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• W2073525626 abstract "Calexcitin/cp20 is a low molecular weight GTP- and Ca2+-binding protein, which is phosphorylated by protein kinase C during associative learning, and reproduces many of the cellular effects of learning, such as the reduction of potassium currents in neurons. Here, the secondary structure of cloned squid calexcitin was determined by circular dichroism in aqueous solution and by Fourier transform infrared spectroscopy both in solution and on dried films. The results obtained with the two techniques are in agreement with each other and coincide with the secondary structure computed from the amino acid sequence. In solution, calexcitin is one-third in α-helix and one-fifth in β-sheet. The conformation of the protein in solid state depends on the concentration of the starting solution, suggesting the occurrence of surface aggregation. The secondary structure also depends on the binding of calcium, which causes an increase in α-helix and a decrease in β-sheet, as estimated by circular dichroism. The conformation of calexcitin is independent of ionic strength, and the calcium-induced structural transition is slightly inhibited by Mg2+ and low pH, while favored by high pH. The switch of calexcitin's secondary structure upon calcium binding, which was confirmed by intrinsic fluorescence spectroscopy and nondenaturing gel electrophoresis, is reversible and occurs in a physiologically meaningful range of Ca2+concentration. The calcium-bound form is more globular than the apoprotein. Unlike other EF-hand proteins, calexcitin's overall lipophilicity is not affected by calcium binding, as assessed by hydrophobic liquid chromatography. Preliminary results from patch-clamp experiments indicated that calcium is necessary for calexcitin to inhibit potassium channels and thus to increase membrane excitability. Therefore the calcium-dependent conformational equilibrium of calexcitin could serve as a molecular switch for the short term modulation of neuronal activity following associative conditioning. Calexcitin/cp20 is a low molecular weight GTP- and Ca2+-binding protein, which is phosphorylated by protein kinase C during associative learning, and reproduces many of the cellular effects of learning, such as the reduction of potassium currents in neurons. Here, the secondary structure of cloned squid calexcitin was determined by circular dichroism in aqueous solution and by Fourier transform infrared spectroscopy both in solution and on dried films. The results obtained with the two techniques are in agreement with each other and coincide with the secondary structure computed from the amino acid sequence. In solution, calexcitin is one-third in α-helix and one-fifth in β-sheet. The conformation of the protein in solid state depends on the concentration of the starting solution, suggesting the occurrence of surface aggregation. The secondary structure also depends on the binding of calcium, which causes an increase in α-helix and a decrease in β-sheet, as estimated by circular dichroism. The conformation of calexcitin is independent of ionic strength, and the calcium-induced structural transition is slightly inhibited by Mg2+ and low pH, while favored by high pH. The switch of calexcitin's secondary structure upon calcium binding, which was confirmed by intrinsic fluorescence spectroscopy and nondenaturing gel electrophoresis, is reversible and occurs in a physiologically meaningful range of Ca2+concentration. The calcium-bound form is more globular than the apoprotein. Unlike other EF-hand proteins, calexcitin's overall lipophilicity is not affected by calcium binding, as assessed by hydrophobic liquid chromatography. Preliminary results