Regularity and Synchrony in Motor Proteins

We investigate the origin of the regularity and synchrony which have been observed in numerical experiments of two realistic models of molecular motors, namely Fisher–Kolomeisky’s model of myosin V for vesicle transport in cells and Duke’s model of myosin II for sarcomere shortening in muscle contraction. We show that there is a generic organizing principle behind the emergence of regular gait for a motor pulling a large cargo and synchrony of action of many motors pulling a single cargo. These results are surprising in that the models used are inherently stochastic, and yet they display regular behaviors in the parameter range relevant to experiments. Our results also show that these behaviors are not tied to the particular models used in these experiments, but rather are generic to a wide class of motor protein models.     

On the NMR structure determination of a 44n RNA pseudoknot: Assignment strategies and derivation of torsion angle restraints

The complete T- and pseudoknotted acceptor arm of the tRNA-like structure of turnip yellow mosaic virus (TYMV) genomic RNA has been studied by NMR spectroscopy. Resonance assignment and the gathering of restraints of the 44-mer are impeded by spectral complexity as well as by line broadening. The latter is caused by local dynamical effects in the pseudoknot domain in the molecule. These specific problems could be solved by using different field strengths and selectively ¹³C/¹⁵ labeled samples. Experiments for assigning the sugar spin systems were adjusted to satisfy the requirements of this system. Furthermore, the quality of the structure could be improved by determining the backbone torsion angles , and , using new approaches that were tailored for use in large RNA molecules.     

Conformational analysis of protein and nucleic acid fragments with the new grid search algorithm FOUND

The new computer algorithm FOUND, which is implemented as an integrated module of the DYANA structure calculation program, is capable of performing systematic local conformation analyses by exhaustive grid searches for arbitrary contiguous fragments of proteins and nucleic acids. It uses torsion angles as the only degrees of freedom to identify all conformations that fulfill the steric and NMR-derived conformational restraints within a contiguous molecular fragment, as defined either by limits on the maximal restraint violations or by the fragment-based DYANA target function value. Sets of mutually dependent torsion angles, for example in ribose rings, are treated as a single degree of freedom. The results of the local conformation analysis include allowed torsion angle ranges and stereospecific assignments for diastereotopic substituents, which are then included in the input of a subsequent structure calculation. FOUND can be used for grid searches comprising up to 13 torsion angles, such as the backbone of a complete -helical turn or dinucleotide fragments in nucleic acids, and yields a significantly higher number of stereospecific assignments than the precursor grid search algorithm HABAS.     

Magnetoreception in microorganisms and fungi

The ability to respond to magnetic fields is ubiquitous among the five kingdoms of organisms. Apart from the mechanisms that are at work in bacterial magnetotaxis, none of the innumerable magnetobiological effects are as yet completely understood in terms of their underlying physical principles. Physical theories on magnetoreception, which draw on classical electrodynamics as well as on quantum electrodynamics, have greatly advanced during the past twenty years, and provide a basis for biological experimentation. This review places major emphasis on theories, and magnetobiological effects that occur in response to weak and moderate magnetic fields, and that are not related to magnetotaxis and magnetosomes. While knowledge relating to bacterial magnetotaxis has advanced considerably during the past 27 years, the biology of other magnetic effects has remained largely on a phenomenological level, a fact that is partly due to a lack of model organisms and model responses; and in great part also to the circumstance that the biological community at large takes little notice of the field, and in particular of the available physical theories. We review the known magnetobiological effects for bacteria, protists and fungi, and try to show how the variegated empirical material could be approached in the framework of the available physical models.     

Binding kinetics of calmodulin with target peptides of three nitric oxide synthase isozymes

Efficient electron transfer from reductase domain to oxygenase domain in nitric oxide synthase (NOS) is dependent on the binding of calmodulin (CaM). Rate constants for the binding of CaM to NOS target peptides was only determined previously by surface plasmon resonance (SPR) (Biochemistry 35, 8742-8747, 1996) suggesting that the binding of CaM to NOSs is slow and does not support the fast electron transfer in NOSs measured in previous and this studies. To resolve this contradiction, the binding rates of holo Alexa 350 labeled T34C/T110W CaM (Alexa-CaM) to target peptides from three NOS isozymes were determined using fluorescence stopped-flow. All three target peptides exhibited fast k_on constants at 4.5 °C: 6.6 × 10⁸ M^− 1 s^− 1 for nNOS_726-749, 2.9 × 10⁸ M^− 1 s^− 1 for eNOS_492-511 and 6.1 × 10⁸ M^− 1 s^− 1 for iNOS_507-531, 3-4 orders of magnitude faster than those determined previously by SPR. Dissociation rates of NOS target peptides from Alexa-CaM/peptide complexes were measured by Ca²⁺ chelation with ETDA: 3.7 s^− 1 for nNOS_726-749, 4.5 s^− 1 for eNOS_492-511, and 0.063 s^− 1 for iNOS_507-531. Our data suggest that the binding of CaM to NOS is fast and kinetically competent for efficient electron transfer and is unlikely rate-limiting in NOS catalysis. Only iNOS_507-531 was able to bind apo Alexa-CaM, but in a very different conformation from its binding to holo Alexa-CaM.     

