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• W1972237784 abstract "The specificity of interaction of the isolated N- and C-terminal domains of calmodulin with peptide WFFp (Ac-KRRWKKNFIAVSAANRFK-amide) and variants of the target sequence of skeletal muscle myosin light chain kinase was investigated using CD and fluorescence. Titrations show that two molecules of either domain bind to 18-residue target peptides. For WFFp, the C-domain binds with 4-fold higher affinity to the native compared with the non-native site; the N-domain shows similar affinity for either site. The selectivity of the C-domain suggests that it promotes occupancy of the correct binding site for intact calmodulin on the target sequence. Far UV CD spectra show the extra helicity induced in forming the 2:1 C-domain-peptide or the 1:1:1 C-domain-N-domain-peptide complex is similar to that induced by calmodulin itself; binding of the C-domain to the Trp-4 site is essential for developing the full helicity. Calmodulin-MLCK-peptide complexes show an approximate two-fold rotational relationship between the two highly homologous domains, and the 2:1 C (or N)-domain-peptide complexes evidently have a similar rotational symmetry. This implies that a given domain can bind sequences with opposite peptide polarities, significantly increasing the possible range of conformations of calmodulin in its complexes, and extending the versatility and diversity of calmodulin-target interactions. The specificity of interaction of the isolated N- and C-terminal domains of calmodulin with peptide WFFp (Ac-KRRWKKNFIAVSAANRFK-amide) and variants of the target sequence of skeletal muscle myosin light chain kinase was investigated using CD and fluorescence. Titrations show that two molecules of either domain bind to 18-residue target peptides. For WFFp, the C-domain binds with 4-fold higher affinity to the native compared with the non-native site; the N-domain shows similar affinity for either site. The selectivity of the C-domain suggests that it promotes occupancy of the correct binding site for intact calmodulin on the target sequence. Far UV CD spectra show the extra helicity induced in forming the 2:1 C-domain-peptide or the 1:1:1 C-domain-N-domain-peptide complex is similar to that induced by calmodulin itself; binding of the C-domain to the Trp-4 site is essential for developing the full helicity. Calmodulin-MLCK-peptide complexes show an approximate two-fold rotational relationship between the two highly homologous domains, and the 2:1 C (or N)-domain-peptide complexes evidently have a similar rotational symmetry. This implies that a given domain can bind sequences with opposite peptide polarities, significantly increasing the possible range of conformations of calmodulin in its complexes, and extending the versatility and diversity of calmodulin-target interactions. Calmodulin is a regulatory protein involved in a variety of Ca2+-dependent cellular signaling pathways. Its importance as a mediator of the second messenger Ca2+ is reflected in its high conservation throughout evolution. This apparently contrasts with its unique ability to interact strongly with and to regulate selectively a variety of proteins (at least 30) without any obvious sequence homology in their calmodulin binding region (see Refs. 1Vogel H.J. Biochem. Cell Biol. 1994; 72: 357-376Crossref PubMed Scopus (222) Google Scholar, 2Crivici A. Ikura M. Annu. Rev. Biophys. Struct. 1995; 24: 85-116Crossref PubMed Scopus (695) Google Scholar, 3Klee C.B. Cohen P. Klee C.B. Calmodulin. Elsevier, Amsterdam1985: 35-56Google Scholar, 4O'Neil K.T. DeGrado W.F. Trends Biochem. Sci. 1990; 15: 59-64Abstract Full Text PDF PubMed Scopus (715) Google Scholar, 5James P. Vorherr T. Carafoli E. Trends Biochem. Sci. 1995; 20: 38-42Abstract Full Text PDF PubMed Scopus (347) Google Scholar for reviews). Recently, structures of calmodulin and calmodulin-peptide complexes at atomic resolution have been determined (reviewed in Refs. 2Crivici A. Ikura M. Annu. Rev. Biophys. Struct. 