N-terminal Myristoylation Regulates Calcium-induced Conformational Changes in Neuronal Calcium Sensor-1

Abstract

Neuronal calcium sensor-1 (NCS-1), a Ca2+-binding protein, plays an important role in the modulation of neurotransmitter release and phosphatidylinositol signaling pathway. It is known that the physiological activity of NCS-1 is governed by its myristoylation. Here, we present the role of myristoylation of NSC-1 in governing Ca2+ binding and Ca2+-induced conformational changes in NCS-1 as compared with the role in the nonmyristoylated protein. The 45Ca binding and isothermal titration calorimetric data show that myristoylation increases the degree of cooperativity; thus, the myristoylated NCS-1 binds Ca2+ more strongly (with three Ca2+ binding sites) than the non-myristoylated one (with two Ca2+ binding sites). Both forms of protein show different conformational features in far-UV CD when titrated with Ca2+. Large conformational changes were seen in the near-UV CD with more changes in the case of nonmyristoylated protein than the myristoylated one. Although the changes in the far-UV CD upon Ca2+ binding were not seen in E120Q mutant (disabling EF-hand 3), the near-UV CD changes in conformation also were not influenced by this mutation. The difference in the binding affinity of myristoylated and non-myristoylated proteins to Ca2+ also was reflected by Trp fluorescence. Collisional quenching by iodide showed more inaccessibility of the fluorophore in the myristoylated protein. Mg2+-induced changes in near-UV CD are different from Ca2+-induced changes, indicating ion selectivity. 8-Anilino-1-naphthalene sulfonic acid binding data showed solvation of the myristoyl group in the presence of Ca2+, which could be attributed to the myristoyl-dependent conformational changes in NCS-1. These results suggest that myristoylation influences the protein conformation and Ca2+ binding, which might be crucial for its physiological functions. Neuronal calcium sensor-1 (NCS-1), a Ca2+-binding protein, plays an important role in the modulation of neurotransmitter release and phosphatidylinositol signaling pathway. It is known that the physiological activity of NCS-1 is governed by its myristoylation. Here, we present the role of myristoylation of NSC-1 in governing Ca2+ binding and Ca2+-induced conformational changes in NCS-1 as compared with the role in the nonmyristoylated protein. The 45Ca binding and isothermal titration calorimetric data show that myristoylation increases the degree of cooperativity; thus, the myristoylated NCS-1 binds Ca2+ more strongly (with three Ca2+ binding sites) than the non-myristoylated one (with two Ca2+ binding sites). Both forms of protein show different conformational features in far-UV CD when titrated with Ca2+. Large conformational changes were seen in the near-UV CD with more changes in the case of nonmyristoylated protein than the myristoylated one. Although the changes in the far-UV CD upon Ca2+ binding were not seen in E120Q mutant (disabling EF-hand 3), the near-UV CD changes in conformation also were not influenced by this mutation. The difference in the binding affinity of myristoylated and non-myristoylated proteins to Ca2+ also was reflected by Trp fluorescence. Collisional quenching by iodide showed more inaccessibility of the fluorophore in the myristoylated protein. Mg2+-induced changes in near-UV CD are different from Ca2+-induced changes, indicating ion selectivity. 8-Anilino-1-naphthalene sulfonic acid binding data showed solvation of the myristoyl group in the presence of Ca2+, which could be attributed to the myristoyl-dependent conformational changes in NCS-1. These results suggest that myristoylation influences the protein conformation and Ca2+ binding, which might be crucial for its physiological functions. Neuronal Ca2+ sensor-1 (NCS-1) 1The abbreviations used are: NCS-1, neuronal calcium sensor-1; CD, circular dichroism; ANS, 8-anilino-1-naphthalene sulfonic acid; DTT, dithiothreitol; ITC, isothermal titration calorimetry; POPC, palmitoyl oleyl phosphatidylcholine; POPS, palmitoyl oleyl phosphatidylserine. belongs to a growing family of EF-hand Ca2+-binding proteins, which is conserved from yeast to human (1Braunewell K.H. Gundelfinger E.D. Cell Tissue Res. 1999; 295: 1-12Crossref PubMed Scopus (239) Google Scholar, 2Burgoyne R.D. Weiss J.L. Biochem. J. 2001; 353: 1-12Crossref PubMed Scopus (379) Google Scholar). The EF-hand is formed by a loop of 12 amino acids