The hypothesis of simultaneous binding of one calmodulin molecule to two neighboring N-terminal helix segments of the Orai oligomer, proposed by Liu et al. , is very interesting. Unfortunately, the structural evidence for this hypothesis is incomplete, while the isothermal titration calorimetry (ITC) data or the evaluation of these data appear to be incorrect or inadequate. In the structural analyses of the co-crystals of calmodulin and the calmodulin-binding segment of Orai1, Liu et al. found the stretched form of calmodulin. However, they could structurally resolve only one of the two bound peptides. The data, therefore, allow for the hypothesis that two peptides are bound, but they are no proof. In isothermal titration calorimetry, Liu et al. found a two-step binding behaviors. I was given the task of introducing a cysteine residue in the calmodulin at a site which is uncritical for target binding. Due to the fact that no cysteine is found in the wild type of human calmodulin, an artificially introduced cysteine enables the selective labeling with a maleimide dye. In this way, all calmodulin molecules should have exactly one dye molecule at the same position. If a two-step binding behavior of the target peptide would be still observed, this would be a clear proof for bivalent binding of one calmodulin molecule to two N-terminal Orai segments. Major problems occurred in the expression and purification of the CaM-cys mutant. Thus we wanted to exchange the role of the molecules for the MST measurements. Now the CaM-binding peptides were to be labeled with a fluorophore and titrated with unlabeled CaM. Two CaM-binding peptides with a C-terminal cysteine were chosen: The CaM-binding segment of Orai3 (termed Orai3NT47-62Caa) and a simple model of a CaM-binding peptide where all hydropbobic residues (except the tryptophane) consisted of leucine and all cationic residues of lysine (termed CMBD1ggcaa) [O'Neill and DeGrado, 1993]. These peptides had to be reacted with maleimide derivatives of fluorophores and purified by reversed-phase chromatography. Our work with the fluorescently labeled CaM-binding peptide segments has not led to quantitative binding studies. The reason for this was the very unusual structure of these peptides. It caused severe self-association after the coupling of Atto488. Nevertheless, our work has been useful because we have discovered the double benefit of NMF: (i) It improves the solubility of hydrophobic peptides, like DMSO or ethanol. (ii) It increases the dielectric constant and thus the kinetics of the labeling reactions, unlike DMSO or ethanol which are hydrophobic and lower the dielectric constant. Both effects of NMF help in the labeling reaction. This is not only important when labeling hydrophobic peptides (as in our case). It is also interesting when we want to couple DNA or RNA (which are very hydrophilic) with rather hydrophobic molecules. A study on the interaction of calmodulin with calmodulin-binding peptides was conducted in the workgroup of the advisor. For this purpose, the peptides had to be biotinylated. The biotinylated peptides had to be separated from free biotin and from unlabeled peptides on a reversed phase column. However, a chromatogram with many peaks was obtained where it was not clear which of the peaks were the biotinylated peptides. Here a biotin assay was expected to help. Because of the small amounts of biotinylated peptides, a sensitive biotin assay was needed. The method described by Ebner et al. , enabled us to detect the HPLC fractions with biotin and to gave a good estimation of the amount. For this, my colleague Eva Krenn created a simple version of this assay. This assay was further optimized by Eva Krenn and myself for a publication and tested in several examples.