1995; 24: 85-116Crossref PubMed Scopus (695) Google Scholar, 6Ikura M. Trends Biochem. Sci. 1996; 21: 14-17Abstract Full Text PDF PubMed Scopus (599) Google Scholar, and 7Chazin W.J. Nat. Struct. Biol. 1995; 2: 707-710Crossref PubMed Scopus (68) Google Scholar), showing two similar domains with two Ca2+ binding sites each, which for calmodulin in solution are connected by a flexible linker (8Bayley P. Martin S. Jones G. FEBS Lett. 1988; 238: 61-66Crossref PubMed Scopus (29) Google Scholar, 9Torok K. Lane A.N. Martin S.R. Janot J.M. Bayley P.M. Biochemistry. 1992; 31: 3452-3462Crossref PubMed Scopus (44) Google Scholar, 10Bayley P.M. Martin S.R. Biochim. Biophys. Acta. 1992; 1160: 16-21Crossref PubMed Scopus (32) Google Scholar, 11Barbato G. Ikura M. Kay L.E. Pastor R.W. Bax A. Biochemistry. 1992; 31: 5269-5278Crossref PubMed Scopus (890) Google Scholar, 12Heidorn D.B. Trewhella J. Biochemistry. 1988; 27: 909-915Crossref PubMed Scopus (282) Google Scholar, 13Persechini A. Kretsinger R.H. J. Cardiovasc. Pharmacol. 1988; 12: S1-S12Crossref PubMed Google Scholar, 14Persechini A. Blumenthal D.K. Jarrett H.W. Klee C.B. Hardy D.O. Kretsinger R.H. J. Biol. Chem. 1989; 264: 8052-8058Abstract Full Text PDF PubMed Google Scholar, 15Persechini A. Kretsinger R.H. Davis T.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 449-452Crossref PubMed Scopus (22) Google Scholar). The conformational change upon Ca2+ binding to calmodulin exposes those residues that create the binding site for most target proteins (1Vogel H.J. Biochem. Cell Biol. 1994; 72: 357-376Crossref PubMed Scopus (222) Google Scholar, 6Ikura M. Trends Biochem. Sci. 1996; 21: 14-17Abstract Full Text PDF PubMed Scopus (599) Google Scholar, 7Chazin W.J. Nat. Struct. Biol. 1995; 2: 707-710Crossref PubMed Scopus (68) Google Scholar, 16Houdusse A. Cohen C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10644-10647Crossref PubMed Scopus (101) Google Scholar). Binding of the Ca2+ saturated form of calmodulin to the target protein triggers its activation. Despite the wide target range, target affinities are strong (K d ≈ 1 nm) (2Crivici A. Ikura M. Annu. Rev. Biophys. Struct. 1995; 24: 85-116Crossref PubMed Scopus (695) Google Scholar, 3Klee C.B. Cohen P. Klee C.B. Calmodulin. Elsevier, Amsterdam1985: 35-56Google Scholar, 4O'Neil K.T. DeGrado W.F. Trends Biochem. Sci. 1990; 15: 59-64Abstract Full Text PDF PubMed Scopus (715) Google Scholar). The interaction is apparently mediated by both hydrophobic and electrostatic forces (17Afshar M. Caves L.S.D. Guimard L. Hubbard R.E. Calas B. Grassy G. Haiech J. J. Mol. Biol. 1994; 244: 554-571Crossref PubMed Scopus (52) Google Scholar). NMR (18Ikura M. Clore G.M. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1179) Google Scholar) and x-ray structures (19Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (941) Google Scholar) of two related Ca2+-calmodulin-target peptide complexes show that the α-helical MLCK target peptide lies in a hydrophobic channel composed by the two domains, with the predominant interactions being those between the N- and C-terminal domains and the C- and N-terminal portions of the target, respectively. Calmodulin can be cleaved by trypsin to generate two half-molecules,i.e. the C-terminal and the N-terminal Ca2+binding domain (20Drabikowski W. Kuznicki J. Grabarek Z. Biochim. Biophys. Acta. 1977; 485: 124-133Crossref PubMed Scopus (76) Google Scholar, 21Walsh M. Stevens F.C. Kuznicki J. Drabikowski W. J. Biol. Chem. 1977; 252: 7440-7443Abstract Full Text PDF PubMed Google Scholar). The equilibrium (22Minowa O. Yagi K. J. Biochem. (Tokyo). 