Figure 1:

Constructs and crosslinking strategy for the covalent attachment of Smt3p^Cys to Hsp82p^K178C.

(A) Full-length Hsp82p construct harboring a cysteine in place of lysine at residue 178, and mature form of Smt3p harboring a cysteine downstream of the diglycine motif. (B) Using the homo-bifunctional maleimide crosslinker, BMOE, singly derivatized Hsp82p^K178C will crosslink to itself to form Hsp82p^K178C-Hsp82p^K178C dimers. However, if derivatized with a vast excess of BMOE, every molecule of Hsp82p^K178C will be conjugated to BMOE thereby preventing the formation of Hsp82p^K178C-Hsp82p^K178C crosslinked dimers. When all molecules of Hsp82p^K178C are derivatized then near-complete crosslinking to Smt3p^Cys can be achieved.