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Kinetics of proton uptake and dye binding by photoactive yellow protein in wild type and in the E46Q and E46A mutants.
Biochemistry. 2002 Aug 06; 41(31):10026-37.B

Abstract

We studied the kinetics of proton uptake and release by photoactive yellow protein (PYP) from Ectothiorhodospira halophila in wild type and the E46Q and E46A mutants by transient absorption spectroscopy with the pH-indicator dyes bromocresol purple or cresol red in unbuffered solution. In parallel, we investigated the kinetics of chromophore protonation as monitored by the rise and decay of the blue-shifted state I(2) (lambda(max) = 355 nm). For wild type the proton uptake kinetics is synchronized with the fast phase of I(2) formation (tau = 500 micros at pH 6.2). The transient absorption signal from the dye also contains a slower component which is not due to dye deprotonation but is caused by dye binding to a hydrophobic patch that is transiently exposed in the structurally changed and partially unfolded I(2) intermediate. This conclusion is based on the wavelength, pH, and concentration dependence of the dye signal and on dye measurements in the presence of buffer. SVD analysis, moreover, indicates the presence of two components in the dye signal: protonation and dye binding. The dye binding has a rise time of about 4 ms and is coupled kinetically with a transition between two I(2) intermediates. In the mutant E46Q, which lacks the putative internal proton donor E46, the formation of I(2) is accelerated, but the proton uptake kinetics remains kinetically coupled to the fast phase of I(2) formation (tau = 100 micros at pH 6.3). For this mutant the protein conformational change, as monitored by the dye binding, occurs with about the same time constant as in wild type but with reduced amplitude. In the alkaline form of the mutant E46A the formation of the I(2)-like intermediate is even faster as is the proton uptake (tau = 20 micros at pH 8.3). No dye binding occurred in E46A, suggesting the absence of a conformational change. In all of the systems proton release is synchronized with the decay of I(2). Our results support mechanisms in which the chromophore of PYP is protonated directly from the external medium rather than by the internal donor E46.

Authors+Show Affiliations

Biophysics Group, Department of Physics, Freie Universität Berlin, Arnimallee 14, D-14195 Berlin, Germany.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

12146967

Citation

Borucki, Berthold, et al. "Kinetics of Proton Uptake and Dye Binding By Photoactive Yellow Protein in Wild Type and in the E46Q and E46A Mutants." Biochemistry, vol. 41, no. 31, 2002, pp. 10026-37.
Borucki B, Devanathan S, Otto H, et al. Kinetics of proton uptake and dye binding by photoactive yellow protein in wild type and in the E46Q and E46A mutants. Biochemistry. 2002;41(31):10026-37.
Borucki, B., Devanathan, S., Otto, H., Cusanovich, M. A., Tollin, G., & Heyn, M. P. (2002). Kinetics of proton uptake and dye binding by photoactive yellow protein in wild type and in the E46Q and E46A mutants. Biochemistry, 41(31), 10026-37.
Borucki B, et al. Kinetics of Proton Uptake and Dye Binding By Photoactive Yellow Protein in Wild Type and in the E46Q and E46A Mutants. Biochemistry. 2002 Aug 6;41(31):10026-37. PubMed PMID: 12146967.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Kinetics of proton uptake and dye binding by photoactive yellow protein in wild type and in the E46Q and E46A mutants. AU - Borucki,Berthold, AU - Devanathan,Savitha, AU - Otto,Harald, AU - Cusanovich,Michael A, AU - Tollin,Gordon, AU - Heyn,Maarten P, PY - 2002/7/31/pubmed PY - 2002/9/6/medline PY - 2002/7/31/entrez SP - 10026 EP - 37 JF - Biochemistry JO - Biochemistry VL - 41 IS - 31 N2 - We studied the kinetics of proton uptake and release by photoactive yellow protein (PYP) from Ectothiorhodospira halophila in wild type and the E46Q and E46A mutants by transient absorption spectroscopy with the pH-indicator dyes bromocresol purple or cresol red in unbuffered solution. In parallel, we investigated the kinetics of chromophore protonation as monitored by the rise and decay of the blue-shifted state I(2) (lambda(max) = 355 nm). For wild type the proton uptake kinetics is synchronized with the fast phase of I(2) formation (tau = 500 micros at pH 6.2). The transient absorption signal from the dye also contains a slower component which is not due to dye deprotonation but is caused by dye binding to a hydrophobic patch that is transiently exposed in the structurally changed and partially unfolded I(2) intermediate. This conclusion is based on the wavelength, pH, and concentration dependence of the dye signal and on dye measurements in the presence of buffer. SVD analysis, moreover, indicates the presence of two components in the dye signal: protonation and dye binding. The dye binding has a rise time of about 4 ms and is coupled kinetically with a transition between two I(2) intermediates. In the mutant E46Q, which lacks the putative internal proton donor E46, the formation of I(2) is accelerated, but the proton uptake kinetics remains kinetically coupled to the fast phase of I(2) formation (tau = 100 micros at pH 6.3). For this mutant the protein conformational change, as monitored by the dye binding, occurs with about the same time constant as in wild type but with reduced amplitude. In the alkaline form of the mutant E46A the formation of the I(2)-like intermediate is even faster as is the proton uptake (tau = 20 micros at pH 8.3). No dye binding occurred in E46A, suggesting the absence of a conformational change. In all of the systems proton release is synchronized with the decay of I(2). Our results support mechanisms in which the chromophore of PYP is protonated directly from the external medium rather than by the internal donor E46. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/12146967/Kinetics_of_proton_uptake_and_dye_binding_by_photoactive_yellow_protein_in_wild_type_and_in_the_E46Q_and_E46A_mutants_ L2 - https://doi.org/10.1021/bi0256227 DB - PRIME DP - Unbound Medicine ER -