Macromolecules Of Interest Research Articles

Preparation of Filamentous Actin for Polarized Total Internal Reflection Fluorescence Microscopy (polTIRFM) Motility Assays

Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. In this protocol, filamentous actin (F-actin) is polymerized from purified, monomeric actin (G-actin) for use in polTIRFM motility assays in which actin interacts with myosin. The procedures include (1) the preparation of unlabeled F-actin from G-actin; (2) the preparation of F-actin that is sparsely labeled with 6'-IATR (6'-iodoacetamidotetramethylrhodamine); and (3) the preparation of F-actin with a combination of unlabeled, biotinylated, and rhodamine-labeled monomers. Rhodamine-phalloidin actin, also used in polTIRFM assays, can be prepared using a procedure similar to the one for unlabeled actin.

Fluorescent Labeling of Myosin V for Polarized Total Internal Reflection Fluorescence Microscopy (polTIRFM) Motility Assays

Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes how to exchange bifunctional rhodamine-calmodulin (BR-CaM) for wild-type calmodulin (WT-CaM) on the lever arm of myosin V. BR-CaM is exchanged at low stoichiometry (∼0.4 BR-CaM per double-headed myosin V) to obtain myosin V molecules with one BR-CaM and to limit the proportion of myosin V molecules with two or more probes. The stoichiometry is very sensitive to the concentration of calcium during the exchange reaction. The labeled myosin V can subsequently be used for investigating the motility of myosin V in vitro with a polTIRFM processive motility assay, which is performed on substrate-attached actin.

Fluorescent Labeling of Calmodulin with Bifunctional Rhodamine: Figure 1.

Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes how to label chicken calmodulin (CaM) with bifunctional rhodamine (BR) at two engineered cysteine (Cys) residues (P66C and A73C) so that it cross-links the two Cys sites. The resulting BR-CaM protein is then purified by high-performance liquid chromatography (HPLC) and concentrated by filter centrifugation. To confirm that the two Cys residues in the labeled CaM are actually cross-linked by BR, a sample of purified BR-CaM is digested by an endoproteinase and analyzed by mass spectrometry. The BR-CaM can then be used to label myosin V, which can in turn be used in a polTIRFM processive motility assay.

Accurate Distance Determination of Nucleic Acids via Förster Resonance Energy Transfer: Implications of Dye Linker Length and Rigidity

In Förster resonance energy transfer (FRET) experiments, the donor (D) and acceptor (A) fluorophores are usually attached to the macromolecule of interest via long flexible linkers of up to 15 Å in length. This causes significant uncertainties in quantitative distance measurements and prevents experiments with short distances between the attachment points of the dyes due to possible dye-dye interactions. We present two approaches to overcome the above problems as demonstrated by FRET measurements for a series of dsDNA and dsRNA internally labeled with Alexa488 and Cy5 as D and A dye, respectively. First, we characterize the influence of linker length and flexibility on FRET for different dye linker types (long, intermediate, short) by analyzing fluorescence lifetime and anisotropy decays. For long linkers, we describe a straightforward procedure that allows for very high accuracy of FRET-based structure determination through proper consideration of the position distribution of the dye and of linker dynamics. The position distribution can be quickly calculated with geometric accessible volume (AV) simulations, provided that the local structure of RNA or DNA in the proximity of the dye is known and that the dye diffuses freely in the sterically allowed space. The AV approach provides results similar to molecular dynamics simulations (MD) and is fully consistent with experimental FRET data. In a benchmark study for ds A-RNA, an rmsd value of 1.3 Å is achieved. Considering the case of undefined dye environments or very short DA distances, we introduce short linkers with a propargyl or alkenyl unit for internal labeling of nucleic acids to minimize position uncertainties. Studies by ensemble time correlated single photon counting and single-molecule detection show that the nature of the linker strongly affects the radius of the dye's accessible volume (6-16 Å). For short propargyl linkers, heterogeneous dye environments are observed on the millisecond time scale. A detailed analysis of possible orientation effects (κ(2) problem) indicates that, for short linkers and unknown local environments, additional κ(2)-related uncertainties are clearly outweighed by better defined dye positions.

