Sherwin S. Lehrer

[10] Solute quenching of protein fluorescence

Publisher Summary This chapter considers various ways of presenting and interpreting data using model calculations based on the modified Stern–Volmer equation. It provides detail information about the experimental methods for the purpose of obtaining meaningful quenching data. A quencher molecule can deactivate an excited fluorophor at the instant of excitation by forming a nonfluorescent, or “dark,” complex in the ground state, or by being present within a certain “sphere of action” at the time of excitation. Before considering heterogeneous systems, certain effects that can cause deviations from the simple Stem–Volmer law are also described in the chapter. Solute quenching of protein fluorescence is a technique that can yield information regarding the exposure of protein-bound intrinsic or extrinsic fluorophors. It is a dynamic method, because the presence of solute alters the fluorescence lifetime and thereby the steady-state intensity, without altering the electronic energy levels that are involved.

The regulatory switch of the muscle thin filament: Ca2+ or myosin heads?

Ca2+ and myosin heads are both involved in regulation, with Ca2+ as the allosteric trigger and the myosin head-induced Tm off-on transition as the switch. The allosteric/cooperative model is a good phenomenological description of part of the process and aspects of the original steric blocking model are involved in the molecular mechanisms.

Effects of an interchain disulfide bond on tropomyosin structure: Intrinsic fluorescence and circular dichroism studies

Abstract An interchain disulfide crosslink was introduced into rabbit skeletal tropomyosin (TM) at Cys190 by two different methods under non-denaturing conditions. The effects of the crosslink on the structure of tropomyosin were investigated by fluorescence and circular dichroism methods as a function of temperature and guanidine · hydrochloride concentration. Four different preparations were studied: Nbs 2 -TM, red-TM crosslinked with Ellmans reagent, 5,5′-dithiobis(2-nitrobenzoate); O 2 -TM, TM whose SH groups were air-oxidized; red-TM, TM reduced with dithiothreitol; IA-TM, red-TM whose SH groups were blocked with iodoacetamide. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis studies indicated that SS crosslinks were quantitatively introduced between the subunits of TM for Nbs 2 -TM and O 2 -TM. In the completely folded state (below 25 °C or in the absence of denaturant) and in the unfolded state (above 65 °C or greater than 4 m -guanidine · hydrochloride) all of the samples had the same Tyr fluorescence quantum yield, accessibility to acrylamide fluorescence quenching, fluorescence polarization and mean residue rotation at 222 nm. Thermal and denaturant-induced unfolding profiles at pH 7.5 were obtained for each sample with measurements of these parameters. The main transition at about 45 °C or 2 m -guanidine · hydrochloride was shifted about +7 deg. C and 0.8 m in guanidine · hydrochloride, respectively, for the crosslinked samples as compared to the uncrosslinked samples. In addition, a destabilizing pretransition was observed in the 30 to 45 °C region or the 0 to 2 m -guanidine · hydrochloride region only for the crosslinked samples when polarization or ellipticity was measured. Studies of the ability of Nbs 2 to crosslink red-TM as a function of guanidine · hydrochloride concentration indicated that the chains separate at Cys190 between 0 and 2 m -guanidine · hydrochloride before they dissociate. Thus, the effect of the SS crosslink at Cys190 on the conformation of TM at physiological temperatures appears to be related to the inherent instability of the molecule in this region of the sequence.

Conserved Asp-137 Imparts Flexibility to Tropomyosin and Affects Function

Tropomyosin (Tm) is an α-helical coiled-coil that controls muscle contraction by sterically regulating the myosin-actin interaction. Tm moves between three states on F-actin as either a uniform or a non-uniform semi-flexible rod. Tm is stabilized by hydrophobic residues in the “a” and “d” positions of the heptad repeat. The highly conserved Asp-137 is unusual in that it introduces a negative charge on each chain in a position typically occupied by hydrophobic residues. The occurrence of two charged residues in the hydrophobic region is expected to destabilize the region and impart flexibility. To determine whether this region is unstable, we have substituted hydrophobic Leu for Asp-137 and studied changes in Tm susceptibility to limited proteolysis by trypsin and changes in regulation. We found that native and Tm controls that contain Asp-137 were readily cleaved at Arg-133 with t½ of 5 min. In contrast, the Leu-137 mutant was not cleaved under the same conditions. Actin stabilized Tm, causing a 10-fold reduction in the rate of cleavage at Arg-133. The actin-myosin subfragment S1 ATPase activity was greater for the Leu mutant compared with controls in the absence of troponin and in the presence of troponin and Ca2+. We conclude that the highly conserved Asp-137 destabilizes the middle of Tm, resulting in a more flexible region that is important for the cooperative activation of the thin filament by myosin. We thus have shown a link between the dynamic properties of Tm and its function.

