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• W2022597101 abstract "Two fragments of the C-terminal tail of the α1 subunit (CT1, amino acids 1538–1692 and CT2, amino acids 1596–1692) of human cardiac L-type calcium channel (CaV1.2) have been expressed, refolded, and purified. A single Ca2+-calmodulin binds to each fragment, and this interaction with Ca2+-calmodulin is required for proper folding of the fragment. Ca2+-calmodulin, bound to these fragments, is in a more extended conformation than calmodulin bound to a synthetic peptide representing the IQ motif, suggesting that either the conformation of the IQ sequence is different in the context of the longer fragment, or other sequences within CT2 contribute to the binding of calmodulin. NMR amide chemical shift perturbation mapping shows the backbone conformation of calmodulin is nearly identical when bound to CT1 and CT2, suggesting that amino acids 1538–1595 do not contribute to or alter calmodulin binding to amino acids 1596–1692 of CaV1.2. The interaction with CT2 produces the greatest changes in the backbone amides of hydrophobic residues in the N-lobe and hydrophilic residues in the C-lobe of calmodulin and has a greater effect on residues located in Ca2+ binding loops I and II in the N-lobe relative to loops III and IV in the C-lobe. In conclusion, Ca2+-calmodulin assumes a novel conformation when part of a complex with the C-terminal tail of the CaV1.2 α1 subunit that is not duplicated by synthetic peptides corresponding to the putative binding motifs. Two fragments of the C-terminal tail of the α1 subunit (CT1, amino acids 1538–1692 and CT2, amino acids 1596–1692) of human cardiac L-type calcium channel (CaV1.2) have been expressed, refolded, and purified. A single Ca2+-calmodulin binds to each fragment, and this interaction with Ca2+-calmodulin is required for proper folding of the fragment. Ca2+-calmodulin, bound to these fragments, is in a more extended conformation than calmodulin bound to a synthetic peptide representing the IQ motif, suggesting that either the conformation of the IQ sequence is different in the context of the longer fragment, or other sequences within CT2 contribute to the binding of calmodulin. NMR amide chemical shift perturbation mapping shows the backbone conformation of calmodulin is nearly identical when bound to CT1 and CT2, suggesting that amino acids 1538–1595 do not contribute to or alter calmodulin binding to amino acids 1596–1692 of CaV1.2. The interaction with CT2 produces the greatest changes in the backbone amides of hydrophobic residues in the N-lobe and hydrophilic residues in the C-lobe of calmodulin and has a greater effect on residues located in Ca2+ binding loops I and II in the N-lobe relative to loops III and IV in the C-lobe. In conclusion, Ca2+-calmodulin assumes a novel conformation when part of a complex with the C-terminal tail of the CaV1.2 α1 subunit that is not duplicated by synthetic peptides corresponding to the putative binding motifs. Calmodulin (CaM), 1The abbreviations used are: CaM, calmodulin; CDI, Ca2+-dependent inactivation; CaV1.2, cardiac L-type voltage-dependent calcium channel; Ca2+-CaM, Ca2+-bound calmodulin; CDF, Ca2+-dependent facilitation; CKI, CaM kinase I; FRET, fluorescence resonance energy transfer; MOPS, 4-morpholinepropanesulfonic acid; DDPM, dimethylamino-3,5-dinitrophenyl)maleimide; 1,5-IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid. a ubiquitous Ca2+ sensor, directly or indirectly regulates excitation-contraction coupling and other important physiological functions in cardiac myocytes (3Yang D. Song L.S. Zhu W.Z. Chakir K. Wang W. Wu C. Wang Y. Xiao R.P. Chen S.R. Cheng H. Circ. Res. 2003; 92: 659-667Crossref PubMed Scopus (31) Google Scholar, 4Walsh K.B. Cheng Q. Am. J. Physiol. Heart Circ. Physiol. 