Fluorescence resonance energy transfer within the complex formed by actin and myosin subfragment 1. Comparison between weakly and strongly attached states

Fluorescence resonance energy transfer measurements have been made between Cys-374 on actin and Cys-177 on the alkali light chain of myosin subfragment 1 (S1) using several pairs of donor-acceptor chromophores. The labeled light chain was exchanged into subfragment 1 and the resulting fluorescently labeled subfragment 1 isolated by ion-exchange chromatography on SP-Trisacryl. The efficiency of energy transfer was measured by steady-state fluorescence in a strong binding complex of acto-S1 and found to represent a spatial separation between the two probes of 5.6-6.3 nm. The same measurements were then made with weak binding acto-S1 complexes generated in two ways. First, actin was complexed with p-phenylenedimaleimide-S1, a stable analogue of S1-adenosine 5'-triphosphate (ATP), obtained by cross-linking the SH1 and SH2 heavy-chain thiols of subfragment 1 [Greene, L. E., Chalovich, J. M., & Eisenberg, E. (1986) Biochemistry 25, 704-709]. Large increases in transfer efficiency indicated that the two probes had moved closer together by some 3 nm. Second, weak binding complexes were formed between subfragment 1 and actin in the presence of the regulatory proteins troponin and tropomyosin, the absence of calcium, and the presence of ATP [Chalovich, J. M., & Eisenberg, E. (1982) J. Biol. Chem. 257, 2432-2437]. The measured efficiency of energy transfer again indicated that the distance between the two labeled sites had moved closer by about 3 nm. These data support the idea that there is a considerable difference in the structure of the acto-S1 complex between the weakly and strongly bound states.     

Kinetic and spectroscopic evidence for three actomyosin:ADP states in smooth muscle

Smooth muscle myosin II undergoes an additional movement of the regulatory domain with ADP release that is not seen with fast skeletal muscle myosin II. In this study, we have examined the interactions of smooth muscle myosin subfragment 1 with ADP to see if this additional movement corresponds to an identifiable state change. These studies indicate that for this myosin:ADP, both the catalytic site and the actin-binding site can each assume one of two conformations. Relatively loose coupling between these two binding sites leads to three discrete actin-associated ADP states. Following an initial, weakly bound state, binding of myosin:ADP to actin shifts the equilibrium toward a mixture of two states that each bind actin strongly but differ in the conformation of their catalytic sites. By contrast, fast myosins, including Dictyostelium myosin II, have reciprocal coupling between the actin- and ADP-binding sites, so that either actin or nucleotide, but not both, can be tightly bound. This uncoupling, which generates a second strongly bound actomyosin ADP state in smooth muscle, would prolong the fraction of the ATPase cycle time that this actomyosin spends in a force-generating conformation and may be central to explaining the physiologic differences between this and other myosins.     

New states of actomyosin

Unstained frozen hydrated samples of myosin subfragment 1 (S-1) cross-linked to actin with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide have been examined by electron microscopy in an effort to probe structural states of the attached cross-bridge. The cross-linked complex in the absence of ATP has a rigor-like appearance. In contrast, both in the presence of ATP and after the N, N'-p-phenylenedimaleimide (pPDM) bridging of the reactive thiols of S-1, the covalently attached cross-bridges of the acto X S-1 complex appear more disordered and no longer assume the characteristic rigor 45 degrees angle with the actin filaments. The images both in the presence and absence of ATP bear a striking resemblance to those obtained by negative staining of the cross-linked acto X S-1 complex (Craig, R., Greene, L. E. & Eisenberg, E. (1985) Proc. Natl. Acad. Sci. U.S. A. 82, 3247-3251). The actin-bound pPDM S-1 complex, formed by treating the cross-linked complex with pPDM in the presence of ATP, is an expected analog of the weakly bound cross-bridge state. The disordered appearance of S-1 molecules of the cross-linked complex in the presence of ATP and after pPDM treatment may reflect the structural state of the weakly bound cross-bridge.     