1984; 96: 1175-1182Crossref PubMed Scopus (71) Google Scholar, 23Linse S. Helmersson A. Forsen S. J. Biol. Chem. 1991; 266: 8050-8054Abstract Full Text PDF PubMed Google Scholar, 24Martin S.R. Bayley P.M. Biochem. J. 1986; 238: 485-490Crossref PubMed Scopus (84) Google Scholar) and kinetic properties (25Martin S.R. Andersson Teleman A. Bayley P.M. Drakenberg T. Forsen S. Eur. J. Biochem. 1985; 151: 543-550Crossref PubMed Scopus (119) Google Scholar, 26Suko J. Pidlich J. Bertel O. Eur. J. Biochem. 1985; 153: 451-457Crossref PubMed Scopus (27) Google Scholar) of intact calmodulin in the Ca2+binding and dissociation reactions, as well as the secondary structure (24Martin S.R. Bayley P.M. Biochem. J. 1986; 238: 485-490Crossref PubMed Scopus (84) Google Scholar, 27Drabikowski W. Brzeska H. Venyaminov S.Yu. J. Biol. Chem. 1982; 257: 11584-11590Abstract Full Text PDF PubMed Google Scholar), are well represented by a summation of the properties of these fragments, suggesting that the two domains are effectively independent structures. The isolated domains are capable of activating target proteins, but the degree of activation varies with the target protein (26Suko J. Pidlich J. Bertel O. Eur. J. Biochem. 1985; 153: 451-457Crossref PubMed Scopus (27) Google Scholar, 28Newton D. Klee C. Woodgett J. Cohen P. Biochim. Biophys. Acta. 1985; 845: 533-539Crossref PubMed Scopus (35) Google Scholar, 29Wolff J. Newton D.L. Klee C.B. Biochemistry. 1986; 25: 7950-7955Crossref PubMed Scopus (10) Google Scholar, 30Kuznicki J. Grabarek Z. Brzeska H. Drabikowski W. Cohen P. FEBS Lett. 1981; 130: 141-145Crossref PubMed Scopus (55) Google Scholar, 31Persechini A. McMillan K. Leakey P. J. Biol. Chem. 1994; 269: 16148-16154Abstract Full Text PDF PubMed Google Scholar, 32Guerini D. Krebs J. Carafoli E. J. Biol. Chem. 1984; 259: 15172-15177Abstract Full Text PDF PubMed Google Scholar, 33Klumpp S. Guerini D. Krebs J. Schultz J.E. Biochem. Biophys. Res. Commun. 1987; 142: 857-864Crossref PubMed Scopus (6) Google Scholar, 34Medvedeva M.V. Kolobova E.A. Wang P. Gusev N.B. Biochem. J. 1996; 315: 1021-1026Crossref PubMed Scopus (13) Google Scholar, 35Newton D.L. Oldewurtel M.D. Krinks M.H. Shiloach J. Klee C.B. J. Biol. Chem. 1984; 259: 4419-4426Abstract Full Text PDF PubMed Google Scholar). In particular, skeletal muscle myosin light chain kinase (sk-MLCK) 1The abbreviations used are: sk-MLCK, skeletal muscle myosin light chain kinase; FFFp, Ac-KKRFKKNFIAVSAANRFK-NH2; FFWp, Ac-KKRFKKNFIAVSAANRWK-NH2; FFWu, unprotected version of FFWp; FW10p, Ac-IAVSAANRWK-NH2; FW10u, unprotected version of FW10p; sm-MLCK, smooth muscle myosin light chain kinase; WF10p, Ac-KKRWKKNFIA-NH2; WF10u, unprotected version of WF10p; WFFp, Ac-KKRWKKNFIAVSAANRFK-NH2; WFFu, unprotected version of WFFp; X-WFFp-Y and related abbreviations, domain-WFFp-complex in which domain X interacts with the Trp-containing N-terminal portion of WFFp and domain Y with the C-terminal portion; M13 and RS20, target sequences of sk-MLCK and sm-MLCK, respectively; HPLC, high performance liquid chromatography. is activated best by a 1:1 mixture of the domains (85% activation compared with calmodulin), but less by either the C-domain (65%) or the N-domain (20%) (31Persechini A. McMillan K. Leakey P. J. Biol. Chem. 1994; 269: 16148-16154Abstract Full Text PDF PubMed Google Scholar). This activation pattern is reproduced when calmodulin chimeras consisting of two linked N-domains or two C-domains are used (36Persechini A. Gansz K.J. Paresi R.J. Biochemistry. 1996; 35: 224-228Crossref PubMed Scopus (46) Google Scholar). Although the C-domain activates target enzymes better than the N-domain in several cases (29Wolff J. Newton D.L. Klee C.B. Biochemistry. 