Adaptation of the gut pathobiont Enterococcus faecalis to deoxycholate and taurocholate bile acids.

Enterococcus faecalis is a natural inhabitant of the human gastrointestinal tract. This bacterial species is subdominant in a healthy physiological state of the gut microbiota (eubiosis) in adults, but can become dominant and cause infections when the intestinal homeostasis is disrupted (dysbiosis). The relatively high concentrations of bile acids deoxycholate (DCA) and taurocholate (TCA) hallmark eubiosis and dysbiosis, respectively. This study aimed to better understand how E. faecalis adapts to DCA and TCA. We showed that DCA impairs E. faecalis growth and possibly imposes a continuous adjustment in the expression of many essential genes, including a majority of ribosomal proteins. This may account for slow growth and low levels of E. faecalis in the gut. In contrast, TCA had no detectable growth effect. The evolving transcriptome upon TCA adaptation showed the early activation of an oligopeptide permease system (opp2) followed by the adjustment of amino acid and nucleotide metabolisms. We provide evidence that TCA favors the exploitation of oligopeptide resources to fuel amino acid needs in limiting oligopeptide conditions. Altogether, our data suggest that the combined effects of decreased DCA and increased TCA concentrations can contribute to the rise of E. faecalis population during dysbiosis.

The gut microbiota, bile acids and their correlation in primary sclerosing cholangitis associated with inflammatory bowel disease.

BACKGROUND: Patients with primary sclerosing cholangitis associated with inflammatory bowel disease (PSC-IBD) have a very high risk of developing colorectal neoplasia. Alterations in the gut microbiota and/or gut bile acids could account for the increase in this risk. However, no studies have yet investigated the net result of cholestasis and a potentially altered bile acid pool interacting with a dysbiotic gut flora in the inflamed colon of PSC-IBD. AIM: The aim of this study was to compare the gut microbiota and stool bile acid profiles, as well as and their correlation in patients with PSC-IBD and inflammatory bowel disease alone. METHODS: Thirty patients with extensive colitis (15 with concomitant primary sclerosing cholangitis) were prospectively recruited and fresh stool samples were collected. The microbiota composition in stool was profiled using bacterial 16S rRNA sequencing. Stool bile acids were assessed by high-performance liquid chromatography tandem mass spectrometry. RESULTS: The total stool bile acid pool was significantly reduced in PSC-IBD. Although no major differences were observed in the individual bile acid species in stool, their overall combination allowed a good separation between PSC-IBD and inflammatory bowel disease. Compared with inflammatory bowel disease alone, PSC-IBD patients demonstrated a different gut microbiota composition with enrichment in Ruminococcus and Fusobacterium genus compared with inflammatory bowel disease. At the operational taxonomic unit level major shifts were observed within the Firmicutes (73%) and Bacteroidetes phyla (17%). Specific microbiota-bile acid correlations were observed in PSC-IBD, where 12% of the operational taxonomic units strongly correlated with stool bile acids, compared with only 0.4% in non-PSC-IBD. CONCLUSIONS: Patients with PSC-IBD had distinct microbiota and microbiota-stool bile acid correlations as compared with inflammatory bowel disease. Whether these changes are associated with, or may predispose to, an increased risk of colorectal neoplasia needs to be further clarified.

Chenodeoxycholic acid stimulated fibroblast growth factor 19 response - a potential biochemical test for bile acid diarrhoea.

BACKGROUND: Bile acid diarrhoea is underdiagnosed and better diagnostic tests are needed. Fasting serum fibroblast growth factor-19 (FGF19) has insufficient diagnostic value, but this may be improved by stimulation. AIM: To explore if an impaired FGF19 response identifies primary bile acid diarrhoea. METHODS: Eight patients with primary bile acid diarrhoea and eight healthy volunteers ingested (i) a meal plus 1250 mg chenodeoxycholic acid (CDCA), (ii) 1250 mg CDCA or (iii) the meal. Blood was sampled at fasting and repeatedly after stimulation. We analysed FGF19 by enzyme-linked immunosorbent assay and bile acids including 7α-hydroxy-4-cholesten-3-one by liquid chromatography-tandem mass spectrometry. RESULTS: Stimulation with the meal plus CDCA increased median FGF19 in healthy volunteers from fasting 62 pg/mL [interquartile range (IQR): 41-138] to 99 pg/mL (IQR: 67-147; P = 0.012) after 90 min and peaked after 150 min at 313 pg/mL (IQR: 54-512). This response was impaired in primary bile acid diarrhoea patients [fasting 56 pg/mL (IQR: 42-79); 90 min: 48 pg/mL [IQR: 37-63); 150 min: 57 pg/mL (48-198)]. Receiver operating characteristics (ROCAUC ) for fasting FGF19 was 0.55 (P = 0.75) and at 90 min 0.84 (P = 0.02). The difference in FGF19 from fasting to 90 min after the meal plus CDCA separated the groups (ROCAUC 1.0; P = 0.001). 7α-hydroxy-4-cholesten-3-one was elevated in primary bile acid diarrhoea (P = 0.038) and not significantly affected by stimulation. CONCLUSIONS: The FGF19 response following chenodeoxycholic acid plus meal is impaired in primary bile acid diarrhoea. This may provide a biochemical diagnostic test.