X-ray diffraction evidence for the lack of stereospecific protein interactions in highly activated actomyosin complex

The structure of actomyosin complex while hydrolyzing ATP was investigated by recording X-ray diffraction patterns from rabbit skeletal muscle fibers, in which exogenously introduced rabbit skeletal subfragment-1 (S1) was covalently cross-linked to the endogenous actin filaments in rigor by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Approximately two-thirds of the introduced S1 was cross-linked. The cross-linking procedure did not affect the profile of the S1-induced enhancement of the actin-based layer line reflections in rigor, indicating that the acto-S1 interactions remained highly stereospecific. In the presence of ATP, the MgATPase of the S1 was highly activated regardless of calcium levels, presumably because the availability of the stereospecific binding sites for both proteins was maximized by the cross-linking. However, the diffraction pattern in the presence of ATP was striking in that the intensity profile of the strong 1/5.9 nm(-1) layer lines was indistinguishable from that from bare actin filaments, despite the fact that the majority of the S1 was still associated with actin. The change of the intensity profiles upon addition of ATP was completely reversible. Model calculations showed that this result can be explained if the S1 is not only swinging around its pivoting point, but the pivoting point itself is also moving on the actin surface in a range of a few nanometers. The results suggest that the stereospecific binding sites, which have been considered important for actomyosin cycling, are paradoxically left unoccupied for most of the time in this highly activated actomyosin complex.     

Dissecting the free energy of formation of the 1:1 actomyosin complex

The behaviour of solutions of pure myosin, of pure F-actin and of the equimolar mixture of myosin and of F-actin is studied. It is found that the chemical potential of the two proteins, in separate solutions, increases monotonically with the increase of protein osmotic pressure. A method is presented to determine the chemical potential of the 1:1 actin-myosin complex formed from equimolar solutions of myosin and of F-actin (as monomer).This is the first evaluation of the chemical potential of actomyosin under conditions similar to those of skeletal muscle. It is found that the filament suspensions of myosin and of the 1:1 actin-myosin complex display a high non-ideal behavior as well as distinctly different energy profiles as a function of protein osmotic pressure. This supports the hypothesis that, in muscle: (a) detached cross-bridge change significantly their free energy when sarcomere is shifting from the relaxed to the active or to the rigor state; and (b) the cross-bridge attachment-detachment process is accompanied by changes of muscle protein osmotic pressure.     

Resonance energy transfer study of lysozyme-lipid interactions

Resonance energy transfer (RET) between the tryptophan residues of lysozyme as donors and anthrylvinyl-labeled phosphatidylcholine (AV-PC) or phosphatidylglycerol (AV-PG) as acceptors has been examined to gain insight into molecular level details of the interactions of lysozyme with the lipid bilayers composed of PC with 10, 20, or 40 mol% PG. Energy transfer efficiency determined from the enhanced acceptor fluorescence was found to increase with content of the acidic lipid and surface coverage. The results of RET experiments performed with lipid vesicles containing 40 mol% PG were quantitatively analyzed in terms of the model of energy transfer in two-dimensional systems taking into account the distance dependence of orientation factor. Evidence for an interfacial location of the two predominant lysozyme fluorophores, Trp62 and Trp108, was obtained. The RET enhancement observed while employing AV-PG instead of AV-PC as an energy acceptor was interpreted as arising from the ability of lysozyme to bring about local demixing of the neutral and charged lipids in PC/PG model membranes.     

Resonance energy transfer study of lysozyme-lipid interactions

Resonance energy transfer (RET) between the tryptophan residues of lysozyme as donors and anthrylvinyl-labeled phosphatidylcholine (AV-PC) or phosphatidylglycerol (AV-PG) as acceptors has been examined to gain insight into molecular level details of the interactions of lysozyme with the lipid bilayers composed of PC with 10, 20, or 40 mol% PG. Energy transfer efficiency determined from the enhanced acceptor fluorescence was found to increase with content of the acidic lipid and surface coverage. The results of RET experiments performed with lipid vesicles containing 40 mol% PG were quantitatively analyzed in terms of the model of energy transfer in two-dimensional systems taking into account the distance dependence of orientation factor. Evidence for an interfacial location of the two predominant lysozyme fluorophores, Trp62 and Trp108, was obtained. The RET enhancement observed while employing AV-PG instead of AV-PC as an energy acceptor was interpreted as arising from the ability of lysozyme to bring about local demixing of the neutral and charged lipids in PC/PG model membranes.     

