Fluorescence calmodulin

Eisner You could address this by putting in one of the fluorescent calmodulins, which change fluorescence when they bind Ca2+. 

Although intended for the biochemistry lab, this experiment provides analytical students with a practical characterization analysis. Of particular interest is the use of Job s method to determine the number of TNS (2-p-toludinylnaphthalene-6-sulfonate) binding sites on calmodulin, fluorescence is measured at 475 nm using an excitation wavelength of 330 nm. 

Romoser, V. A., Hinkle, P. M. and Persechini, A. (1997). Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J. Biol. Chem. 272, 13270-4. 

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). 

P. K. Lambooy, R. F. Steiner, and H. Sternberg, Molecular dynamics of calmodulin as monitored by fluorescence anisotropy, Arch. Biochem. Biophys, 217, 517-528 (1982). 

Toeroek, K. Cowley, D.J. Brandmeier, B.D. Howell, S. Aitken, A. Tren-tham, D.R. Inhibition of calmodulin-activated smooth-muscle myosin light-chain kinase by calmodulin-binding peptides and fluorescent (phosphodiesterase-activating) calmodulin derivatives. Biochemistry, 37, 6188-6198 (1998)... 

ChapmanER, Alexander K, VorherrT, CarafoliE, StormDR. Fluorescence energy-transfer analysis of calmodulin-peptide complexes. Biochemistry 1992, 31, 12819-12825. 

Fig. 10. Highly schematic representation of the orientation of several tryptophan-containing peptides with respect to calmodulin. (A) With tryptophan in position 1, the indole is located on the hydrophilic side of the helix and is exposed to solvent. Peptides with tryptophan on this face of the helix should exhibit emission maxima near that of indole in water ( 350 nm), a small anisotropy, and a high accessibility for acrylamide quenching. (B) In position 2, the tryptophan is partially exposed at the interface between the peptide and calmodulin. Peptides with a tryptophan in this location should have fluorescence properties that are intermediate between example A and C. (C) The tryptophan is on the hydrophobic side of the helix and is almost entirely buried. The emission maximum should be strongly blue-shifted, the anisotropy should be large, and the accessibility to acrylamide quenching low. Taken from O Neil et al. (1987).

A novel pump-damp-probe method (PDPM), which allows the characterization of solvation dynamics of a fluorescence probe not only in excited but also in the ground states has been recently developed (Changenet-Barret, 2000 and references therein). In PDPM, a pump produces a nonequilibrium population of the probe excited, which, after media relaxation, is simulated back to the ground states. The solvent relaxation of the nonequlibrium ground state is probed by monitoring with absorption technique. The inramolecular protein dynamics in a solvent-inaccessible region of calmodulin labeled with coumarin 343 peptide was examined by PDPM. In the pump-dump-probe experiments, part of a series of laser output pulses was frequency-doubled and softer beams were used as the probe. The delay of the probe with respect to the pump was fixed at 500 ps. 

CAM, Crassulacean acid metabolism CaM—Ca2+-Mg2+-ATPase, Ca2+-CaM-activated Ca2+-Mg2+-ATPase CaM—Ca2+-ATPase, Ca2+—calmodulin-dependent Ca2+-ATPase CaM-FC, Ca2+-dependent calmodulin fluorescence change... 

---