Interplay between bile acid metabolism and microbiota in irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) physiopathology is multifactorial and roles for both microbiota and bile acid (BA) modifications have been proposed. We investigated role of dysbiosis, transit pattern and BA metabolism in IBS. METHODS: Clinical data, serum, and stool samples were collected in 15 healthy subjects (HS), 16 diarrhea-predominant (IBS-D) and 15 constipation-predominant IBS (IBS-C). Fecal microbiota composition was analyzed by real-time PCR. Sera and fecal BA profiles, 7α-C4 levels, and in vitro BA transformation activity by fecal microbiota were measured by mass spectrometry. Serum Fibroblast Growth Factor 19 (FGF19) was assayed by ELISA. KEYS RESULTS: Dysbiosis was present in IBS patients with an increase in Escherichia coli in IBS-D patients (p = 0.03), and an increase in Bacteroides (p = 0.01) and Bifidobacterium (p = 0.04) in IBS-C patients. Sera primary and amino-conjugated BA were increased in IBS-D (63.5 ± 5.5%, p = 0.01 and 78.9 ± 6.3%, p = 0.03) and IBS-C patients (55.9 ± 5.5%, p = 0.04 and 65.3 ± 6.5%, p = 0.005) compared to HS (37.0 ± 5.8% and 56.7 ± 8.1%). Serum 7α-C4 and FGF19 levels were not different among all three groups. Fecal primary BA were increased in IBS-D patients compared to HS, including chenodeoxycholic acid which has laxative properties (25.6 ± 8.5% vs 3.5 ± 0.6%, p = 0.005). Bile acid deconjugation activity was decreased in IBS-D (p = 0.0001) and IBS-C (p = 0.003) feces. Abdominal pain was positively correlated with serum (R = 0.635, p < 0.001) and fecal (R = 0.391, p = 0.024) primary BA. CONCLUSIONS & INFERENCES: Different sera and fecal BA profiles in IBS patients could be secondary to dysbiosis and further differences between IBS-C and IBS-D could explain stool patterns. This study opens new fields in IBS physiopathology and suggests that modification of BA profiles could have therapeutic potential.

Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn's disease.

BACKGROUND: Crohn's disease (CD)-associated dysbiosis is characterised by a loss of Faecalibacterium prausnitzii, whose culture supernatant exerts an anti-inflammatory effect both in vitro and in vivo. However, the chemical nature of the anti-inflammatory compounds has not yet been determined. METHODS: Peptidomic analysis using mass spectrometry was applied to F. prausnitzii supernatant. Anti-inflammatory effects of identified peptides were tested in vitro directly on intestinal epithelial cell lines and on cell lines transfected with a plasmid construction coding for the candidate protein encompassing these peptides. In vivo, the cDNA of the candidate protein was delivered to the gut by recombinant lactic acid bacteria to prevent dinitrobenzene sulfonic acid (DNBS)-colitis in mice. RESULTS: The seven peptides, identified in the F. prausnitzii culture supernatants, derived from a single microbial anti-inflammatory molecule (MAM), a protein of 15 kDa, and comprising 53% of non-polar residues. This last feature prevented the direct characterisation of the putative anti-inflammatory activity of MAM-derived peptides. Transfection of MAM cDNA in epithelial cells led to a significant decrease in the activation of the nuclear factor (NF)-κB pathway with a dose-dependent effect. Finally, the use of a food-grade bacterium, Lactococcus lactis, delivering a plasmid encoding MAM was able to alleviate DNBS-induced colitis in mice. CONCLUSIONS: A 15 kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model.

