Tyrosine fluorescence calmodulin

The way in which calcium binding changes the tyrosine fluorescence of calmodulin was initially a controversial issue. In 1980, Seamon(n5) examined the binding of Ca2+ and Mg2+ by NMR and concluded that both Tyr-99 and Tyr-138 were perturbed by the first two calcium ions. Although Tyr-138 was also perturbed by the binding of the fourth calcium, both residues appeared to be associated with high-affinity domains (III and IV). 

The fluorescence of the two tyrosine residues in bovine testes calmodulin was investigated by Pundak and Roche.(123) Upon excitation at 278 nm, a second emission, in addition to tyrosine fluorescence, was observed at 330-355 nm, which they characterized as being due to tyrosinate fluorescence. The tyrosinate fluorescence appeared to be from Tyr-99, which has an anomalously low pKa of about 7 for the phenol side chain. Pundak and Roche(123) reasoned that since tyrosinate emission is apparently not being seen in other species of calmodulin, it is possible that the bovine protein contains a carboxylate side chain in domain III which is amidated in other species. They further argued that the tyrosinate emission from bovine testes calmodulin arises from direct excitation of an ionized tyrosine residue. This tyrosinate fluorescence is discussed in more detail in Section 1.5.2. 

The physical dimensions and dynamics of calmodulin have also been investigated by tyrosine fluorescence. To learn about the internal mobility of calmodulin, Lambooy et al 1 and Steiner et al measured the steady-state fluorescence anisotropy of the tyrosine. Since the average correlation... 

The tyrosinate fluorescence observed with bovine testes calmodulin is argued to be due to tyrosinate in the ground state.(123) Of the two tyrosine residues in this calmodulin, Tyr-99 apparently has a low pKa near 7 for the formation of tyrosinate, which is most likely due to nearby side chains that are involved in calcium binding. These groups could then also account for the complex pH dependence of the 345-nm emission intensity. Besides the tyrosine and tyrosinate emissions at 305 and 345 nm, respectively, Pundak and Roche(123) also reported the existence of a third emission band between 312 and 320 nm. This band was similar in its pH and calcium dependence to the other residue, Tyr-138, and was speculated to be a result of a combination of contributions from the tyrosine and tyrosinate emissions. Since this band has its excitation profile shifted to the red, however, it could be that a hydrogen-bonded tyrosine exists in this calmodulin. Alternatively, it has also been found that the presence of the 345-nm emission depends upon the method of preparation (G. Sanyal, personal communication). 

M.-C. Kilhoffer, J. G. Demaille, and D. Gerard, Tyrosine fluorescence of ram testis and octopus calmodulins. Effects of calcium, magnesium, and ionic strength, Biochemistry 20, 4407 1414 (1981). 

K. P. Kohse and L. M. Heilmeyer, The effects of Mg2+ on the Ca2+-binding properties and Ca2 +-induced tyrosine-fluorescence changes of calmodulin isolated from rabbit skeletal muscle, Eur. J. Biochem. 117, 507-513 (1981). 

S. Pundak and R. S. Roche, Tyrosine and tyrosinate fluorescence of bovine testes calmodulin Calcium and pH dependence, Biochemistry 23, 1549-1555 (1984). 

It has been suggested that tyrosine fluorescence measurements conducted in conjunction with terbium binding to calmodulin allow the determination of the sequences of ion binding to the four domains (36). Through comparison of mammalian calmodulin, which has a tyrosyl residue at position 99 (domain III) and at position 138 (domain IV), with invertebrate calmodulin, which has only one tyrosyl residue (analogous to the mammalian position 138), the sequence of binding by this technique was reported to occur first at domains I and II (no tyrosyl residues), then to III, and then to IV. 

Some other applications of the temperature jump method to the kinetics of the association of calcium to control sites are discussed by Tsuruta Sano (1990), who use fluorescent dyes as well as the tyrosine fluorescence of calmodulin as an indicator of conformation changes resulting from calcium binding. 

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