Stopped-flow fluorescence

The role of Ca2+ in inducing refolding of a-lactalbumin is reflected in clean two-stage kinetics, with rate constants 6.0 and 1.3 s-1. The maximum concentration of the intermediate, monitored by stopped-flow fluorescence and time-resolved photo-CIDNP NMR, occurs at about 200 ms (327). 

Lin, C.-E, Houng, L.-M. and Lo, K.S. (1994) Kinetics of copper complexation with dissolved organic matter using stopped-flow fluorescence quenching. Toxicol. Environ. 

The interpretation of 2AP fluorescence in terms of the folding pathway of the ribozyme indicates that there are at least three states in the process (Fig. 13.2). Stopped-flow fluorescence experiments over the folding time-course of 10 s show that the initial increase in 2AP fluorescence occurs rapidly (feDbs = 106 s and is followed by three slower phases that greatly decrease its fluorescence. 

AP insertions. (B) Fluorescence emission intensity of 2AP62 as a function of added TPP. (C) Stopped-flow fluorescence of 2AP62 riboswitch upon addition of 5 x TPP. [RNA] = 0.3 pMin 50 mMKMOPS, 100 mMKCl, 2 mMMgClj, 25 °C, pH 7.5. Data are described by a first-order exponential, and the apparent rate constants as a function of added TPP are shown to the right (Lang et al., 2007). 

Circular polarization of luminiscence, stopped-flow fluorescence, fluorescence-monitored chemical relaxation, the evaluation of relative orientation by polarized excitation energy transfer, time-resolved fluorescent polarization ( nanosecond polarization ), and other new techniques have become valuable means for studying protein structures, their interactions and structural changes in relation to various treatments (e.g. denaturation). New fluorescent probes and quenchers have enabled the research field to expand from isolated proteins to more complicated systems such as membranes, muscle and nerve components and other subcellular structures (see also 7.3). 

Stopped-flow fluorescence spectroscopy was used to obtain insight into the interaction of fluoride with tyrosinase (Ty) under pseudo-first-order conditions, as well as to validate the use of fluorescence quenching in studying ligand-binding kinetics. ... 

In addition, many examples of binding systems were investigated by stopped-flow fluorescence spectroscopy, such as the binding of calmodulin to calcineuiin, the binding of guanine to calf spleen purine nucleoside phosphorylase, the nucleotide cofactor binding to the Escherichia coli PriA Helicase, etc. 

The inhibitor could be displaced from Factor Xa by substrates and, based on steady-state assumptions, the dissociation constant for (19) was found to be 14 pM (87). However, the reaction progress curves indicated a slow-binding process, probably by mechanism B. Stopped-flow fluorescence studies, combined with kinetic analysis, showed that the isomerization step (E. I -I- E. I ) is unusually fast and that the formation of E I is, at least, partially rate limiting. 

Stopped-flow fluorescence studies of ES complexes provided a direct comparison of the peptide binding aflBnities of the zinc and cadmium enzymes and, simultaneously, an explanation for the different roles of metals in peptide and ester hydrolysis (48). Cadmium carboxypeptidase binds the peptide Dns-(Gly)3-L-Phe as readily as does [(CPD)Zn] but catalyzes its hydrolysis at a rate that is reduced considerably (Figure 8). Initial rate studies of oligopeptides are in agreement with this observation. For all peptides examined, the catalytic rate constants of the cadmium enzyme are decreased markedly, but the association constants (1) (Km values) of the cadmium enzyme are identical to those of the zinc enzyme (48,51,57). However, in marked contrast, for all esters examined the catalytic rate constants of the cadmium enzyme are nearly the same as those of the zinc enzyme, but the association constants are decreased greatly. 

Figure 8. Tracings of stopped-flow fluorescence assays of the hydrolym of Dns-(Gly)g-ia-Phe, 2.5 X catalyzed by zinc and cadmium carboxypeptidase A, 5 X J0-5M, at pH = 7.5 and 25 C in 0.03M Tris-l.OM NaCl (48). Enzyme tryptophans were excited at 285 nm and their emission was measured by means of a bandpass filter peaking at 360 nm.

Figure 9. Stopped-flow fluorescence measurements of Dns-(Giy)s-L.-Phe, 1 X and Dns-(Gly)g-L.-OPhe, 1 X 10 M,...

Haloalkane dehalogenase from Xanthobacter autotrophicus GJIO exhibits a solvent isotope effect of 3 and linear proton inventories for k for hydrolysis of dibrome-thane and 1,2-dichloroethane [58]. Stopped-flow fluorescence experiments under single-turnover conditions suggest that halide release limits k. This isotope effect was interpreted to reflect a conformational change of the protein that allows departure of halide. 

Fig. 5. Kinetics of folding of murine PrP( 121-231) (variant F175W) at pH 7.0 and 4°C, measured by stopped-flow fluorescence. PrP(l21-231 )-F175W was unfolded with 8 M urea and diluted with refolding buffer to a final concentration of 3.7 M urea. The extrapolated value at t = 0 s corresponds to the expected value from the urea-induced equilibrium transition (excitation 280 nm emission > 320 nm).

Figure 10 Superimposition of rapid chemical quench (open circles O) and stopped-flow fluorescence (blue) assays. In (a) tryptophan emission was detected, and in (b) the fluorescence change from 2-AP was monitored. Insets show the dNTP binding-induced conformational change In the presence of dideoxy-terminated DNA substrate. Adapted with permission from A. K. Showalter B. J. Lamarche M. Bakhtina M. I. Su K. H. Tang M. D. Tsai, Chem. Rev. 2006, 106, 340-360. Copyright 2006 American Chemical Society.

Previous stopped-flow fluorescence assays investigating matched dNTP incorporation showed that both the fast and the slow fluorescence transitions demonstrated a hyperbolic dependence on dNTP concentra-tion. " " " Similarly, the dNTP dependence of both the fast and the slow fluorescence phases during mismatched dNTP incorporation in stopped-flow has been examined. The observed rate constants for the fast and the slow phases, individually plotted as a function of dNTP concentration, reveal that both phases demonstrate a hyperbolic dependence on dNTP concentration (parameters obtained for k2, K, k o, and d,app as described in Section 8.10.4.2.3 and reported in Table 1). The observed hyperbolic dependence of the fast phase on mismatched dNTP largely indicates that this phase originates from a conformational change induced by mismatched dNTP binding. 

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