Increase in fecal primary bile acids and dysbiosis in patients with diarrhea-predominant irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) is a multifactorial disease for which a dysbiosis of the gut microbiota has been described. Bile acids (BA) could play a role as they are endogenous laxatives and are metabolized by gut microbiota. We compared fecal BA profiles and microbiota in healthy subjects (HS) and patients with diarrhea-predominant IBS (IBS-D), and we searched for an association with symptoms. METHODS: Clinical features and stool samples were collected in IBS-D patients and HS. Fecal BA profiles were generated using HPLC coupled to tandem mass spectrometry. The fecal microbiota composition was assessed by q-PCR targeting dominant bacterial groups and species implicated in BA transformation. KEY RESULTS: Fourteen IBS-D patients and 18 HS were included. The two groups were comparable in terms of age and sex. The percentage of fecal primary BA was significantly higher in IBS-D patients than in HS, and it was significantly correlated with stool consistency and frequency. Fecal counts of all bacteria, lactobacillus, coccoides, leptum and Faecalibacterium prausnitzii were similar. There was a significant increase of Escherichia coli and a significant decrease of leptum and bifidobacterium in IBS-D patients. CONCLUSIONS & INFERENCES: We report an increase of primary BA in the feces of IBS-D patients compared to HS, correlated with stool consistency and frequency. A dysbiosis of different bacterial groups was detected, some of them involved in BA transformation. As the gut microbiota is the exclusive pathway to transform primary into secondary BA, this suggests a functional consequence of dysbiosis, leading to lower BA transformation.

Lipidomics of the red cell in diagnosis of human disorders.

Applications of tandem mass spectrometry in the field of lipid clinical chemistry are considered. Haemato-logical and biochemical advantages are presented favoring the choice of red blood cell membranes as a starting material in a wide variety of biomedical fields. Practical considerations are discussed with respect to methods of sampling, storage, and lipid extraction of red blood cells. The chapter describes the capabilities of a direct infusion of raw lipid extracts in the electro-spray ionization source compared with the more sophisticated method of high-performance liquid chromatography coupled with hybrid tandem mass spectrometry. Both methods have been evaluated and have been shown to be suitable for diagnosis and/or monitoring for a variety of human disorders.

Building up of the liquid-ordered phase formed by sphingomyelin and cholesterol.

The long-range and molecular orders and dynamics in codispersions of egg sphingomyelin-cholesterol have been investigated by synchrotron x-ray diffraction and electron spin resonance using phosphatidylcholine spin-labeled at several positions on the sn-2 chain. Mixtures containing 0, 17, 33, 41, 50 mol% cholesterol exhibited a single phase by x-ray diffraction methods. The temperature dependence of the d-spacing between 20 and 50 degrees C is attenuated with increasing proportions of cholesterol, becoming invariant for cholesterol contents of 41 and 50 mol% on completion of the liquid-ordered phase. Electron spin resonance revealed two sites for 17 and 33 mol% cholesterol. One site is highly ordered and the other is less ordered than the fluid phase of pure sphingomyelin as shown by the molecular and the intramolecular order parameters reflecting the segmental motions of the probe. The two-sites exchange rate indicates a mean lifetime of the sites of approximately 0.1 micros during which the lipid displacement is approximately 1 nm. The short lifetime of the sites probed by ESR and the single phase detected by x-ray diffraction support in this binary mixture, the building up of the Lo phase by a progressive accumulation of randomly distributed sphingomyelin-cholesterol condensed complexes rather than by diffusional exchange between extended domains.

Establishment of plasma membrane polarity in mammary epithelial cells correlates with changes in prolactin trafficking and in annexin VI recruitment to membranes.

