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Proceedings Paper

Fluorescence spectroscopic studies of tyrosine environment and ligand binding of plant calmodulin
Author(s): Gautam Sanyal; Faith Thompson; David Puett
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Paper Abstract

Recent studies in our laboratories have focused on using tyrosine (Tyr) fluorescence of calmodulin (CaM) and tryptophan (Trp) fluorescence of CaM-bound peptdies as intrinsic probes of structure and interactions of this Ca2+ regulatory protein. Plant CaM contains a single Tyr (Tyr.-l38) and vertebrate CaM contains two (Tyr-99 and Tyr-.l38). Neither protein contains Trp. The fluorescence properties of Tyr-138 of wheat-germ CaM is sensitive to conformational changes induced by perturbations such as Ca2+ ligation or depletion, and pH changes. Effects of these perturbations on quantum yield, lifetime and dynamic quenching of Tyr-l38 fluorescence are reported. We have also studied binding of amphiphilic peptides to wheat-germ CaM. A comparison of wheat CaM induced changes in the fluorescence properties of a single Trp of these peptides with those induced by bovine testes CaM indicate general similarities of the peptide binding surfaces of plant and mammalian CaMs. Frequency domain measurements of decay of intensity and anisotropy have suggested some orientational freedom and local motion of the Trp residue of CaM-bound peptide, independent of the overall protein motion, even when the Trp is expected to be buried in the doubly apolar protein-peptide interface. Calmodulin (CaM) is a ubiquitous calcium binding protein which is believed to regulate several different enzymes in diverse cells (Klee et al., 1982). Much of the structural work to date has been carried out on mammalian CaM. However, CaM has also been isolated from plant and invertebrate sources, and a high degree of sequence homology with vertebrate CaM has been found. The amino acid sequence of wheat germ CaM shows eleven substitutions, two insertions and one deletion compared with the 148.-residue bovine brain CaM (Toda et al., 1985). Specific differences with mammalian CaM at two sites make plant CaM attractive for fluorescence spectroscopic studies. These are: (1) The presence of a single tyrosine residue (Tyr-138) as the only unambiguous source of its intrinsic fluorescence as opposed to two (Tyr-99 and Tyr-l38) in mammalian CaM, (2) The presence of a single cysteine, Cys-27 (as opposed to none in mammalian CaM) which can be specifically labeled by extrinsic fluorescent probes (Mills et al., 1988). He have found that the fluorescence quantum yield and lifetime of Tyr-l38 as well as its accessibility to polar quenchers are especially sensitive to binding of Ca2+ to wheat germ CaM. In the presence of Ca2+ wheat CaM structure becomes significantly helical and amphiphilic peptides bind with high affinity. Ca2+ dependent binding of amphiphilic peptides to bovine CaM has been thoroughly studied and this provides a viable model for interaction of CaM with its dependent enzymes (Malencik and Anderson, 1983; McDowell et al., 1985; O'Neil et al., 1987). The data presented here suggest general similarities between the peptide binding surfaces of mammalian and plant Cam.

Paper Details

Date Published: 1 May 1990
PDF: 17 pages
Proc. SPIE 1204, Time-Resolved Laser Spectroscopy in Biochemistry II, (1 May 1990); doi: 10.1117/12.17754
Show Author Affiliations
Gautam Sanyal, Merck Sharp & Dohme Research Labs. (United States)
Faith Thompson, Hamilton College (United States)
David Puett, Univ. of Miami (United States)

Published in SPIE Proceedings Vol. 1204:
Time-Resolved Laser Spectroscopy in Biochemistry II
Joseph R. Lakowicz, Editor(s)

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