Well-defined graft copolymers: from controlled synthesis to multipurpose applications

Graft copolymers with a large number of side chains chemically attached onto a linear backbone are endowed with unusual properties thanks to their confined and compact structures, including wormlike conformation, compact molecular dimensions and notable chain end effects. Growing attention has been paid to these interesting macromolecules due to their importance in understanding the correlation between architectures and properties, as well as their potential applications. To date, the synthesis and properties of graft copolymers in both solution and bulk have been extensively investigated, along with their applications. In this tutorial review, recent advances in synthetic approaches towards the construction of well-defined graft copolymers are discussed in detail and applications of these interesting macromolecules are highlighted by selected examples.

Application of flow field-flow fractionation for the characterization of macromolecules of biological interest: a review

An overview is given of the recent literature on (bio) analytical applications of flow field-flow fractionation (FlFFF). FlFFF is a liquid-phase separation technique that can separate macromolecules and particles according to size. The technique is increasingly used on a routine basis in a variety of application fields. In food analysis, FlFFF is applied to determine the molecular size distribution of starches and modified celluloses, or to study protein aggregation during food processing. In industrial analysis, it is applied for the characterization of polysaccharides that are used as thickeners and dispersing agents. In pharmaceutical and biomedical laboratories, FlFFF is used to monitor the refolding of recombinant proteins, to detect aggregates of antibodies, or to determine the size distribution of drug carrier particles. In environmental studies, FlFFF is used to characterize natural colloids in water streams, and especially to study trace metal distributions over colloidal particles. In this review, first a short discussion of the state of the art in instrumentation is given. Developments in the coupling of FlFFF to various detection modes are then highlighted. Finally, application studies are discussed and ordered according to the type of (bio) macromolecules or bioparticles that are fractionated.

Analytical Ultracentrifugation

Analytical Ultracentrifugation

Determining the distribution of probes between different subcellular locations through automated unmixing of subcellular patterns

Many proteins or other biological macromolecules are localized to more than one subcellular structure. The fraction of a protein in different cellular compartments is often measured by colocalization with organelle-specific fluorescent markers, requiring availability of fluorescent probes for each compartment and acquisition of images for each in conjunction with the macromolecule of interest. Alternatively, tailored algorithms allow finding particular regions in images and quantifying the amount of fluorescence they contain. Unfortunately, this approach requires extensive hand-tuning of algorithms and is often cell type-dependent. Here we describe a machine-learning approach for estimating the amount of fluorescent signal in different subcellular compartments without hand tuning, requiring only the acquisition of separate training images of markers for each compartment. In testing on images of cells stained with mixtures of probes for different organelles, we achieved a 93% correlation between estimated and expected amounts of probes in each compartment. We also demonstrated that the method can be used to quantify drug-dependent protein translocations. The method enables automated and unbiased determination of the distributions of protein across cellular compartments, and will significantly improve imaging-based high-throughput assays and facilitate proteome-scale localization efforts.

Adaptive Anisotropic Kernels for Nonparametric Estimation of Absolute Configurational Entropies in High-Dimensional Configuration Spaces

The quasiharmonic approximation is the most widely used estimate for the configurational entropy of macromolecules from configurational ensembles generated from atomistic simulations. This method, however, rests on two assumptions that severely limit its applicability, (i) that a principal component analysis yields sufficiently uncorrelated modes and (ii) that configurational densities can be well approximated by Gaussian functions. In this paper we introduce a nonparametric density estimation method which rests on adaptive anisotropic kernels. It is shown that this method provides accurate configurational entropies for up to 45 dimensions thus improving on the quasiharmonic approximation. When embedded in the minimally coupled subspace framework, large macromolecules of biological interest become accessible, as demonstrated for the 67-residue coldshock protein.

