate of ATP and the off-rate of adenosine diphosphate (ADP) strongly depend on the rotary angle of the γ subunit (Hirono-Hara et al. 2005; Iko et al.

Correlation between the conformational states of F1-ATPase as determined from its crystal structure and single-molecule rotation.

F(1)-ATPase is a rotary molecular motor driven by ATP hydrolysis that rotates the gamma-subunit against the alpha(3)beta(3) ring. The crystal structures of F(1), which provide the structural basis for the catalysis mechanism, have shown essentially 1 stable conformational state. In contrast, single-molecule studies have revealed that F(1) has 2 stable conformational states: ATP-binding dwell state and catalytic dwell state.

Structural studies reveal that the diverse morphology of beta(2)-microglobulin aggregates is a reflection of different molecular architectures.

Amyloid deposition accompanies over 20 degenerative diseases in human, including Alzheimer's, Parkinson's, and prion diseases. Recent studies revealed the importance of other type of protein aggregates, e.g.

Control of iron(III) spin-state in the model complexes of azide hemoprotein by porphycene, corrphycene, and hemiporphycene macrocycles.

Spin states of the iron(III) complexes of porphyrin, porphycene, hemiporphycene, and corrphycene bearing both 1-methylimidazole and azide as axial ligands were analyzed with infrared (IR) spectroscopy at 20 degrees C. The IR stretching band of coordinating azide split into two peaks around 2047 and 2017 cm(-1) reflecting an equilibrium between the high- (S = 5/2) and low- (S = 1/2) spin states. The high-spin fraction changed over a 0-90% range among the macrocycles, demonstrating that the tetrapyrrole array is essential to control the equilibrium.

FTIR detection of protonation/deprotonation of key carboxyl side chains caused by redox change of the Cu(A)-heme a moiety and ligand dissociation from the heme a3-Cu(B) center of bovine heart cytochrome c oxidase.

FTIR spectral changes of bovine cytochrome c oxidase (CcO) upon ligand dissociation from heme a(3)() and redox change of the Cu(A)-heme a moiety (Cu(A)Fe(a)()) were investigated. In a photosteady state under CW laser illumination at 590 nm to carbonmonoxy CcO (CcO-CO), the C-O stretching bands due to Fe(a3)()(2+)CO and Cu(B)(1+)CO were identified at 1963 and 2063 cm(-)(1), respectively, for the fully reduced (FR) state [(Cu(A)Fe(a)())(3+)Fe(a3)()(2+)Cu(B)(1+)] and at 1965 and 2061 cm(-)(1) for the mixed valence (MV) state [(Cu(A)Fe(a)())(5+)Fe(a3)()(2+)Cu(B)(1+)] in H(2)O as well as in D(2)O. For the MV state, however, another band due to Cu(B)(1+)CO was found at 2040 cm(-)(1), which was distinct from the alpha/beta conformers in the spectral behaviors, and therefore was assigned to the (Cu(A)Fe(a)())(4+)Fe(a3)()(3+)Cu(B)(1+)CO generated by back electron transfer.

Iron hemiporphycene as a functional prosthetic group for myoglobin.

The iron complex of hemiporphycene, a molecular hybrid of porphyrin with porphycene, was incorporated into the apomyoglobin pocket to examine ligand binding ability of the iron atom in the novel porphyrinoid. Apomyoglobin was successfully coupled with a stoichiometric amount of ferric hemiporphycene to afford the reconstituted myoglobin equipped with the iron coordination structure of native protein. Cyanide, imidazole, and fluoride coordinated to the ferric protein with affinities comparable with those for native myoglobin.