muramidase and benzamidine

Protein-ligand recognition is dynamic and complex. A key approach in deciphering the mechanism underlying the recognition process is to capture the kinetic process of the ligand in its act of binding to its designated protein cavity. Toward this end, ultralong all-atom molecular dynamics simulation has recently emerged as a popular method of choice because of its ability to record these events at high spatial and temporal resolution. However, success via this route comes at an exorbitant computational cost. Herein, we demonstrate that coarse-grained models of the protein, when systematically optimized to maintain its tertiary fold, can capture the complete process of spontaneous protein-ligand binding from bulk media to the cavity at crystallographic precision and within wall clock time that is orders of magnitude shorter than that of all-atom simulations. The exhaustive sampling of ligand exploration in protein and solvent, harnessed by coarse-grained simulation, leads to elucidation of new ligand recognition pathways and discovery of non-native binding poses.

Topics: Bacterial Proteins; Bacteriophage T4; Benzamidines; Benzene; Camphor; Catalytic Domain; Cytochrome P-450 Enzyme System; Ligands; Molecular Dynamics Simulation; Muramidase; Protein Binding; Pseudomonas putida; Trypsin; Viral Proteins

It was found that the crystals of at least a dozen different proteins could be thoroughly stained to an intense color with a panel of dyes. Many, if not most, of the stained protein crystals retained the dyes almost indefinitely when placed in large volumes of dye-free mother liquor. Dialysis experiments showed that most of the dyes that were retained in crystals also bound to the protein when free in solution; less frequently, some dyes bound only in the crystal. The experiments indicated a strong association of the dyes with the proteins. Four protein crystals were investigated by X-ray diffraction to ascertain the mode of binding. These were crystals of lysozyme, thaumatin, trypsin inhibited with benzamidine and satellite tobacco mosaic virus. In 30 X-ray analyses of protein crystal-dye complexes, in only three difference Fourier maps was any difference electron density present that was consistent with the binding of dye molecules, and even in these three cases (thaumatin plus thioflavin T, xylene cyanol and m-cresol purple) the amount of dye observed was inadequate to explain the intense color of the crystals. It was concluded that the dye molecules, which are clearly inside the crystals, are disordered but are paradoxically tightly bound to the protein. It is speculated that the dyes, which exhibit large hydrophobic cores and peripheral charged groups, may interact with the crystalline proteins in the manner of conventional detergents.

Topics: Animals; Benzamidines; Binding Sites; Cattle; Chickens; Coloring Agents; Crystallization; Crystallography, X-Ray; Models, Molecular; Muramidase; Plant Proteins; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Staining and Labeling; Tobacco mosaic satellite virus; Trypsin

Biopharmaceutical and biotechnology companies and regulatory agencies require novel methods to determine the structural stabilities of proteins and the integrity of protein-protein, protein-ligand, and protein-membrane interactions that can be applied to a variety of sample states and environments. Infrared spectroscopy is a favorable method for a number of reasons: it is adequately sensitive to minimal sample amounts and is not limited by the molecular weight of the sample; yields spectra that are simple to evaluate; does not require protein modifications, a special supporting matrix, or internal standard; and is applicable to soluble and membrane proteins. In this paper, we investigate the application of infrared spectroscopy to the quantification of protein structural stability by measuring the extent of amide hydrogen/deuterium exchange in buffers containing D(2)O for proteins in solution and interacting with ligands and lipid membranes. We report the thermodynamic stability of several protein preparations, including chick egg-white lysozyme, trypsin bound by benzamidine inhibitors, and cytochrome c interacting with lipid membranes of varying net-negative surface charge density. The results demonstrate that infrared spectroscopy can be used to compare protein stability as determined by amide hydrogen/deuterium exchange for a variety of cases.

Topics: Amides; Benzamidines; Cytochromes c; Deuterium; Kinetics; Muramidase; Protein Binding; Protein Stability; Protein Structure, Tertiary; Protons; Spectrophotometry, Infrared; Thermodynamics; Trypsin

A novel approach for the quantification of ligand-protein interactions is presented. Electrospray ionization mass spectrometry (ESI-MS) is used to monitor the diffusion behavior of noncovalent ligands in the presence of their protein receptors. These data allow the fraction of free ligand in solution to be determined, such that the corresponding dissociation constants can be calculated. A set of conditions is developed that provides an "allowable range" of concentrations for this type of assay. The method is tested by applying it to two different inhibitor-enzyme systems. The dissociation constants measured for benzamidine-trypsin and for N,N',N' '-triacetylchitotriose-lysozyme are (50 +/- 10) and (6 +/- 1) mM, respectively. Both of these results are in good agreement with previous data from the literature. In contrast to traditional ESI-MS-based methods, the approach used in this work does not rely on the preservation of specific solution-type noncovalent interactions in the gas phase. It is shown that this method allows an accurate determination of dissociation constants, even in cases in which the ion abundance ratio of free to ligand-bound protein in ESI-MS does not reflect the corresponding concentration ratio in solution.

Topics: Benzamidines; Diffusion; Enzyme Inhibitors; Ligands; Muramidase; Protein Binding; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Structure-Activity Relationship; Time Factors; Trisaccharides; Trypsin