Abstract
The low-resolution three-dimensional structure of purified native beef heart mitochondrial cytochrome c oxidase (COX) in asolectin unilamellar liposomes has been measured by small-angle neutron scattering under the conditions where the protein remains fully functional. From a neutron scattering perspective, the use of mixed-lipid liposomes provided for a more homogeneous matrix than can be achieved using a single lipid. As a result, the measurements were able to be performed under conditions where the liposome scattering was essentially eliminated (contrast-matched conditions). The protein structure in the membrane was modeled as a simple parallelepiped with side lengths of (59 × 70 × 120) Å with uncertainties, respectively, (11, 12, 20 Å). The molecular mass calculated for a typical protein with this volume is estimated to be (410 ± 124) kDa, which indicates the mass of a COX dimer. The longest dimension has some uncertainty due to intermolecular scattering contributing to the data. Nevertheless, that length was estimated using an average protein density and the known dimer molecular mass. Using the same cross sectional dimensions for the structure, the length is estimated to be 120 Å. However, the measured scattering curve of the dimer in the liposome differs significantly from that calculated from the X-ray structure of the dimer in a crystal of mixed micelles (PDB 3AG1). The calculated SANS scattering from the crystal structure was fit with a parallelepiped, measuring (59 × 101 × 129) Å with fitting uncertainties, respectively, (2, 3, 3 Å). Our results suggest that COX is a functional dimer when reconstituted into mixed-lipid liposomes.
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Abbreviations
- COX:
-
Cytochrome c oxidase
- DMPC:
-
Dimyristoylphosphatidylcholine
- DPPC:
-
Dipalmitoylphosphatidylcholine
- EM:
-
Electron microscopy
- H/D:
-
Hydrogen-deuterium ratio
- HEPES:
-
N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid
- NCNR:
-
NIST Center for Neutron Research
- PDB:
-
Protein data bank
- PAGE:
-
Polyacrylamide gel electrophoresis
- Pt/C:
-
Platinum-carbon
- SANS:
-
Small-angle neutron scattering
- SDS:
-
Sodium dodecyl sulfate
References
Althoff T, Mills DJ, Popot J-L, Kühlbrandt W (2011) EMBO J 1–13
Antonini G, Brunori M, Malatesta E, Sarti P, Wilson MT (1987) J Biol Chem 262(21):10077–10079
Blasie JK, Erecińska M, Samuels S, Leigh JS (1978) Biochem Biophys Acta 501:33–52
Carroll RC, Racker E (1977) J Biol Chem 252(10):6981–6990
Dudkina NV, Kudryashev M, Stahlberg H, Boekema EJ (2011) Proc Nat Acad Sci US 108:15196–15200
Estey LA, Prochaska LJ (1993) Biochem 32:13270–13276
Frey TG, Costello MJ, Chan SHP (1984) Ultramicros 13:85–92
Glinka CJ, Barker JG, Hammouda B, Krueger S, Moyer JJ, Orts WJ (1998) J Appl Cryst 31(3):430–445
Green D, Wharton D (1963) Biochem Z 338:335–342
Gregory L, Ferguson-Miller S (1989) Biochemistry 28:2655–2662
Hansen FB, Nicholls P (1978) Biochim Biophys Acta 502:400–408
Harpaz Y, Gerstein M, Chothia C (1994) Structure 2:641–649
Heidorn DB, Trewhella J (1988) Biochem 27:909–915
Helling S, Vogt S, Rhiel A, Ramzan R, Wen L, Marcus K, Kandnbach B (2008) Mol Cell Proteomics 7(9):1714–1724