Resonance energy transfer study of peptide-lipid complexes

Resonance energy transfer involving tryptophan as a donor and anthrylvinyl-labeled phosphatidylcholine (AV-PC), 3-methoxybenzanthrone (MBA) and 8-anilino-1-naphthalene sulfonic acid (ANS) as acceptors has been examined to obtain information on the structure of peptide-lipid systems consisting of 18A or Ac-18A-NH(2) peptides and large unilamellar phosphatidylcholine vesicles. The lower and upper limits for the tryptophan distance from the bilayer midplane have been assessed in terms of the models of energy transfer in two-dimensional systems, taking into account orientational effects. Evidence for the existence of preferential orientations of Ac-18A-NH(2) with respect to the lipid-water interface has been obtained.     

Resonance energy transfer for assessing the molecular integrity of proteins for local delivery

It remains unclear whether the limitations to the therapeutic potential of angiogenic growth factors stem from pharmacokinetic concerns related to inadequate delivery or from a reduced sensitivity of target tissues. Here, we report a novel method using resonance energy transfer to assess the molecular integrity of proteins after local delivery. As an example, we labeled fibroblast growth factor-2 with a fluorescent donor and nonfluorescent acceptor pair, tetramethylrhodamine and QSY-7, and demonstrate in an ex vivo bovine carotid artery model that this growth factor is not limited by proteolytic constraints imposed by the tissue. Our data indicate that FGF-2 is unlikely to be degraded within the arterial wall and suggest that pharmacokinetic limitations alone cannot fully explain the muted response seen thus far in therapeutic angiogenesis. In general, resonance energy transfer may serve as a novel approach to assess the molecular integrity of protein-based therapies in local delivery.     

Resonance energy transfer,pH‐induced folded states and the molecular interaction of human serum albumin and icariin

Icariin is a flavonol glycoside with a wide range of pharmacological and biological activities. The pharmacological and biological functions of flavonoid compounds mainly originate from their binding to proteins. The mode of interaction of icariin with human serum albumin (HSA) has been characterized by fluorescence spectroscopy and far‐ and near‐UV circular dichroism (CD) spectroscopy under different pH conditions. Fluorescence quenching studies showed that the binding affinity of icariin with HSA in the buffer solution at different pH values is: K_a (pH 4.5) > K_a (pH 3.5) > K_a (pH 9.0) > K_a (pH 7.0). Red‐edge excitation shift (REES) studies revealed that pH had an obvious effect on the mobility of the tryptophan microenvironment and the addition of icariin made the REES effect more distinct. The static quenching mechanism and number of binding sites (n ≈ 1) were obtained from fluorescence data at three temperatures (298, 304 and 310 K). Both ?H⁰ < 0 and ??⁰ < 0 suggested that hydrogen bonding and van der Waal's interaction were major driving forces in the binding mechanism, and this was also confirmed by the molecular simulation results. The distance r between the donor (HSA) and the acceptor (icariin) was calculated based on Förster non‐radiation energy transfer theory. We found that pH had little impact on the energy transfer between HSA and icariin. Far‐ and near‐UV CD spectroscopy studies further indicated the influence of pH on the complexation process and the alteration in the protein conformation upon binding. Copyright © 2015 John Wiley & Sons, Ltd.     

Intermediate states of actomyosin adenosine triphosphatase.

The early kinetic steps of actomyosin subfragment 1 (acto-S1) adenosine triphosphatase have been investigated by simultaneous monitoring of fluorescence and light scattering and also by observation of the time course of the production of phosphate. The results show that fluorescence enhancement occurs after the dissociation of actomyosin and that the rate of enhancement is similar to the maximum rate of enhancement for S1 alone, under similar conditions of pH and temperature. The maximum rate of the phosphate burst for acto S1 is also approximately the same as that for S1 alone. The maximum rates for fluorescence enhancement or phosphate formation are reached at much lower adenosine triphosphate concentrations for acto-S1 than for S1. An extension of the actomyosin scheme is presented which accounts for these results.     