1986; 25: 7950-7955Crossref PubMed Scopus (10) Google Scholar, 30Kuznicki J. Grabarek Z. Brzeska H. Drabikowski W. Cohen P. FEBS Lett. 1981; 130: 141-145Crossref PubMed Scopus (55) Google Scholar, 31Persechini A. McMillan K. Leakey P. J. Biol. Chem. 1994; 269: 16148-16154Abstract Full Text PDF PubMed Google Scholar, 35Newton D.L. Oldewurtel M.D. Krinks M.H. Shiloach J. Klee C.B. J. Biol. Chem. 1984; 259: 4419-4426Abstract Full Text PDF PubMed Google Scholar), this is not always so (31Persechini A. McMillan K. Leakey P. J. Biol. Chem. 1994; 269: 16148-16154Abstract Full Text PDF PubMed Google Scholar, 33Klumpp S. Guerini D. Krebs J. Schultz J.E. Biochem. Biophys. Res. Commun. 1987; 142: 857-864Crossref PubMed Scopus (6) Google Scholar). Therefore, differences in domain sequence and structure may contribute to the versatility of calmodulin's regulatory functions. Binding of the domains to the target enzyme is not necessarily sufficient for activation since isolated domains can inhibit calmodulin-induced activation of enzymes which are not activated by the domain itself (28Newton D. Klee C. Woodgett J. Cohen P. Biochim. Biophys. Acta. 1985; 845: 533-539Crossref PubMed Scopus (35) Google Scholar, 29Wolff J. Newton D.L. Klee C.B. Biochemistry. 1986; 25: 7950-7955Crossref PubMed Scopus (10) Google Scholar, 31Persechini A. McMillan K. Leakey P. J. Biol. Chem. 1994; 269: 16148-16154Abstract Full Text PDF PubMed Google Scholar, 35Newton D.L. Oldewurtel M.D. Krinks M.H. Shiloach J. Klee C.B. J. Biol. Chem. 1984; 259: 4419-4426Abstract Full Text PDF PubMed Google Scholar). Although the overall structure of the two domains show marked similarities (37Kuboniwa H. Tjandra N. Grzesiek S. Ren H. Klee C.B. Bax A. Nat. Struct. Biol. 1995; 2: 768-776Crossref PubMed Scopus (618) Google Scholar), differences between them in sequence and Ca2+ affinity have apparently been well conserved during evolution (38Klee C.B. Vanaman T.C. Adv. Protein Chem. 1982; 35: 213-321Crossref PubMed Scopus (737) Google Scholar). This points toward possible functional differences of the two domains, which can be further amplified by variations in target sequences. Studies of enhancement of Ca2+ affinities of calmodulin by target sequences suggest that calmodulin which is half-saturated with Ca2+ could bind to the target protein by only one domain without activation, allowing a rapid response in enzyme activity to an increase in Ca2+ concentration (39Bayley P.M. Findlay W.A. Martin S.R. Protein Sci. 1996; 5: 1215-1228Crossref PubMed Scopus (142) Google Scholar). In the present work, we address on a molecular level the specificity of the interaction of the individual calmodulin domains with target peptides derived from the target sequence of sk-MLCK. Previously, spectroscopic studies have been reported on the interaction of the domains with the short peptides melittin and mastoparan (40Steiner R.F. Marshall L. Needleman D. Arch. Biochem. Biophys. 1986; 246: 286-300Crossref PubMed Scopus (27) Google Scholar, 41Sanyal G. Richard L.M. Carraway K.L. Puett D. Biochemistry. 1988; 27: 6229-6236Crossref PubMed Scopus (24) Google Scholar, 42Itakura M. Iio T. J. Biochem. (Tokyo). 1992; 112: 183-191Crossref PubMed Scopus (28) Google Scholar). Here, the binding affinities, the molecular interactions in the complex, and the effects of domain binding on the conformation of the target peptides are investigated using a variety of spectroscopic techniques. The striking finding is that two molecules of either domain bind with good affinity to the 18-residue target peptides. The symmetry of the resulting complexes is considered by comparison with the calmodulin-MLCK peptide structure (2Crivici A. Ikura M. Annu. Rev. Biophys. Struct. 