Bulk charge transport in liquid-crystalline polymer semiconductors based on poly(2,5-bis(3-alkylthiophen-2-yl)thieno[3,2-b]thiophene)

The class of liquid-crystalline semiconducting polymers based on poly(2,5-bis(3-alkylthiophen-2-yl)thieno[3,2-b]thiophene) recently has attracted significant interest in the field of organic electronics, predominantly due to their promising performance in field-effect transistor (FET) structures with device mobilities reaching—if not exceeding—those of amorphous silicon architectures. Less is known, however, about the bulk charge-transport properties of these interesting macromolecules. We therefore conducted time-of-flight (TOF) photoconductivity measurements on one particular material of this class of organic semiconductors—i.e. poly(2,5-bis(3-dodecylthiophen-2-yl)thieno[3,2-b]thiophene), pBTTT-C12—and attempted to correlate the electronic bulk properties of this polymer with its microstructural development with temperature. The effect of annealing was also investigated.

Adaptive anisotropic kernels for nonparametric estimation of absolute configurational entropies in high-dimensional configuration spaces

The quasiharmonic approximation is the most widely used estimate for the configurational entropy of macromolecules from configurational ensembles generated from atomistic simulations. This method, however, rests on two assumptions that severely limit its applicability, (i) that a principal component analysis yields sufficiently uncorrelated modes and (ii) that configurational densities can be well approximated by Gaussian functions. In this paper we introduce a nonparametric density estimation method which rests on adaptive anisotropic kernels. It is shown that this method provides accurate configurational entropies for up to 45 dimensions thus improving on the quasiharmonic approximation. When embedded in the minimally coupled subspace framework, large macromolecules of biological interest become accessible, as demonstrated for the 67-residue coldshock protein.

Star-shaped oligofluorene nanostructured blend materials: controlled micro-patterning and physical characteristics

Star-shaped oligofluorene consists of highly-fluorescent macromolecules of considerable interest for organic electronics. Here, we demonstrate controlled micro-patterning of these organic nanostructured molecules by blending them with custom-synthesized photo-curable aliphatic polymer matrices to facilitate solventless inkjet printing. The printed microstructures are spherical with minimum dimensions of 12 μm diameter and 1 μm height when using a cartridge delivering ∼1 pL droplets. We evaluate the physical characteristics of the printed structures. Photoluminescence studies indicate that the blend materials possess similar fluorescence properties to neat materials in solid films or toluene solution. The fluorescence lifetime consists of two components, respectively 0.68±0.01 ns (τ1) and 1.23±0.12 ns (τ2). This work demonstrates that inkjet printing of such blends provides an attractive method of handling fluorescent nano-scaled molecules for photonic and optoelectronic applications.

Diels–Alder “Click” Chemistry in Designing Dendritic Macromolecules

In the current decade, design of dendritic macromolecules including dendrimers and polymers has reached a new era, mainly due to the remarkably successful and elegant synthetic routes that have been developed recently. This survey aims at demonstrating the potential of the Diels-Alder "click" chemistry, a very effective, simple and versatile synthetic tool in the formation of complex and functional nanometer-sized assemblies. The role of retro Diels-Alder reaction is also explored to highlight the increasing competitive potential of this strategy in the design of dendritic macromolecules of topical interest. The potential "green" nature of this methodology in constructing macromolecular assemblies is also evaluated.

