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• W2034261645 abstract "The acidic, bilobed protein calmodulin (CaM; molecular mass of 16.7 kDa) can activate some 40 distinct proteins in a calcium-dependent manner. The majority of the CaM-binding domain regions of the target proteins are basic and hydrophobic in nature, are devoid of multiple negatively charged residues, and have a propensity to form an α-helix. The CaM-binding domain in the C-terminal region of petunia glutamate decarboxylase (PGD) is atypical because it contains five negatively charged residues. Therefore, we chose to study the binding of calcium-CaM to a 26-residue synthetic peptide encompassing the C-terminal region of PGD. Gel band shift assays, fluorescence spectroscopy, and NMR titration studies showed that a single unique complex of calcium-CaM with two PGD peptides is formed. The formation of a 1:2 protein-peptide complex is unusual; normally, calcium-CaM forms 1:1 complexes with the majority of its target proteins. Circular dichroism spectroscopy showed that the bound PGD peptides have an α-helical structure. NMR studies of biosynthetically [methyl-13C]methionine-labeled CaM revealed that all the Met side chains in CaM are involved in the binding of the PGD peptides. Analysis of fluorescence spectra showed that the single Trp residue of the two peptides becomes bound to the N- and C-terminal lobes of CaM. These results predict that binding of calcium-CaM to PGD will give rise to dimerization of the protein, which may be necessary for activation. Possible models for the structure of the protein-peptide complex, such as a dimeric peptide structure, are discussed. The acidic, bilobed protein calmodulin (CaM; molecular mass of 16.7 kDa) can activate some 40 distinct proteins in a calcium-dependent manner. The majority of the CaM-binding domain regions of the target proteins are basic and hydrophobic in nature, are devoid of multiple negatively charged residues, and have a propensity to form an α-helix. The CaM-binding domain in the C-terminal region of petunia glutamate decarboxylase (PGD) is atypical because it contains five negatively charged residues. Therefore, we chose to study the binding of calcium-CaM to a 26-residue synthetic peptide encompassing the C-terminal region of PGD. Gel band shift assays, fluorescence spectroscopy, and NMR titration studies showed that a single unique complex of calcium-CaM with two PGD peptides is formed. The formation of a 1:2 protein-peptide complex is unusual; normally, calcium-CaM forms 1:1 complexes with the majority of its target proteins. Circular dichroism spectroscopy showed that the bound PGD peptides have an α-helical structure. NMR studies of biosynthetically [methyl-13C]methionine-labeled CaM revealed that all the Met side chains in CaM are involved in the binding of the PGD peptides. Analysis of fluorescence spectra showed that the single Trp residue of the two peptides becomes bound to the N- and C-terminal lobes of CaM. These results predict that binding of calcium-CaM to PGD will give rise to dimerization of the protein, which may be necessary for activation. Possible models for the structure of the protein-peptide complex, such as a dimeric peptide structure, are discussed. calmodulin glutamate decarboxylase a 26-residue CaM-binding domain peptide derived from the C-terminal end of petunia glutamate decarboxylase polyacrylamide gel electrophoresis selenomethionine heteronuclear multiple quantum coherence nuclear Overhauser effect nuclear Overhauser effect spectroscopy myosin light chain kinase trifluoroethanol 5-dimethylaminonaphthalene-1-sulfonyl. Calmodulin (CaM)1 is a 148-amino acid acidic protein that acts as a ubiquitous calcium regulatory protein in eukaryotic cells. It can bind and activate >40 different target proteins (for recent reviews, see Refs. 1Vogel H.J. Biochem. Cell Biol. 1994; 72: 357-376Crossref PubMed Scopus (222) Google Scholar, 2Crivici A. Ikura M. