Biochemists extract the contents of cells and separate the constituents based on differences in their chemical or physical properties, a process called fractionation. Of particular interest are proteins, the workhorses of many cellular processes. A typical fractionation scheme involves use of various separation techniques in a sequential fashion. These separation techniques commonly are based on differences in the size of molecules or the electrical charge on their surface (Chapter 3). To purify a particular protein of interest, a purification scheme is designed so that each step yields a preparation with fewer and fewer contaminating proteins, until finally only the protein of interest remains (Figure 1-22).
The initial purification of a protein of interest from a cell extract often is a tedious, time-consuming task. Once a small amount of purified protein is obtained, antibodies to it can be produced by methods discussed in Chapter 6. For a biochemist, antibodies are near-perfect tools for isolating larger amounts of a protein of interest for further analysis. In effect, antibodies can "pluck out" the protein they specifically recognize and bind from a semipure sample containing numerous different proteins. An increasingly common alternative is to engineer a gene that encodes a protein of interest with a small attached protein "tag," which can be used to pull out the protein from whole cell extracts.
Purification of a protein is a necessary prelude to studies on how it catalyzes a chemical reaction or carries out other functions and how its activity is regulated. Some enzymes are made of multiple protein chains (subunits) with one chain catalyzing a chemical reaction and other chains regulating when and where that reaction occurs. The molecular machines that perform many critical cell processes constitute even larger assemblies of proteins. By separating the individual proteins composing such assemblies, their individual catalytic or other activities can be assessed. For example, purification and study of the activity of the individual proteins composing the DNA replication machine provided clues about how they work together to replicate DNA during cell division (Chapter 4).
The folded, three-dimensional structure, or conformation, of a protein is vital to its function. To understand the relation between the function of a protein and its form, we need to know both what it does and its detailed structure. The most widely used method for determining the complex structures of proteins, DNA, and RNA is x-ray crystallography. Computer-assisted analysis of the data often permits the location of every atom in a large, complex molecule to be determined. The double-helix structure of DNA, which is key to its role in heredity, was first proposed based on x-ray crys-tallographic studies. Throughout this book you will encounter numerous examples of protein structures as we zero in on how proteins work.
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