from patch-clamp experiments indicated that calcium is necessary for calexcitin to inhibit potassium channels and thus to increase membrane excitability. Therefore the calcium-dependent conformational equilibrium of calexcitin could serve as a molecular switch for the short term modulation of neuronal activity following associative conditioning. Pavlovian conditioning in animal models has been used to study associative learning and memory on a cellular and molecular basis. Following this strategy, a 22-kDa, low abundance G protein, designated calexcitin (CE) 1The abbreviations used are: CE, calexcitin; FT-IR, Fourier transform infrared spectroscopy; IMAC, immobilized metal-affinity chelation; HPLC, high performance liquid chromatography; Iz, imidazole; PMSF, phenylmethylsulfonyl fluoride. or cp20, was identified in the past 15 years as an extremely promising substrate for associative conditioning (1Neary J.T. Crow T. Alkon D.L. Nature. 1981; 293: 658-660Crossref PubMed Scopus (56) Google Scholar, 2Nelson T.J. Collin C. Alkon D.L. Science. 1990; 247: 1479-1483Crossref PubMed Scopus (74) Google Scholar, 3Nelson T.J. Cavallaro S. Yi C.-L. McPhie D.L. Schreus B.G. Gusev P.A. Favit A. Zohar O. Kim J.-H. Beushausen S. Ascoli G. Olds J.L. Neve R. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13808-13813Crossref PubMed Scopus (52) Google Scholar, 4Nelson T.J. Alkon D.L. Mol. Neurobiol. 1991; 5: 315-328Crossref PubMed Scopus (6) Google Scholar, 5Nelson T.J. Yoshioka T. Toyoshima S. Han Y.-F. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9287-9291Crossref PubMed Scopus (17) Google Scholar, 6Kim C.S. Han Y.-F. Etcheberrigaray R. Nelson T.J. Olds J.L. Yoshioka T. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3060-3064Crossref PubMed Scopus (29) Google Scholar, 7Alkon D.L. Nelson T.J. FASEB J. 1990; 4: 1567-1576Crossref PubMed Scopus (76) Google Scholar, 8Nelson T.J. Sanchez-Andres J.-V. Schreus B.G. Alkon D.L. J. Neurochem. 1994; 57: 2065-2069Crossref Scopus (9) Google Scholar, 9Alkon D.L. Ikeno H. Dworkin J. McPhie D.L. Olds J.L. Lederhendler I. Matzel J. Schreus B.G. Kuziran A. Collin C. Yamoah E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1611-1614Crossref PubMed Scopus (47) Google Scholar, 10Moshiah S. Nelson T.J. Sanchez-Andres J.-V. Sakakibara M. Alkon D.L. Brain Res. 1993; 605: 298-304Crossref PubMed Scopus (7) Google Scholar, 11Nelson T.J. Alkon D.L. J. Neurochem. 1995; 65: 2350-2357Crossref PubMed Scopus (25) Google Scholar). CE is a protein kinase C substrate whose phosphorylation state increases 2–3-fold in the eye of the marine snail Hermissenda crassicornis after Pavlovian conditioning produced by the association of visual and vestibular stimuli. CE, as determined by immunoreactivity, has also been found in the central nervous systems of squid (5Nelson T.J. Yoshioka T. Toyoshima S. Han Y.-F. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9287-9291Crossref PubMed Scopus (17) Google Scholar), rabbit and rat (4Nelson T.J. Alkon D.L. Mol. Neurobiol. 1991; 5: 315-328Crossref PubMed Scopus (6) Google Scholar), and in human fibroblasts (6Kim C.S. Han Y.-F. Etcheberrigaray R. Nelson T.J. Olds J.L. Yoshioka T. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3060-3064Crossref PubMed Scopus (29) Google Scholar). The phosphorylation state of CE is also increased in rabbit CA1 hippocampus after associative conditioning (8Nelson T.J. Sanchez-Andres J.-V. Schreus B.G. Alkon D.L. J. Neurochem. 1994; 57: 2065-2069Crossref Scopus (9) Google Scholar). Upon phosphorylation by protein kinase C (which is also inhibited in vitro by CE's binding of GTP), calexcitin translocates from the cytosol to the membrane. Microinjection of purifiedHermissenda or squid CE into Hermissenda or rabbit neurons causes the same increase in membrane excitability previously related to memory formation and retention (2Nelson T.J. Collin C. Alkon D.L. Science. 