2004; 286: H186-H194Crossref PubMed Google Scholar, 5Wu Y. Kimbrough J.T. Colbran R.J. Anderson M.E. J. Physiol. 2004; 554: 145-155Crossref PubMed Scopus (31) Google Scholar, 6Wu Y. Colbran R.J. Anderson M.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2877-2881Crossref PubMed Scopus (77) Google Scholar, 7Mouton J. Ronjat M. Jona I. Villaz M. Feltz A. Maulet Y. FEBS Lett. 2001; 505: 441-444Crossref PubMed Scopus (21) Google Scholar). Cardiac L-type Ca2+ channels (CaV1.2) are modulated by the interaction of the channel α1 subunit C-terminal tail with CaM (8Ivanina T. Blumenstein Y. Shistik E. Barzilai R. Dascal N. J. Biol. Chem. 2000; 275: 39846-39854Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), such that CaM binding to this region is required for both Ca2+-dependent inactivation (CDI) and Ca2+-dependent facilitation (CDF) of cardiac L-type Ca2+ channels (9Pitt G.S. Zuhlke R.D. Hudmon A. Schulman H. Reuter H. Tsien R.W. J. Biol. Chem. 2001; 276: 30794-30802Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 10Zu ̈hlke R.D. Pitt G.S. Tsien R.W. Reuter H. J. Biol. Chem. 2000; 275: 21121-21129Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 11Zu ̈hlke R.D. Pitt G.S. Deisseroth K. Tsien R.W. Reuter H. Nature. 1999; 399: 159-162Crossref PubMed Scopus (740) Google Scholar, 12Peterson B.Z. DeMaria C.D. Adelman J.P. Yue D.T. Neuron. 1999; 22: 549-558Abstract Full Text Full Text PDF PubMed Google Scholar, 13Qin N. Olcese R. Bransby M. Lin T. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2435-2438Crossref PubMed Scopus (249) Google Scholar, 14Soldatov N.M. Zuhlke R.D. Bouron A. Reuter H. J. Biol. Chem. 1997; 272: 3560-3566Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). CDI is the process whereby the entry of Ca2+ enhances channel closing during a maintained depolarization (15McDonald T.F. Pelzer S. Trautwein W. Pelzer D.J. Physiol. Rev. 1994; 74: 365-507Crossref PubMed Google Scholar, 16Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1953) Google Scholar), whereas CDF is the process whereby increased basal Ca2+ or repeated transient depolarizations leads to increased channel opening (17Anderson M.E. J. Mol. Cell. Cardiol. 2001; 33: 639-650Abstract Full Text PDF PubMed Scopus (50) Google Scholar). CDI of CaV1.2 appears to be driven by Ca2+ binding to the C-lobe of CaM and is unaltered by the presence of intracellular Ca2+ buffers (18Liang H. DeMaria C.D. Erickson M.G. Mori M.X. Alseikhan B.A. Yue D.T. Neuron. 2003; 39: 951-960Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Although a number of sequences within the C-terminal tail of the α1 subunit appear to be capable of binding CaM (1Pate P. Mochca-Morales J. Wu Y. Zhang J.Z. Rodney G.G. Serysheva I.I. Williams B.Y. Anderson M.E. Hamilton S.L. J. Biol. Chem. 2000; 275: 39786-39792Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 9Pitt G.S. Zuhlke R.D. Hudmon A. Schulman H. Reuter H. Tsien R.W. J. Biol. Chem. 2001; 276: 30794-30802Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 19Tang W. Halling D.B. Black D.J. Pate P. Zhang J.Z. Pedersen S. Altschuld R.A. Hamilton S.L. Biophys. J. 2003; 85: 1538-1547Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 20Soldatov N.M. Oz M. O'Brien K.A. Abernethy D.R. Morad M. J. Biol. Chem. 1998; 273: 957-963Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 21Zu ̈hlke R.D. Reuter H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3287-3294Crossref PubMed Scopus (160) Google Scholar), it is unclear which actually contribute to CaM binding in the native channel. A sequence designated the IQ motif is required for both CDI and CDF, but the precise roles of other neighboring sequences remain to be elucidated. Peterson et al. (22Peterson B.Z. Lee J.S. Mulle J.G. Wang Y. de Leon M. Yue D.T. Biophys. J. 2000; 78: 1906-1920Abstract Full Text Full Text PDF PubMed Google Scholar) and De Leon et al. (23De Leon M. Wang Y. Jones L. Perez-Reyes E. Wei X. Soong T.W. Snutch T.P. Yue D.T. Science. 