1995; 24: 85-116Crossref PubMed Scopus (695) Google Scholar), and is discussed in relation to the known versatility of calmodulin in its specific interactions with a range of target proteins. The feasibility of calmodulin domains to bind with reversed polarity and alternative positions on a target sequence greatly extends the potential range of conformations of calmodulin in its bound form, and provides added diversity to the calcium sensitivity of calmodulin-dependent activation processes. Drosophila melanogastercalmodulin expressed in Escherichia coli was purified as described previously (43Maune J.F. Klee C.B. Beckingham K. J. Biol. Chem. 1992; 267: 5286-5295Abstract Full Text PDF PubMed Google Scholar). The purified protein ran as a single band on an SDS-polyacrylamide gel electrophoresis (15% gel; Laemmli system). The tryptic fragments of calmodulin were prepared as described in Ref.31Persechini A. McMillan K. Leakey P. J. Biol. Chem. 1994; 269: 16148-16154Abstract Full Text PDF PubMed Google Scholar with an additional gel filtration step (G75 column) included after the cleavage and before the anion exchange chromatography. For each fragment, no impurities could be detected on SDS-polyacrylamide gel electrophoresis. However, HPLC chromatography showed an impurity of unknown origin in the C-domain preparation corresponding to 2.4% of the protein mass. Mass spectroscopy showed that the predominant fragment for the N-domain preparation was residues 1–75 of calmodulin and, for the C-domain preparation, residues 78–148. WFFp (Ac-KKRWKKNFIAVSAANRFK-NH2), WF10p (Ac-KKRWKKNFIA-NH2), FFFp (Ac-KKRFKKNFIAVSAANRFK-NH2), FFWp (Ac-KKRFKKNFIAVSAANRWK-NH2), and FW10p (Ac-IAVSAANRWK-NH2) were synthesized on an Applied Biosystems 430A peptide synthesizer and purified by reverse-phase HPLC on a C18 column. All peptides were protected at both termini. Peptide purity was assessed by mass spectrometry, reverse-phase HPLC, and ion exchange chromatography. Concentrations of proteins and peptides were determined spectrophotometrically using the following extinction coefficients: 5690 m−1 cm−1 at 280 nm for Trp-containing peptides (44Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5058) Google Scholar); 585 m−1cm−1 at 259 nm for FFFp (44Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5058) Google Scholar) and 1578m−1 cm−1 at 279 nm forDrosophila calmodulin in the presence of excess Ca2+ (43Maune J.F. Klee C.B. Beckingham K. J. Biol. Chem. 1992; 267: 5286-5295Abstract Full Text PDF PubMed Google Scholar). The same extinction coefficient was used for the C-domain of calmodulin assuming that the absorption of the single Tyr-138 in this domain is unaffected by the tryptic cleavage. For the N-domain, 975 m−1 cm−1 at 259 nm was used based on its five Phe residues (44Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5058) Google Scholar). It was confirmed that the extinction coefficient of the N-domain changed by less than 10% upon denaturation with 6 m guanidine HCl. Uncorrected fluorescence emission spectra were recorded in UV-transmitting plastic cuvettes or in quartz cuvettes using a SPEX FluoroMax fluorimeter. Excitation was at 300 nm for measurements at ≥50 μm peptide concentration, otherwise at 290 or 295 nm for the C-domain and at 280 or 300 nm for the N-domain (bandwidth 0.9 nm). The emission was scanned from 300 or 310 nm to 400 nm (bandwidth 4.3 nm). The temperature was 20 °C and the buffer 25 mm Tris/HCl, 100 mm KCl, 1 mm CaCl2 at pH 8.0. Data for the fluorescence titrations were obtained either by integrating the spectra in the region of the largest fluorescence change (300–330 nm) or by measurements of 30 s in duration at a wavelength in the range 322–334 nm with bandwidth 17 nm. Unless otherwise