1990; 247: 1479-1483Crossref PubMed Scopus (74) Google Scholar, 3Nelson T.J. Cavallaro S. Yi C.-L. McPhie D.L. Schreus B.G. Gusev P.A. Favit A. Zohar O. Kim J.-H. Beushausen S. Ascoli G. Olds J.L. Neve R. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13808-13813Crossref PubMed Scopus (52) Google Scholar, 7Alkon D.L. Nelson T.J. FASEB J. 1990; 4: 1567-1576Crossref PubMed Scopus (76) Google Scholar). CE injection also causes other cellular effects of conditioning, such a modified dendritic arborization (9Alkon D.L. Ikeno H. Dworkin J. McPhie D.L. Olds J.L. Lederhendler I. Matzel J. Schreus B.G. Kuziran A. Collin C. Yamoah E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1611-1614Crossref PubMed Scopus (47) Google Scholar) and an altered retrograde axonal transport of vesicles (10Moshiah S. Nelson T.J. Sanchez-Andres J.-V. Sakakibara M. Alkon D.L. Brain Res. 1993; 605: 298-304Crossref PubMed Scopus (7) Google Scholar). Although different proteins, such as cAMP-dependent protein kinase, protein kinase C, calmodulin, and others, have been implicated in learning-related phenomena (13Dudai Y. FEBS Lett. 1985; 191: 165-170Crossref PubMed Scopus (17) Google Scholar, 14Sakakibara M. Alkon D.L. De Lorenzo R. Goldenring J.R. Neary J.T. Heldman E. Biophys. J. 1986; 50: 319-327Abstract Full Text PDF PubMed Scopus (56) Google Scholar, 15Silva A. Paylor R. Wehner J.M. Tonegawa S. Science. 1992; 257: 206-211Crossref PubMed Scopus (1085) Google Scholar, 16Schwartz H. Greenberg S. Annu. Rev. Neurosci. 1987; 10: 459-476Crossref PubMed Scopus (93) Google Scholar, 17Olds J.L. Anderson M.L. McPhie D.L. Staten L.D. Alkon D.L. Science. 1989; 245: 866-869Crossref PubMed Scopus (240) Google Scholar), calexcitin is thus far the only protein capable of reproducing the electrophysiological and cytomorphological effects of learning when injected in the same neurons of the species from which it was extracted. Calexcitin presents the interesting property of binding both Ca2+ and GTP, whose signaling pathways are known to interact (18De Matteis M.A. Santini G. Kahn R.A. Di Tullio G. Luini A. Nature. 1993; 364: 818-821Crossref PubMed Scopus (132) Google Scholar, 19Jeng A.Y. Srivastava S.K. Lacal J.C. Blumberg P.M. Biochem. Biophys. Res. Commun. 1987; 145: 782-788Crossref PubMed Scopus (33) Google Scholar, 20Sahyoun N. McDonald O. Farrel F. Lapetina E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2643-2647Crossref PubMed Scopus (50) Google Scholar). Several other post-translational modification consensus domains are also present on the amino acid sequence of CE, such as myristoylation, isoprenylation, and glycosylation (3Nelson T.J. Cavallaro S. Yi C.-L. McPhie D.L. Schreus B.G. Gusev P.A. Favit A. Zohar O. Kim J.-H. Beushausen S. Ascoli G. Olds J.L. Neve R. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13808-13813Crossref PubMed Scopus (52) Google Scholar). It is therefore tempting to propose that CE may be implicated in the molecular mechanisms of associative memory acquisition (in response to the temporal association of two or more sensory inputs) by providing a convergence between different cell signaling pathways (21Alkon D.L. Rasmussen H. Science. 1988; 239: 998-1005Crossref PubMed Scopus (262) Google Scholar, 22Alkon, D. L. (1989) Sci. Am., July 42–50Google Scholar, 23Alkon D.L. Behav. Brain Res. 1995; 66: 151-160Crossref PubMed Scopus (7) Google Scholar). The molecular study of calexcitin has always been difficult because of the scarcity of the protein in neural tissues. After CE was recently cloned (3Nelson T.J. Cavallaro S. Yi C.