Mammary epithelial cells (MEC) of lactating animals ferry large amounts of milk constituents in vesicular structures which have mostly been characterized by morphological approaches (Ollivier-Bousquet, 1998). Recently, we have shown that under conditions of lipid deprivation, perturbed prolactin traffic paralleled changes in the membrane phospholipid composition and in the cytosol versus membrane distribution of annexin VI (Ollivier-Bousquet et al., 1997). To obtain additional information on the membrane events involved in the vesicular transport of the hormone to the apical pole of the cell, we conducted a biochemical study on prolactin-containing vesicles in MEC at two different stages of differentiation. We first showed that MEC of pregnant and lactating rabbits exhibited membrane characteristics of non-polarized and polarized cells respectively, using annexin IV and the alpha-6 subunit of integrin as membrane markers. Incubation of both cell types with biotinylated prolactin for 1 h at 15 degrees C, followed by a 10-min chase at 37 degrees C revealed that prolactin transport was activated upon MEC membrane polarization. This was confirmed by subcellular fractionation of prolactin-containing vesicles on discontinuous density gradients. In non-polarized MEC, (125)I-prolactin was mainly recovered in gradient fractions enriched with endocytotic vesicles either after incubation at 15 degrees C or after a 10-min chase at 37 degrees C. In contrast, in polarized MEC, the hormone switched from endocytotic compartments to a fraction enriched in exocytotic clathrin-coated vesicles during the 10-min chase at 37 degrees C. Association of annexin VI to prolactin carriers was next studied in both non-polarized and polarized cells. Membrane compartments collected at each gradient interface were solubilized under mild conditions by Triton X-100 (TX100) and the distribution of annexin VI in TX100-insoluble and TX100-soluble fractions was analyzed by Western blotting. Upon MEC polarization, the amount of annexin VI recovered in TX100-insoluble fractions changed. Quite interestingly, it increased in a membrane fraction enriched with endocytotic clathrin-coated vesicles, suggesting that annexin VI may act as a sorting signal in prolactin transport.

Lipid-depleted diet perturbs membrane composition and intracellular transport in lactating mammary cells.

When rats were fed a control or a lipid-depleted diet for five generations, reproduction was not disturbed but pup growth was affected. The membrane organization and the secretory activity of mammary epithelial cells from these lactating rats were investigated. This diet induced a large decrease in the level of polyunsaturated fatty acids of membrane phospholipids (26.6% versus 44.0%). The level of 20:4 (n-6) was strongly decreased, mainly in phosphatidylethanolamine. Annexin VI, which interacts preferentially with this phospholipid, accumulated at the periphery of the cell and was largely associated to the hydrophobic region of the bilayer as compared to control membranes. Casein synthesis and casein secretion measured in incubated explants, after pulse-chase metabolic labeling, were both reduced by about 60% in lipid-deprived cells. The secretory ratio (radioactive secreted caseins in %) was not modified, suggesting that the mechanism of basal secretion was not mainly affected. On the contrary, the secretagogue effect of prolactin disappeared. The intracellular transport of the hormone was considerably slowed down by the diet and prolactin did not reach the lumen of the acini after 1 h of chase, in contrast to what occurred in control cells. Addition of 20:4 (n-6), in vitro, to mammary fragments from lipid-deprived rats restored the localization of annexin VI, increased synthesis and secretion of caseins as well as intracellular transport of PRI. Together, these data underline the importance of the level of 20:4 (n-6) in membrane phospholipids for exocytic and endocytic transport in lactating mammary epithelial cells.

Characterization and ultrastructural localization of annexin VI from mitochondria.

Annexin VI, a member of a family of related intracellular proteins that associate reversibly with membrane phospholipids in a Ca(2+)-dependent manner, has been purified from bovine liver mitochondria and characterized. Moreover, biochemical and immunocytochemical lines of evidence are presented which strongly suggest that annexin VI is closely associated with the cristae in the inner membrane of mitochondria. These findings are consistent with a calcium channel activity of annexin VI in mitochondria.

Annexins in rat enterocyte and hepatocyte: an immunogold electron-microscope study.

In the present study, immunogold labeling of ultrathin sections of rat small intestine and liver has been used to obtain insights into the ultrastructural localization and possible functions of annexins. In enterocytes, annexins II, IV, and VI are found at the periphery of the core of each microvillus and of the rootlets, but are absent from the interrootlet space. Annexins II, IV, and VI are also observed close to the interdigitated plasma membrane. In hepatocytes, only annexin VI is found to be concentrated within the microvilli in the bile canaliculi, on the inner face of the sinusoidal cell surface, particularly in the space of Disse, and all along the plasma membrane. Annexin VI is also detected in mitochondria of enterocytes and hepatocytes. These localizations are in agreement with the concept of a close calcium-dependent association of annexins with membranes and cytoskeletal proteins, particularly with actin. Moreover, they support the hypothesis of an involvement of annexins in exocytotic and endocytotic processes, which take place in epithelial cells.

Calmodulin during human sperm incorporation into hamster oocyte: an immunogold electron microscope study.