1995; 270: 1502-1506Crossref PubMed Google Scholar) identified critical determinants of CDI within the consensus Ca2+ binding motif (the EF hand) of the cardiac L-type Ca2+ channel. Specifically, Peterson et al. (22Peterson B.Z. Lee J.S. Mulle J.G. Wang Y. de Leon M. Yue D.T. Biophys. J. 2000; 78: 1906-1920Abstract Full Text Full Text PDF PubMed Google Scholar) found a four amino acid cluster (VVTL) within the EF hand to be essential for CDI. However, Zhou et al. (24Zhou J. Olcese R. Qin N. Noceti F. Birnbaumer L. Stefani E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2301-2305Crossref PubMed Scopus (85) Google Scholar) and Qin et al. (13Qin N. Olcese R. Bransby M. Lin T. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2435-2438Crossref PubMed Scopus (249) Google Scholar) maintained that CDI of CaV1.2 did not require the EF hand motif. Zühlke and Reuter (21Zu ̈hlke R.D. Reuter H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3287-3294Crossref PubMed Scopus (160) Google Scholar) suggested that CDI was a cooperative process involving three noncontiguous amino acid sequences: the EF hand motif, two hydrophilic residues (asparagine and glutamic acid, residues 1630 and 1631), and the IQ motif. To complicate matters further, two other sequences (amino acids 1609–1628, designated A motif; amino acids 1627–1652, designated C or CB motif) between the EF hand and the IQ motif have also been implicated in CaM binding (1Pate P. Mochca-Morales J. Wu Y. Zhang J.Z. Rodney G.G. Serysheva I.I. Williams B.Y. Anderson M.E. Hamilton S.L. J. Biol. Chem. 2000; 275: 39786-39792Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 9Pitt G.S. Zuhlke R.D. Hudmon A. Schulman H. Reuter H. Tsien R.W. J. Biol. Chem. 2001; 276: 30794-30802Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 19Tang W. Halling D.B. Black D.J. Pate P. Zhang J.Z. Pedersen S. Altschuld R.A. Hamilton S.L. Biophys. J. 2003; 85: 1538-1547Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 20Soldatov N.M. Oz M. O'Brien K.A. Abernethy D.R. Morad M. J. Biol. Chem. 1998; 273: 957-963Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 21Zu ̈hlke R.D. Reuter H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3287-3294Crossref PubMed Scopus (160) Google Scholar). Additional data to define the CaM binding site on the C-terminal tail of the α1 subunit and to elucidate the mechanisms of CDI and CDF of CaV1.2 are needed. The molecular details of the interactions and the stoichiometry of this interaction remain to be determined. In this report, we describe the expression of fragments of the α1 subunit C-terminal domain and provide new details of its interaction with CaM. Materials—[15N]NH4Cl and d-[U-13C]glucose were obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA). 5-((((2-Iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) was purchased from Molecular Probes, Inc. (Eugene, OR). N-(4-Dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) and other chemicals were purchased from Sigma. Peptides were synthesized in the core facility at Baylor College of Medicine under the direction of Dr. Richard Cook. Expression and Purification of CT1, CT2, Wild-type CaM, N-CaM, C-CaM, E12Q CaM, E34Q CaM, E1234Q CaM, and T34C/T110C CaM—We made two constructs of the C-terminal tail using the human CaV1.2 α1 subunit cDNA as a template. One construct codes for