stated, the dissociation constants are derived from at least three independent titrations. CD spectra were recorded in fused silica cuvettes using a Jasco J-600 spectropolarimeter. The measurements were made at 20 °C in 25 mm Tris/HCl, 100 mm KCl, 1 mmCaCl2 at pH 8.0. Far UV-CD measurements (200–280 nm) with calmodulin and all peptides as well as with WFFp and isolated domains were made using 1-mm cuvettes with peptide and protein concentrations in the range 7–25 μm. With isolated domains and FFFp or FFWp the measurements were made in a 0.1-mm demountable cuvette at 10-fold higher concentrations. Spectra are presented as the molar CD absorption coefficient (ΔεM) using the molar concentration of the protein rather than the mean residue weight for the normalization. In the case of the domain mixture complexes, the concentration of the complex was used for the normalization to facilitate the comparison with the spectra of calmodulin (46Findlay W.A. Martin S.R. Beckingham K. Bayley P.M. Biochemistry. 1995; 34: 2087-2094Crossref PubMed Scopus (49) Google Scholar). The difference between the CD absorption coefficient at 222 nm in the presence and the absence of the peptide was used to estimate the number of residues adopting a helical structure upon complex formation. The results of three experiments were averaged. The difference was expressed as molar in peptide (not protein) concentration and compared with the ΔεM value of fully helical peptides of different lengths, which were calculated according to Ref. 45Scholtz J.M. Qian H. York E.J. Stewart J.M. Baldwin R.L. Biopolymers. 1991; 31: 1463-1470Crossref PubMed Scopus (480) Google Scholar. Near UV-CD spectra (255–340 nm) were measured using 15–30 repetitive scans for peptide and protein concentrations in the range 80–380 μm with 10 mm cuvettes. The spectral range of 310–340 nm was used to zero the curves. A base-line spectrum was recorded, smoothed over 10 nm, and subtracted, and the resulting spectra were slightly smoothed. The spectra are the average of at least two independent measurements. For near UV CD titrations, spectra of 10–20 scans were recorded between 280 and 320 nm, and were zeroed using the data between 308 and 320 nm. A base-line spectrum was smoothed over 10 nm, subtracted, and the CD signal of the resulting spectrum integrated between 280 and 293 nm for titrations with the N-domain and between 292 and 299 nm for titrations with the C-domain. At least four independent titrations were evaluated. Under the conditions of the CD experiments with the N-domain and the short peptides WF10p and FW10p, incomplete complex formation is expected owing to the low affinity of the interaction. Using the dissociation constants of 60 and 75 μm (see below), it is calculated that 53% (N-WF10p) and 48% (FW10p-N) of the peptides are bound under the conditions of the experiment, and these values were used to subtract the free peptide contribution to the spectra and to normalize them to 1 mcomplex concentration. Titrations were performed by addition of the domain solution to the peptide solution, and recording changes in fluorescence or CD signals deriving from the Trp chromophore of the peptide. Titration curves of WF10p and FW10p peptide with either domain were fitted with a stoichiometry of 1:1, using standard fitting procedures (46Findlay W.A. Martin S.R. Beckingham K. Bayley P.M. Biochemistry. 