Abstract
Cytochrome b5 binds spontaneously to lipid vescles and also self-associates in aqueous solution. Two mutant proteins have been generated, one has a self-association constant which is less than that of the native protein, while the other has a larger self-association constant. All three proteins have Trp in the membrane-binding domain but as aqueous solutions of these proteins contain differing amounts of monomeric protein, the kinetics of fluorescence enhancement, when the proteins are mixed with lipid vesicles, are complex. Similar complex kinetics are seen when the Trp are quenched by the addition of bromolipid vesicles. The mutant which has Trp 108 and 112 both replaced by Leu does not self-associate and shows monoexponential stopped-flow fluorescence kinetics. Identical rate constants are seen with this mutant for fluorescence enhancement by POPC and fluorescence quenching by three bromolipids with bromines at the 6,7-, 9,10-, and 11,12-positions of thesn-2 acyl chain. This rate constant is only 1% of the calculated collisional rate constant and it is suggested that the reduced rate is caused by a reduction in the number of productive collisions rather than by a slow rate of penetration of the membrane-binding domain into the bilayer.
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References
J. P. Hendrick and F.-U. Hartl (1993)Annu. Rev. Biochem. 62, 349–384.
D. J. Anderson, K. E. Mostov, and G. blobel (1983)Proc. Natl. Acad. Sci. USA 80, 7249–7253.
W. L. C. Vaz, H. Vogel, F. Jahnig, and R. H. Austin (1978)FEBS Lett. 87, 269–272.
T. L. Leto and P. W. Holloway (1979)J. Biol. Chem. 254, 5015–5019.
V. G. Tretyachenko-Ladokhina, A. S. Ladokhin, L. Wang, A. W. Steggles, and P. W. Holloway (1993)Biochim. Biophys. Acta 1153, 163–169.
N. Krishnamachary and P. W. Holloway (1989)Biophys. J. 55, 129a.
A. S. Ladokhin, L. Wang, A. W. Steggles, H. Malak, and P. W. Holloway (1993)Biochemistry 32, 6951–6956.
G. Vergères and L. Waskell (1992)J. Biol Chem. 267, 12583–12591.
T. Markello, A. Zlotnick, J. Everett, J. Tennyson, and P. W. Holloway (1985)Biochemistry 24, 2895–2901.
N. Dariush, C. W. Fisher, and A. W. Steggles (1988)Prot. Seq. Data Anal. 1, 351–353.
T. A. Kunkel (1985)Proc. Natl. Acad. Sci. USA 82, 488–492.
M. L. Johnson and S. G. Frasier (1985) in C. H. W. Hirs and S. N. Timasheff (eds.),Methods in Enzymology, Academic Press, New York, pp. 301–342.
G. Schwarz, H. Gerke, V. Rizzo, and S. Stankowski (1987)Biophys. J. 52, 685–692.
G. Cevc and D. Marsh (1987).Phospholipid Bilayers: Physical Principles and Models, Wiley, New York.
M. A. Roseman, B. R. Lentz, B. Sears, D. Gibbs, and T. E. Thompson (1978)Chem. Phys. Lipids 21, 205–222.
M. A. Calabro, J. T. Katz and P. W. Holloway (1976).J. Biol. Chem. 251, 2113–2118.
T. J. McIntosh and P. W. Holloway (1987)Biochemistry 26, 1783–1788.
J. T. Mason and C. Huang (1978)Ann. N.Y. Acad. Sci. 308, 29–49.
J. M. González-Mañas, J. H. Lakey, and F. Pattus (1992)Biochemistry 31, 7294–7300.
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Krishnamachary, N., Stephenson, F.A., Steggles, A.W. et al. Stopped-flow fluorescence studies of the interaction of a mutant form of cytochrome b5 with lipid vesicles. J Fluoresc 4, 227–233 (1994). https://doi.org/10.1007/BF01878455
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DOI: https://doi.org/10.1007/BF01878455