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 85-116Crossref PubMed Scopus (693) Google Scholar, 3Vogel H.J. Zhang M. Mol. Cell Biochem. 1995; 149/150: 3-15Crossref Scopus (70) Google Scholar, 4Ikura M. Trends Biochem. Sci. 1996; 21: 14-17Abstract Full Text PDF PubMed Scopus (597) Google Scholar). These proteins include several protein kinases, phosphatase 2B (calcineurin), multiple components in smooth muscle contraction, ion channels, etc. In the x-ray structure, Ca2+-CaM adopts a dumbbell-shaped structure, with the two lobes connected by a 26-residue-long α-helix (5Babu Y.S. Bugg C.E. Cook W.J. J. Mol. Biol. 1988; 204: 191-204Crossref PubMed Scopus (972) Google Scholar). Each lobe contains two helix-loop-helix Ca2+-binding motifs that are interconnected by a small β-sheet between the two Ca2+-binding loops. In solution, the middle portion of the central α-helical linker region is flexible, as demonstrated by NMR spectroscopy (6Barbato G. Ikura M. Kay L.E. Pastor R.W. Bax A. Biochemistry. 1992; 31: 5269-5278Crossref PubMed Scopus (889) Google Scholar). Methionine-rich hydrophobic patches become exposed on each lobe of CaM following the binding of four Ca2+ ions (5Babu Y.S. Bugg C.E. Cook W.J. J. Mol. Biol. 1988; 204: 191-204Crossref PubMed Scopus (972) Google Scholar), whereas these two hydrophobic patches are absent or not fully exposed in apo-CaM structures (7Zhang M. Tanaka T. Ikura M. Nat. Struct. Biol. 1995; 2: 758-767Crossref PubMed Scopus (645) Google Scholar, 8Kuboniwa H. Tjandra N. Grzesiek S. Ren H. Klee C.B. Bax A. Nat. Struct. Biol. 1995; 2: 768-776Crossref PubMed Scopus (613) Google Scholar). When the concentration of the secondary messenger Ca2+increases inside cells from ∼10−7 to ∼10−5m, four Ca2+ ions will bind to CaM, and CaM will expose its two hydrophobic patches for target protein binding. This sequence of events is confirmed by high resolution x-ray and NMR structures of the complexes between Ca2+-CaM and peptides derived from CaM-binding domains of target proteins (9Ikura M. Clore G.M. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1176) Google Scholar, 10Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (939) Google Scholar, 11Meador W.E. Means A.R. Quiocho F.A. Science. 1993; 262: 1718-1721Crossref PubMed Scopus (611) Google Scholar). CaM-binding domains in typical target proteins do not have any amino acid sequence homology. They often comprise a stretch of ∼20–25 amino acid residues; this region has the potential to form a positively charged amphipathic α-helix (2Crivici A. Ikura M. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 85-116Crossref PubMed Scopus (693) Google Scholar, 3Vogel H.J. Zhang M. Mol. Cell Biochem. 1995; 149/150: 3-15Crossref Scopus (70) Google Scholar, 12O'Neil K.T. DeGrado W.F. Trends Biochem. Sci. 1990; 15: 59-64Abstract Full Text PDF PubMed Scopus (712) Google Scholar). CaM-binding domains usually do not contain a significant number of negatively charged residues such as Asp or Glu, which could give rise to unfavorable electrostatic repulsion with CaM. CaM is highly acidic, as it carries a net negative charge of 17 even after it has bound four Ca2+ ions. Synthetic peptides encompassing CaM-binding domains of distinct target proteins usually bind to Ca2+-CaM in a 1:1 complex with an amphipathic α-helical motif (9Ikura M. Clore G.M. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1176) Google Scholar, 10Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (939) Google Scholar, 11Meador W.E. Means A.R. Quiocho F.A. Science. 