-L. McPhie D.L. Schreus B.G. Gusev P.A. Favit A. Zohar O. Kim J.-H. Beushausen S. Ascoli G. Olds J.L. Neve R. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13808-13813Crossref PubMed Scopus (52) Google Scholar), larger amounts could be expressed in Escherichia coli, allowing a spectroscopic analysis of the protein. To characterize the structure-activity relationship for CE, we studied its secondary structure by CD, FT-IR, intrinsic fluorescence, hydrophobic chromatography, and nondenaturing gel electrophoresis. We report here that calcium binding causes a conformational switch for calexcitin and suggest that such a structural transition may be relevant for the biochemical mechanisms underlying the cellular pathways of memory acquisition. Twenty liters of Luria-Bertani broth containing ampicillin (100 mg/liter) were incubated with E. coli BL21(DE3) cells which had been transformed with a pET-16b expression vector (Novagen, Inc., New York, NY) containing the sequence for the CE protein, a factor X proteolytic site and an oligohistidine tail (3Nelson T.J. Cavallaro S. Yi C.-L. McPhie D.L. Schreus B.G. Gusev P.A. Favit A. Zohar O. Kim J.-H. Beushausen S. Ascoli G. Olds J.L. Neve R. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13808-13813Crossref PubMed Scopus (52) Google Scholar). Cells were grown at 37 °C to mid-log phase, and CE expression was induced by adding the activator isopropyl-β-d-thiogalactopyranoside (1 mmfinal). After 3 h, bacteria were collected by gentle centrifugation (3000 × g, Sorvall), suspended in 100 ml of loading buffer (0.5 m NaCl, 20 mmTris/HCl, pH 7.9, 0.5 mm Iz, 20 mg/liter pepstatin, 20 mg/liter leupeptin, 10 mg/liter aprotinin A, 0.5 mm PMSF), and stored at −80 °C. Upon partial thawing, the cells were homogenized at 0 °C by sonication and centrifuged (100,000 × g, Beckman) at 4 °C. Pellets were resuspended in 100 ml of loading buffer and recentrifuged. Pooled supernatants were divided into three batches and loaded onto a His-Tag column (15 ml, Novagen Inc., water-jacketed at 8 °C, flow rate 2.5 ml/min), previously charged with an excess of NiCl2. The column was washed with 150 ml of loading buffer followed by 150 ml of the same buffer with 25 mm Iz. The column was developed with eluting buffer (same as above with 0.5m Iz) and stripped with 0.1 m EDTA. Fractions were subjected to gel electrophoresis and Western blot analysis with standard procedures (6Kim C.S. Han Y.-F. Etcheberrigaray R. Nelson T.J. Olds J.L. Yoshioka T. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3060-3064Crossref PubMed Scopus (29) Google Scholar, 24Sambrook J. Fritsch E.F. Mariatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), using a polyclonal antibody raised against a 26-amino acid (see Fig. 2) synthetic peptide (3Nelson T.J. Cavallaro S. Yi C.-L. McPhie D.L. Schreus B.G. Gusev P.A. Favit A. Zohar O. Kim J.-H. Beushausen S. Ascoli G. Olds J.L. Neve R. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13808-13813Crossref PubMed Scopus (52) Google Scholar). The antibody, recognizing natural squid and cloned E. coli CE, was tested by antigen preabsorption and prebleed antiserum controls (24Sambrook J. Fritsch E.F. Mariatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Fractions containing CE were pooled together, desalted at 8 °C by ultrafiltration and concentrated down to 10 ml. For all the ultrafiltration and desalting steps described in this report, the filtration membrane (Centriprep/Centricon, Amicon, Beverly, MA) was preincubated with bovine serum albumin and extensively washed with water prior to usage. Upon concentration, some precipitation occurred, and the sample was centrifuged and filtered before the next chromatographic step. IMAC-HPLC was performed with a