amino acids 1538–1692 (includes the EF hand regions and the A, C, and IQ motifs) and is designated CT1. A second construct codes for amino acids 1596–1692 (includes the A, C, and IQ motifs) and is designated CT2. We subcloned the cDNA of these sequences into pET23a(+) and pET28a(+) vectors (Novagen, Madison, WI) between NdeI and HindIII sites and used these constructs for the expression of the fragments with and without a His tag. We transformed the subcloned vectors into BL21(DE3) host (Novagen) for expression and grew the cells in LB media containing the suitable antibiotics at 37 °C. Cells were induced with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside at an OD600 of 0.6 and incubated another 3 h at 37 °C before harvesting. The cells were lysed with lysozyme and nuclease and soluble material was removed by sedimentation of the insoluble material. The insoluble pellet was washed with urea and Triton X-100 to obtain an insoluble fraction enriched in inclusion body proteins. Wild-type CaM, N-CaM, and C-CaM were expressed and purified as described previously (25Xiong L.W. Newman R.A. Rodney G.G. Thomas O. Zhang J.Z. Persechini A. Shea M.A. Hamilton S.L. J. Biol. Chem. 2002; 277: 40862-40870Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The Ca2+ binding site mutants of CaM (E12Q CaM, E34Q CaM, and E1234Q CaM) were expressed and purified as described previously (26Rodney G.G. Krol J. Williams B. Beckingham K. Hamilton S.L. Biochemistry. 2001; 40: 12430-12435Crossref PubMed Scopus (37) Google Scholar). Expression and purification of T34C/T110C CaM (cysteine substitution for threonine at positions 34 and 110 of CaM) is the same as that of wild-type CaM. Refolding of CT1 (Amino Acids 1538–1692) and CT2 (Amino Acids 1596–1692)—The inclusion bodies were solubilized with Inclusion Body solubilization reagent (Pierce) and refolded by dialyzing against 90 mm Tris-HCl, pH 7.4, 6 m urea, 6 mm calcium in the presence of CaM (wild-type CaM or CaM mutants). Water was added gradually to the dialysis buffer to lower the urea concentration to 2 m. The remaining urea was removed by dialysis against 30 mm Tris-HCl, pH 7.4 and 2 mm calcium. Soluble and insoluble proteins were then separated by centrifugation at 48,000 × g for 1 h at 4 °C. His tags were removed using a Thrombin Cleavage Capture kit (Novagen). The refolding in the presence of CaM was also examined at three different Ca2+ concentrations. Preparation of CT1 for Analysis of CaM Binding by Nondenaturing Gel Electrophoresis—The untagged, solubilized CT1 was further purified using chelating Sepharose (Amersham Biosciences). To avoid CT1 precipitation, all buffers (equilibration, binding, wash, and elution buffer) included a 50% solution of the Inclusion Body solubilization reagent. CT1 was eluted with 10 mm imidazole. The purified CT1 was refolded by dialysis against 90 mm Tris-HCl, pH 7.4, 6 m urea, 3% Triton X-100, gradually adding water into the dialysis buffer until 2 m urea was reached. At this stage CT1 was dialyzed against 50 mm MOPS, pH 7.4, 1% Triton X-100. Increasing amounts of refolded CT1 were incubated with CaM (2 μg in 20 μl (6 μm) per sample) for 30 min at room temperature in 50 mm MOPS (pH 7.4), 1% Triton X-100, and 2 mm CaCl2 or 2 mm EGTA. Nondenaturing PAGE (15%) was performed to assess complex formation of CT1 and CaM. Protein concentrations were determined by the method of Lowry (27Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as a standard. Preparation of CT1, CT2, and Donor/Acceptor-labeled CaM for Fluorescence Resonance Energy Transfer (FRET)—The purified complex of CT1 or CT2-CaM was obtained by refolding CT1 or CT2 in the presence of CaM. His tags were cleaved with thrombin. The