flanked by helices E and F (named after helices in parvalbumin), which are positioned like the forefinger and thumb of the right hand (3Kretsinger R.H. Carafoli E. Clementi F. Drabikowski W. Margeth A. Calcium Transport in Contraction and Secretion. North Holland Publishing Co., Amsterdam1975: 469-478Google Scholar). This three-dimensional arrangement of the EF-hand motifs in various Ca2+-binding proteins is conserved. The proteins of calcium sensor family have in common a fatty acid-modified N terminus, one pseudo or primitive non-canonical EF-hand as the first Ca2+ binding site, and three canonical EF-hands. NCS-1, a 22-kDa protein (theoretical pI 4.7), is a mammalian homologue of frequenin in Drosophila and Xenopus. Frequenin has been shown to enhance synaptic efficacy at the neuromuscular junctions (4Pongs O. Lindemeier J. Zhu X.R. Theil T. Endelkamp D. Krah-Jentgens I. Lambrecht H.-G. Koch K.W. Schwemer J. Rivosecchi R. Mallart A. Galceran J. Canal I. Barbas J.A. Ferrus A. Neuron. 1993; 11: 15-28Abstract Full Text PDF PubMed Scopus (285) Google Scholar, 5Olafsson P. Wang T. Lu B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8001-8005Crossref PubMed Scopus (87) Google Scholar). NCS-1 is expressed in neurons and neuromuscular junctions. It also is expressed in other secretory cell types similar to chromaffin cells and in the kidney, suggesting a more generalized role in exocytosis (6McFerran B.W. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 7McFerran B.W. Weiss J.L. Burgoyne R.D. J. Biol. Chem. 1999; 274: 30258-30265Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 8Weisz O.A. Gibson G.A. Leung S.M. Roders J. Jeromin A. J. Biol. Chem. 2000; 275: 24341-24347Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 9Werle M. Roder J. Jeromin A. Neurosci. Lett. 2000; 284: 33-36Crossref PubMed Scopus (28) Google Scholar) that is dependent critically on the spatial and temporal regulation of the intracellular Ca2+ concentration (for example see Ref. 10Rettig J. Neher E. Science. 2002; 298: 781-785Crossref PubMed Scopus (282) Google Scholar). The NCS family includes several proteins such as recoverin, neurocalcin, hippocalcin, frequenin, p22, GCAP1, GCAP2, VILIP, and the budding yeast homologue, Frq1. All of them are myristoylated and possess four EF-hand units. The first EF-hand (EF-1) does not bind to Ca2+ because of the presence of Gly in place of invariant Asp at the +x position (11Tanaka T. Ames J.B. Harvey T.S. Stryer S. Ikura M. Nature. 1995; 376: 444-447Crossref PubMed Scopus (286) Google Scholar). Of all of the aforementioned NCS proteins, recoverin is the most studied protein. It is a Ca2+ sensor in retinal rod and cone cells wherein, besides the EF-1, EF-4 is also non-functional and Ca2+ binds only to EF-2 and EF-3 sites, leading to the exposure of the buried N-terminal myristoyl group known as Ca2+-myristoyl switch (12Ames J.B. Ishima R. T. Stryer Ikura M. Nature. PubMed Scopus Google Scholar). a of the myristoyl group is to a that might with proteins and (12Ames J.B. Ishima R. T. Stryer Ikura M. Nature. PubMed Scopus Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The role of myristoylation is to be because the the physiological role of NCS-1 and its with proteins such as phosphatidylinositol are myristoylation is for the of NCS-1 and phosphatidylinositol and of its activity P. A. Roder J. Jeromin A. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The physiological role of myristoylation the various of the Ca2+ sensor the of with is dependent on Ca2+ and myristoylation S. B. M. Biol. 1999; PubMed Scopus Google in GCAP1, GCAP2, and VILIP, is the Ca2+ binding that K.H. T. Gundelfinger E.D. J. PubMed Scopus Google Scholar, J.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). neurocalcin, and NCS-1, all of Ca2+ sensor myristoyl group NCS-1 is to its myristoyl Weiss J.L. Burgoyne R.D. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). recoverin, the N-terminal myristoylation the conformation of the protein, suggesting a role of the myristoyl group as an J. Koch K.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). on and has been shown that myristoylation does not the and the Ca2+ binding of yeast J.B. T. J. 2000; PubMed Scopus Google Scholar). myristoylated NCS-1 was to be to bind from chromaffin cells in the of Ca2+ (6McFerran B.W. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). It plays a more role in the regulation of neurotransmitter release and in synaptic a (4Pongs O. Lindemeier J. Zhu X.R. Theil T. Endelkamp D. Krah-Jentgens I. Lambrecht H.