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Protein

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FIGURE 26.01 Denaturation of Protein by SDS

(A) The structure of SDS is amphipathic, that is, the long hydrocarbon tail is hydrophobic and the sulfate is hydrophilic. (B) The first step to separate a mixture of proteins by size is to boil the sample. Boiling proteins in a solution of SDS destroys the tertiary structure of the protein. The hydrophobic portion of SDS coats the polypeptide backbone and prevents the protein from refolding. The hydrophilic group of SDS keeps the protein soluble in water, and the negative charges repel each other, which also helps to keep the protein from refolding. The result is an unfolded protein with a net negative charge that is proportional to its molecular weight.

approach. Generic methods for purification of proteins rely on attaching the same tag molecule (e.g., His-tag, FLAG or GST) to many different proteins and then binding the tag.

When proteins are separated by size using gel electrophoresis, they are first denatured and coated with negatively charged detergent molecules.

Gel Electrophoresis of Proteins

Because nucleic acids are all negatively charged they all move towards the positive electrode during electrophoresis (Ch. 21). However, proteins are not so convenient. Some of the amino acids from which proteins are built have a positive charge, some have a negative charge, and most are neutral. So, depending on its overall amino acid composition, a protein may be positive, negative or neutral.

If a mixture of native (i.e. non-denatured) proteins is run on a gel, some move towards the positive electrode, others towards the negative electrode whereas neutral proteins scarcely move at all. Consequently, the samples are usually started in the middle of the gel, rather than at the end. This is referred to a native protein elec-trophoresis and is sometimes used to purify proteins without inactivating them. After running, the gel is stained with reagents specific to the protein of interest. For example, an enzyme that gives a colored product may be located in this manner.

To separate proteins on the basis of molecular weight, they are first boiled in a solution of the detergent sodium dodecyl sulfate (SDS). Boiling in detergent destroys the folded 3-D structure of the protein; that is, the protein is denatured. The SDS molecule has a hydrophobic tail with a negative charge at the end. The tail wraps around the backbone of the protein and the negative charge dangles in the water. The protein is unrolled and covered from head to toe with SDS molecules, which give it an overall negative charge (Fig. 26.01). In addition, the disulfide bonds that help maintain the tertiary structure of some proteins and/or hold protein subunits together must be disrupted for proper denaturation. This is done by adding small sulfhydryl reagents, usually b-mercaptoethanol (HS—CH2CH2OH):

sodium dodecyl sulfate (SDS) Detergent used to unfold proteins and cover them with negative charges for electrophoresis

SDS treated protein samples

Gel slab of polyacrylamide

SDS treated protein samples

Gel slab of polyacrylamide

Apply power

FIGURE 26.02 SDS Polyacrylamide Gel Electrophoresis

Apply power

Larger proteins (slow moving)

Smaller proteins (fast moving)

Larger proteins (slow moving)

Smaller proteins (fast moving)

FIGURE 26.02 SDS Polyacrylamide Gel Electrophoresis

Proteins treated with SDS can be separated by size using gel electrophoresis. Since all the proteins have a net negative charge, the proteins are repelled by the negative cathode and attracted to the positive anode. As the proteins move toward the anode, the polyacrylamide meshwork obstructs and slows the larger proteins but allows the smaller proteins to move faster. In consequence, the distance traveled in a given time is proportional to the log of the molecular weight. After separation, the proteins are visualized with a dye such as Coomassie blue or a silver compound.

Protein-S—S-Protein + 2 SH—CH2CH2OH ^ 2 Protein-SH + HO—CH2CH2—S—S—CH2CH2OH

Furthermore, the number of negative charges bound is proportional to the length of the protein. Thus proteins can be separated according to size by running them through a gel (Fig. 26.02). Because proteins are a lot smaller on average than DNA or RNA, the gel is made of the artificial polymer polyacrylamide, which gives smaller gaps in its meshwork than agarose. The technique is thus known as PAGE or polyacrylamide gel electrophoresis. After running, the gel is stained to visualize the protein bands. The two favorite choices are Coomassie Blue, a blue dye that binds strongly to proteins, or silver compounds. Silver atoms bind very tightly to proteins and yield black or purple complexes. Silver staining is more sensitive and, of course, more expensive.

Much larger numbers of proteins can be separated by gel electrophoresis in two directions.

Two Dimensional PAGE of Proteins

Separation of large numbers of proteins is normally done by two-dimensional poly-acrylamide gel electrophoresis (2D PAGE). The proteins are separated by charge in the first dimension and then by size in the second dimension. Isoelectric focusing is used in the first dimension and separates native proteins based on their original charge. A pH gradient is set up along a cylindrical gel and proteins migrate until they find a position where their native charges are neutralized—the isoelectric point. Standard SDS-PAGE as described above is used in the second dimension and separates denatured proteins based on their molecular weight (Fig. 26.03).

Early 2D gels were able to resolve a 1,000 or so protein spots and were used to characterize the protein complement of bacteria such as E. coli where about 1,000 of the 4,000 genes are expressed at any given time (see Ch. 9 Fig. 9.01). More recently, large 2D gels with higher resolution have been developed that allow separation of over 10,000 spots and can be used to analyze the proteome of higher organisms (Fig. 26.04). After separation, the protein spots are cut out from the gel, digested by protease

Coomassie Blue A blue dye used to stain proteins isoelectric focusing Technique for separating proteins according to their charge by means of electrophoresis through a pH gradient PAGE Polyacrylamide gel electrophoresis. Technique for separating proteins by electrophoresis on a gel made from polyacrylamide polyacrylamide gel electrophoresis (PAGE) Technique for separating proteins by electrophoresis on a gel made from polyacrylamide

FIGURE 26.03 Two-Dimensional Polyacrylamide Gel Electrophoresis

The first step in separating large numbers of proteins in two dimensions is to separate them according to their inherent charge. The mixture of proteins is loaded onto a gel that has a gradient of increasing pH. An electric field is applied and the proteins move along the pH gradient until they reach the point at which their charges are neutralized. At this point, each band in the gel contains several different proteins with the same (or very similar) isoelectric point. The tube gel is removed from its tube and exposed to SDS to denature the proteins. It is then placed on a slab of polyacrylamide gel and traditional SDS-PAGE is run in the second dimension to separate the proteins by size. After staining, the result of 2D-PAGE is a square with small scattered dots representing individual proteins.

FIGURE 26.04 2D Protein Gel of Mouse Brain Tissue i

FIGURE 26.04 2D Protein Gel of Mouse Brain Tissue

Soluble proteins were extracted from mouse brain and separated by 2D PAGE. The first dimension used isoelectric focusing and the second dimension used standard SDS-PAGE. The proteins were visualized by silver staining. Each spot on the gel is due to a separate protein. However, because proteins are frequently modified after synthesis, multiple spots sometimes arise from variants of the same original protein. Courtesy of Prof. Dr. Joachim Klose, Institut für Humangenetik, HumboldtUniversität, Berlin.

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