Because NMR studies are performed in solution, this method also offers a convenient means of monitoring changes in protein structure, for example during protein folding or when the protein binds to another molecule. NMR is also used widely to investigate molecules other than proteins and is valuable, for example, as a method to determine the three-dimensional structures of RNA molecules and the complex carbohydrate side chains of glycoproteins.
A third major method for the determination of protein structure, and particularly the structure of large protein complexes, is single-particle analysis by electron microscopy. We discuss this approach in Chapter 9.
Having discussed methods for purifying and analyzing proteins, we now turn to a common situation in cell and molecular biology: an investigator has identified a gene important for a biological process but has no direct knowledge of the biochemical properties of its protein product.
Thanks to the proliferation of protein and nucleic acid sequences that are cataloged in genome databases, the function of a gene—and its encoded protein— can often be predicted by simply comparing its sequence with those of previously characterized genes. Because amino acid sequence determines protein structure, and structure dictates biochemical function, proteins that share a similar amino acid sequence usually have the same structure and usually perform similar biochemical functions, even when they are found in distantly related organisms. In modern cell biology, the study of a newly discovered protein usually begins with a search for previously characterized proteins that are similar in their amino acid sequences.
Searching a collection of known sequences for similar genes or proteins is typically done over the Internet, and it simply involves selecting a database and entering the desired sequence. A sequence-alignment program—the most popular is BLAST—scans the database for similar sequences by sliding the submitted sequence along the archived sequences until a cluster of residues falls into full or partial alignment (Figure 8–23). Such comparisons can predict the functions of individual proteins, families of proteins, or even most of the protein complement of a newly sequenced organism.
As was explained in Chapter 3, many proteins that adopt the same conformation and have related functions are too distantly related to be identified from a comparison of their amino acid sequences alone (see Figure 3–13). Thus, an ability to reliably predict the three-dimensional structure of a protein from its amino acid sequence would improve our ability to infer protein function from the sequence information in genomic databases. In recent years, major progress has been made in predicting the precise structure of a protein. These predictions are based, in part, on our knowledge of the thousands of protein structures that have already been determined by x-ray crystallography and NMR spectroscopy and, in part, on computations using our knowledge of the physical forces acting on the atoms. However, it remains a substantial and important challenge to predict the structures of proteins that are large or have multiple domains, or to predict structures at the very high levels of resolution needed to assist in computer-based drug discovery.
While finding related sequences and structures for a new protein will provide many clues about its function, it is usually necessary to test these insights through direct experimentation. However, the clues generated from sequence comparisons typically point the investigator in the correct experimental direction, and their use has therefore become one of the most important strategies in modern cell biology.
Many methods exist for identifying proteins and analyzing their biochemical properties, structures, and interactions with other proteins. Small-molecule inhibitors allow the functions of proteins they act upon to be studied in living cells. Because proteins with similar structures often have similar functions, the biochemical activity of a protein can often be predicted by searching databases for previously characterized proteins that are similar in their amino acid sequences.
Until the early 1970s, DNA was the most difficult biological molecule for the biochemist to analyze. Enormously long and chemically monotonous, the string of nucleotides that forms the genetic material of an organism could be examined only indirectly, by protein sequencing or by genetic analysis. Today, the situation has changed entirely. From being the most difficult macromolecule of the cell to
Figure 8–23 Results of a bLAST search.