Correction for incomplete labeling in the measurement of distance distributions by frequency-domain fluorometry

Joseph R. Lakowicz; Ignacy Gryczyński; Wieslaw Wiczk; Józef Kuśba; Michael L. Johnson

doi:10.1016/0003-2697(91)90324-M

Correction for incomplete labeling in the measurement of distance distributions by frequency-domain fluorometry

Joseph R. Lakowicz, Ignacy Gryczyński, Wieslaw Wiczk, Józef Kuśba, Michael L. Johnson

Microbiology, Immunology & Genetics

Research output: Contribution to journal › Article › peer-review

13 Scopus citations

Abstract

Measurements of time-resolved fluorescence are now being used to recover conformational distributions of biological macromolecules. The fluorescence data of the donor are easily corrupted by incomplete labeling of the macromolecules by the acceptor. In the present paper we describe a general procedure to correct for incomplete acceptor labeling in the determination of distance distributions from frequency-domain measurements of the donor fluorescence decay kinetics. The method can also be used to determine the extent of acceptor labeling. Simulated data were used to determine the effect of incomplete labeling on resolution of the distance distribution and the effect on the recovered distributions if one fails to account for incomplete labeling by the acceptor. The expressions and implemented algorithm were verified using known mixtures of donor-control and donor-acceptor pair molecules, which simulated the presence of a donor population lacking the acceptor. Finally, we present data on the distance distributions between two labeled sites in myosin S1 (Cys-697 to Cys-707) where it was not possible to obtain complete labeling of the acceptor site.

Original language	English
Pages (from-to)	243-254
Number of pages	12
Journal	Analytical Biochemistry
Volume	195
Issue number	2
DOIs	https://doi.org/10.1016/0003-2697(91)90324-M
State	Published - Jun 1991

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@article{6990fea716b4432789c9e19a67ba1865,

title = "Correction for incomplete labeling in the measurement of distance distributions by frequency-domain fluorometry",

abstract = "Measurements of time-resolved fluorescence are now being used to recover conformational distributions of biological macromolecules. The fluorescence data of the donor are easily corrupted by incomplete labeling of the macromolecules by the acceptor. In the present paper we describe a general procedure to correct for incomplete acceptor labeling in the determination of distance distributions from frequency-domain measurements of the donor fluorescence decay kinetics. The method can also be used to determine the extent of acceptor labeling. Simulated data were used to determine the effect of incomplete labeling on resolution of the distance distribution and the effect on the recovered distributions if one fails to account for incomplete labeling by the acceptor. The expressions and implemented algorithm were verified using known mixtures of donor-control and donor-acceptor pair molecules, which simulated the presence of a donor population lacking the acceptor. Finally, we present data on the distance distributions between two labeled sites in myosin S1 (Cys-697 to Cys-707) where it was not possible to obtain complete labeling of the acceptor site.",

author = "Lakowicz, {Joseph R.} and Ignacy Gryczy{\'n}ski and Wieslaw Wiczk and J{\'o}zef Ku{\'s}ba and Johnson, {Michael L.}",

note = "Funding Information: i This work was supported by grants from the National Institutes of Health (GM 35154) and from the National Science Foundation (DMB-8804931), with additional support from the National Science Foundation for instrumentation (DMB-8511065) and the Center for Fluorescence Spectroscopy (DIR-8710401). J.R.L. and W.W. acknowledge support from Medical Biotechnology Center at the University of Maryland. authors thank Professor Herb Cheung for use of the myosin which was obtained under NIH Grant AR-31239, and Ms. for her careful editing of this manuscript. {\textquoteright} To whom correspondence should be addressed. 3 Permanent address: University of Gdansk, Institute try, Gdansk, Sobieskiego 18, Poland. {\textquoteright} Permanent address: Technical University of Gdansk, Depart ment of Technical Physics and Applied Mathematics, SO-952 Gdansk, Poland.",

year = "1991",

month = jun,

doi = "10.1016/0003-2697(91)90324-M",

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journal = "Analytical Biochemistry",

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T1 - Correction for incomplete labeling in the measurement of distance distributions by frequency-domain fluorometry

AU - Lakowicz, Joseph R.

AU - Gryczyński, Ignacy

AU - Wiczk, Wieslaw

AU - Kuśba, Józef

AU - Johnson, Michael L.

N1 - Funding Information: i This work was supported by grants from the National Institutes of Health (GM 35154) and from the National Science Foundation (DMB-8804931), with additional support from the National Science Foundation for instrumentation (DMB-8511065) and the Center for Fluorescence Spectroscopy (DIR-8710401). J.R.L. and W.W. acknowledge support from Medical Biotechnology Center at the University of Maryland. authors thank Professor Herb Cheung for use of the myosin which was obtained under NIH Grant AR-31239, and Ms. for her careful editing of this manuscript. ’ To whom correspondence should be addressed. 3 Permanent address: University of Gdansk, Institute try, Gdansk, Sobieskiego 18, Poland. ’ Permanent address: Technical University of Gdansk, Depart ment of Technical Physics and Applied Mathematics, SO-952 Gdansk, Poland.

PY - 1991/6

Y1 - 1991/6

N2 - Measurements of time-resolved fluorescence are now being used to recover conformational distributions of biological macromolecules. The fluorescence data of the donor are easily corrupted by incomplete labeling of the macromolecules by the acceptor. In the present paper we describe a general procedure to correct for incomplete acceptor labeling in the determination of distance distributions from frequency-domain measurements of the donor fluorescence decay kinetics. The method can also be used to determine the extent of acceptor labeling. Simulated data were used to determine the effect of incomplete labeling on resolution of the distance distribution and the effect on the recovered distributions if one fails to account for incomplete labeling by the acceptor. The expressions and implemented algorithm were verified using known mixtures of donor-control and donor-acceptor pair molecules, which simulated the presence of a donor population lacking the acceptor. Finally, we present data on the distance distributions between two labeled sites in myosin S1 (Cys-697 to Cys-707) where it was not possible to obtain complete labeling of the acceptor site.

AB - Measurements of time-resolved fluorescence are now being used to recover conformational distributions of biological macromolecules. The fluorescence data of the donor are easily corrupted by incomplete labeling of the macromolecules by the acceptor. In the present paper we describe a general procedure to correct for incomplete acceptor labeling in the determination of distance distributions from frequency-domain measurements of the donor fluorescence decay kinetics. The method can also be used to determine the extent of acceptor labeling. Simulated data were used to determine the effect of incomplete labeling on resolution of the distance distribution and the effect on the recovered distributions if one fails to account for incomplete labeling by the acceptor. The expressions and implemented algorithm were verified using known mixtures of donor-control and donor-acceptor pair molecules, which simulated the presence of a donor population lacking the acceptor. Finally, we present data on the distance distributions between two labeled sites in myosin S1 (Cys-697 to Cys-707) where it was not possible to obtain complete labeling of the acceptor site.

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Correction for incomplete labeling in the measurement of distance distributions by frequency-domain fluorometry

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