different are different for functional reasons or just because they don't matter."

But his late colleague's mutant lysozymes reinforced his doubts. Although a few of the mutations destroyed the protein's structure, most did virtually nothing to change it. Nonetheless, Matthews knew that it would have been dangerous to generalize from his results since Streisinger's techniques had created mutations in but a small fraction of lysozyme's 164 amino acid positions. "For large parts of the protein there was no information at all."

The advent of recombinant DNA made it possible to extend this research to the rest of the protein. One could now substitute any amino acid at any position and crank out copious copies of the mutant. By the mid-1980s, lysozyme had been cloned, and Matthews' studies would soon take off on a wave of technical progress. When Alber came to Matthews's laboratory in 1982, Matthews had studied five mutants. By the time he left in 1987, 50 had been characterized. The total has now reached 500. And as the experimental record grew, it continued to support the hypothesis that most single mutations do not change the structure of proteins.

In one of his first experiments to take advantage of recombinant DNA, in 1984, Alber took one of Streisinger's mutants, which had an amino acid substitution at position 157, and created a series of additional mutants by inserting virtually every other amino acid into that position, in turn. He performed the first x-ray crystallography that had ever been done on any of these variants, and the surprising result was that all were very similar to one another. The stability of each mutant was then measured by heating it until it melted. The mutants were all quite stable.

To further test these ideas, Matthews's postdoctoral student Xue-Jun Zhang systematically substituted the amino acid alanine at each position in the protein, one by one. Alanine is one of the simplest of all the amino acids and lacks the reactive chemical groups that interact with those on other amino acids. In subsequent experiments, Zhang substituted 10 alanines at 10 consecutive positions at once, repeating the process all over the protein. The structure of lysozyme was impervious to these changes at all but a few sites.

The experiments with alanine, and extensive substitutions with other amino acids, such as those at position 157, led to two general conclusions. First, "Probably less than 50 percent of the amino acids in the protein are critical to structure," says Matthews. And different proteins can have the same basic three-dimensional structures despite large differences in amino acid sequence.



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