-G. Koch K.W. Schwemer J. Rivosecchi R. Mallart A. Galceran J. Canal I. Barbas J.A. Ferrus A. Neuron. 1993; 11: 15-28Abstract Full Text PDF PubMed Scopus (285) Google Scholar). NCS-1 not only the activity and of Ca2+ T. Jeromin A. Roder T. Science. 2002; 295: PubMed Scopus Google Scholar, M. T. S. Jeromin A. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) also has a on T. A. Jeromin A. Neurosci. PubMed Scopus Google Scholar). NCS-1 is in T. Jeromin A. Roder T. Science. 2002; 295: PubMed Scopus Google Scholar, M. T. S. Jeromin A. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the role of myristoylation and the presence of the Ca2+-myristoyl switch in NCS-1 is not is to the Ca2+ binding of NCS-1 and the myristoylated and non-myristoylated forms of the protein. the data for affinity of NCS-1 in a of are not and results for non-myristoylated NCS-1 J.A. I. M. S. P. Gundelfinger E.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and yeast J.B. T. J. 2000; PubMed Scopus Google Scholar). of the role of myristoylation on the and of NCS-1 is not This to an of the of NCS-1 in the role of myristoylation. this we the of myristoylation on Ca2+ binding and Ca2+-induced conformational changes in NCS-1 on various that myristoylation and the Ca2+ binding affinity and conformational changes by the for ion This is of physiological because Ca2+ binding proteins for by conformational results suggest the presence of a Ca2+-myristoyl switch and that myristoylation influences the conformational changes in NCS-1, which might have NCS-1 has three Ca2+ binding with a them and a affinity compared with non-myristoylated NCS-1, which is to have two ion binding the Ca2+-myristoyl switch is in the E120Q other such as were by this mutation. of and NCS-1 and of myristoylated and non-myristoylated NCS-1 was the S. D. 1995; PubMed Scopus Google Scholar, S. D. J. Roder J. Jeromin A. 2000; PubMed Scopus Google Scholar). The NCS-1 was by S. and or with by to cells the of NCS-1 from the the of in is dependent on the NCS-1 was by growing cells NCS-1 in at to an of and the to NCS-1 was by growing cells with NCS-1 in and at to an of and acid and were was expressed by the of when the cells were for the myristoylated NCS-1 as were by and in of of to the of protein as as the of the was in myristoylated NCS-1, the was with the for was as The data were with proteins and in different E120Q EF-hand was by a of at position by with the The was in the myristoylated the was with to was as S. D. J. Roder J. Jeromin A. 2000; PubMed Scopus Google Scholar). by was and from as S. D. J. Roder J. Jeromin A. 2000; PubMed Scopus Google Scholar). All of the were on a protein with for data were on a The were in DTT, and was DTT, and and a was we have proteins on of and the protein with after The protein was on and in case of in the a was on a 12 to a protein myristoylation of the protein was by the of acid and not of the protein was by calcium and by mammalian was and by as S. D. J. Roder J. Jeromin A. 2000; PubMed Scopus Google Scholar). concentration was by its at the of for NCS-1 J.A. I. M. S. P. Gundelfinger E.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were in the were or by which was by a and in protein was a of that been with by the by binding to myristoylated and non-myristoylated NCS-1 was and compared by the T. Koch K.W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus (64) Google Scholar). concentration was in calcium concentration was from to in and were on and for from the and were used for for the of Ca2+ concentration in a and Ca2+ in the were by an of protein. of calcium were in the and This was several and data were as shown in Ca2+ is the in the is in protein and and are the and Ca2+ The were at three with the proteins in different were as by T. Koch K.W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and the by with non-myristoylated and myristoylated NCS-1 were a titration were and to titration and for after the the case of non-myristoylated NCS-1, a titration of of the protein of of were the case of myristoylated NCS-1, the titration was by of the protein of of were a the of the were the the to the of All of the were at The data were with were on a at near-UV CD of proteins were in the of proteins were titrated with Ca2+ or and CD were were expressed in of of the were on a were in the and also for the quenching the of iodide and were in and the