MC-Poros system (PerSeptive Biosystems, Cambridge, MA) at room temperature with a flow rate of 5 ml/min. The perfusion column (100 × 4.6 mm, 20-mm particle diameter) was charged with 25 column volumes of 0.2 m NiSO4or NiCl2, and equilibrated in loading buffer (until the base line was stable) according to the manufacturer's recommendations. The column was tested before each preparation by injecting two solutions containing, respectively, 20 and 100 μg of commercial horse heart myoglobin, and washing the column with 10 column volumes of 0.5 mm Iz followed by 5 column volumes of eluting buffer (50 mm Iz). The calexcitin-containing samples from the previous step were injected onto the column in loop (2 ml), and loading buffer was passed through until the base line stabilized. The column was developed with a linear gradient from loading to eluting buffer in 15 min, using optically pure Iz (Sigma) to avoid interference with the following spectroscopic analyses. Fractions containing CE were pooled together, concentrated to 0.5 mg/ml, and aliquoted (50 μl) for prolonged storage at −80 °C. For several experiments, calexcitin was proteolyzed to remove the oligohistidine chain from the protein N terminus. Fifty micrograms of purified CE in 180 μl of water were mixed with 5 μg of activated factor X (bovine ImmunoPure grade, Pierce) in 3 μl of buffer (5 mm Tris/HCl, pH 6.0, 0.5 m NaCl, 1 mm benzamidine hydrochloride). The solution was incubated at room temperature for 2 h, and the reaction was stopped by dilution with protease inhibitor mixture (20 mg/liter leupeptin, 10 mg/liter aprotinin A, 0.5 mm PMSF) and two passages through a 50-kDa cutoff ultrafiltration membrane to eliminate factor Xa (25Aurell L. Friberger P. Karsson G. Claeson G. Thromb. Res. 1977; 11: 595-609Abstract Full Text PDF PubMed Scopus (110) Google Scholar,26Nagai K. Th⊘gersen H.C. Nature. 1984; 309: 810-812Crossref PubMed Scopus (327) Google Scholar). The resulting mixture was repurified by IMAC-HPLC. Reacted CE (without the oligohistidine tail) was recovered in the void volume, while unreacted CE eluted on the Iz gradient. Samples of CE were collected, desalted, concentrated, and stored at −80 °C. Optic lobes from fresh squid (Loligo pealei, Calamari Inc., Woods Hole, MA) were dissected, frozen in liquid nitrogen, and stored at −80 °C. Two batches of 400 squid optic lobes (approximately 120 g) were high speed homogenized (Polytron, Switzerland, setting 5) at 4 °C in 150 ml of Hermissenda buffer (50 mm Tris/HCl, pH 7.4, 1 mm EDTA, 1 mm EGTA, 20 mg/liter leupeptin, 10 mg/liter aprotinin A, 0.5 mm PMSF, 0.2m dithiothreitol), followed by sonication. The suspension was centrifuged for 2 h at 4 °C (150,000 × g, Beckman), and the pellets were resuspended in 150 ml ofHermissenda buffer and centrifuged. Combined supernatants were passed through a 50-kDa cutoff ultrafiltration membrane and applied onto a medium pressure, preparative weak anion-exchange column (Pharmacia, Uppsala, Sweden, 50 × 3 cm, fast-flow DEAE-Sepharose, Sigma), water-jacketed at 8 °C. The flow rate was maintained at 1.5 ml/min during the loading and the washing (water, 12 h). The retained fraction was eluted isocratically at 6 ml/min with 1m potassium acetate buffer (pH 7.4), extensively desalted (final ionic strength <30 mm), and loaded onto a Cibacron blue-agarose column (25 × 2 cm, constant flow rate of 2.5 ml/min, Sigma), water-jacketed at 8 °C. After 7 h of washing, the column was developed with 1 m potassium acetate buffer (pH 7.4), and the retained fraction was desalted and concentrated down to 10 ml. This sample was divided in two aliquots and injected in loop (5 ml) on a semipreparative AX-300 HPLC (1 × 25 cm, 30-mm gel particle diameter, Thomson, Springfield, VA), equipped