Proteins molecular probe/ dynamics

Lectins, sugar-binding proteins, have become powerful molecular probes to investigate the structure, topography and dynamics of cell-surface saccharide determinants (1). The utility of these proteins in the study of the surface properties of a variety of cell types has stimulated renewed interest in the determination of the molecular basis of their saccharide specificity. Furthermore lectins provide relatively simple models for the investigation of noncovalent interactions between saccharides and proteins. 

Finally, dynamic structure-based pharmacophore models can be derived through a method first described by Carlson et al that uses multiple conformations of the target protein, which are obtained either by molecular dynamics simulation or by the use of multiple experimentally determined conformations. The binding sites of the respective snapshots are flooded with small molecular probes (e.g., methanol for hydrogen-bond interactions and benzene for aromatic hydrophobic interactions) and while the protein structure is held rigid the probe molecules are subjected to a low-temperature Monte Carlo minimization where they undergo multiple, simultaneous gas-phase... 

Detection methods based on the post-electrophoretic staining of proteins with fluorescent compounds have the potential of increased sensitivity combined with an extended dynamic range for improved quantitation. The most commonly used reagents are the SYPRO series of dyes from Molecular Probes (Patton, 2000). Extensive studies have been carried out to evaluate the sensitivity of these fluorescent dyes compared with silver staining (Berggren et al., 2002 Lopez et al., 2000). In our laboratory (Yan et al.,... 

Figure 18.4. Confocal time-lapse microscopy of a microinjected Drosophila embryo. Dynamics of the actin (green) and microtubule (red) cytoskeleton are visualized during the syncytial mitotic divisions (A-D). An embryo that expresses the actin-binding protein GFP-moesin was microinjected with rho-damine-labeled a-tubulin (Molecular Probes), and the actin and microtubule dynamics were visualized as the mitotic cycle progressed from interphase (A) to anaphase (D).

Example Molecular dynamics simulations of selected portions of proteins can demonstrate the motion of an amino acid sequence while fixing the terminal residues. These simulations can probe the motion of an alpha helix, keeping the ends restrained, as occurs n atiirally m transmembrane proteins. You can also investigate the conformations of loops with fixed endpoints. 

For 25 years, molecular dynamics simulations of proteins have provided detailed insights into the role of dynamics in biological activity and function [1-3]. The earliest simulations of proteins probed fast vibrational dynamics on a picosecond time scale. Fifteen years later, it proved possible to simulate protein dynamics on a nanosecond time scale. At present it is possible to simulate the dynamics of a solvated protein on the microsecond time scale [4]. These gains have been made through a combination of improved computer processing (Moore s law) and clever computational algorithms [5]. 

The use of computer simulations to study internal motions and thermodynamic properties is receiving increased attention. One important use of the method is to provide a more fundamental understanding of the molecular information contained in various kinds of experiments on these complex systems. In the first part of this paper we review recent work in our laboratory concerned with the use of computer simulations for the interpretation of experimental probes of molecular structure and dynamics of proteins and nucleic acids. The interplay between computer simulations and three experimental techniques is emphasized (1) nuclear magnetic resonance relaxation spectroscopy, (2) refinement of macro-molecular x-ray structures, and (3) vibrational spectroscopy. The treatment of solvent effects in biopolymer simulations is a difficult problem. It is not possible to study systematically the effect of solvent conditions, e.g. added salt concentration, on biopolymer properties by means of simulations alone. In the last part of the paper we review a more analytical approach we have developed to study polyelectrolyte properties of solvated biopolymers. The results are compared with computer simulations. 

The aim of this Chapter is to review a method by which fluorescence properties of organic dyes can, in general, be predicted and understood at a microscopic (nm scale) by interfacing quantum methods with classical molecular dynamics (MD) methods. Some review of our extensive applications [1] of this method to the widely exploited intrinsic fluorescence probe in proteins, the amino acid tryptophan (Trp) will be followed by a discussion of electrochromic membrane voltagesensing dyes. 

When compared to fluorescent proteins, fluorophores and quenchers of fluorescence (short quenchers) are small molecules with sizes varying from 1 to 10 A. They are the main building blocks for constructing small molecule FRET probes. As molecular entities, they might influence the performance of the probe to a great extent. Their fluorescent properties will determine the sensitivity and dynamic range of the sensor. The success of the probe for a specific application will depend on the selection of the right fluorophores... 

It is not uncommon for protons to be taken up or released upon formation of a biomolecular complex. Experimental data on such processes can be compared to computational results based on, for example, Poisson-Boltzmann calculations.25 There is a need for methods that automatically probe for the correct protonation state in free energy calculations. This problem is complicated by the fact that proteins adapt to and stabilize whatever protonation state is assigned to them during the course of a molecular dynamics simulation.19 When the change in protonation state is known, equations are available to account for the addition or removal of protons from the solvent in the overall calculation of the free energy change.11... 

Fluorescence is also a powerful tool for investigating the structure and dynamics of matter or living systems at a molecular or supramolecular level. Polymers, solutions of surfactants, solid surfaces, biological membranes, proteins, nucleic acids and living cells are well-known examples of systems in which estimates of local parameters such as polarity, fluidity, order, molecular mobility and electrical potential is possible by means of fluorescent molecules playing the role of probes. The latter can be intrinsic or introduced on purpose. The high sensitivity of fluo-rimetric methods in conjunction with the specificity of the response of probes to their microenvironment contribute towards the success of this approach. Another factor is the ability of probes to provide information on dynamics of fast phenomena and/or the structural parameters of the system under study. 

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