An Optical Torque Wrench For Studying Kinesin Dynamics

We constructed an optical tweezers instrument capable of exerting torque and measuring the angular motions of small trapped particles, based on the rotation of linear polarization of the trapping laser beam. To change polarization, we employed an electro-optic modulator (EOM), which allows for a much simpler setup than a previous design (La Porta, A. and Wang, M.D. 2004. Optical torque wrench: angular trapping, rotation, and torque detection of quartz microparticles. Phys. Rev. Lett. 19:190801). Torque is monitored by measuring the difference between circularly left-handed and right-handed components of the transmitted beam: constant torque is implemented by feeding this angular signal back into a custom-designed electronic servo loop. The limited dynamic range of the EOM (±180°) is extended by monitoring the drive signal with a microcontroller, which triggers a switch to flip the output polarization by ±180° once a pre-set threshold is reached (within 10 μs). These features enable us to maintain constant torque over unlimited rotations at high bandwidth (∼100 kHz). In addition, we developed optically birefringent, non-spherical particles suitable for this instrument using nanofabrication techniques. The polarization-sensitive method employed by the apparatus precludes the use of Wollaston prisms to perform differential interference contrast (DIC) imaging. However, by exploiting conventional video-enhancement techniques (including background subtraction, contrast enhancement, and frame averaging), we report that individual microtubules (∼25 nm in diameter) can be visualized without DIC optics at ∼5 frames per second. Altogether, our instrument allows for the simultaneous application of force and torque to the study of macromolecules of interest. We are presently extending our previous studies (Gutierrez-Medina, B., et al. 2339-Pos. Torsional properties of kinesin. Biophys. J. 2008. 94:2339-Pos) on the torsional properties of the molecular motor kinesin to investigate the effect of torque on its stepwise motion.

A molecular ruler for measuring quantitative distance distributions.

We report a novel molecular ruler for measurement of distances and distance distributions with accurate external calibration. Using solution X-ray scattering we determine the scattering interference between two gold nanocrystal probes attached site-specifically to a macromolecule of interest. Fourier transformation of the interference pattern provides a model-independent probability distribution for the distances between the probe centers-of-mass. To test the approach, we measure end-to-end distances for a variety of DNA structures. We demonstrate that measurements with independently prepared samples and using different X-ray sources are highly reproducible, we demonstrate the quantitative accuracy of the first and second moments of the distance distributions, and we demonstrate that the technique recovers complex distribution shapes. Distances measured with the solution scattering-interference ruler match the corresponding crystallographic values, but differ from distances measured previously with alternate ruler techniques. The X-ray scattering interference ruler should be a powerful tool for relating crystal structures to solution structures and for studying molecular fluctuations.

Feasibility Investigation of NbN Nanowires as Detector in Time-of-Flight Mass Spectrometers for Macromolecules of Interest in Biology (Proteins)

We are investigating the possibility of using NbN nanowires as detectors in time-of-flight mass spectrometers for investigation of macromolecules of interest in biology (proteins). NbN nanowires could overcome the two major drawbacks encountered so far by cryogenic detectors, namely the low working temperature in the mK region and the slow temporal response. In fact, NbN nanowires can work at 5 K and the response time is at least a factor 10–100 better than that of other cryogenic detectors. We present a feasibility study based on a numerical code to calculate the response of a NbN nanowire. The parameter space is investigated at different energies from IR to macromolecules (i.e. from eV to keV) in order to understand if larger value of film thickness and width can be used for the keV energy region. We also present preliminary experimental results of irradiation with X-ray photons of NbN to simulate the effect of macromolecules of the same energy.

Overcoming the Inadequacies or Limitations of Experimental Structures as Drug Targets by Using Computational Modeling Tools and Molecular Dynamics Simulations