In the present study, immunogold labeling of ultrathin sections of human sperm, before and after incorporation into hamster oocyte, was used to obtain insight into the ultrastructural localization and possible function of calmodulin during fertilization. In heads of ejaculated, capacitated, and acrosome-reacted fixed human sperm, calmodulin was mainly found in two compartments, the subacrosomal layer and the postacrosome. After sperm-egg fusion, the subacrosomal calmodulin was unaltered and surrounded by the fertilization cone in which actin was abundant. There was no co-localization of calmodulin and actin. In contrast, postacrosomal calmodulin disappeared as soon as the sperm head was incorporated into egg cytoplasm. These unique localizations and redistributions are in agreement with the concept of a calmodulin targeting from acrosome toward postacrosome through the subacrosomal layer during spermatogenesis (Weinman et al., 1986b: J Histochem Cytochem 34:118). Moreover, they strongly suggest a role for calmodulin both in sperm-egg fusion and in the initial pulse of Ca2+ occurring during fertilization.

Calmodulin in rat enterocyte: an immunogold electron-microscope study.

Immunogold labeling of ultrathin sections of the epithelium of rat small intestine has been used to obtain insights into the ultrastructural localization and possible function of calmodulin in the enterocyte. Calmodulin is found mainly overlying the periphery of the microvillous core, in agreement with the location of the 110-kDa calmodulin complex. Extremely small amounts of calmodulin can be detected along the interdigitating basolateral membrane. This immunogold electron-microscope study suggests that calmodulin plays an important role in regulating the mechanochemical activity of myosin I but not in processes associated with the basolateral membrane of rat enterocyte.

Annexins from bovine adrenal cortex exhibit specific cytosol/membrane solvation.

Cytosol/membrane localization of annexins I to VI was analyzed in tissue extracts from bovine adrenal cortex. Based on their solubility in either aqueous or detergents solutions, they were subfractionated in three groups named cytosolic (C), membrane-bound (MB) and membrane-inserted (MI). Less than 1% of the total annexins present in the tissue were recovered in the C fraction when as much as 76.5 and 22.5% were obtained respectively in the MB and the MI fractions. By immunoblotting after SDS-PAGE, it was shown that the various members of the annexin family were not equally recovered in the different fractions. A-V and A-VI were found present in the three fractions whereas the distribution of A-I, A-II, A-III and A-IV was distinct, suggesting different cellular functions.

Localization of calmodulin in perinuclear structures of spermatids and spermatozoa: a comparison of six mammalian species.

The distribution of Calmodulin was examined during spermiogenesis and sperm epididymal maturation in rabbit, hamster, mouse, rat, monkey, and human. An affinity-purified antibody to Calmodulin was used to characterize this protein in sperm extracts by immunoblot analysis. Post-embedding immunogold procedures were used to localize Calmodulin at the ultrastructural level. The pattern of Calmodulin distribution was similar in the six species studied. A diffuse labeling was observed in round spermatids. Gold particles accumulated first in the subacrosomal layer of elongating spermatids. The perinuclear ring was also labeled. During the maturation phase of spermatids, Calmodulin labeling extended to the postacrosomal sheath. Dramatic changes occurred at spermiation so that in testicular sperm Calmodulin immunostaining was predominant in the postacrosomal sheath. Some labeling was still detected in restricted areas of the subacrosomal layer. This feature varied from species to species. Calmodulin location did not change during sperm epididymal maturation. A role for Calmodulin in the control of manchette development and regulation of subacrosomal actin aggregation state during spermiogenesis is proposed. The unique location of Calmodulin in the postacrosomal sheath of all species that have been studied in this work, together with the known presence of calcium in this area suggest a pivotal role for Calmodulin in sperm-egg fusion process.

Differential localization of annexins in ram germ cells: a biochemical and immunocytochemical study.

We used antibodies that specifically bind annexins on Western blots to determine the distribution and abundance of these proteins in ram spermatids and sperm by immunogold electron microscopy. Annexins I and II were found essentially within the entire acrosome of spermatids. During epididymal maturation, they concentrated in the postacrosomal region or the acrosomal equatorial segment, respectively. They were also present in sperm flagellum, on the surface of the coarse fibers and fibrous sheath. These findings show that during ram germ cell maturation, annexins I and II are exported from the spermatid acrosome towards structurally and functionally defined parts of the sperm. Annexins III, IV, and V were not found in ram germ cells. Annexin VI was isolated from testis and sperm. In spermatids, it was found to be associated with endoplasmic reticulum and the mitochondria but was absent from the acrosome. In sperm, it was confined to the flagellum, the mitochondria, and on the coarse fibers and fibrous sheath. The presence of three annexins, in addition to calmodulin, in functional areas may indicate differential ways for sperm to control and regulate events that are known to be calcium dependent, such as flagellar motility, acrosome reaction, and fertilization.