1995; 34: 2087-2094Crossref PubMed Scopus (49) Google Scholar). In the case of the long peptides WFFp and FFWp, the titration curves clearly indicated that saturation of the optical signal was achieved close to a stoichiometry of two molecules of domain per molecule of peptide. The simplest model for binding assumes that the optical signal monitors binding of a domain to the Trp-containing portion of the peptide; binding of a second molecule of the domain is revealed only indirectly via the competition of the domain between the two sites. The analysis is based on the known structure of the complex of calmodulin with the sk-MLCK target M13 peptide, in which the C-domain of calmodulin, interacting exclusively with the Trp residue of the peptide, binds predominantly with the (Trp containing) N-terminal portion of the peptide, and the N-domain binds predominantly with the C-terminal portion of the peptide. The two sites on the peptide are therefore designated according to their position in the peptide sequence as site N and site C; in the case of binding to peptide WFFp, binding at site N withK d N, produces an optical signal, and binding at site C with K d C is optically “silent,” whereas for peptide FFWp, the optical properties of sites N and C are reversed. The mechanism for the binding of two molecules of a given domain (D) per molecule of peptide with sites N and C(understood to be oriented N-peptide-C) is shown in Scheme ins;1857s1}1. According to the scheme, in the course of a titration of a fixed amount of peptide (P) with a single domain, D, the optical signal at any point is determined by ΣX i ·S i, where X i is the mole fraction of various peptide-containing species (and ΣX i = 1), together with S i, their intrinsic spectroscopic properties. On the above model, for WFFp peptide, and indices 1–4 referring to free peptide, D-peptide-, -peptide-D, and D-peptide-D, respectively, then S 1 = S 3 (known from free peptide) and S 2 =S 4, (known from, or fitted to, the plateau titration value). To evaluate the values of X i for a given concentration of peptide (fixed) and variable concentration of added domain [D], the analysis requires the values ofK d C, K d N(and parameter f when f ≠ 1) with the solution of a cubic equation. The unknowns (K d C,K d N, and f) are then refined by least squares methods in fitting to the experimental titration data. Conversely, simulations were made, using chosen values of these parameters to examine their individual effects on model titration curves. To simplify the analysis, the determination of the two binding constants K d N andK d C was done in two steps. First, in titrations of the fluorescence and the CD signals at high peptide concentration, the ratioK d C/K d Nwas determined. This value was then used as a fixed parameter in the fits to the fluorescence titrations at low concentration, which finally resulted in the individual K d values. The rationale behind this approach, demonstrated by simulation, is that titration curves at high concentration (i.e. >K d C andK d N) depend strongly on the ratioK d C/K d Nand less on their absolute values, whereas the opposite is true at lower concentrations. The simplest analysis is where the two sites are non-interacting,i.e. there is no thermodynamic co-operativity in the binding of the second copy of the domain. For this case, the co-operativity factor in the scheme is given by f = 1. Interaction can be included with f > 1 (negative co-operativity) orf < 1 (positive co-operativity). In titrations of a full-length peptide with a single domain, it was found that the inclusion of the additional co-operativity parameter f gave no significant improvement to the numerical fitting, and thus fits were generally done with the simplest model with f = 1. In calculations based on studies of the 1:1:1 complex of C-domain plus N-domain plus WFFp peptide, the inclusion of an f ∼ 0.5 was deduced, suggesting a small degree of positive co-operativity; in one out of four cases, evidence was found for a spectroscopic interaction. These analyses are discussed under “Results.” Solutions of the free Trp-containing peptides have a