1993; 262: 1718-1721Crossref PubMed Scopus (611) Google Scholar). The hydrophobic face of the peptide interacts with the two Met-rich hydrophobic patches in Ca2+-CaM, and the positively charged Lys and Arg residues of the peptide bind to several negatively charged residues (Asp and Glu) in Ca2+-CaM (9Ikura M. Clore G.M. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1176) Google Scholar, 10Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (939) Google Scholar, 11Meador W.E. Means A.R. Quiocho F.A. Science. 1993; 262: 1718-1721Crossref PubMed Scopus (611) Google Scholar). The flexible central linker region of CaM can bend to a different extent to accommodate the binding of diverse peptides, and the two Met-rich hydrophobic patches also create a highly adjustable binding surface to allow binding of peptides of distinct amino acid sequence (9Ikura M. Clore G.M. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1176) Google Scholar, 10Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (939) Google Scholar, 11Meador W.E. Means A.R. Quiocho F.A. Science. 1993; 262: 1718-1721Crossref PubMed Scopus (611) Google Scholar). The interactions between Ca2+-CaM and the peptides are predominantly hydrophobic in nature, and the interface exclusively involves amino acid side chain-side chain interactions (9Ikura M. Clore G.M. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1176) Google Scholar, 10Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (939) Google Scholar, 11Meador W.E. Means A.R. Quiocho F.A. Science. 1993; 262: 1718-1721Crossref PubMed Scopus (611) Google Scholar). Here we characterize an unusual CaM-binding domain derived from petunia glutamate decarboxylase. Glutamate decarboxylase catalyzes the conversion of glutamate to CO2 and γ-aminobutyric acid. γ-Aminobutyric acid is an important inhibitory neurotransmitter in mammalian cells, and the γ-aminobutyric acid level is crucial for the development of plant cells (13Baum G. Lev-Yadun S. Fridmann Y. Arazi T. Katsnelson H. Zik M. Fromm H. EMBO J. 1996; 15: 2988-2996Crossref PubMed Scopus (243) Google Scholar). Petunia GAD is a 58-kDa cytosolic protein that has a 34-amino acid extension at its C terminus that is absent in mammalian or Escherichia coli GAD (14Baum G. Chen Y. Arazi T. Takatsuji H. Fromm H. J. Biol. Chem. 1993; 268: 19610-19617Abstract Full Text PDF PubMed Google Scholar). This part of the protein has been shown to interact with petunia CaM as well as with mammalian [35S]Met-labeled CaM, CaM-Sepharose, or biotinylated CaM (14Baum G. Chen Y. Arazi T. Takatsuji H. Fromm H. J. Biol. Chem. 1993; 268: 19610-19617Abstract Full Text PDF PubMed Google Scholar). The CaM-binding domain was further mapped to a 26-residue peptide (15Arazi T. Baum G. Snedden W.A. Shelp B.J. Fromm H. Plant Physiol. (Bethesda). 1995; 108: 551-561Crossref PubMed Scopus (108) Google Scholar). Thus, in contrast to mammalian GADs, plant GADs are CaM-binding proteins, and the CaM-binding properties of plant GAD have now been demonstrated in different plant species such as petunia, Arabidopsis thaliana, fava bean, and soybean (14Baum G. Chen Y. Arazi T. Takatsuji H. Fromm H. J. Biol. Chem. 1993; 268: 19610-19617Abstract Full Text PDF PubMed Google Scholar, 15Arazi T. Baum G. Snedden W.A. Shelp B.J. Fromm H. Plant Physiol. (Bethesda). 1995; 108: 551-561Crossref PubMed Scopus (108) Google Scholar, 16Ling V. Snedden W.A. Shelp B.J. Assmann S.M. Plant Cell. 1994; 6: 1135-1143PubMed Google Scholar, 17Snedden W.A. Arazi T. Fromm H. Shelp B.J. Plant Physiol. (Bethesda). 