Structural aspects of the Ca2+-ATPase from sarcoplasmic reticulum

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Jan 5, 1994 - Heim, K., Vogel, G., Mathews, S., Strehler-Page,. M. A,, James, P., Vorherr, T., Krebs, J. and Carafoli, E. (1988) J. Biol. Chem. 263, 14152-14159.
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Received 5 January 1994

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Structural aspects of the Ca2+-ATPasefrom sarcoplasmic reticulum Juan C. Gomez-Fernandez*, Senena Corbalan-Garcia, Jose Villalain and Jose A. Teruel Departamento de Bioquimica y Biologia Molecular (A), Facultad de Veterinaria, Universidad de Murcia, Apdo. 4021, E-30080 Murcia, Spain

Introduction CaL+-ATPase from sarcoplasmic reticulum is an integral membrane protein with a single polypeptide chain of 110000 M,, and it has been widely used as an example of a membrane transport protein [ 1]. The three-dimensional structure of this protein has been elucidated at low resolution by means of X-ray diffraction and studies using electron microscopy. A pear-shaped globular head extends from the plane of the membrane, with a stalk portion connecting the globular head with the intramembranal portion [ 2,3]. The secondary structure of the Ca’ -ATPase was predicted on the basis of its amino acid sequence and hydrophobicity [ 41. According to these predictions, the globular head contains a /?sheet-rich domain and other portions with alternating /?-sheet and a-helices where the ATP-binding site and the phosphorylation site are located. W e have studied the extramembrane protein portion by specific labelling of selective residues with fluorescence probes to determine distances between the probes and localize different domains in the three-dimensional structure of the globular region of the Ca’ -ATPase. In addition, the secondary structure of the intramembranal portion has been investigated using Fourier transform i.r. spec+

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Abbreviations used: e-ATP, l,Nh-ethenoadenosine5’-triphosphate; ATK, attenuated total reflectance; FITC, fluorescein S’-isothiocyanate;FTIR, Fourier transform i.r. spectroscopy; IAEL)ANS, 5-[(2-iodoacetyl)aminoethyl]amino-naphthalene-1-sulphonic acid; NBD-CI. 7-chloro4-nitro-2,1,3-benzoxadiazole. ‘To whom correspondence should be addressed.

troscopy (FTIR) after extensive proteolysis of the Ca’+-ATPase to eliminate the extramembranal domain. Native sarcoplasmic reticulum vesicles were prepared from rabbit hind leg muscle as previously described by Eletr and Inesi [S], and Ca”-ATPase was purified using method no. 2 of Meissner et al. [6]. Reconstitution of Ca’+-ATPase with fluorescent phospholipids was carried out by the method of detergent dilution as described by Johannson et al. [7). Ca”-ATPase was covalently labelled with the following probes: 7-chlor0-4-nitro-2,1,3-benzodiazole (NRD-C 1) [HI, 5-[(2-iodoacetyl)amino-ethyl]amino-napthalene- 1-sulphonic acid (IAEIIANS) [9], maleimides [ lo] and fluorescein 5’-isothiocyanate (FITC) [9]. Proteolysis of the Ca’+-A’TPase was performed by incubation with trypsin at an ATPase/trypsin ratio of 4:l (w/w) for 30 min at 35°C. Further proteolysis was carried out by incubating the ATPase with proteinase K at an ATPaseIproteinase K ratio of 16:l (w/w) for 30 min at 25°C. Amino acid composition determination was accomplished, after acid hydrolysis, of the phenylthiocarbamyl derivatives essentially as described by Heinrikson and Meredith [ 111. FTIR measurements were performed in €120 buffer using a Philips PU9800 FTIR spectrometer equipped with a deuterated triglycine sulphate detector. Quantitative analysis of the secondary structure was made by a partial least squares method [ 121. The FTIR spectra of 23 proteins were taken into account to create the calibration set; their secondary structures were estimated by X-ray diffraction.

Membrane Dynamics and Transport

For attenuated total reflectance (ATR) measurements the protein was laid on a ZnSe ATR crystal, and it was cut with an incident angle of 45". The orientation of the sample was calculated as previously described [ 131. The distance between two probes located at sites in the protein was calculated from the efficiency of fluorescence energy transfer within a single donor-acceptor pair [ 141, and the distance between a probe bound to the protein and the phospholipid polar headgroup was calculated assuming a random distribution of acceptors on the surface of a planar membrane [ 15 1.

Figure I 1.r. spectra of (A) purified Ca2+-ATPase and (B) trypsin/proteinase K digested Ca2+-ATPase after subtraction of water and normalization t o the absorbance of the amide I bands

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Infrared studies Proteins show two strong i.r. absorption bands corresponding to the amide bonds which are called amide I and amide I1 bands. The amide I (C=O stretching) band has been used to monitor the secondary structure of proteins because it originates from absorption of amide bonds in a-helical structure, B-sheets, p-turns and random structures. In order to study the secondary structure of the intramembranal portion of the Ca"-ATPase only, the extramembranal portion was previously eliminated by extensive digestion using trypsin and proteinase K. Under these conditions, -50% of the protein mass is solubilized, and the remaining peptides inserted in the membrane having a total mass of