protein was The were and the data were by the of Google Scholar) as shown in or by the as shown in and F are the in the and presence of is the concentration of is the quenching and the of the NCS-1 fluorophore to 8-Anilino-1-naphthalene binding were by of myristoylated and non-myristoylated proteins with were by at in the Ca2+ or was and the was The were for in protein. binding of myristoylated and non-myristoylated NCS-1 to of in of palmitoyl oleyl and palmitoyl oleyl in the and the presence of Ca2+ was as by S. S. PubMed Scopus Google Scholar) or The binding was by the in upon the binding of the protein to after for and the from the The binding data were this and of and NCS-1 and NCS-1 and its myristoylated were in the of a NCS-1 was by of by a The protein was at various with a of S. D. J. Roder J. Jeromin A. 2000; PubMed Scopus Google Scholar). All of the protein were by and mammalian S. D. J. Roder J. Jeromin A. 2000; PubMed Scopus Google Scholar). The of myristoylation was by an of for the myristoylated NCS-1 and an of for the non-myristoylated protein, which are in with of and the degree of myristoylation of NCS-1, we of of NCS-1 in the with This was more a was for myristoylated and nonmyristoylated NCS-1. this the myristoylation was to was by a on a The E120Q was by position in the by which this The mutant proteins also were by the with the that of by on protein was The was to the role of myristoylation on Ca2+ binding and protein this we studied Ca2+ and binding and the and Mg2+-induced changes in conformation of myristoylated NCS-1 and compared the data with non-myristoylated protein the role of N-terminal myristoylation in E120Q (with EF-3 in the Ca2+-myristoyl switch and Ca2+-induced conformational changes also was and for Ca2+, 45Ca binding, and the Ca2+ binding affinity the by T. Koch K.W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus (64) Google Scholar). All of the such as protein and concentration were by the The binding data showed that the of Ca2+ was for myristoylated NCS-1 than for non-myristoylated protein The binding data were and as by T. Koch K.W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The binding for myristoylated and non-myristoylated NCS-1 were and NCS-1 showed in the suggesting a degree of of NCS-1 by results of 45Ca binding were by The of calorimetric upon the of of Ca2+ to non-myristoylated NCS-1 is shown in and the the is shown in The calorimetric were to of a of by T. S. Biochem. Scopus Google Scholar). the of the was that the was and that other could be that have a this could the with the The are in I. The binding the release of upon Ca2+ binding to the protein. the results in the of of binding sites) and the binding These results the presence of two Ca2+ binding with The of binding and to a of the of the two from of non-myristoylated NCS-1 of binding of binding in a of NCS-1 by calorimetric of the myristoylated NCS-1 upon the of of the Ca2+ to myristoylated NCS-1 is shown in and the the protein is shown in The calorimetric were with the of a of three binding by T. S. Biochem. Scopus Google Scholar). this could the with the The are in The with two of and one of with the three binding and the of binding The binding is than the first binding The first and Ca2+ binding is indicating in Ca2+ from of myristoylated NCS-1 binding in a in CD of Both Ca2+ titration with myristoylated and non-myristoylated NCS-1 and E120Q and the changes in far-UV the the of of myristoylated and non-myristoylated NCS-1 binding to the NCS-1 showed two different myristoylated NCS-1, the Ca2+-induced changes were more which are to a J.A. P. J. Chem. Scopus Google Scholar, I. J. Chem. Scopus Google Scholar). non-myristoylated NCS-1, the changes are more at which are because of of the and of Google Scholar). was a and in the at upon binding Ca2+ in myristoylated and non-myristoylated NCS-1, a and These results suggest that myristoylation the affinity as as the of Ca2+ have also studied the of on far-UV CD to myristoylated and non-myristoylated NCS-1 and the in non-myristoylated and in myristoylated NCS-1 at The in by was than the by Ca2+ and case of the E120Q Ca2+ not in the far-UV CD not indicating that a is for conformational in CD upon near-UV CD of forms of NCS-1 are shown in NCS-1 is in amino acid and and the for the amino acid were in near-UV The of NCS-1 showed a of Trp at by a at the for Trp and The were with the at and The of Ca2+ changes in the CD of myristoylated and nonmyristoylated