1995; 108: 543-549Crossref PubMed Scopus (138) Google Scholar). The amino acid sequences of the CaM-binding domains of petunia and A. thaliana have been reported (13Baum G. Lev-Yadun S. Fridmann Y. Arazi T. Katsnelson H. Zik M. Fromm H. EMBO J. 1996; 15: 2988-2996Crossref PubMed Scopus (243) Google Scholar, 14Baum G. Chen Y. Arazi T. Takatsuji H. Fromm H. J. Biol. Chem. 1993; 268: 19610-19617Abstract Full Text PDF PubMed Google Scholar). The most interesting feature of these CaM-binding domains is that they contain four (for A. thaliana) or five (for petunia) Asp or Glu residues at conserved positions (Fig. 1) (15Arazi T. Baum G. Snedden W.A. Shelp B.J. Fromm H. Plant Physiol. (Bethesda). 1995; 108: 551-561Crossref PubMed Scopus (108) Google Scholar). The presence of this many negatively charged residues in a CaM-binding domain peptide is unique. CaM is an acidic protein, and hence, it is not designed to interact with negatively charged residues in the peptide. We have studied the interaction of Ca2+-CaM and a 26-residue PGD peptide derived from GAD by nondenaturing urea-PAGE, circular dichroism, steady-state Trp fluorescence, and two-dimensional NMR. The peptide is a monomer in aqueous solution in the absence of CaM. However, we have found that two α-helical peptides bind simultaneously to Ca2+-CaM. To the best of our knowledge, the Ca2+-CaM-induced dimerization of plant GAD represents a new Ca2+-CaM·protein-binding motif. A synthetic gene encoding the mammalian CaM sequence was cloned and expressed in E. coli as described previously (18Waltersson Y. Linse S. Brodin P. Grundström T. Biochemistry. 1993; 32: 7866-7871Crossref PubMed Scopus (95) Google Scholar, 19Zhang M. Vogel H.J. J. Biol. Chem. 1993; 268: 22420-22428Abstract Full Text PDF PubMed Google Scholar). The purification of wild-type CaM and [methyl-13C]]Met-labeled CaM followed established procedures (19Zhang M. Vogel H.J. J. Biol. Chem. 1993; 268: 22420-22428Abstract Full Text PDF PubMed Google Scholar, 20Zhang M. Vogel H.J. Biochemistry. 1994; 33: 1163-1171Crossref PubMed Scopus (52) Google Scholar). The purification of CT-CaM (a CaM mutant with all four Met residues in the C-terminal lobe of CaM mutated to Leu residues), SeMet-CaM (a CaM variant in which all nine Met residues are substituted with the unnatural amino acid SeMet), and SeMet-CT-CaM (CT-CaM with the remaining five Met residues substituted with SeMet) has been described in detail elsewhere (21Yuan T. Weljie A.M. Vogel H.J. Biochemistry. 1998; 37: 3187-3195Crossref PubMed Scopus (359) Google Scholar); SeMet was incorporated to 87%. A 26-residue peptide (NH2-HKKTDSEVQLEMITAWKKFVEEKKKK-CONH2) that corresponds to the CaM-binding domain in petunia GAD (amino acid residues 470–495) (15Arazi T. Baum G. Snedden W.A. Shelp B.J. Fromm H. Plant Physiol. (Bethesda). 1995; 108: 551-561Crossref PubMed Scopus (108) Google Scholar) was chemically synthesized by the Peptide Synthesis Core Facility at the University of Calgary (Calgary, Canada). The composition and purity of the peptide were confirmed by analytical high pressure liquid chromatography, amino acid analysis, and mass spectrometry. Ultrapure CsCl was purchased from Life Technologies, Inc. The soluble nitroxide spin label TEMPOL (4-hydroxyl-2,2,6,6-tetramethylpiperidinyl-1-oxy; also called HyTEMPO) was purchased from Sigma. The concentration of CaM was determined by ultraviolet spectroscopy using an extinction coefficient ε2761% = 1.8. The concentration of the PGD peptide was determined by weight and by using a molar extinction coefficient ε280 = 5500m−1 cm−1, which was consistent with the outcome of quantitative amino acid analysis. Nondenaturing urea-PAGE gel band shift assays were performed using a published procedure (22Yuan T. Mietzner T.A. Montelaro R.C. Vogel H.J. Biochemistry. 