with an AX-300 Guardagel precolumn (Thomson, 2.5 × 1 cm). The column was eluted at 2 ml/min and 8 °C with a linear gradient of 0–0.6 mpotassium acetate buffer (pH 7.4) for 20 min followed by 0.6m buffer for 30 min and 1 m buffer for 10 min. The chromatograms were analyzed by Western blot of 1-ml fractions. CE-containing fractions were pooled, desalted, concentrated, and aliquoted at −80 °C. Spectra were acquired with the ion-spray ionization technique on a Perkin-Elmer API III Plus mass spectrometer (Sciex Co., Thornill, ONT, Canada). The experimental parameters for cation mode were: ion-spray voltage, 5.5 kV; orifice voltage, 90 V; scan range (m/z), 500–2000; scan rate, 10.5 ms/atomic mass unit; no interscan delay; resolution, >1 atomic mass unit. The stock solution of purified cloned CE was diluted 1:1 with acetonitrile containing 0.1% formic acid and 1% HCl, and delivered to the ion-spray source by infusion using a syringe pump (model 22, Harvard Apparatus, Inc., South Natick, MA). Computation for secondary structure prediction was performed remotely using a multiple sequence alignment algorithm (Sopma) available on the internet (e.g. http://dot.imgen.bcm.tmc; [email protected]). Default parameters were selected, and a simple query of percentage of α, β, and other structures was submitted to maximize reliability and accuracy (evaluated as 81% for each component) (27Geourjon C. Deleage G. Protein Eng. 1994; 7: 157-164Crossref PubMed Scopus (310) Google Scholar, 28Geourjon C. Deleage G. Comput. Appl. Biosci. 1995; 11: 681-684PubMed Google Scholar). Infrared spectra were recorded by using a Perkin-Elmer 1760 FT-IR spectrophotometer, equipped with a TGS detector and a Perkin-Elmer model 7300 computer. The instrument was continuously purged with dry air for 15 min before data collection and during measurements to eliminate water vapor absorption. For each sample, 128 interferograms were recorded, averaged, and Fourier transformed to produce a spectrum with a nominal resolution of 4 cm−1. Dried films were prepared by spreading 10 μl of the protein solutions (1.7, 0.85, 0.43, and 0.21 mg/ml) on BaF2 supports and slowly evaporating the water in a dry chamber at 25 °C for 3 h. The analysis of the protein aqueous solution (5% w/w) was performed by placing 4 μl between two discs of BaF2 using a Teflon spacer 9.3 mm thick. Spectra of the buffer were recorded in the same cell under the same instrumental conditions as the sample spectra. Difference spectra were generated by an interactive difference routine to subtract the appropriate solvent spectrum from the spectrum of each protein solution. Subtraction of water was considered appropriate when it yielded a flat baseline from 1900 to 1720 cm−1, avoiding negative side lobes, and when it removed the water band near 2130 cm−1. The procedure of spectra processing for quantitative analysis by the deconvolution method has been described previously (29Bramanti E. Bramanti R. Stiavetti P. Benedetti E. J. Chemometrics. 1994; 8: 409-421Crossref Scopus (20) Google Scholar, 30Bramanti E. Benedetti E. Biopolymers. 1996; 38: 639-654Crossref PubMed Google Scholar, 31Bramanti, E., Benedetti, E., Sagripanti, A., Papineschi, F., and Benedetti, E. (1997) Biopolymers , 41, in pressGoogle Scholar); the fit standard errors estimated between x-ray and infrared secondary structure values are 2.5% for α-helix, 7.16% for β structures, and 5.1% for other components (turns and coils). Vacuum UV-CD spectra were recorded over the range of 178 to 260 nm using either Jasco J720, Jasco J700, or Jasco J600 spectropolarimeter (Jasco Instruments, Tokyo, Japan). Measurements were made in triplicate using a 0.01-cm quartz disassemblable cell (V = 20 μl), or a 0.02 cm quartz