X-ray crystallography, NMR spectroscopy, and cryoelectron microscopy stand out as powerful tools that enable us to obtain atomic detail about biomolecules that can be potentially targeted by drugs. This knowledge is essential if virtual screening or structure-based ligand-design methods are going to be used in drug discovery. However, the macromolecule of interest is not always amenable to these types of experiment or, as is often the case, the conformation found experimentally cannot be used directly for docking studies because of significant changes between apo and bound forms. Furthermore, sometimes the desired insight into the binding mechanism cannot be gained because the structure of the ligand-receptor complex, not having been time-resolved, represents the endpoint of the binding process and therefore retains little or no information about the intermediate stages that led to its creation. Molecular dynamics (MD) simulations are routinely applied these days to the study of biomolecular systems with the aims of sampling configuration space more efficiently and getting a better understanding of the factors that determine structural stability and relevant biophysical and biochemical processes such as protein folding, ligand binding, and enzymatic reactions. This field has matured significantly in recent years, and strategies have been devised (for example activated, steered, or targeted MD) that allow the calculated trajectories to be biased in attempts to properly shape a ligand binding pocket or simulate large-scale motions involving one or more protein domains. On the other hand, low-frequency motions can be simulated quite inexpensively by calculation of normal modes which allow the investigation of alternative receptor conformations. Selected examples in which these methods have been applied to several medicinal chemistry and in silico pharmacology endeavors are presented.

Cryo‐fluorescence microscopy facilitates correlations between light and cryo‐electron microscopy and reduces the rate of photobleaching

Fluorescence light microscopy (LM) has many advantages for the study of cell organization. Specimen preparation is easy and relatively inexpensive, and the use of appropriate tags gives scientists the ability to visualize specific proteins of interest. LM is, however, limited in resolution, so when one is interested in ultrastructure, one must turn to electron microscopy (EM), even though this method presents problems of its own. The biggest difficulty with cellular EM is its limited utility in localizing macromolecules of interest while retaining good structural preservation. We have built a cryo-light microscope stage that allows us to generate LM images of vitreous samples prepared for cryo-EM. Correlative LM and EM allows one to find areas of particular interest by using fluorescent proteins or vital dyes as markers within vitrified samples. Once located, the sample can be placed in the EM for further study at higher resolution. An additional benefit of the cryo-LM stage is that photobleaching is slower at cryogenic temperatures (-140 degrees C) than at room temperature.

Solvent-Mediated Proton Transfer in Catalysis by Carbonic Anhydrase

Considerable attention has been focused on proton transfer through intervening water molecules in complex macromolecules of biological interest, such as bacteriorhodopsin, cytochrome c oxidase, and many others. Proton transfer in catalysis by carbonic anhydrase provides a useful model for the study of the properties of such proton translocations. High-resolution X-ray crystallography in combination with measurements of catalysis have revealed new details of this process. A prominent proton shuttle residue His64 shows evidence of structural mobility, which appears to enhance proton transfer between the active site and bulk solvent. Moreover, the properties of the imidazole side chain of His64, including its conformations and pK(a), are finely tuned by surrounding residues of the active-site cavity. The structure of a network of ordered solvent molecules located between His64 and the active site are also sensitive to surrounding residues. These features combine to provide efficient proton-transfer rates as great as 10(6) s(-1) necessary to sustain rapid catalysis.

Modelling of growth and product formation of Porphyridium purpureum

In this contribution experimental data and simulations of growth and product formation of the unicellular microalgae Porphyridium purpureum are presented. A mathematical model has been developed for a better understanding of growth and product formation in production plants. The model has been refined with the results of several cultivations in a new photobioreactor designed especially for the study of microalgal kinetics under highly defined illumination conditions. In this photobioreactor light is generated by an external light source and then distributed by means of optical fibres into an internal draft tube which also serves as irradiation element. All cultivations were performed in turbidostate mode. The influence of different light intensity changes, including stepwise change and light–dark cycles in the range from millisecond to second, has been investigated and the results were integrated into the mathematical model. The structured mathematical model consists of three levels: metabolic flux, control of macromolecules and the reactor level. A new linear optimization approach has been realized, enabling the model to describe even very different cultivation conditions. Output variables are among others the commercially interesting macromolecules of the microalgae, e.g. polysaccharides, pigments and polyunsaturated fatty acids. Thus, reliable predictions of the specific production rates of these products are possible for the production in a larger scale.