samples were dialyzed against 30 mm Tris-HCl buffer, pH 7.4, with EGTA to precipitate CT1 or CT2. The pellet was washed with 30 mm Tris-HCl buffer, pH 7.4, to obtain purified CT1 or CT2 (insoluble). Samples were solubilized in the Inclusion Body solubilization reagent. Donor/acceptor-labeled CaM (D/A CaM) was obtained by labeling T34C/T110C CaM with 1,5-IAEDANS (the donor) and DDPM (the acceptor). To obtain T34C/T110C that was only labeled with the donor, T34C/T110C CaM was labeled with 0.4 mol of donor/mol of protein in 20 mm Tris-HCl, pH 7.5, 100 mm KCl for 2 h at 20 °C in the dark. Free donor was removed by a desalting column packed with size exclusion medium, Bio-Gel P-6DG (Bio-Rad). The product had 0.31 mol of the donor per mol of protein. A portion of this partially labeled protein was saved as the donor-alone protein, while the rest was labeled with excess acceptor to give D/A CaM saturated by acceptor at the same reaction conditions. Free acceptor was removed by the same desalting column as above. FRET—D/A CaM (0.5 μm) was incubated with increasing concentrations of CT1, CT2, A-peptide, C-peptide, or IQ-peptide for 5 min at 20 °C in 20 mm MOPS pH 7.5, 100 mm KCl, 2 mm CaCl2, or 2 mm EGTA. Fluorescence data were collected on a PTI QuantaMaster spectrofluorometer. The excitation wavelength was set at 334 nm and emission spectra were scanned from 400 nm to 600 nm with a 5-nm slit width for excitation and a 10-nm slit width for emission. Spectra were processed by subtracting the background of buffer and/or additives where appropriate and by averaging three sets of scans. To ensure that the fluorescence changes were due to the changes of distance between donor and acceptor and not to the interaction of these fragments with the donor, parallel experiments were performed with donor-alone-labeled CaM. Data were compared with the FRET obtained with a CaM kinase KII peptide (FNARRKLKGAILTTMLATRN, designated FNA-peptide) that is known to bring the two lobes of CaM in close proximity (28Meador W.E. Means A.R. Quiocho F.A. Science. 1993; 262: 1718-1721Crossref PubMed Google Scholar). To calculate the apparent affinity of CT2 and the synthetic peptides for CaM, we titrated D/A CaM in 20 mm MOPS pH 7.5, 100 mm KCl, 2 mm CaCl2 with increasing CT1-, CT2-, IQ-, A-, or C-peptide. Fluorescence emission data at 490 nm were read on an ISS PC1 Photon Counting Spectrofluorometer (ISS, Champaign, Illinois), with a 1-mm slit for excitation and 2-mm slit for emission, at a 334 nm excitation wavelength. Data were processed by subtracting the background of buffer and/or additives where appropriate. The data were fit with a four parameter-Hill equation in SigmaPlot. Preparation of CT1 or CT2/15N,13C-labeled CaM Complex for Nuclear Magnetic Resonance Spectroscopy (NMR)—15N,13C-labeled CaM was created by growing cells in M9 minimal medium using an expression plasmid coding for CaM. [15N]NH4Cl and d-[U-13C]glucose were the only nitrogen and carbon sources for the cells. Other steps involved in expression and purification of 15N,13C-labeled CaM were the same as for wild-type CaM. The complex of 15N,13C-labeled CaM/CT1 or CT2 for NMR was obtained by refolding the solubilized CT1 or CT2 in the presence of 15N,13C-labeled CaM. The His tag was cleaved with biotinylated thrombin, and the thrombin was removed from solution with streptavidin agarose (Novagen). The cleaved His tag was removed from the complex solution during dialysis against the final buffer (10 mm imidazole pH 6.4, 2 mm CaCl2) with a 10,000 MWCO cassette (Pierce). NMR Methodology—NMR experiments were collected on a DRX 600 MHz spectrometer instrument using a 5-mm TXI probe or cryoprobe at 47 °C. Complexes of Ca2+-loaded 15N,13C-labeled CaM bound to CT1 or CT2 were prepared as described above and then concentrated to 0.5–1 mm. Peptide-CaM complexes were prepared by adding the appropriate peptide directly to the Ca2+-CaM NMR sample. Final NMR samples contained 10 mm imidazole, and 2 mm CaCl2 in 95% H2O, 5% D2OatpH 6.4. Amide chemical shifts for free Ca2+-CaM in the absence of salt were assigned by comparison to reported chemical shifts for free Ca2+-CaM in 100 mm KCl (29Ikura M. Spera S. Barbato G. Kay L.E. Krinks M. Bax A. Biochemistry. 