water-jacketed cell (V = 15 μl) at 15 °C. Cells were rinsed with water extensively before each measurement, and the chamber was equilibrated with dry N2 (15 liters/min). The instrumental parameters were: scan rate, 10 nm/min; time constant (J600 only), 3 s; spectral slit width, 1 nm. All blanks were recorded in the same conditions and subtracted from the sample spectra. Solutions were prepared at 0 °C by diluting 5 μl of a freshly thawed purified CE aliquot with 5 μl of protease inhibitor mixture and 5 μl of water or calcium buffer (final protein concentration around 0.15 mg/ml). The pH was checked after every addition of the calcium buffer, and adjusted to pH 7.5 (Tris/HCl) in the few instances when this was necessary. Calcium buffer concentrations were calculated (taking into account all the interactions between Ca2+, Mg2+, H+, and EGTA) (32Portzehl I.L. Caldwell P.C. Ruegg J.C. Biochim. Biophys. Acta. 1964; 79: 581-591PubMed Google Scholar) using the EGTA software written by T. J. N. and available by anonymous FTP in binary mode at las1.ninds.nih.gov in the pub/dos directory. The exact protein concentration of the solutions (necessary to obtain secondary structures from CD spectra) was determined both spectrophotometrically (A 279 measured on a Varian Cary 4E double-ray spectrophotometer) and with the bicinchoninic acid assay (33Smith P.K. Krohn R.I. Hermason G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18713) Google Scholar) (Pierce) according to the manufacturer's specifications, using bovine serum albumin and horse heart myoglobin as standards. Spectra were cut at 180 or 182 nm and analyzed by single-value decomposition with the Varsel software (34Hennessey Jr., J.P. Johnson Jr., W.C. Biochemistry. 1981; 20: 1085-1094Crossref PubMed Scopus (574) Google Scholar, 35Johnson Jr., W.C. Methods Enzymol. 1992; 210: 426-447Crossref PubMed Scopus (60) Google Scholar) running on a Unix machine (SGI PI-4D20 with Irix 4.01). The complete basis of 33 proteins was used in 528 subgroups of 31 elements each, and the search was optimized to reduce fit standard errors using protein concentration as a variable parameter. Intrinsic fluorescence measurements were obtained in triplicate at room temperature using a Spex Fluorolog-2 spectrofluorimeter with dm-3000 software (Spex Industries, Inc., Edison, NJ). The experimental parameters were excitation wavelength, 279 nm; scan range, 300–400 nm; speed, 10 nm/min. The instrument was calibrated with 1 mmfluorescein, according to the manufacturer's protocol. Protein solutions were recovered from CD analysis, diluted with protease inhibitor mixture, and buffered with 1 mm calcium to 3 ml in a 1-cm quartz cell (final protein concentration, 0.03 mg/ml). Phenyl-Sepharose chromatography was performed at room temperature using 2 ml of CL4-B resin (Sigma) packed in a 5-ml polypropylene column (Pharmacia, Uppsala, Sweden). Purified CE (85 μl, 112 ng) was loaded and eluted at a constant flow rate of 0.5 ml/min, collecting 1-ml fractions for dot-blot analysis. A pair of parallel elutions were run in triplicate, with Tris-buffered saline modified with 2 mm calcium and 2 mmEDTA, respectively. The column was washed with 20 ml of 6 murea and equilibrated with native mobile phase. Nondenaturing polyacrylamide gel electrophoresis was performed using a precasted 4–20% gradient gel (Novex, San Diego, CA), with SDS- and β-mercaptoethanol-free running and sample buffers. The gel was electroblotted onto an unmodified nitrocellulose membrane (Pierce) with a semidry apparatus (6Kim C.S. Han Y.-F. Etcheberrigaray R. Nelson T.J. Olds J.L. Yoshioka T. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3060-3064Crossref PubMed Scopus (29) Google Scholar) (Bio-Rad). The membrane was incubated with a polyclonal primary antibody (3Nelson T.J. Cavallaro S. Yi C.