Hirai M, Iwase H, Hayakawa T, Koizumi M, Takahashi H (2003) Biophys J 85:1600–1610
Hüttemann M, Lee I, Smavati L, Yu H, Doan JW (2007) Biochim Biophys Acta 1773:1701–1720
IUPAC (2011) Schulz-Zimm distribution. IUPAC Gold Book
Jacrot B (1976) Rep Prog Phys 39:911–953
Kiselev MA, Zemlyanaya EV, Aswal VK (2004) Cryst Reports 49:S136–S141
Kline SR (2006) J Appl Cryst 39:895–900
Krab K, Wikström M (1978) Biochim Biophys Acta 504:200–214
Krueger S, Groshkova I, Brown J, Hoskins J, McKenney KH, Schwarz FP (1998) J Biol Chem 273(32):20001–20008
Kučerka N, Pencer J, Sachs JN, Nagle JF, Katsaras J (2007) Langmuir 23:1292–1299
Mochizuki M, Aoyama H, Shinzawa-Itoh K, Usui T, Tsukihara T, Yoshikawa S (1999) J Biol Chem 274(19):33403–33411
Müller M, Azzi A (1985) J Bioenerg Biomemb 17(6):385
Muramoto K, Ohta K, Shinzawa-Itoh K, Kanda K, Taniguchi M, Nabekura H, Yamashita E, Tsukihara T, Yoshikawa S (2010) Proc Nat Acad Sci USA 107(17):7740–7745
Musatov A, Robinson NC (2002) Biochem 41:4371–4376
Musatov A, Ortega-Lopez J, Robinson NC (2000) Biochem 39:12996–13004
Nguyen X-T, Pabarue HA, Geyer RR, Shroyer LA, Estey LA, Parilo M, Wilson KS, Prochaska LJ (2002) Prot Exp Purif 26:122–130
Nicholls P, Hildebrandt V, Wrigglesworth JM (1980) Arch Biochem Biophys 204:533–543
Parsegian VA, Fuller N, Rand RP (1979) Proc Natl Acad Sci USA 76(6):2750–2754
Parsons DF, Ninham BW (2009) J Phys Chem A 113:1141–1150
Pencer J, Mills T, Anghel V, Krueger S, Epand RM, Katsaras J (2005) Eur Phys J E 18:447–458
Ramzan R, Staniek K, Kadenbach B, Vogt S (2010) Biochim Biophys Acta 1797:1672–1680
Rich PR, Maréchal A (2010) Essays Biochem 47:1–23
Robinson NC, Talbert L (1986) Biochem 25:2328–2335
Schmiedel H, Jörchel P, Kiselev M, Klose G (2001) J Phys Chem 105:111–117
Squire PG, Himmel ME (1979) Arch Biochem Biophys 196(1):165–177
Suarez MD, Revzin A, Narlock R, Kempner ES, Thompson DA, Ferguson-Miller S (1984) J Biol Chem 259:13791–13799
Tsukihara T, Aoyama H, Yamashita E, Tomizaki T, Yamaguchi H, Shinzawa-Itoh K, Nakashima R, Yaono R, Yoshikawa S (1996) Science 272:1136–1144
Valpuesta JM, Henderson R (1990) J Mol Biol 214:237–251
Van Gelder BF (1966) Biochim Biophys Acta 118:35–46
Wilson KS, Prochaska LJ (1990) Arch Biochem Biophys 282:413–420
Wittig I, Schägger H (2009) Biochim Biophys Acta 1787:672–680
Yonetani T (1967) Methods Enz 10:332–335
Acknowledgments
Small-angle neutron scattering experiments were performed on the NG3 and NG7 30 m SANS instruments at the NIST Center for Neutron Research. This equipment was partially supported by the National Science Foundation under agreement No. DMR-0454672. Funding at Wright State was provided by the Wright State University Boonshoft School of Medicine, the Emily Webb Foundation, and the American Heart Association, Ohio Affiliate.
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Rubinson, K.A., Pokalsky, C., Krueger, S. et al. Structure Determination of Functional Membrane Proteins using Small-Angle Neutron Scattering (SANS) with Small, Mixed-Lipid Liposomes: Native Beef Heart Mitochondrial Cytochrome c Oxidase Forms Dimers. Protein J 32, 27–38 (2013). https://doi.org/10.1007/s10930-012-9455-0
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DOI: https://doi.org/10.1007/s10930-012-9455-0