Analogues containing the paramagnetic amino acid TOAC as substrates for angiotensin I-converting enzyme

The angiotensin I-converting enzyme (ACE) converts the decapeptide angiotensin I (Ang I) into angiotensin II by releasing the C-terminal dipeptide. A novel approach combining enzymatic and electron paramagnetic resonance (EPR) studies was developed to determine the enzyme effect on Ang I containing the paramagnetic 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) at positions 1, 3, 8, and 9. Biological assays indicated that TOAC(1)-Ang I maintained partly the Ang I activity, and that only this derivative and the TOAC(3)-Ang I were cleaved by ACE. Quenching of Tyr(4) fluorescence by TOAC decreased with increasing distance between both residues, suggesting an overall partially extended structure. However, the local bend known to be imposed by the substituted diglycine TOAC is probably responsible for steric hindrance, not allowing the analogues containing TOAC at positions 8 and 9 to act as substrates. In some cases, although substrates and products differ by only two residues, the difference between their EPR spectral lineshapes allows monitoring the enzymatic reaction as a function of time.     

Electron paramagnetic resonance of single crystal deoxycobaltohemoglobin

Single crystals of horse ^CoHb were obtained by reduction of ^CoHb⁺ crystals with dithionite. Epr measurements showed that the g? and ^Coà tensors are both axial and share the same principal axis systems. Of the four subunits, the “heme” normals of C? and d? subunits ãb?plane 29 ± 1° from b?; they have the same orientation as the hemes in methemoglobin. The normals of “hemes” à and B? are 47 above the ãb? plane as compared to 16° in methemoglobin.     

A dual inhibitor against prolyl isomerase Pin1 and cyclophilin discovered by a novel real-time fluorescence detection method

Pin1, a peptidyl prolyl cis/trans isomerase (PPIase), is a potential target molecule for cancer, infectious disease, and Alzheimer’s disease. We established a high-throughput screening method for Pin1 inhibitors, which employs a real-time fluorescence detector. This screening method identified 66 compounds that inhibit Pin1 out of 9756 compounds from structurally diverse chemical libraries. Further evaluations of surface plasmon resonance methods and a cell proliferation assay were performed. We discovered a cell-active inhibitor, TME-001 (2-(3-chloro-4-fluoro-phenyl)-isothiazol-3-one). Surprisingly, kinetic analyses revealed that TME-001 is the first compound that exhibits dual inhibition of Pin1 (IC₅₀ = 6.1 μM) and cyclophilin, another type of PPIase, (IC₅₀ = 13.7 μM). This compound does not inhibit FKBP. This finding suggests the existence of similarities of structure and reaction mechanism between Pin1 and cyclophilin, and may lead to a more complete understanding of the active sites of PPIases.     

Fluorescent metal nanoshell and CK19 detection on single cell image

In this article, we report the synthesis strategy and optical properties of a novel type of fluorescence metal nanoshell when it was used as imaging agent for fluorescence cell imaging. The metal nanoshells were made with 40 nm silica cores and 10 nm silver shells. Unlike typical fluorescence metal nanoshells which contain the organic dyes in the cores, novel metal nanoshells were composed of Cy5-labelled monoclonal anti-CK19 antibodies (mAbs) on the external surfaces of shells. Optical measurements to the single nanoparticles showed that in comparison with the metal free labelled mAbs, the mAb-Ag complexes displayed significantly enhanced emission intensity and dramatically shortened lifetime due to near-field interactions of fluorophores with metal. These metal nanoshells were found to be able to immunoreact with target cytokeratin 19 (CK19) molecules on the surfaces of LNCAP and HeLa cells. Fluorescence cell images were recorded on a time-resolved confocal microscope. The emissions from the metal nanoprobes could be clearly isolated from the cellular autofluorescence backgrounds on the cell images as either individuals or small clusters due to their stronger emission intensities and shorter lifetimes. These emission signals could also be precisely counted on single cell images. The count number may provide an approach for quantifying the target molecules in the cells.     

RhoA-ROCK-Myosin pathway regulates morphological plasticity of cultured olfactory ensheathing cells

Olfactory ensheathing cells (OECs) are glial cells in the olfactory system with morphological and functional plasticity. Cultured OECs have the flattened and process-bearing shape. Reversible changes have been found between these two morphological phenotypes. However, the molecular mechanism underlying the regulation of their morphological plasticity remains elusive. Using RhoA FRET biosensor, we found that the active RhoA signal mainly distributed in the lamellipodia and/or filopodia of OECs. Local disruption of these active RhoA distributions led to the morphological change from the flattened into process-bearing shape and promoted process outgrowth. Furthermore, RhoA pathway inhibitors, Toxin-B, C3, Y-27632 or over-expression of DN-RhoA blocked serum-induced morphological change of OECs from the process-bearing into flattened shape, whereas the activation of RhoA pathway by lysophosphatidic acid (LPA) promoted the morphological change from the process-bearing into flattened shape. Finally, ROCK–Myosin–F-actin as a downstream of RhoA pathway was involved in morphological plasticity of OECs. Taken together, these results suggest that RhoA–ROCK–Myosin pathway mediates the morphological plasticity of cultured OECs in response to extracellular cues.