The labeling of muscle fiber proteins with iodoacetamido)tetramethylrhodamine (IATR) was reinvestigated with the purified 5' or 6' isomers of IATR. Both isomers modify the myosin heavy chain within the 20-kDa fragment of myosin subfragment 1 (S1) but with different rates, and only the 5'-IATR alters K(+)-EDTA- and Ca(2+)-activated ATPases. Absorption spectroscopic and ATPase studies of probe stoichiometry indicate that for 5'-IATR there are two probes per myosin sulfhydryl 1 (SH1). Quantitative fluorograms of the SDS-PAGE gels confirm that there are one covalent and one noncovalent probe per SH1 when S1 is labeled with 5'-IATR (5'-IATR-S1) and that there are one covalent and two noncovalent probes per S1 when S1 is labeled with 6'-IATR (6'-IATR-S1). The 5'- and 6'-IATR probes have similar fluorescent lifetimes when bound to S1, but quenching studies with potassium iodide show that 5'-IATR-S1 has a single class of strongly bound chromophores while 6'-IATR-S1 has two or more classes of chromophores. It is possible that 5'-IATR labels SH1 as a dimer. The polarization anisotropies of 5'- and 6'-IATR-S1 indicate that 5'-IATR is immobilized, while 6'-IATR is moving independently, on the surface of S1. The emission spectrum from 5'-IATR-S1 is unaffected by the addition of MgATP, while 6'-IATR-S1 shows a spectral shift and total intensity change. When labeling muscle fibers, 5'-IATR labels myosin SH1 and differentiates between the fiber physiological states by indicating cross-bridge rotation in quantitative agreement with previous results [Burghardt et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7515]. 6'-IATR reacts preferentially with actin in muscle fibers and does not differentiate between fiber physiological states as expected for an actin probe. The stereospecificity of the rhodamine isomers for SH1 indicates features of the local protein structure. The experimental results are used with theoretical methods for determining molecular structure to suggest a qualitative scheme for the specific interaction of 5'-IATR with its binding pocket on the surface of S1.
Calmodulin binds to amphiphilic, helical peptides of a variety of amino-acid sequences. These peptides are usually positively charged, although there is spectroscopic evidence that at least one neutral peptide binds. The complex between calmodulin and one of its natural target peptides, the binding site for calmodulin on smooth muscle myosin light-chain kinase (RS20), has been investigated by crystallography and NMR which have characterized the interactions between the ligand and the protein. From these data, it appears that the calmodulin-binding surface is sterically malleable and van der Waals forces probably dominate the binding. To explore further this apparently permissive binding, we investigated the chiral selectivity of calmodulin using synthesized analogues of melittin and RS20 that consisted of only D-amino acids. Fluorescence and NMR measurements show that D-melittin and D-RS20 both bind avidly to calmodulin, probably in the same general binding site as that for peptides having all L-amino acids. The calmodulin-peptide binding surface is therefore remarkably tolerant sterically. Our results suggest a potentially useful approach to the design of non-hydrolysable or slowly hydrolysable intracellular inhibitors of calmodulin.
This novel approach to the analysis of multiexponential functions is based on the combined use of the Laplace transform and Padé approximants (Yeramian, E., and P. Claverie. 1987. Nature (Lond.). 326:169-174). It is similar in principle to the well-known Isenberg method of moments (Isenberg, I. 1983. Biophys. J. 43:141-148) traditionally applied to the analysis of fluorescence decay. The advantage of the Padé-Laplace method lies in its ability to detect the number of components in a multiexponential function as well as their parameters. In this paper we modified the original method so that it can be applied to the analysis of multifrequency phase/modulation measurements of fluorescence decay. The method was tested first on simulated data. It afforded recovery up to four distinct lifetime components (and their fractional contributions). In the case of simulated data corresponding to continuous lifetime distributions (nonexponential decay), the results of the analysis by the Padé-Laplace method indicated the absence of discrete exponential components. The method was also applied to real phase/modulation data gathered on known fluorophores and their mixtures and on tryptophan fluorescence in phospholipase A2. The lifetime and fraction recoveries were consistent with those obtained from standard methods involving nonlinear least-square fitting.
The interpretation of fluorescence intensity decay times in terms of protein structure and dynamics depends on the accuracy and sensitivity of the methods used for data analysis. The are many methods available for the analysis of fluorescence decay data, but justification for choosing any one of them is unclear. In this paper we generalize the recently proposed Padé-Laplace method to include deconvolution with respect to the instrument response function. In this form the method can be readily applied to the analysis of time-correlated single photon counting data. By extensive simulations we have shown that the Padé-Laplace method provides more accurate results than the standard least squares method with iterative reconvolution under the condition of closely spaced lifetimes. The application of the Padé-Laplace method to several experimental data sets yielded results consistent with those obtained by use of the least squares analysis.
Measurements of homogeneous and heterogeneous fluorescence intensity decays using a hybrid time-correlated single photon counting/multifrequency phase fluorometer are reported. A trio of fluorophores exhibiting a range of decay profiles was selected. p-Terphenyl, 1,4-bis[2-(4-methyl-5-phenyloxazolyl)]benzene [(Me)2POPOP], and p-bis[2-(5-phenyloxazolyl)]benzene (POPOP), commonly used reference fluorophores, were analyzed initially; their emissions were characterized by monoexponential decay functions. Additionally, emissions from two single tryptophan proteins with different decay profiles were measured. Scorpion neurotoxin variant 3 required three exponentials to fit the emission decay properly (average lifetime approximately 500 ps). At pH 5.5, the fluorescence emission of ribonuclease T1 showed a monoexponential decay with a measured lifetime of approximately 4.0 ns. Thus, in each case, the results from both measurements were consistent between the two detection systems, confirming the view that the two approaches for measuring fluorescence lifetimes are equivalent.
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