-L. McPhie D.L. Schreus B.G. Gusev P.A. Favit A. Zohar O. Kim J.-H. Beushausen S. Ascoli G. Olds J.L. Neve R. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13808-13813Crossref PubMed Scopus (52) Google Scholar) and an alkaline phosphatase-conjugated secondary antibody before staining with the 5-Br-4-Cl-3-indoyl phosphate reaction (Pierce). Line 3652 of human skin fibroblasts from young control (Coriell Cell Repositories, Camden, NJ) was seeded (∼5 cells/mm2) in 35-mm Nunc Petri dishes in Dulbecco's modified Eagle's medium (Life Technologies, Inc.), supplemented with 10% calf serum. The cells were used for patch-clamp experiments (at 21–23 °C) 3–5 days after seeding (3Nelson T.J. Cavallaro S. Yi C.-L. McPhie D.L. Schreus B.G. Gusev P.A. Favit A. Zohar O. Kim J.-H. Beushausen S. Ascoli G. Olds J.L. Neve R. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13808-13813Crossref PubMed Scopus (52) Google Scholar, 12Etcheberrigaray R. Ito E. Kim C.S. Alkon D.L. Science. 1994; 264: 276-279Crossref PubMed Scopus (178) Google Scholar). Bath solutions contained 150 mm NaCl, 5 mm KCl, 0 or 2 mm CaCl2, 1 mm MgCl2, 10 mm HEPES/NaOH, pH = 7.4. Pipettes were made from Blue Tip capillary tubes (inside diameter, 1.1–1.2 mm) and filled with 140 mm KCl, 0.01 EGTA, 1 mm MgCl2, 10 mm HEPES/Na, pH = 7.4. Pipette resistances were ∼7 megohms. Records were obtained using an Axopatch-1D amplifier, stored on tape (Toshiba, PCM video recorder), and digitized at 2 kHz (interface Digidata 1200). Single-channel data acquisition and analysis were performed with pClamp 6. Amplifier, interface, and software were obtained from Axon Instruments (Foster City, CA). To study the secondary structure of a protein by spectroscopic methods such as CD, fluorescence or FT-IR, it is necessary to obtain an extremely pure sample to eliminate incorrect deconvolution of the experimental data due to impurities or optical interferences. The standard isolation procedure for cloned calexcitin (3Nelson T.J. Cavallaro S. Yi C.-L. McPhie D.L. Schreus B.G. Gusev P.A. Favit A. Zohar O. Kim J.-H. Beushausen S. Ascoli G. Olds J.L. Neve R. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13808-13813Crossref PubMed Scopus (52) Google Scholar) was therefore optimized by adding an extra IMAC-HPLC purification step. The overall protocol yielded an average of 0.5 mg of CE from 20 liters of medium. The protein was over 99% pure by densitometric analysis of silver stained gel electrophoresis (Fig.1 A). When the sample was left for several hours at room temperature (in conditions similar to those used for spectroscopic analysis), a faint, new band (∼16 kDa) appeared on the gel below the CE band (∼25 kDa, in good agreement with the calculated value of 24.5 kDa for the cloned fused protein). This low molecular weight band, which accounted for less than 5% of the main CE band, possibly corresponded to a degradation product of CE, as suggested by the immunoreactivity on Western blot (Fig.1 B). The purification protocol for squid CE yielded only 1–10 μg of protein per preparation, with a purity of ∼60%. Therefore this sample could not be used for spectroscopic studies. When the fused oligohistidine tail was enzymatically removed from cloned CE, the product had a molecular weight corresponding to the natural squid protein (Fig. 1, C and D). The reaction was stopped after 2 h to avoid CE degradation, with 50% of the protein remainin" @default.
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• W2073525626 title "Secondary Structure and Ca2+-induced Conformational Change of Calexcitin, a Learning-associated Protein" @default.
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