Equilibrium unfolding of neuronal calcium sensor-1: N-terminal myristoylation influences unfolding and reduces protein stiffening in the presence of calcium.
Neuronal calcium sensor-1 (NCS-1), a Ca(2+)-binding protein of the calcium sensor family, modulates various functions in intracellular signaling pathways. The N-terminal glycine in this protein is myristoylated, which is presumably necessary for its physiological functions. In order to understand the structural role of myristoylation and calcium on conformational stability, we have investigated the equilibrium unfolding and refolding of myristoylated and non-myristoylated NCS-1. The unfolding of these two forms of NCS-1 in the presence of calcium is best characterized by a five-state equilibrium model, and multiple intermediates accumulate during unfolding. Calcium exerts an extrinsic stabilizing effect on both forms of the protein. In the absence of calcium, the stability of both forms is dramatically decreased, and the unfolding follows a four-state equilibrium model. The equilibrium transitions are fully reversible in the presence of calcium. Myristoylation affects the pattern of equilibrium transitions substantially but not the number of intermediates, suggesting a structural role. Our data suggest that myristoylation reduces the stiffening of the protein during initial unfolding in the presence of calcium. The effects of myristoylation are more pronounced when calcium is present, suggesting a relationship between them. Inactivating the third EF-hand motif (E120Q mutant) drastically affects the equilibrium unfolding transitions, and calcium has no effect on these transitions of the mutants. The unfolding transitions of both forms of the mutant are similar to the transitions followed by the apo forms of myristoylated and non-myristoylated NCS-1. These results suggest that the role of myristoylation in unfolding/refolding of the protein is largely dependent on the presence of calcium.
Muralidhar
Dasari
D
Centre for Cellular and Molecular Biology, Hyderabad-500007, India.
Jobby
Maroor K
MK
Krishnan
Kannan
K
Annapurna
Vallabhaneni
V
Chary
Kandala V R
KV
Jeromin
Andreas
A
Sharma
Yogendra
Y
D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019791:Guanidine; D009154:Mutation; D019814:Myristic Acid / Q000378:metabolism*; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation / Q000502:physiology; D051381:Rats; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism; D014364:Tryptophan
D002135:Calcium-Binding Proteins; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; C101464:frequenin calcium sensor proteins; D019814:Myristic Acid; D014364:Tryptophan; D014357:Trypsin; D019791:Guanidine; D002118:Calcium