fluorescence emission maximum at ∼358 nm. The maximum of the enhanced fluorescence emission of all the peptide-domain complexes (Table I) lies between 335 and 338 nm, except for the N-WF10p complex (348 nm), suggesting that the N-domain is less effective than the C-domain in burying the Trp residue of this short peptide in a hydrophobic environment. As discussed in more detail below, two molecules of either domain were found to bind to one molecule of full-length peptide (WFFp or FFWp). Complexes are represented as e.g. X-WFFp-Y, indicating that domain X binds to the N-terminal portion of the peptide and domain Y to the C-terminal portion.Table IProperties of the complexes formed between target peptides and calmodulin domainsComplexStoichiometryλemK N dK C dK C d/K N dΔG/kJ · mol−1nmC-WFFp-C2:133518 ( ± 8) nm1-aCalculated using the K d ratio determined from CD experiments.45 ( ± 25) nm 1-aCalculated using the K d ratio determined from CD experiments.2.5 ( ± 0.5)−43.4, −41.210 ( ± 4) nm1-bCalculated using the K d ratio determined from fluorescence experiments.80 ( ± 35) nm 1-bCalculated using the K d ratio determined from fluorescence experiments.8.0 ( ± 0.5)−44.9, −39.8C-WF10p1:1335560 ( ± 30) nm−35.1N-WFFp-N2:1335143 ( ± 30) nm180 ( ± 45) nm1.26 ( ± 0.05)−38.4, −37.8N-WF10p1:134860 ( ± 14) μm−23.7C-FFWp-C2:13372.0 ( ± 1.1) μm370 ( ± 150) nM0.19 ( ± 0.05)−32.0, −36.1FW10p-C1:13386.4 ( ± 3.5) μM−29.1N-FFWp-N2:13355.5 ( ± 2.6) μm2.1 ( ± 0.5) μM0.38 ( ± 0.09)−29.5, −31.8FW10p-N1:133775 ( ± 18) μM−23.11-a Calculated using the K d ratio determined from CD experiments.1-b Calculated using the K d ratio determined from fluorescence experiments. Open table in a new tab Near UV CD spectra of the domains in the absence and the presence of a target peptide, as well as spectra of the free peptides are shown in Fig. 1. The free peptides WFFp (Fig. 1 A), FFWp (Fig. 1 B), and WF10p (data not shown) have similar near UV CD spectra; below 290 nm, the signal increases steadily and without fine structure to a ΔεM = 0.4 m−1cm−1 at 255 nm. Only the FW10p peptide (Fig.1 C) shows a signal above 290 nm. The spectrum of the N-domain is composed of the Phe signals below 270 nm (Fig. 1 B, bold line). The spectrum of the C-domain (bold line in Fig. 1 C) shows the additional contribution of the single Tyr residue above 270 nm. Spectra of the isolated domains of bovine testis calmodulin (24Martin S.R. Bayley P.M. Biochem. J. 1986; 238: 485-490Crossref PubMed Scopus (84) Google Scholar) and of the C-domain of Drosophila calmodulin (39Bayley P.M. Findlay W.A. Martin S.R. Protein Sci. 1996; 5: 1215-1228Crossref PubMed Scopus (142) Google Scholar) have been reported elsewhere. The spectrum of a 1:1 domain mixture (Fig. 1 A) is closely similar to that of intact calmodulin (39Bayley P.M. Findlay W.A. Martin S.R. Protein Sci. 1996; 5: 1215-1228Crossref PubMed Scopus (142) Google Scholar, 46Findlay W.A. Martin S.R. Beckingham K. Bayley P.M. Biochemistry. 1995; 34: 2087-2094Crossref PubMed Scopus (49) Google Scholar) confirming earlier reports that the near UV CD spectrum of calmodulin can be represented by a summation of the CD spectra of the individual domains (24Martin S.R. Bayley P.M. Biochem. J. 1986; 238: 485-490Crossref PubMed Scopus (84) Google Scholar). Fig. 1 (D–F) shows the spectral change induced by binding of a given target peptide to the domai" @default.
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• W1972237784 title "Specificity and Symmetry in the Interaction of Calmodulin Domains with the Skeletal Muscle Myosin Light Chain Kinase Target Sequence" @default.
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• W1972237784 doi "https://doi.org/10.1074/jbc.273.4.2174" @default.
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