PYRENE EXCIMER FLUORESCENCE IN RABBIT SKELETAL ALPHA-ALPHATROPOMYOSIN LABELED WITH N-(1-PYRENE)MALEIMIDE - PROBE OF SULFHYDRYL PROXIMITY AND LOCAL CHAIN SEPARATION

Abstract

Rabbit skeletal .alpha..alpha.tropomyosin [.alpha..alpha.Tm] was specifically labeled at cysteine 190 with the fluorescent reagent, N-(1-pyrene)maleimide. Spectroscopically different products were obtained by labeling at pH 6.0 (PyrI-.alpha..alpha.Tm) or pH 7.5 (PyrII-.alpha..alpha.Tm). PyrII-.alpha..alpha.Tm results from a secondary reaction between the N-(1-pyrene)succinimido moiety at cysteine 190 of PyrI-.alpha..alpha.Tm and a lysine group on the same chain, probably lysine 189. Pyrene excimer fluorescence was present in the native state but absent in the unfolded state of both products, thus verifying the proximity of the -SH [sulfhydryl] groups and the chain register model for the structure of tropomyosin. Studies of the guanidinium chloride-dependent unfolding of PyrII-.alpha..alpha.Tm showed that loss of excimer fluorescence precedes unfolding, providing evidence for a region of preferential inability in the molecule near cysteine 190. N-(1-pyrene)maleimide may be used to probe both -SH proximity and local conformation in any protein if the presence of 2 or more proximal -SH groups is suspected.