1991; 30: 9216-9228Crossref PubMed Google Scholar) and confirmed by the following triple resonance experiments: CBCANH, CBCA(CO)NH, HNCA, HN(CO)CA, and 15N-edited NOESYHSQC. CaM complexes were assigned using the above mentioned experiments and in addition three-dimensional HNCO and HN(CA)CO experiments were also collected. All data were processed using Felix 2002 software from Accelrys. Expression of Fragments of the C-terminal Tail of the CaV1.2 α1 Subunit and Analysis of Their Ability to Bind CaM—To elucidate the determinants of the molecular interactions of CaM with the C-terminal tail of the CaV1.2 α1 subunit, we expressed or synthesized fragments of this region containing all or part of the putative binding motifs (Fig. 1). For these studies we used three synthetic peptides designated A (amino acids 1609–1628), C (amino acids 1627–1652), and IQ (amino acids 1665–1685). We expressed two fragments of the C-terminal tail: CT1 (amino acids 1538–1692, containing the putative EF hand as well as the A, C, and IQ motifs) and CT2 (amino acids 1596–1692, containing the A, C, and IQ motifs but missing the putative EF hand). We analyzed the interactions of these fragments and peptides with CaM using nondenaturing gel electrophoresis. When expressed in Escherichia coli, both CT1 and CT2 were found in inclusion bodies. A small amount of soluble CT1 or CT2 can be obtained by extraction of the inclusion bodies and a subsequent refolding step (see “Experimental Procedures”). The interaction of refolded CT1 with CaM as assessed by nondenaturing gel electrophoresis is shown in Fig. 2. CT1 is highly positively charged (isoelectric point 10.5) and does not enter the nondenaturing gel at either high or low Ca2+ concentrations (lane 1 of Fig. 2, A and B, respectively). The interaction of CT1 with CaM was assessed by the appearance of a more slowly migrating complex CT1/CaM or by a decrease in the intensity of CaM band. A stable CT1-CaM complex was formed at high Ca2+ concentrations (Fig. 2A). The densitometric analysis of the disappearance of the CaM band at different fragment concentrations is shown in Fig. 2C. A very limited and low affinity interaction of this fragment was detected with apoCaM (assessed by the disappearance of the apoCaM band on the gel, Fig. 2, B and C).Fig. 2Calcium-dependent complex formation of CT1 with CaM in 15% polyacrylamide nondenaturing gel. CT1 was incubated with 2 μg of CaM (in 20 μl, final 6 μm) were incubated for 30 min at room temperature in 50 mm MOPS pH 7.4, 1% Triton X-100 with 2 mm Ca2+ (A)or 2mm EGTA (B) before electrophoresis. Lane 1, 2 μg CT1 alone; lanes 2–9, the molar ratio of CT1:CaM is 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, respectively. I/I0 designates the ratio of the intensity of the CaM band in the presence of CT1 versus that of CaM alone (C), and intensities were obtained by densitometric analysis of Coomassie Blue-stained CaM bands from three independent nondenaturing gels. The curve with filled squares is for the low Ca2+ condition and the one with filled circles is for the high Ca2+ condition.