Intersubunit contacts made by tryptophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin.

In natural streptavidin, tryptophan 120 of each subunit makes contacts with the biotin bound by an adjacent subunit through the dimer-dimer interface. To understand quantitatively the role of tryptophan 120 and its intersubunit communication in the properties of streptavidin, a streptavidin mutant in which tryptophan 120 is converted to phenylalanine was produced and characterized. The streptavidin mutant forms a tetrameric molecule and binds one biotin per subunit, as does natural streptavidin, indicating that the mutation of tryptophan 120 to phenylalanine has no significant effect on the basic properties of streptavidin. However, its biotin-binding affinity was reduced substantially, to approximately 10(8) M-1, indicating that the contact made by tryptophan 120 to biotin has a considerable contribution to the extremely tight biotin binding by streptavidin. The mutant retained bound biotin over a wide pH range or with the addition of urea up to 6 M at neutral pH. However, bound biotin was efficiently released by the addition of excess free biotin due, presumably, to exchange reactions. Electrophoretic analysis revealed that the intersubunit contact made by tryptophan 120 to biotin through the dimer-dimer interface is the major interaction responsible for the biotin-induced, tighter subunit association of streptavidin. In addition, the mutant has weaker subunit association than natural streptavidin even in the absence of biotin, indicating that tryptophan 120 also contributes to the subunit association of tetramers in the absence of biotin.

Strong natural selection causes microscale allozyme variation in a marine snail.

Natural selection is one of the most fundamental processes in biology. However, there is still a controversy over the importance of selection in microevolution of molecular traits. Despite the general lack of data most authors hold the view that selection on molecular characters may be important, but at lower rates than selection on most phenotypic traits. Here we present evidence that natural selection may contribute substantially to molecular variation on a scale of meters only. In populations of the marine snail Littorina saxatilis living on exposed rocky shores, steep microclines in allele frequencies between splash and surf zone groups are present in the enzyme aspartate aminotransferase (allozyme locus Aat; EC. 2.6.1.1). We followed one population over 7 years, including a period of strong natural perturbation. The surf zone part of the population dominated by the allele Aat100 was suddenly eliminated by a bloom of a toxin-producing microflagellate. Downshore migration of splash zone snails with predominantly Aat120 alleles resulted in a drastic increase in surf zone frequency of Aat120, from 0.4 to 0.8 over 2 years. Over the next four to six generations, however, the frequency of Aat120 returned to the original value. We estimated the coefficient of selection of Aat120 in the surf zone to be about 0.4. Earlier studies show similar or even sharper Aat clines in other countries. Thus, we conclude that microclinal selection is an important evolutionary force in this system.