Experiments of proteins

In our AFM-FS experiments of protein-DNA complexes, the respective DNA target sequences were covalently coupled to Si3N4 AFM cantilevers via monolayer activation and a functional heterobifunctional spacer molecule. The corresponding (His)6ExpG protein was immobilized on a silanized mica surfaces by a short linker molecule coupled to one of the five ExpG lysines. We used a commercial AFM instrument at 25°C that has been modified with a home-built electronics and data acquisition (Bartels et al. 2003). 

A period in the United States in 1939 as a Rockefeller Fellow in the Harvard laboratory of E. J. Cohn widened his experience of proteins and knowledge of their physical chemistry and led to his first crystallization of a muscle jirotein, a myogen from rabbit muscle which was later shown hy other workers to be identical with triosephosphate dehydrogenase. 

Third, there is no NMR evidence for rapid tumbling of the macromolecules or for rapid motion of their sidechains. However, these motions would be difficult to detect. Careful experiments of protein relaxation in concentrated solutions and gels are needed before accurate conclusions can be drawn with regard to motions of macromolecules. 

Transient relaxation experiments of protein adsorption layers were published by Miller et al. (1993a, c, d). The experiments were performed using a modified pendent drop technique described in Section 6.3.4. The surface tension response to three subsequent square pulse perturbations of 0.1 mg/ml HA adsorbed at the aqueous solution/air interfaces (Miller et al. 1993a) are shown in Fig. 6.21. 

Sampling limitations have more severe consequences in the case of indirectly sampled dimensions, where acquisition of each sampling point takes up to a few seconds. Even in 3D NMR experiments of proteins, featuring relatively fast transverse relaxation, it is almost impossible to reach the natural (determined by relaxation) line width in a reasonable experimental time. Limited experiment duration causes signal tmncation and results in broadened spectral peaks, according to the Fourier Uncertainty Principle [11]. 

The data described above suggest that the ADP-ribosylated arginine may be sequenced near serine residue 38 which is phosphorylated by cAMP-dependent protein kinase (Fig. 3). In the experiment of protein kinase-catalyzed phosphorylation of synthetic peptide, Zetterqvist et al. [9] and Kemp et al. [10] proposed that two arginine residues are necessary at the N-terminal side of phosphate-accepting serine residue. Therefore, we speculated that the ADP-ribosylation may influence the cAMP-dependent protein kinase activity. Actually, we found that when whole histones from calf thymus were... 

Based on the observations in this chapter about resolution and band spreading in SEC and on the experiences of protein chemists over the past decades with protein-matrix interactions, the ideal HPSEC support should be ... 

Apart from tliese mainstream metliods enabling one to gain a comprehensive and detailed stmctural picture of proteins, which may or may not be in tlieir native state, tliere is a wide variety of otlier metliods capable of yielding detailed infonnation on one particular stmctural aspect, or comprehensive but lower resolution infonnation while keeping tlie protein in its native environment. One of tlie earliest of such metliods, which has recently undergone a notable renaissance, is analytical ultracentrifugation [24], which can yield infonnation on molecular mass and hence subunit composition and their association/dissociation equilibria (via sedimentation equilibrium experiments), and on molecular shape (via sedimentation velocity experiments), albeit only at solution concentrations of at least a few tentlis of a gram per litre. 

Abstract. Molecular dynamics (MD) simulations of proteins provide descriptions of atomic motions, which allow to relate observable properties of proteins to microscopic processes. Unfortunately, such MD simulations require an enormous amount of computer time and, therefore, are limited to time scales of nanoseconds. We describe first a fast multiple time step structure adapted multipole method (FA-MUSAMM) to speed up the evaluation of the computationally most demanding Coulomb interactions in solvated protein models, secondly an application of this method aiming at a microscopic understanding of single molecule atomic force microscopy experiments, and, thirdly, a new method to predict slow conformational motions at microsecond time scales. 

The previous application — in accord with most MD studies — illustrates the urgent need to further push the limits of MD simulations set by todays computer technology in order to bridge time scale gaps between theory and either experiments or biochemical processes. The latter often involve conformational motions of proteins, which typically occur at the microsecond to millisecond range. Prominent examples for functionally relevant conformatiotial motions... 

The amount of computation necessary to try many conformers can be greatly reduced if a portion of the structure is known. One way to determine a portion of the structure experimentally is to obtain some of the internuclear distances from two-dimensional NMR experiments, as predicted by the nuclear Over-hauser effect (NOE). Once a set of distances are determined, they can be used as constraints within a conformation search. This has been particularly effective for predicting protein structure since it is very difficult to obtain crystallographic structures of proteins. It is also possible to define distance constraints based on the average bond lengths and angles, if we assume these are fairly rigid while all conformations are accessible. 

This experiment provides a nice example of the application of spectroscopy to biochemistry. After presenting the basic theory for the spectroscopic treatment of protein-ligand interactions, a procedure for characterizing the binding of methyl orange to bovine serum albumin is described. 

Weber, P. L. Buck, D. R. Capillary Electrophoresis A Past and Simple Method for the Determination of the Amino Acid Composition of Proteins, /. Chem. Educ. 1994, 71, 609-612. This experiment describes a method for determining the amino acid composition of cyctochrome c and lysozyme. The proteins are hydrolyzed in acid, and an internal standard of a-aminoadipic acid is added. Derivatization with naphthalene-2,3-dicarboxaldehyde gives derivatives that absorb at 420 nm. Separation is by MEKC using a buffer solution of 50 mM SDS in 20 mM sodium borate. 

The units of [77] reveal the concentration units in this experiment to be grams of protein per cubic centimer of solution. Dividing this concentration unit by the density of the unsolvated protein converts these concentration units to volume fractions ... 

Design code Design for Recycling Design of experiments Design of proteins Design patents Designs Desipramine Desipramine [50-47-5]... 

Biosynthesis of Protein. The dynamic equilibrium of body protein was confirmed by animal experiments using A/-labeled amino acids in 1939 (104). The human body is maintained by a continuous equilibrium between the biosynthesis of proteins and their degradative metabolism where the nitrogen lost as urea (about 85% of total excreted nitrogen) and other nitrogen compounds is about 12 g/d under ordinary conditions. The details of protein biosynthesis in living cells have been described (2,6) (see also Proteins). 

Protein engineering encompasses a vast amount and wide variety of research. At least two textbooks (1,2) have been devoted exclusively to this topic, and several excellent reviews have been pubHshed (3,4). Herein, an overview of principles, an introduction to basic techniques, and a summary of results of representative experiments on protein engineering are provided. 

Research and clinical experience on dmg resistance suggests that tumor cells are particularly adept at genetic selections leading to alterations in the stmcture, function, or synthesis of proteins involved in the antitumor dmg action and detoxification. Multiple mechanisms of resistance have been shown to account for the resistance seen in the clinic (46). 

A dynamic transition in the internal motions of proteins is seen with increasing temperamre [22]. The basic elements of this transition are reproduced by MD simulation [23]. As the temperature is increased, a transition from harmonic to anharmonic motion is seen, evidenced by a rapid increase in the atomic mean-square displacements. Comparison of simulation with quasielastic neutron scattering experiment has led to an interpretation of the dynamics involved in terms of rigid-body motions of the side chain atoms, in a way analogous to that shown above for the X-ray diffuse scattering [24]. 

RA Laskowski, MW MacArthur, JM Thornton. Validation of protein models derived from experiment. Curr Opm Struct Biol 5 631-639, 1998. 

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