Abstract
The Ca2+-binding protein oncomodulin was altered by cassette mutagenesis of the CD site (CDOM33) with a sequence that was derived by a consensus method using over 250 known Ca2+-binding loop sequences. This mutant was studied using time-resolved and steady-state fluorescence from the Trp residue included at position 7 of the loop (position 57 of the protein sequence). The fluorescence characteristics of this species in the absence and presence of metal ions were compared to those of a tetradecapeptide containing the loop and the single Trp mutant of oncomodulin, Y57W. The fluorescence properties of CDOM33 were quite different from the peptide, both in the apo form and in response to metal binding. The consensus CD loop in CDOM33 exhibited the characteristics of a Ca2+/Mg2+ site in contrast to the Ca2+ specificity of the wild-type CD loop. The Trp analogue, 5-hydroxytryptophan (5HW), was incorporated into both oncomodulin mutants to produce Y75(5HW) and 5HW-CDOM33. Results showed that this intrinsic probe was relatively insensitive to structural changes in the mutants upon metal binding compared to Trp itself.
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N. C. J. Strynadka and M. N. G. James (1989)Annu. Rev. Biochem. 58, 951–998.
B. J. Marsden, G. S. Shaw, and B. D. Sykes (1989)Biochem. Cell Biol. 68, 587–601.
J. P. MacManus, C. W. Hogue, B. J. Marsden, M. Sikorska, and A. G. Szabo (1990)J. Biol. Chem. 265, 10358–10366.
C. V. W. Hogue, J. P. MacManus, D. Banville, and A. G. Szabo (1992)J. Biol. Chem. 267, 13340–13347.
C. M. L. Hutnik, J. P. MacManus, D. Banville, and A. G. Szabo (1991)Biochemistry 30, 7652–7660.
R. C. Hapak, P. J. Lammers, W. A. Palmisano, E. R. Birnbaum, and M. T. Henzl (1989)J. Biol. Chem. 264, 18751–18760.
J. J. Cox, M. Milos, and J. P. MacManus (1990).J. Biol. Chem. 265, 6633–6637.
J. J. Falke, E. E. Snyder, K. C. Thatcher, and C. S. Voertler (1991)Biochemistry 30, 8690–8697.
C. W. V. Hogue, I. Rasquinha, A. G. Szabo, and J. P. MacManus (1992)FEBS Lett. 310, 269–272.
J. P. MacManus, C. M. L. Hutnik, B. D. Sykes, A. G. Szabo, T. C. Williams, and D. Banville (1989)J. Biol. Chem. 264, 3470–3477.
C. M. Hutnik, J. P. MacManus, D. Banville, and A. G. Szabo (1990)J. Biol. Chem. 265, 11456–11464.
K. J. Willis and A. G. Szabo (1989)Biochemistry 28, 4902–4908.
K. J. Willis, A. G. Szabo, and D. T. Krajcarski (1990)Photochem. Photobiol. 51, 375–377.
D. W. Marquardt (1963)J. Soc. Ind. Appl. Math. 11, 431–441.
J. Durbin and G. S. Watson (1971)Biometrika 58, 1–19.
M. Zuker, A. G. Szabo, L. Bramall, D. T. Krajcarski, and B. Selinger (1985)Rev. Sci. Instrum. 56, 14–22.
J. R. Knutson, J. M. Beechem, and L. Brand (1983)Chem. Phys. Lett. 102, 501–507.
J. R. Knutson, D. G. Walbridge, and L. Brand (1982)Biochemistry 21, 4671–4679.
S. R. Meech and D. Phillips (1983)J. Photochem. 23, 193–217.
M. Goodman, J.-F. Pechère, J. Haiech, and J. Demaille (1979)J. Mol. Evol. 13, 331–352.
J. Haiech, J. Derancourt, J.-F. Pechère, and J. G. Demaille (1981)Biochemistry 20, 3890–3897.
J. B. A. Ross, D. F. Senear, E. Waxman, B. B. Kombo, E. Rusinova, Y. T. Huang, W. R. Laws, and C. A. Hasselbacher (1992)Proc. Natl. Acad. Sci. USA 89, 12023–12027.
C. W. V. Hogue and A. G. Szabo (1993)Biophys. Chem. 48, 158–169.
A. G. Szabo and D. M. Rayner (1980)J. Am. Chem. Soc. 102, 554–563.
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Clark, I.D., Bruckman, A.J., Hogue, C.W.V. et al. Effects of metal ion binding on an oncomodulin mutant containing a novel calcium-binding loop. J Fluoresc 4, 235–241 (1994). https://doi.org/10.1007/BF01878456
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DOI: https://doi.org/10.1007/BF01878456