1995; 34: 10690-10696Crossref PubMed Scopus (43) Google Scholar). The urea is normally included in the gel to prevent the formation of nonspecific interactions. CD spectra were acquired on a Jasco J-715 spectropolarimeter. All experiments were performed at room temperature (22 °C) using a 1-mm path length cylindrical quartz cuvette. The parameters used were as follows: 0.2-nm step resolution, 50-nm/min scanning speed, 2-s response time, 1-nm bandwidth, and 20-millidegree sensitivity. All spectra shown were the average over 10 scans. The concentration of the CaM or PGD peptide used was 10 μm in 10 mm Tris-HCl, pH 7.2, with 2 mmCaCl2 or 2 mm EDTA. The total volume of each sample was 200 μl. The background signals from the buffer were subtracted, and each spectrum was smoothed and converted to molar ellipticity using Jasco software. The CD spectra were reported either as molar ellipticity or as mean residue molar ellipticity. The α-helical content of the peptide was calculated according to Scholtzet al. (23Scholtz J.M. Qian H. York E.J. Stewart J.M. Baldwin R. Biopolymers. 1991; 31: 1463-1470Crossref PubMed Scopus (478) Google Scholar). Steady-state Trp fluorescence and CsCl quenching experiments were carried out on a Hitachi 2000 spectrofluorometer as described previously (21Yuan T. Weljie A.M. Vogel H.J. Biochemistry. 1998; 37: 3187-3195Crossref PubMed Scopus (359) Google Scholar). The Trp fluorescence was excited at 295 nm to reduce the excitation of the two Tyr residues in CaM. Emission wavelength scans were recorded from 300 to 450 nm. Fluorescence samples contained 10 μm PGD peptide in 10 mm Tris-HCl, pH 7.2, and 100 mm KCl in the presence of 1 mm CaCl2 or 5 mmEDTA. The total sample volume was 1 ml. The CaM concentration was 5 or 10 μm for a PGD peptide/CaM ratio of 2:1 or 1:1, respectively. The fluorescence titration of the PGD peptide with Ca2+-CaM was performed by continuously adding 2 μl of a 200 μm CaM stock solution into 1 ml of the PGD peptide in 10 mm Tris-HCl, pH 7.2, 100 mm KCl, and 1 mm CaCl2. The total volume of CaM added to the cuvette was 20 μl. Since we monitored only the shift of the maximum emission wavelength in these experiments, the increase in the total sample volume could be neglected. All NMR experiments were carried out on a Bruker AMX-500 spectrometer using a 5-mm broadband, z axis gradient-shielded probe at 298 K. Two-dimensional1H-13C heteronuclear multiple quantum coherence (HMQC) spectra were acquired with pulsed field gradient selection (24Wider G. Wüthrich K. J. Magn. Reson. 1993; 102: 239-241Crossref Scopus (98) Google Scholar). Quadrature detection in the F1 dimension was obtained using the time-proportional phase incrementation technique. The sweep width was 8 ppm in the 1H dimension and 6 ppm in the13C dimension, with the 1H carrier set at 500.1388 MHz and the 13C carrier at 125.7613 MHz. The size of the HMQC spectra was a 1024 × 128 real data matrix with eight scans for each experiment. The TEMPOL titration experiments were carried out as described. 2T. Yuan, H. Ouyang, and H. J. Vogel, submitted for publication. Two-dimensional homonuclear1H-1H NMR spectra were acquired for the spectral assignment of the PGD peptide in H2O. The peptide sample contained 2 mm PGD peptide in 90% H2O and 10% D2O, pH 5.0 (not corrected for the isotope effects). COSY and NOESY spectra were acquired with pulsed field gradient experiments (25Jeener J. Meier H. Bachman P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4825) Google Scholar, 26Davis A.L. Laue E.D. Keeler J. Moskau D. Lohman J. J. Magn. Reson. 