A RaPID Macrocyclic Peptide That Inhibits the Formation of α-Synuclein Amyloid Fibrils.

There is considerable interest in drug discovery targeting the aggregation of α-synuclein (αSyn) since this molecular process is closely associated with Parkinson's disease. However, inhibiting αSyn aggregation remains a major challenge because of its highly dynamic nature which makes it difficult to form a stable binding complex with a drug molecule. Here, by exploiting Random non-standard Peptides Integrated Discovery (RaPID) system, we identified a macrocyclic peptide, BD1, that could interact with immobilized αSyn and inhibit the formation of fibrils.

Structural dynamics of the chromo-shadow domain and chromodomain of HP1 bound to histone H3K9 methylated peptide, as measured by site-directed spin-labeling EPR spectroscopy.

The structural dynamics of the chromo-shadow domain (CSD) and chromodomain (CD) of human HP1 proteins essential for heterochromatin formation were investigated at the nanosecond and nanometer scales by site-directed spin labeling electron paramagnetic resonance and pulsed double resonance spectroscopy. Distance measurements showed that the spin-labeled CSD of human HP1α and HP1γ tightly dimerizes. Unlike CD-CD interaction observed in fission yeast HP1 in an inactivated state (Canzio et al.

Structural analysis of cross-linked poly(vinyl alcohol) using high-field DNP-NMR.

Poly(vinyl alcohol) (PVOH) is a water-soluble synthetic polymer, widely used in materials for functional films and moldings, fiber fabric sizing agents, paper coating resins, and adhesives. PVOH is mainly applied in the form of an aqueous solution, yet after its application, insolubility (water resistance) is required. To achieve this, additives are introduced.

Myosin and Other Energy-Transducing ATPases: Structural Dynamics Studied by Electron Paramagnetic Resonance.

The objective of this article was to document the energy-transducing and regulatory interactions in supramolecular complexes such as motor, pump, and clock ATPases. The dynamics and structural features were characterized by motion and distance measurements using spin-labeling electron paramagnetic resonance (EPR) spectroscopy. In particular, we focused on myosin ATPase with actin-troponin-tropomyosin, neural kinesin ATPase with microtubule, P-type ion-motive ATPase, and cyanobacterial clock ATPase.

Structural Dynamics of the N-Extension of Cardiac Troponin I Complexed with Troponin C by Site-Directed Spin Labeling Electron Paramagnetic Resonance.

The secondary structure of the N-extension of cardiac troponin I (cTnI) was determined by measuring the distance distribution between spin labels attached to the i and i + 4 residues: 15/19, 23/27, 27/31, 35/39, and 43/47. All of the EPR spectra of these regions in the monomeric state were broadened and had a amplitude that was reduced by two-thirds of that of the single spin-labeled spectra and was fit by two residual distance distributions, with a major distribution one spreading over the range from 1 to 2.5 nm and the other minor peak at 0.

Difference in the hydration water mobility around F-actin and myosin subfragment-1 studied by quasielastic neutron scattering.

Hydration water is essential for a protein to perform its biological function properly. In this study, the dynamics of hydration water around F-actin and myosin subfragment-1 (S1), which are the partner proteins playing a major role in various cellular functions related to cell motility including muscle contraction, was characterized by incoherent quasielastic neutron scattering (QENS). The QENS measurements on the DO- and HO-solution samples of F-actin and S1 provided the spectra of hydration water, from which the translational diffusion coefficient (D), the residence time (τ), and the rotational correlation time (τ) were evaluated.

Internal dynamics of F-actin and myosin subfragment-1 studied by quasielastic neutron scattering.

Various biological functions related to cell motility are driven by the interaction between the partner proteins, actin and myosin. To obtain insights into how this interaction occurs, the internal dynamics of F-actin and myosin subfragment-1 (S1) were characterized by the quasielastic neutron scattering measurements on the solution samples of F-actin and S1. Contributions of the internal motions of the proteins to the scattering spectra were separated from those of the global macromolecular diffusion.

Site-directed spin labeling-electron spin resonance mapping of the residues of cyanobacterial clock protein KaiA that are affected by KaiA-KaiC interaction.