Resonance energy transfer in a calcium concentration-dependent cameleon protein

We report investigations of resonance energy transfer in the green fluorescent protein and calmodulin-based fluorescent indicator constructs for Ca(2+) called cameleons using steady-state and time-resolved spectroscopy of the full construct and of the component green fluorescent protein mutants, namely ECFP (donor) and EYFP (acceptor). EYFP displays a complicated photophysical behavior including protonated and deprotonated species involved in an excited-state proton transfer. When EYFP is excited in the absorption band of the protonated species, a fast nonradiative deactivation occurs involving almost 97% of the excited protonated population and leading to a low efficiency of excited-state proton transfer to the deprotonated species. ECFP displays a multiexponential fluorescence decay with a major contributing component of 3.2 ns. The time-resolved fluorescence data obtained upon excitation at 420 nm of Ca(2+)-free and Ca(2+)-bound YC3.1 cameleon constructs point to the existence of different conformations of calmodulin dependent on Ca(2+) binding. Whereas steady-state data show only an increase in the efficiency of energy transfer upon Ca(2+) binding, the time-resolved data demonstrate the existence of three distinct conformations/populations within the investigated sample. Although the mechanism of the interconversion between the different conformations and the extent of interconversion are still unclear, the time-resolved fluorescence data offer an estimation of the rate constants, of the efficiency of the energy transfer, and of the donor-acceptor distances in the Ca(2+)-free and Ca(2+)-bound YC3.1 samples.     

Electron microscopy and x-ray diffraction evidence for two Z-band structural states

In vertebrate muscles, Z-bands connect adjacent sarcomeres, incorporate several cell signaling proteins, and may act as strain sensors. Previous electron microscopy (EM) showed Z-bands reversibly switch between a relaxed, “small-square” structure, and an active, “basketweave” structure, but the mechanism of this transition is unknown. Here, we found the ratio of small-square to basketweave in relaxed rabbit psoas muscle varied with temperature, osmotic pressure, or ionic strength, independent of activation. By EM, the A-band and both Z-band lattice spacings varied with temperature and pressure, not ionic strength; however, the basketweave spacing was consistently 10% larger than small-square. We next sought evidence for the two Z-band structures in unfixed muscles using x-ray diffraction, which indicated two Z-reflections whose intensity ratios and spacings correspond closely to the EM measurements for small-square and basketweave if the EM spacings are adjusted for 20% shrinkage due to EM processing. We conclude that the two Z-reflections arise from the small-square and basketweave forms of the Z-band as seen by EM. Regarding the mechanism of transition during activation, the effects of Ca²⁺ in the presence of force inhibitors suggested that the interconversion of Z-band forms was correlated with tropomyosin movement on actin.     

Microspectrophotometric evidence for two photointerconvertible states of visual pigment in the barnacle lateral eye

Microspectrophotometrically derived difference spectra from the barnacles Balanus amphitrite and B. eburneus show that a blue illumination after an orange illumination causes a decrease in absorption in the blue region and an increase in absorption in the green-yellow region, with an isosbestic point around 535 nm. Orange-following-blue illumination causes the reverse changes. The dark time between the adapting and measuring lights has no influence on the data. The results confirm previously reported ERP measurements which indicate that the barnacle visual pigment has two photointerconvertible dark-stable states. If one assumes a Dartnall nomogram shape for the two absorption spectra, a best fit to the observed difference spectra is obtained with nomograms peaking at 492 nm and 532 nm, with a peak absorbance ratio around 1.6:1. These two nomograms fit very well the ERP action spectra of metarhodopsin and rhodopsin, respectively, thus indicating that the ERP is a reliable measure of visual-pigment changes in the barnacle. The existence of a photostable blue pigment is demonstrated in B. eburneus and in some of B. amphitrite receptors, and the possible influence of this photostable pigment on the various action spectra measured in the barnacle is discussed.