1991; 94: 637-644Google Scholar). Total correlation spectra were acquired according to Griesinger et al. (27Griesinger C. Otting G. Wüthrich K. Ernst R.R. J. Am. Chem. Soc. 1988; 110: 7870-7872Crossref Scopus (1193) Google Scholar). The typical size of a spectrum was a 2048 × 400 real data matrix with 64∼128 scans for each experiment. NOESY spectra were acquired with two different mixing times (100 and 250 ms) to check for spin diffusion effects. All spectra were acquired at two different temperatures (289 and 298 K) for cross-checking the assignments. The assignment obtained at 298 K is reported under “Results.” NMR spectra were processed using nmrPipe and nmrDraw software (28Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11441) Google Scholar). All spectra were zero-filled once in both dimensions, and 90° and 75° sine square shifted window functions were applied to the F2 and F1 dimensions, respectively, before Fourier transformation. Proton chemical shifts are referenced to 2,2-dimethyl-2-silapentane-5-sulfonate as 0 ppm. Carbon-13 chemical shifts are referenced indirectly to 2,2-dimethyl-2-silapentane-5-sulfonate using the converting ratio13C/1H = 0.251449530 as suggested by Wishart et al. (29Wishart D. Bigam C.G. Yao J. Abildgaard F. Dyson H.J. Oldfield E. Markley J.L. Sykes B.D. J. Biomol. NMR. 1995; 6: 135-140Crossref PubMed Scopus (2058) Google Scholar). The α-1H and α-13C random coil chemical shifts for the amino acid residues are taken from Wishart and Sykes (30Wishart D.S. Sykes B.D. Methods Enzymol. 1994; 239: 363-392Crossref PubMed Scopus (933) Google Scholar). The α-helical content of the peptide was calculated using the Agadir program, which is available on the web site of EMBL (31Muñoz V. Serrano L. Biopolymers. 1997; 41 (, and references therein): 495-509Crossref PubMed Google Scholar). This program has been developed for predicting peptide rather than protein secondary structure, and it has had considerable success when compared with experimental determinations of secondary structure (31Muñoz V. Serrano L. Biopolymers. 1997; 41 (, and references therein): 495-509Crossref PubMed Google Scholar). First, we used a gel band shift assay to assess the interaction between Ca2+-CaM and the PGD peptide. At increasing ratios of the PGD peptide to Ca2+-CaM, we observed a large band shift due to the formation of a complex between the PGD peptide and Ca2+-CaM (Fig. 2). This phenomenon is normally observed for high affinity CaM-binding peptides (1Vogel H.J. Biochem. Cell Biol. 1994; 72: 357-376Crossref PubMed Scopus (222) Google Scholar, 3Vogel H.J. Zhang M. Mol. Cell Biochem. 1995; 149/150: 3-15Crossref Scopus (70) Google Scholar). We found that the band for CaM did not disappear until 2 eq of the PGD peptide were added (Fig. 2). No changes occurred when >2 eq of PGD peptide were added. These results suggest that two PGD peptide molecules bind simultaneously to one CaM molecule. This observation is surprising in view of the fact that most of the well characterized CaM-binding peptides, such as peptides derived from myosin light chain kinase (MLCK), constitutive nitric-oxide synthase, and CaM-dependent protein kinase I, form only a 1:1 complex with Ca2+-CaM (Fig. 2 and data not shown) (1Vogel H.J. Biochem. Cell Biol. 1994; 72: 357-376Crossref PubMed Scopus (222) Google Scholar, 3Vogel H.J. Zhang M. Mol. Cell Biochem. 