Methods of determining polypeptide structure and function

A method of determining ligand binding to one or more domains of calpain or isoform thereof, comprising:

• (a) providing isolated crystallized domain I and domain II of said calpain or isoform thereof, said domains having protease activity;
• (b) providing a ligand;
• (c) mixing said ligand with said isolated crystallized domain I and domain II in the presence of a cation to form a mixture;
• (d) illuminating said mixture with light at a wavelength of about 260 to 300 nm;
• and
• (e) measuring the amount of fluorescence emitted by said mixture at a wavelength of about 320 to 360 nm;
• wherein a reduction in emission by said ligand-domain mixture relative to a control crystallized domain I and domain II illuminated in the absence of said ligand indicates binding of said ligand to said domain I domain II.

A method of determining ligand binding to one or more domains of calpain or isoform thereof, comprising:

• (a) co-crystallizing a ligand with domain I and domain II of said calpain or isoform thereof in the presence of a cation, said domains having protease activity;
• (b) illuminating said co-crystallized ligand and domain I and domain II with light at a wavelength of about 260 to 300 nm;
• and
• (c) measuring the amount of fluorescence emitted by said co-crystallized ligand and domain I domain II at a wavelength of about 320 to 360 nm;
• wherein a reduction in emission by said co-crystallized ligand and domain I and domain II relative to a control crystallized domain I and domain II illuminated in the absence of said ligand indicates binding of said ligand to said domain I and domain II.