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although only a small amount of soluble CT1 or CT2 can be obtained as described above, the presence of CaM during the refolding step greatly enhances the amount of CT1 and CT2 that can be isolated in a soluble form. As shown in the SDS-polyacrylamide gel of the soluble fraction in Fig. 3, the amount of soluble CT1 is directly proportional to the CaM added (lane 2 of Fig. 3A). Other proteins and excess CT1 are found in the insoluble pellet (lane 3 of Fig. 3A). The CT1-CaM complex remains soluble even when concentrated to 100 mg of protein per ml. The complex of CT1 and CaM showed evidence of 2 complexes in nondenaturing gels (Fig. 3B, lane 2). Using N-terminal sequencing we found that some proteolysis after amino acid 1595 had occurred during the isolation. This observation led us to create CT2. CT2 also required the presence of CaM in the refolding step (lane 4 of Fig. 3A), and the amount of soluble protein obtained was proportional to the amount of CaM added (lane 5 of Fig. 3A). The presence of free CaM in nondenaturing gels of CT1 but not CT2 (lane 2 of Fig. 3B) suggests that the N-terminal half of CT1 either decreases the affinity for CaM or inhibits the refolding of the fragment. To examine the effect of Ca2+ concentration on the facilitated refolding the amount of soluble protein obtained when the refolding was performed in <10 nm, 100 nm, 5 μm, 100 μm, and 2 mm free Ca2+ was examined (Fig. 4). Some facilitated refolding of both CT1 (Fig. 4A) and CT2 (Fig. 4B) could be detected at all Ca2+ concentrations in the presence of CaM (lanes 1–5), but was maximal at concentrations above 100 μm. Without CaM, no facilitated refolding was detected at any Ca2+ concentration for either CT1 and CT2 (lanes 6–10). The molecular equivalents of CaM required to refold CT1 and CT2 suggests that only one CaM is binding to CT1 and CT2. Ca2+Binding to Both Lobes of CaM Is Required for Maximal CaM-assisted Refolding—To evaluate the effects of Ca2+ binding to the N- and C-lobes of CaM on the facilitated refolding we examined the ability of Ca2+ binding site mutants of CaM to promote refolding at high Ca2+ concentrations. For these experiments we used: 1) N-CaM, a CaM composed of amino acids 1–75, missing the C-lobe, 2) C-CaM, a CaM composed of amino acids 76–148, missing the N-lobe, 3) E12Q CaM which cannot bind Ca2+ at the N-lobe, 4) E34Q which cannot bind Ca2+ at the C-lobe, and 5) E1234Q which cannot bind Ca2+ at either the N- or C-lobes. These CaM mutants have been described previously (25Xiong L.W. Newman R.A. Rodney G.G. Thomas O. Zhang J.Z. Persechini A. Shea M.A. Hamilton S.L. J. Biol. Chem. 2002; 277: 40862-40870Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 26Rodney G.G. Krol J. Williams B. Beckingham K. Hamilton S.L. Biochemistry. 2001; 40: 12430-12435Crossref PubMed Scopus (37) Google Scholar). Although none of the five CaM mutants were as effective as the wild-type CaM for the refolding, N-CaM (lane 4 of Fig. 5, A and B), C-CaM (lane 6 of Fig. 5, A and B), E12Q (lane 8 of Fig. 5, A and B) and E34Q (lane 10 of Fig. 5B) produced some facilitated refolding. In contrast, the E1234Q did not facilitate refolding. This suggests that both lobes of Ca2+-CaM contribute to the facilitated refolding of the CaV1.2 α1 C-terminal tail fragments. FRET Analysis to Determine the Effects of CT1, CT2, and Synthetic Peptides on the Conformation of CaM—To determine the effects of binding to the C-terminal tail of the CaV1.2 α1 subunit on the conformation of CaM, we examined the ability of a double-labeled CaM (1,5-IAEDANS and DDPM) bound to the fragments or the peptides to produce FRET at high and low Ca2+ concentrations. Since the efficiency of FRET depends upon the distance between the donor and the acceptor in CaM, the emission fluorescence