1995; 149/150: 3-15Crossref Scopus (70) Google Scholar). Also the band for the Ca2+-CaM·PGD peptide complex runs much slower on the gel than the bands of either the Ca2+-CaM·MLCK peptide complex (Fig. 2) or the Ca2+-CaM·constitutive nitric-oxide synthase peptide complex (data not shown). Although there is no direct correlation between the band migration distance and the molecular mass of proteins on the nondenaturing urea-polyacrylamide gel, the mobility of the band on such a gel usually suggests either that the Ca2+-CaM·PGD peptide complex has a higher molecular mass or that this complex has a very different shape compared with the Ca2+-CaM·MLCK peptide complex (Fig. 2). We also performed the band shift assay without including urea in the gel and samples and obtained exactly the same results (data not shown). We acquired CD spectra to study the secondary structural changes that occur upon binding of the PGD peptide to Ca2+-CaM. The PGD peptide itself has ∼31% α-helix in aqueous solution, and this percentage agrees very well with the 38% predicted by the Agadir program (Fig. 3). The observation of such an extent of α-helix formation is not uncommon in 20∼25-residue-long monomeric peptides that contain potential ion pairs in (i, i + 3) or (i, i + 4) positions (32Kuhlman B. Yang H.Y. Boice J.A. Fairman R. Raleigh D.P. J. Mol. Biol. 1997; 270: 640-647Crossref PubMed Scopus (43) Google Scholar, 33Smith J.S. Scholtz J.M. Biochemistry. 1998; 37: 33-40Crossref PubMed Scopus (94) Google Scholar). When the PGD peptide was added to a Ca2+-CaM solution, the negative molar ellipticity at 208 and 222 nm increased dramatically upon adding 1 eq of the PGD peptide. This suggests an increase in the α-helical content of the Ca2+-CaM·PGD peptide complex (Fig. 3). Since Ca2+-CaM usually does not gain any α-helical structure upon peptide binding (9Ikura M. Clore G.M. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1176) Google Scholar, 10Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (939) Google Scholar, 11Meador W.E. Means A.R. Quiocho F.A. Science. 1993; 262: 1718-1721Crossref PubMed Scopus (611) Google Scholar), the increase in α-helix in the Ca2+-CaM·peptide complex can be attributed to the bound peptide (1Vogel H.J. Biochem. Cell Biol. 1994; 72: 357-376Crossref PubMed Scopus (222) Google Scholar, 3Vogel H.J. Zhang M. Mol. Cell Biochem. 1995; 149/150: 3-15Crossref Scopus (70) Google Scholar). Therefore, we conclude that the PGD peptide has ∼72% α-helix when complexed with Ca2+-CaM. Interestingly, when we added a second equivalent of peptide, we observed a further increase in the amount of α-helix in the complex, which is more than would be obtained from addition of unbound PGD peptide (Fig. 3). Our CD results are consistent with the idea that Ca2+-CaM is able to bind 2 eq of the PGD peptide, both in an α-helical conformation. In addition, isotope-filtered Fourier transform infrared spectroscopy (34Zhang M. Fabian H. Mantsch H.H. Vogel H.J. Biochemistry. 1994; 33: 10883-10888Crossref PubMed Scopus (68) Google Scholar) also confirmed that the PGD peptide adopts an α-helix when bound to Ca2+-CaM. 3H. Fabian, T. Yuan, and H. J. Vogel, unpublished results. We next performed a trifluoroethanol (TFE) titration experiment to study the α-helix-forming potential of the PGD peptide. The α-helical content of the PGD peptide increases with increasing TFE concentration up to 20% TFE, after which it levels off (data not shown). The percentage α-helix formed in the PGD peptide is ∼68% in 20% TFE aqueous solution. Since TFE is a well known α-helix-stabilizing solvent (35Nelson J.W. Kallenbach N.R. Proteins Struct. Funct. Genet. 1986; 1: 211-217Crossref PubMed Scopus (404) Google Scholar, 36Zhang M. Yuan T. Vogel H.J. Protein Sci. 1993; 2: 1931-1937Crossref PubMed Scopus (41) Google Scholar), the observation that the α-helix of the PGD peptide can be further stabilized by TFE is as expected. In many of our studies concerning peptide binding to CaM, we have observed that the extent of α-helix formation induced in the peptide by binding to CaM or by addition of TFE is identical (36Zhang M. Yuan T. Vogel H.J. Protein Sci. 1993; 2: 1931-1937Crossref PubMed Scopus (41) Google Scholar). Also for the PGD peptide, these two values are in close