Ca2+-Induced Movement of Tropomyosin in Skeletal Muscle Thin Filaments Observed by Multi-Site FRET

To obtain information on Ca(2+)-induced tropomyosin (Tm) movement in Ca(2+)-regulated muscle thin filaments, frequency-domain fluorescence energy transfer data were collected between 5-(2-iodoacetyl-amino-ethyl-amino)naphthalene-1-sulfonic acid at Cys-190 of Tm and phalloidin-tetramethylrhodamine B isothiocyanate bound to F-actin. Two models were used to fit the experimental data: an atomic coordinate (AC) model coupled with a search algorithm that varies the position and orientation of Tm on F-actin, and a double Gaussian distance distribution (DD) model. The AC model showed that little or no change in transfer efficiency is to be expected between different sites on F-actin and Tm if Ca(2+) causes azimuthal movement of Tm of the magnitude suggested by structural data (C. Xu, R. Craig, L. Tobacman, R. Horowitz, and W. Lehman. 1999. Biophys. J. 77:985-992). However, Ca(2+) produced a small but significant change in our phase/modulation versus frequency data, showing that changes in lifetime decay can be detected even when a change of the steady-state transfer efficiency is very small. A change in Tm azimuthal position of 17 on the actin filament obtained with the AC model indicates that solution data are in reasonable agreement with EM image reconstruction data. In addition, the data indicate that Tm also appears to rotate about its axis, resulting in a rolling motion over the F-actin surface. The DD model showed that the distance from one of the two chains of Tm to F-actin was mainly affected, further verifying that Ca(2+) causes Tm to roll over the F-actin surface. The width of the distance distributions indicated that the position of Tm in absence and in presence of Ca(2+) is well defined with appreciable local flexibility.

A Modulatory Role for the Troponin T Tail Domain in Thin Filament Regulation

In striated muscle the force generating acto-myosin interaction is sterically regulated by the thin filament proteins tropomyosin and troponin (Tn), with the position of tropomyosin modulated by calcium binding to troponin. Troponin itself consists of three subunits, TnI, TnC, and TnT, widely characterized as being responsible for separate aspects of the regulatory process. TnI, the inhibitory unit is released from actin upon calcium binding to TnC, while TnT performs a structural role forming a globular head region with the regulatory TnI- TnC complex with a tail anchoring it within the thin filament. We have examined the properties of TnT and the TnT1 tail fragment (residues 1–158) upon reconstituted actin-tropomyosin filaments. Their regulatory effects have been characterized in both myosin S1 ATPase and S1 kinetic and equilibrium binding experiments. We show that both inhibit the actin-tropomyosin-activated S1 ATPase with TnT1 producing a greater inhibitory effect. The S1 binding data show that this inhibition is not caused by the formation of the blocked B-state but by significant stabilization of the closed C-state with a 10-fold reduction in the C- to M-state equilibrium, K T , for TnT1. This suggests TnT has a modulatory as well as structural role, providing an explanation for its large number of alternative isoforms.

The binding of fluorescent phallotoxins to actin in myofibrils

SummaryFluorescence microscope observation of myofibrils incubated with rhodamine-phalloidin and coumarine-phallacidin showed an initial appearance of fluorescence bands at the Z-lines and near the middle of the sarcomeres indicating preferential binding of dye to actin subunits located at both actin filament ends. After long incubation times (1–3 h) however, a final pattern is reached which consists of fluorescent Z-lines in the center of uniformly labelled actin bands, with greater fluorescence in the Z-lines than in the uniform region outside the Z-lines. Increasing the temperature or the ionic strength increased the rate of change to the final pattern. These data indicate: (1) that the ends of the actin filament are kinetically more accessible to phallotoxins; (2) at long times when equilibrium binding presumably occurs, the concentration of actin subunits in the Z-band is greater than in the rest of the sarcomere.

Terbium binding to troponin C: binding stoichiometry and structural changes induced in the protein.

Abstract Terbium (Tb3+) binding to skeletal muscle troponin C was studied by fluorescence spectroscopy and circular dichroism. Titrations indicate that Tb3+, like Ca2+, preferentially binds to the two high affinity Ca2+-Mg2+ sites (III and IV) inducing structural changes similar to those induced by Ca2+. Tb3+ readily displaces Ca2+ from these sites suggesting a K(Tb3+) ≥ 109 M−1 In 6 M urea, both Ca2+ and Tb3+ bind preferentially to a single site on troponin C. The spectral changes suggest this to be site III.

The crosslinking of actin and of tropomyosin by glutaraldehyde

Abstract Glutaraldehyde treatment of actin results in intermolecular crosslinked oligomers for the fibrous form (F-actin) but only monomers containing intramolecular crosslinks for the globular form (G-actin). Fluorescence studies on solutions which were dialyzed to remove salt, indicated that both modified actins maintained their native structure and were stabilized against denaturation by EDTA. Glutaraldehyde crosslinks tropomyosin producing oligomers with molecular weights n × 68,000. In the presence of F-actin in 0.1M KCl, tropomyosin is crosslinked end to end giving rise to a polymer of molecular weight > 500,000, indicating the close proximity of tropomyosin molecules on the F-actin filament. Glutaraldehyde modification thus appears to be useful for the study of muscle and other protein interactions.

The binding of Cu2+ to actin without loss of polymerizability: The involvement of the rapidly reacting SH group

Abstract G-or F-actin binds 1 mole of Cu 2+ per mole of actin without denaturation and without replacement of the essential Ca 2+ or Mg 2+ . The association constant is ~10 16 . Denaturation of actin, e.g., by EDTA treatment, abolishes the high-affinity Cu 2+ binding. The absorption spectrum of Cu-actin shows a new intense absorption band centered at 347 nm (ϵ = 3000 M −1 cm −1 ) and a weak band centered at 550 nm (ϵ = 130 M −1 cm −1 ). The tryptophyl fluorescence of actin is strongly (70%) quenched by the binding of Cu 2+ . Circular dichroic spectra show multiple bands associated with each absorption band indicating an asymmetrical environment for the bound Cu 2+ and suggest that histidine is one of the ligands. Electron-spin resonance studies show that Cu 2+ maintains its valence when bound and also suggest that there are approximately three nitrogen ligands. Chemical modification studies of the sulfhydryl groups provide evidence for the involvement of one SH group in the binding of Cu 2+ to actin.