The cyanobacterial clock proteins KaiA, KaiB and KaiC interact with each other to generate circadian oscillations. We have identified the residues of the KaiA homodimer affected through association with hexameric KaiC (KaiC6mer) using a spin-label-tagged KaiA C-terminal domain protein (KaiAc) and performing electron spin resonance (ESR) analysis. Cys substitution and/or the attachment of a spin label to residues located at the bottom area of the KaiAc concave surface, a KaiC-binding groove, hindered the association of KaiAc with KaiC6mer, suggesting that the groove likely mediates the interaction with KaiC6mer.

Nucleotide-dependent displacement and dynamics of the α-1 helix in kinesin revealed by site-directed spin labeling EPR.

In kinesin X-ray crystal structures, the N-terminal region of the α-1 helix is adjacent to the adenine ring of the bound nucleotide, while the C-terminal region of the helix is near the neck-linker (NL). Here, we monitor the displacement of the α-1 helix within a kinesin monomer bound to microtubules (MTs) in the presence or absence of nucleotides using site-directed spin labeling EPR. Kinesin was doubly spin-labeled at the α-1 and α-2 helices, and the resulting EPR spectrum showed dipolar broadening.

Calcium-dependent interaction sites of tropomyosin on reconstituted muscle thin filaments with bound Myosin heads as studied by site-directed spin-labeling.

To identify the interaction sites of Tm, we measured the rotational motion of a spin-label covalently bound to the side chain of a cysteine that was genetically incorporated into rabbit skeletal muscle tropomyosin (Tm) at positions 13, 36, 146, 160, 174, 190, 209, 230, 271, or 279. Most of the Tm residues were immobilized on actin filaments with myosin-S1 bound to them. The residues in the mid-portion of Tm, namely, 146, 174, 190, 209, and 230, were mobilized when the troponin (Tn) complex bound to the actin-Tm-S1 filaments.

Difference in hydration structures between F-actin and myosin subfragment-1 detected by small-angle X-ray and neutron scattering.

Hydration structures around F-actin and myosin subfragment-1 (S1), which play central roles as counterparts in muscle contraction, were investigated by small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS). The radius of gyration of chymotryptic S1 was evaluated to be 41.3±1.

Spatial arrangement of rhodopsin in retinal rod outer segment membranes studied by spin-labeling and pulsed electron double resonance.

We have determined the spatial arrangement of rhodopsin in the retinal rod outer segment (ROS) membrane by measuring the distances between rhodopsin molecules in which native cysteines were spin-labeled at ~1.0 mol/mol rhodopsin. The echo modulation decay of pulsed electron double resonance (PELDOR) from spin-labeled ROS curved slightly with strong background decay.

Substrate-induced conformational changes in sarcoplasmic reticulum Ca2+-ATPase probed by surface modification using diethylpyrocarbonate with mass spectrometry.

We have identified 15 residues from the surface of sarcoplasmic reticulum Ca(2+)-pump ATPase, by mass spectrometry using diethylpyrocarbonate modification. The reactivity of 9 residues remained high under all the conditions. The reactivity of Lys-515 at the nucleotide site was severely inhibited by ATP, whereas that of Lys-158 in the A-domain decreased by one-half and increased by five-fold in the presence of Ca(2+) and MgF(4), respectively.

Interaction sites of tropomyosin in muscle thin filament as identified by site-directed spin-labeling.

To identify interaction sites we measured the rotational motion of a spin label covalently bound to the side chain of a cysteine genetically incorporated into rabbit skeletal muscle tropomyosin (Tm) at positions 13, 36, 146, 160, 174, 190, 209, 230, 271, and 279. Upon the addition of F-actin, the mobility of all the spin labels, especially at position 13, 271, or 279, of Tm was inhibited significantly. Slow spin-label motion at the C-terminus (at the 230th and 271st residues) was observed upon addition of troponin.

Switch action of troponin on muscle thin filament as revealed by spin labeling and pulsed EPR.

We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to measure the distance between spin labels at Cys(133) of the regulatory region of TnI (TnI133) and a native or genetically substituted cysteine of TnC (TnC44, TnC61, or TnC98). In the +Ca(2+) state, the TnC44-TnI133-T distance was 42 A, with a narrow distribution (half-width of 9 A), suggesting that the regulatory region binds the N-lobe of TnC. Distances for TnC61-TnI133 and TnC98-TnI133 were also determined to be 38 A (width of 12 A) and 22 A (width of 3.

Scapinin, the protein phosphatase 1 binding protein, enhances cell spreading and motility by interacting with the actin cytoskeleton.

