Protein : Other info

Methods of study Main article: Protein methods The activities and structures of proteins may be examined in vitro, in vivo, and in silico.In vitrostudies of purified proteins in controlled environments are useful for learning how a protein carries out its function: for example, enzyme kineticsstudies explore the chemical mechanismof an enzyme's catalytic activity and its relative affinity for various possible substrate molecules. By contrast,in vivoexperiments can provide information about the physiological role of a protein in the context of a cellor even a whole organism.In silicostudies use computational methods to study proteins. Protein purification Main article: Protein purification To perform in vitroanalysis, a protein must be purified away from other cellular components. This process usually begins with cell lysis, in which a cell's membrane is disrupted and its internal contents released into a solution known as a crude lysate. The resulting mixture can be purified using ultracentrifugation, which fractionates the various cellular components into fractions containing soluble proteins; membrane lipidsand proteins; cellular organelles, and nucleic acids. Precipitationby a method known as salting outcan concentrate the proteins from this lysate. Various types of chromatographyare then used to isolate the protein or proteins of interest based on properties such as molecular weight, net charge and binding affinity. [ 38 ]The level of purification can be monitored using various types of gel electrophoresisif the desired protein's molecular weight and isoelectric pointare known, by spectroscopyif the protein has distinguishable spectroscopic features, or by enzyme assaysif the protein has enzymatic activity. Additionally, proteins can be isolated according their charge using electrofocusing. [ 39 ] For natural proteins, a series of purification steps may be necessary to obtain protein sufficiently pure for laboratory applications. To simplify this process, genetic engineeringis often used to add chemical features to proteins that make them easier to purify without affecting their structure or activity. Here, a "tag" consisting of a specific amino acid sequence, often a series of histidineresidues (a " His-tag"), is attached to one terminus of the protein. As a result, when the lysate is passed over a chromatography column containing nickel, the histidine residues ligate the nickel and attach to the column while the untagged components of the lysate pass unimpeded. A number of different tags have been developed to help researchers purify specific proteins from complex mixtures. [ 40 ] Cellular localization Proteins in different cellular compartmentsand structures tagged with green fluorescent protein(here, white) The study of proteinsin vivois often concerned with the synthesis and localization of the protein within the cell. Although many intracellular proteins are synthesized in the cytoplasmand membrane-bound or secreted proteins in the endoplasmic reticulum, the specifics of how proteins are targetedto specific organelles or cellular structures is often unclear. A useful technique for assessing cellular localization uses genetic engineering to express in a cell a fusion proteinor chimeraconsisting of the natural protein of interest linked to a " reporter" such as green fluorescent protein(GFP). [ 41 ]The fused protein's position within the cell can be cleanly and efficiently visualized using microscopy, [ 42 ]as shown in the figure opposite. Other methods for elucidating the cellular location of proteins requires the use of known compartmental markers for regions such as the ER, the Golgi, lysosomes or vacuoles, mitochondria, chloroplasts, plasma membrane, etc. With the use of fluorescently tagged versions of these markers or of antibodies to known markers, it becomes much simpler to identify the localization of a protein of interest. For example, indirect immunofluorescencewill allow for fluorescence colocalization and demonstration of location. Fluorescent dyes are used to label cellular compartments for a similar purpose. [ 43 ] Other possibilities exist, as well. For example, immunohistochemistryusually utilizes an antibody to one or more proteins of interest that are conjugated to enzymes yielding either luminescent or chromogenic signals that can be compared between samples, allowing for localization information. Another applicable technique is cofractionation in sucrose (or other material) gradients using isopycnic centrifugation. [ 44 ]While this technique does not prove colocalization of a compartment of known density and the protein of interest, it does increase the likelihood, and is more amenable to large-scale studies. Finally, the gold-standard method of cellular localization is immunoelectron microscopy. This technique also uses an antibody to the protein of interest, along with classical electron microscopy techniques.