of the D/A CaM at 490 nm can be used to compare the relative distances between the two lobes of CaM when bound to the fragments or peptides. At high Ca2+ concentrations, CT2 (Fig. 6A), the IQ-peptide (Fig. 6B), and the A-peptide (Fig. 6C) all decreased the fluorescence of D/A CaM. In contrast, the C-peptide (Fig. 6D) increased the fluorescence of D/A CaM. These findings suggest that the lobes of CaM are closer together when bound to CT2, the IQ-peptide, and the A-peptide compared with the C-peptide. The longer CT1 fragment produces similar FRET changes to those determined with CT2 (not shown). As a control we examined the fluorescence at 490 nm with CaM that was labeled only with the donor compound (D/CaM) and found that the fluorescence did not change upon addition of the peptides or fragments (data not shown). With CT1, CT2, and IQ, saturation of the FRET changes was obtained at 1:1 molar ratios of fragment/CaM (Fig. 6, A and B). A higher molar ratio for saturation of the FRET changes was required for both the A- and C-peptides (Fig. 6, C and D), consistent with our findings that the IQ-peptide has a higher affinity for CaM than either the A- or C-peptide (1Pate P. Mochca-Morales J. Wu Y. Zhang J.Z. Rodney G.G. Serysheva I.I. Williams B.Y. Anderson M.E. Hamilton S.L. J. Biol. Chem. 2000; 275: 39786-39792Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 19Tang W. Halling D.B. Black D.J. Pate P. Zhang J.Z. Pedersen S. Altschuld R.A. Hamilton S.L. Biophys. J. 2003; 85: 1538-1547Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Analysis of the concentration dependence of the FRET changes (Fig. 6E) was used to calculate apparent affinities for CaM of 37 ± 3 nm for CT2 (n = 3), 45 ± 4nm for IQ (n = 3), and 76 ± 5nm (n = 3) for A-peptide). The titration curve of CT1 is essentially identical to that of CT2 (data not shown). Our measured Kd for CaM binding to CT2 is comparable to the value (Kd = 163 nm) reported by Erickson et al. (30Erickson M.G. Liang H. Mori M.X. Yue D.T. Neuron. 2003; 39: 97-107Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) for CaM binding to full-length α1C. The maximal fluorescence decrease of the D/A CaM in the presence of either the IQ-peptide or A-peptide is similar to that obtained with the control FNA-peptide (Fig. 6, B and C), which is known to bring the two lobes of CaM into close proximity (28Meador W.E. Means A.R. Quiocho F.A. Science. 1993; 262: 1718-1721Crossref PubMed Google Scholar). In contrast, the fragments CT1 and CT2 produced an intermediate decrease of D/A CaM fluorescence, suggesting that the conformation of CaM bound to the fragment is different than when bound to either A- or IQ-peptides. This could reflect a difference in conformation of these sequences (IQ or A) within the larger fragment or a contribution of the C sequence to the CaM interaction. At low Ca2+ concentrations, the fragments and peptides did not produce FRET of D/A CaM (data not shown), indicating that the either conformational change in CaM or its interaction with the fragments and peptides are Ca2+-dependent. Effect of CT1 and CT2 on Backbone Amide Chemical Shifts of Ca2+-CaM—We used amide chemical shift perturbation mapping to compare the interactions of CaM with the expressed fragments of the C-terminal tail of the CaV1.2 α1 subunit (CT1 and CT2) versus the synthetic peptides (C and IQ) corresponding to the putative CaM bind" @default.
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• W2022597101 date "2005-02-01" @default.
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• W2022597101 title "Sites on Calmodulin That Interact with the C-terminal Tail of Cav1.2 Channel" @default.
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