agreement (72 and 68%, respectively). The outcome of the steady-state Trp fluorescence experiments are presented in Fig. 4. The PGD peptide has a maximum emission wavelength at 353 nm, which is typical for a solvent-exposed Trp residue in a peptide (Fig. 4 A). When Ca2+-CaM was titrated into a PGD peptide solution, we found a significant blue shift of the maximum emission wavelength (note that there are no Trp residues in CaM). The maximum emission wavelength recorded for the complex is 335 nm (Fig. 4 A). This relatively large blue shift indicates that the Trp residue(s) in the peptide move from a solvent-exposed environment to a hydrophobic environment in the complex, which is quite common for CaM-binding peptides (21Yuan T. Weljie A.M. Vogel H.J. Biochemistry. 1998; 37: 3187-3195Crossref PubMed Scopus (359) Google Scholar). A change in the microenvironment around the Trp residues in the PGD peptides upon binding to Ca2+-CaM was also observed by near-UV CD spectroscopy (data not shown). However, we noted that the fluorescence intensity of the CaM·PGD peptide complex experienced only a small increase; this is quite different from most of the CaM-binding peptides, which typically experience a doubling or tripling of the fluorescence quantum yield upon binding to CaM (21Yuan T. Weljie A.M. Vogel H.J. Biochemistry. 1998; 37: 3187-3195Crossref PubMed Scopus (359) Google Scholar). This could be due to the fact that the PGD peptide contains negatively charged residues (Asp and Glu) as opposed to normal CaM-binding peptides and that the carboxyl side chain of Asp and Glu residues can have quenching effects on the Trp fluorescence. Another possibility is that the two Trp residues in the bound dimeric PGD peptide may sit in a unique environment upon binding to Ca2+-CaM, so the higher Trp fluorescence, which is normally seen when it resides in a hydrophobic environment, is quenched. Titration experiments of the PGD peptide with Ca2+-CaM showed that the maximum emission wavelength already reached a plateau (337 nm) when only 0.5 eq of Ca2+-CaM was added (Fig. 4, A and B). This result indicates again that the PGD peptide binds to Ca2+-CaM in a 2:1 ratio (Fig. 4 B). In addition, we did not find any changes in the Trp fluorescence of the PGD peptide upon adding apo-CaM (with 5 mm EDTA in the sample) (data not shown). Therefore, the interactions between CaM and the PGD peptide take place only in the presence of calcium. Fluorescence quenching experiments with small molecules such as CsCl were also performed. The Trp residue in the PGD peptide was effectively quenched by increasing concentrations of CsCl, indicating a fully solvent-exposed Trp residue in the peptide. At 1:1 and 2:1 ratios of PGD to Ca2+-CaM, the Trp residues in the bound peptide were inaccessible to the quenching agent (Fig. 4 C). These data again indicate that the PGD peptide binds to Ca2+-CaM in a 2:1 ratio and that the indole rings of the Trp residues from both peptides are shielded from the solvent in the complex. To obtain further information about the potential arrangement of the two PGD peptides when bound to Ca2+-CaM, we recorded the fluorescence spectra of the PGD peptide complexed with four CaM variant proteins with different Met substitutions in the presence of calcium. We have successfully used the same approach to determine the orientation" @default.
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• W2034261645 title "Calcium-Calmodulin-induced Dimerization of the Carboxyl-terminal Domain from Petunia Glutamate Decarboxylase" @default.
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• W2034261645 doi "https://doi.org/10.1074/jbc.273.46.30328" @default.
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