The role of Asn14 in the stability and conformation of the reactive-site loop of winged bean chymotrypsin inhibitor: crystal structures of two point mutants Asn14-->Lys and Asn14-->Asp.

A double-headed chymotrypsin inhibitor, WCI, from winged bean seeds was cloned for structural and biochemical studies. The inhibitor was subjected to two point mutations at a conserved position, Asn14. This residue, known to have a pivotal role in stabilizing the first reactive-site loop (Gln63-Phe68) of the inhibitor, is highly conserved in the sequences of the other members of Kunitz (STI) family as well as in the sequences of Kazal family of serine protease inhibitors. The mutants, N14K and N14D, were subjected to biochemical assay and their characteristics were compared with those of the recombinant inhibitor (rWCI). Crystallographic studies of the recombinant and the mutant proteins are discussed. These studies were primarily aimed at understanding the importance of the protein scaffolding towards the conformational rigidity of the reactive-site loop. Our analysis reveals that, as the Lys14 side chain takes an unusual fold in N14K and the Asp14 side chain in N14D interacts with the loop residues by water-mediated hydrogen bonds, the canonical conformation of the loop has remained effectively intact in both the mutant structures. However, minor alterations such as a 2-fold increase in the inhibitory affinity towards the cognate enzyme were observed.     

Genetics of mosquitoes

Beginning in the mid-1950s, much progress has been made in studying various aspects of the genetics of mosquitoes, particularly involving several species of three principal genera,Aedes, Culex andAnopheles, that transmit important human diseases. Here I discuss selected areas of research involving formal genetics; genome structure, organization and evolution at the interspecific and intraspecific level; and evolutionary genetics of theAedes scutellaris group. Information and insights gained from in-depth analyses of these areas, particularly transmission genetics, cytogenetics and genetics of chromosomal rearrangements, and of mutagen-induced sexual sterility, have proved invaluable for the development of the theory and evaluation of feasibility of genetic control of natural populations. As a result, mosquitoes represent some of the best studied taxa at various levels of genetic organization. Recent developments in molecular genetics offer exciting possibilities for extension of these concepts.