NCS-1 a and The of the the of and and when titrated with Ca2+ The in the at was and for myristoylated and nonmyristoylated proteins, The E120Q of non-myristoylated and myristoylated proteins also a in near-UV CD, similar to that seen in proteins and of various binding of myristoylated and non-myristoylated of binding CD in at CD in upon Ca2+ upon Ca2+ the presence of of in a studied the of on near-UV CD of forms of NCS-1. to Ca2+, the of the of the Trp at the of the and at and and Mg2+-induced conformational changes were for myristoylated and non-myristoylated These results show that Mg2+-induced changes in near-UV CD are different from the Ca2+-induced changes and that myristoylation does not and in by Trp and was used to more myristoylated and non-myristoylated proteins and the of Ca2+. are two Trp in the protein, in the E of and in the E of Both myristoylated and non-myristoylated NCS-1 showed the at with for myristoylated protein than for non-myristoylated NCS-1 This is attributed to the on Trp by the myristoyl myristoylated NCS-1 was titrated with of Ca2+, the by and The non-myristoylated protein a similar with the that the in and was than that seen in the case of the myristoylated protein at were and for non-myristoylated and myristoylated proteins, indicating a affinity for Ca2+ for the myristoylated NCS-1. case of the E120Q we not see in Trp upon Ca2+ only a in myristoylated NCS-1 and an in non-myristoylated NCS-1 not not changes to the myristoylated NCS-1. Ca2+ changes in non-myristoylated and myristoylated NCS-1 not The and of the fluorophore in myristoylated and non-myristoylated proteins were compared by iodide and by seen in the the for myristoylated NCS-1 was the indicating that the quenching was in the case of the myristoylated protein. The for was the presence of Ca2+ the of fluorophore in myristoylated NCS-1, and the for myristoylated and non-myristoylated NCS-1 were and suggesting that myristoylation inaccessibility The which does not the protein, was not to of Trp of myristoylated protein from non-myristoylated NCS-1 not most because of the of of have used the to the Ca2+-induced exposure or solvation of the myristoyl with myristoylated and non-myristoylated NCS-1 showed an at indicating a degree of the was in the case of myristoylated NCS-1 The of Ca2+ to non-myristoylated NCS-1 the indicating inaccessibility of to binding than exposure to Ca2+ binding and The of Ca2+ on myristoylated protein was in to that seen in the case of the non-myristoylated protein. myristoylated NCS-1, the upon the of Ca2+ and because of and of binding for This of myristoylated NCS-1 could be attributed to the solvation of the myristoyl which for the of Ca2+ on of E120Q case of non-myristoylated forms of the E120Q was an in when Ca2+ was in case of the myristoylated E120Q The of Ca2+ an in with a in the This be attributed to the of the myristoyl group in the presence of Ca2+. These results suggest that EF-3 does not the or solvation of the myristoyl changes in and of the of the myristoyl the of on was titration with the in myristoylated and non-myristoylated NCS-1 a and with a in the non-myristoylated protein. myristoylated NCS-1, the in was the of E120Q to a than the proteins and These results was to changes in the protein was not to the of the myristoyl group as Ca2+ binding the solvation or of myristoyl group is to Ca2+ and is not protein, the of Ca2+ the the of the solvation of the myristoyl group by Ca2+. NCS-1 to in a the myristoylated and non-myristoylated NCS-1 proteins bind and and to on the binding of Ca2+, we binding with various binding protein, a in was The non-myristoylated protein showed only a with of the Ca2+ concentration It to the with a of the of and (with indicating on Ca2+. this concentration of Ca2+ was to all of the binding sites, and has been that a more titration is to the Ca2+ binding of NCS-1 to various The myristoylated NCS-1 showed binding to in the presence and the of Ca2+ similar also was seen for in the of Ca2+ not in the presence of calcium the myristoylated to with an affinity of myristoylated NCS-1 showed a for phosphatidylinositol when compared with other not non-myristoylated NCS-1 These results show a binding of only myristoylated NCS-1 to The of N-terminal myristoylation in the and conformational of NCS-1 has been and is a that in proteins in 1999; PubMed Scopus Google Scholar). It has been known that the Ca2+-myristoyl switch plays a role in the Ca2+ sensor family of Ca2+-binding proteins upon Ca2+ binding to the buried N-terminal myristoyl and binding (12Ames J.B. Ishima R. T. Stryer Ikura M. Nature. PubMed Scopus Google Scholar, S. O. S. F. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). have used several such as 45Ca binding, Ca2+ titration by ITC, Trp and CD, ANS, and binding, for the role of myristoylation in protein and its on Ca2+ we have the data a by the EF-3 in NCS-1. Ca2+ binding to myristoylated NCS-1 increases the have seen that Ca2+ binding to myristoylated protein conformational changes in the at in near-UV CD that Ca2+ binding a in the conformation of Ca2+-induced changes in near-UV CD are different from Mg2+-induced changes, suggesting sensor role for NCS-1. The of E120Q this the changes in far-UV CD, not the changes in the suggesting that most of the conformational place of This might be attributed to the that most of the are not in the of 45Ca binding and with non-myristoylated and myristoylated NCS-1 the of Ca2+ binding as two and the case of myristoylated NCS-1, with three Ca2+ binding sites, the binding is The seen in the also a degree of in the case of the myristoylated protein. It is to upon the binding of Ca2+ to the first in myristoylated NCS-1, the binding affinity of Ca2+ to the increases which is than the binding affinity for the two in nonmyristoylated NCS-1 for myristoylated protein is in the case of the non-myristoylated protein is These binding and binding by were by 45Ca binding also the difference in the Ca2+ binding affinity of myristoylated and non-myristoylated NCS-1. results the data of J.A. I. M. S. P. Gundelfinger E.D. J. Biol. Chem. Full Text PDF PubMed Google which only two an for non-myristoylated NCS-1. Ames J.B. T. J. 2000; PubMed Scopus Google Scholar) have three for yeast in the of These might be the of the in the of yeast and mammalian NCS-1. The data that myristoylation Ca2+ binding and were by Trp and iodide The two Trp of NCS-1 at and of the of and in the be more by the of the myristoyl of the Trp the of the of different iodide quenching in the presence of Ca2+, myristoylation inaccessibility of quenching was different when Ca2+ was indicating a role by myristoylation in the presence of Ca2+. have used to conformational changes in NCS-1. This has been used to the presence of the switch by the in the of the solvation of recoverin J.B. T. T. Ikura M. Stryer J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). Ca2+ binding the of non-myristoylated NCS-1 and the of the myristoylated protein. This upon binding Ca2+ seen in the case of myristoylated NCS-1 the exposure of the myristoyl which to binding in the presence of Ca2+. was not to the of myristoylated NCS-1 as Ca2+ indicating the conformational changes by Ca2+ and not by non-myristoylated and myristoylated E120Q the was in the presence of Ca2+ with a in the myristoylated This that myristoylation the and in to Ca2+ The of on has not been different from the non-myristoylated NCS-1 indicating that binding were not by the E120Q mutation. binding of myristoylated NCS-1 to also the of myristoyl from the protein the binding to other was not in the presence of Ca2+. of the of the of NCS-1 with this does not to the for The of NCS-1 is not to be by because is on the N we suggest that myristoylation the binding of NCS-1 to and with The of NCS-1 with phosphatidylinositol and its modulation is dependent on myristoylation J.B. 1995; PubMed Scopus Google Scholar). of NCS-1 is important for the with phosphatidylinositol and of its the of myristoylation for the physiological activity of NCS-1. the myristoylation mutant of yeast is also in the of the suggesting that myristoylation is important for the activity of yeast as J. Neher E. Science. 2002; 298: 781-785Crossref PubMed Scopus (282) Google Scholar). data show that the myristoylation influences the Ca2+ that Ca2+-binding proteins show a similar in myristoylated with to Ca2+ D. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Google Scholar) has similar of the conformational changes in myristoylated is similar to the human non-myristoylated NCS-1 J. P. O. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). It be important to the changes in conformation and affinity to the various physiological of calcium the of myristoylated NCS-1 common that the of This is of because the release of the neurotransmitter and exocytosis in is strongly by the intracellular calcium A. for in of the and T. for and for Koch for the for calcium binding