Methods Crystallography: In protein crystallization, it is essential to create large protein crystals so thatits molecules are big enoughto produce a three-dimensional map of its electron density when diffracted by an x-ray beam. It is through the reflections of the diffraction that the map can be created. From there, the molecule’s known sequence is fit on the map.
Courtesy Of" "Structural Basis of Heroin and Cocaine Metabolism by a Promiscuous Human Drug-Processing Enzyme.” Picture Of Enzyme Which Metabolizes Heroin And Cocaine
The crystal is obtained by assembling its molecules into a lattice. The lattice is created with a solution of the protein with a high concentration; reagents are then added that reduce the solubility to spontaneous precipitation. Under the right conditions, the crystals will start to grow. The most common technique used in crystallization experiments is vapor diffusion. Here, a drop of the protein, mixed with stabilizing buffers, crystallization aids, and precipitants equalizes in a large, closed reservoir which contains all of the same chemicals as the drop except for the protein. These chemicals are at a higher concentration than those in the drop so that water evaporates from the drop. This causes an increase in the concentrations of the protein and precipitate, so crystals start to form if under the right conditions.
and the Sitting Drop. In the Hanging Drop, the drop sits on a coverslip which is then inverted used to seal a reservoir in a Linbro plate. Small crystals can be seen growing after a few hours. In the Sitting Drop, the drop is placed in a depression the the Microbridge of a Linbro plate or glass plate, which is put into a larger reservoir. The crystals can again be seen after a few hours. Because the protein used in the experiment must be very pure, it is often important to cleanse it well. There are different tags (His Tags and S-tags) that make purification simpler. Detergents and buffers are also needed in purification to maintain the protein’s proper pH and ionic strength.Detergents are specifically used to keep the protein soluble and prevent it from aggregating. For this experiment, the enzyme hCE1 was crystallized in order to allow reactivity
with the analog cocaine and heroin molecules. The Process Visually:
Atmoic Force Spectroscopy The atomic force microscope is a tool used for imaging surface topography of cells and biomaterials. It is able to measure on a scale from angstroms to 100 microns and is thus very precise in measuring surface topography. It works by using a cantilever, the tip of which can sense a force between it and the surface of the sample on the scale of nano-Newtons. The AFM can be used in two modes: the Contact Mode and the Tapping Mode. In Contact mode, the tip has continuous contact with the sample and raster-scans over the surface, while in Tapping Mode, the contact is intermittent. By scanning the sample and recording the deflectionof the cantilever, the height of the samplecan be measured. From here, three-dimensional topographical maps can be constructed by also using the horizontal probe position. The AFM can also determine chemical and mechanical properties of the sample's surface like adhesion and elasticity. This is done by measuring the force felt by the tip when brought close to or indented into the sample surface as a function of the deflection of the tip on the cantilever. This was also used to map the enzyme hCE1, but was used mainly for volume analysis.
Courtesy Of http://www.physics.ucsb.edu/~hhansma/afm-acs_news.htm
The structure of an AFM machine. 1.
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