Scapinin, also named phactr3, is an actin and protein phosphatase 1 (PP1) binding protein, which is expressed in the adult brain and some tumor cells. At present, the role(s) of scapinin in the brain and tumors are poorly understood. We show that the RPEL-repeat domain of scapinin, which is responsible for its direct interaction with actin, inhibits actin polymerization in vitro.

Nucleotide-induced flexibility change in neck linkers of dimeric kinesin as detected by distance Measurements using spin-labeling EPR.

Using dipolar continuous-wave and pulsed electron paramagnetic resonance methods, we have determined the distribution of the distances between two spin labels placed on the middle of each of the neck linkers of dimeric kinesin. In the absence of microtubules, the distance was centered at 3.3 nm, but displayed a broad distribution with a width of 2.

Degradation of DNA into 5'-monodeoxyribonucleotides in the presence of Mn(2+) ions.

DNA is known to be aggregated by metal ions including Mn(2+) ions, but analysis of the aggregation process from a chemical viewpoint, which means identification of the product yielded during the process, has not been performed yet. On examination of the kinds of degraded materials that were in the supernatant obtained on centrifugation of a DNA mixture aggregated under conditions of 10 mM Mn(2+) ions ([Mn]/[P] = 46.3) at 70 degrees C for 1 h, the degradation products were found to be dAMP, dCMP, dGMP, and TMP.

Conformational dynamics of loops L11 and L12 of kinesin as revealed by spin-labeling EPR.

The EPR spectra of the spin labels attached to loops L11 and L12 of kinesin were resolved into slow (rotational correlation time, tau=10-45 ns) and fast (tau=2 ns) components. The fraction of the slow component increased considerably when kinesin was complexed with a microtubule (MT). On MT binding and in the presence of nucleotides ADP and AMPPNP, the spin labels on L11, particularly at A252C and L249C, significantly decreased the fraction of the slow component.

Structure of the human GINS complex and its assembly and functional interface in replication initiation.

The eukaryotic GINS complex is essential for the establishment of DNA replication forks and replisome progression. We report the crystal structure of the human GINS complex. The heterotetrameric complex adopts a pseudo symmetrical layered structure comprising two heterodimers, creating four subunit-subunit interfaces.

Calcium structural transition of troponin in the complexes, on the thin filament, and in muscle fibres, as studied by site-directed spin-labelling EPR.

We have measured the intersite distance, side-chain mobility and orientation of specific site(s) of troponin (Tn) complex on the thin filaments or in muscle fibres as well as in solution by means of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR). We have examined the Ca(2+)-induced movement of the B and C helices relative to the D helix in a human cardiac (hc)TnC monomer state and hcTnC-hcTnI binary complex. An interspin distance between G42C (B helix) and C84 (D helix) was 18.

Morphology of isolated Gli349, a leg protein responsible for Mycoplasma mobile gliding via glass binding, revealed by rotary shadowing electron microscopy.

Several species of mycoplasmas rely on an unknown mechanism to glide across solid surfaces in the direction of a membrane protrusion at the cell pole. Our recent studies on the fastest species, Mycoplasma mobile, suggested that a 349-kDa protein, Gli349, localized at the base of the membrane protrusion called the neck, forms legs that stick out from the neck and propel the cell by repeatedly binding to and releasing from a solid surface, based on the energy of ATP hydrolysis. Here, the Gli349 protein was isolated from mycoplasma cells and its structure was analyzed.

Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR.

We measured EPR spectra from a spin label on the Cys133 residue of troponin I (TnI) to identify Ca(2+)-induced structural states, based on sensitivity of spin-label mobility to flexibility and tertiary contact of a polypeptide. Spectrum from Tn complexes in the -Ca(2+) state showed that Cys133 was located at a flexible polypeptide segment (rotational correlation time tau=1.9ns) that was free from TnC.

Dynamic structures of motor proteins myosin and kinesin, and switch protein troponin as detected by SDSL-ESR.

We have studied biological nano-machines, motor and switch proteins operating as supramolecular complexes by electron spin resonance (ESR) and found key features of their molecular movements. In all the systems, the specific movements of elements or domains were detected and quite dynamic at nanometer scale. We have observed two broad but distinct orientations, separated by a 25 degrees axial rotation, of a spin label attached specifically to the light chain (LC) domain of myosin motor in the muscle fibers.