FLUORESCENCE OF A TRYPTOPHAN BEARING PEPTIDE FROM SMOOTH-MUSCLE MYOSIN LIGHT-CHAIN KINASE UPON BINDING TO 2 CLOSELY-RELATED CALMODULINS

We have investigated the fluorescence of a calmodulin binding peptide (AS19) based on the sequence of the calmodulin binding domain of smoot h muscle myosin light drain kinase and bearing a tryptophan residue at position 5 upon binding to two closely related calmodulins. The emiss ion maximum of peptide AS19 bound to the engineered SYNCAM calmodulin was 318 nm and a vibrational structure was clearly apparent. The emiss ion maximum of peptide AS19 bound to chicken gizzard calmodulin (ChG C aM) was 327 nm and its spectrum was featureless. Red edge excitation e ffect supports the assumption that the polarity of Trp-5 environment i s larger in the complex with ChG CaM than with SYNCAM, in agreement wi th fluorescence spectra. Time-resolved fluorescence and anisotropy mea surements showed that, in both complexes, the tryptophan emitting stat e was (1)L(a). The X-ray structure of the calmodulin-peptide complex h as been resolved (W. E. Meader, A. R. Means, and F. A. Quiocho, 1992, Science 257, 1251-1255). The Trp binding site has been characterized. It differs by a single-point mutation between the two calmodulins: Met -144 of ChG CaM has been replaced by Val in SYNCAM. This suggests that the spectral relaxation of Trp-5 in the complex with ChG CaM as compa red to SYNCAM is due to the polarizability of the sulfur atom containi ng Met side chain that is higher than that of Val. This provides an id eal system to investigate the origin of the Stokes shift of the indole moiety in proteins. (C) 1995 Academic Press, Inc.