Co-distribution of annexin VI and actin in secretory ameloblasts and odontoblasts of rat incisor.

Annexin VI and actin were detected by immunoblot analysis in the enamel- and dentin-related portions of dental tissues. Annexin VI was found mainly in the particulate fraction whereas actin was detected in both the soluble and particulate fractions. By immunoelectron microscopy, annexin VI antibodies conjugated with colloidal gold were seen to label the mitochondria, the cytosol and the nucleus of secretory ameloblasts and odontoblasts of rat incisor. In the processes of these cells, the plasmalemmal undercoat was labeled. Anti-actin antibodies labeled the desmosome-like junctions, the cytosol, and the mitochondria of the cell bodies. Extensive labeling was seen at the periphery of the Tomes' processes and odontoblast processes. These results suggest that annexin VI may play a role in Ca2(+)-regulation in the cell bodies, especially as a calcium receptor protein in the mitochondria. Moreover, annexin VI and actin seem to be co-distributed in secretory processes. Thus, these proteins might be both involved in exocytotic and endocytotic events.

Annexins I-VI in secretory ameloblasts and odontoblasts of rat incisor.

Immunoblot analyses and ultrastructural immunogold studies have been conducted on annexins in the secretory ameloblasts and odontoblasts of the rat incisor. Annexins I and II were seen in the soluble and particulate fractions of the enamel-related portion but not in the dentin-related portion. These proteins were visualized in the cytosol, near to the plasma membrane of Tomes' processes and in secretory vesicles in the ameloblasts. The forming enamel was also labeled. Annexins III, VI an V were detected in both the soluble and particulate fractions of the enamel-and dentin-related portions. Annexin IV was mainly localized in the proximal and distal areas of the secretory ameloblasts and virtually absent from in the supranuclear area. Annexin V was mainly detected in the cytosol of the cells and to a lesser extent near the plasma membrane. Annexin VI was mainly detected in the particulate fraction of enamel- and dentin-related portions. It was seen in the mitochondria and in the subplasmalemmal undercoat. All these proteins may play a role in exocytosis and endocytosis. They are implied in the regulation of cell calcium, but not in the transfer of calcium through the cells in the direction of the forming enamel and dentin, except annexins I and II since they are both present in the secretory vesicles and in the forming enamel.

Effects of calcium and calcium analogs on calmodulin: a Fourier transform infrared and electron spin resonance investigation.

Fourier transform infrared (FTIR) and electron spin resonance (ESR) spectroscopies have been used to monitor changes in the conformation of calmodulin induced by Ca2+ and Ca2+ analogs. Using FTIR spectroscopy we observe that Ca2+: (i) favors the alpha-helical conformation and decreases the flexibility of the molecule; (ii) multiplies the intramolecular hydrogen bonds (the ratio of freely vibrating NH/hydrogen bound NH groups decreases); (iii) induces changes in the C-terminal tyrosine environment; and (iv) increases compactness of the molecule (less NH groups in the peptide bonds can be deuterated). As proved by ESR, Ca2+ binding induces exposure of hydrophobic domains allowing binding of a spin-labelled phenothiazine on calmodulin. When the experiments are performed in the presence of increasing amounts of Ca2+, both ESR and FTIR provide evidence that major conformational changes result after the filling of only two Ca2+-binding sites. But achievement of the spectroscopical changes is only observed when the four binding sites are filled (Ca2+/calmodulin = 4). The effects of analogs are monitored with the same spectroscopical parameters. Zn+ does not induce structural modifications of calmodulin but all other analogs studied mimic the calcium effects to some extent. As regards the amplitude of the spectroscopical effects, analogs rank in the following order: Ca2+ greater than Cd2+ greater than Tb3+ = Eu3+ greater than Gd3+ greater than La3+ greater than Zn2+ = cation depleted. Except for Zn2+, ranking for their activating potency of MLCK, the analogs can be arranged in a similar order.