High-affinity actin-binding nebulin fragments influence the actoS1 complex.

Human nebulin fragments, NA3 and NA4, corresponding to individual superrepeats display high-affinity interactions with individual actin protomers in cosedimentation and solid-phase binding assays. Stoichiometric analysis of nebulin fragment-induced actin polymerization and inhibition of actin-activated S1 ATPase indicate that one superrepeat influences multiple actin molecules along the F-actin filament, consistent with a combination of strong and weak interactions of nebulin over the length of the actin filament. The mechanisms by which human nebulin fragments affect the interaction between actin and myosin S1 are studied by fluorescence quenching, polarization, and resonance energy transfer. We show that, under strong binding conditions, premixing actin with the NA3 prior to adding myosin subfragment 1 (S1) inhibits the rate of actoS1 association. The nebulin fragments, NA3 and NA4, caused little effect on the extent of actoS1 binding at equilibrium but did alter the nature of the complex as evidenced by an increase in the resonance energy transfer efficiencies between S1 and actin in the absence of ATP. The addition of low concentrations of ATP rapidly dissociates the strong-binding actoS1 irrespective of the presence or absence of nebulin fragment. Interestingly, the strongly bound state reforms rapidly after S1 hydrolyzes all available ATP. These observations are consistent with the notion that nebulin might contribute to optimizing the alignment of actomyosin interactions and inhibit suboptimal actomyosin contacts.

Modulation of troponin T molecular conformation and flexibility by metal ion binding to the NH2-terminal variable region.

Troponin T (TnT) plays an allosteric signal transduction role in the actin thin-filament-based Ca(2+)-regulation of striated muscle contraction. Developmentally regulated alternative RNA splicing produces TnT isoforms differing in their NH(2)-terminal structure. Physical property variations of the NH(2)-terminal hypervariable region of TnT may have a role in tuning the Ca(2+)-sensitivity and overall cooperativity of the muscle. We have previously demonstrated that metal ion or monoclonal antibody binding to the NH(2)-terminal region can modulate the epitopic conformation and troponin I and tropomyosin binding affinity of TnT. To further establish the molecular basis of this conformational and functional modulation, we have characterized the NH(2)-terminal variable region-originated secondary conformational effect in TnT using fluorescence spectral analysis. The chicken fast skeletal muscle TnT isoform, TnT8e16, containing a cluster of transition-metal ion binding sites (Tx) in the NH(2)-terminal variable region was used in this study. TnT8e16 was titrated for Cu(II) binding-induced changes in fluorescence intensity and anisotropy of the COOH-domain Trp residues (W234, W236, and W285), which demonstrated considerable environmental sensitivity in TnT denaturation studies. Nonlinear Stern-Volmer plots of Trp quenching indicated a metal ion binding-induced conformational change in TnT. Fluorescence anisotropy changes upon metal ion binding indicated a decrease in the mobility of the Trp residues and an increase in the flexibility of fluorescein-labeled Cys263 in the COOH domain. These data support a model that the alternatively spliced NH(2)-terminal variable region of TnT modulates conformation and flexibility of other domains of the protein.

Conformational selection during weak binding at the actin and myosin interface.

The molecular mechanism of the powerstroke in muscle is examined by resonance energy transfer techniques. Recent models suggesting a pre-cocking of the myosin head involving an enormous rotation between the lever arm and the catalytic domain were tested by measuring separation distances among myosin subfragment-2, the nucleotide site, and the regulatory light chain in the presence of nucleotide transition state analogs. Only small changes (<0.5 nm) were detected that are consistent with internal conformational changes of the myosin molecule, but not with extreme differences in the average lever arm position suggested by some atomic models. These results were confirmed by stopped-flow resonance energy transfer measurements during single ATP turnovers on myosin. To examine the participation of actin in the powerstroke process, resonance energy transfer between the regulatory light chain on myosin subfragment-1 and the C-terminus of actin was measured in the presence of nucleotide transition state analogs. The efficiency of energy transfer was much greater in the presence of ADP-AlF(4), ADP-BeF(x), and ADP-vanadate than in the presence of ADP or no nucleotide. These data detect profound differences in the conformations of the weakly and strongly attached cross-bridges that appear to result from a conformational selection that occurs during the weak binding of the myosin head to actin.

Native purification of biomolecules with temperature-mediated hydrophobic modulation liquid chromatography.

The high-resolution purification of native enzymes is impeded by the limitations in the mobile-phase choices required for conventional hydrophobic separations such as in reverse-phase chromatography. To avoid problems associated with varying the composition of the mobile phase, we developed a stationary phase with a hydrophobicity that can be modulated by slight variations in temperature to bind and elute biomolecules. This chromatographic matrix was tested on nucleotide analogs, amino acids, and protein samples. Visualization of the temperature-dependent hydrophobic interaction with the chromatographic matrix was performed with fluorescence microscopy of CY3-ATP. Amino acids adsorbed to the column according to their known hydrophobicities, confirming the hydrophobic nature of their interaction with the matrix. Biomolecules were separated by modulating the hydrophobicity of the column matrix with slight adjustments to the running temperature between 22 and 37 degrees C without changing the mobile phase. Freedom in the choice of a mobile phase for both the loading and the elution of samples provides great practical advantages by eliminating the need for buffer-exchange steps and allowing more native conditions for purifying delicate enzymes, such as myosin.

Determination of fluorescent probe orientations on biomolecules by conformational searching: algorithm testing and applications to the atomic model of myosin.

The ability of a localized conformational searching method to predict probe orientation was tested on model nucleic acid and protein structures and applied to the prediction of skeletal myosin integrity upon chemical modification of its reactive thiols. Double-stranded oligonucleotides were chemically labeled with donor and acceptor resonance energy transfer probes at each end for distance determinations. These measurements were made independently using a terbium chelate as a donor to each of four chemically and spectroscopically distinct acceptor probes from the xanthene and cyanine dye groups. The choice of acceptor significantly affected the separation distance measured. Conformational searching algorithms on the atomic model corrected for the differences to within 0.2 nm on average. Verifying its usefulness on proteins, the localized conformational searching method determined the orientation of a fluorescent probe on RNase A that corresponds closely to available crystallographic models of the labeled protein (RMS deviation = 0.1 nm). Also, analysis of the symmetry of the fluorophores' structures suggests why FRET orientation factors are often closer to their dynamic average value than might normally be expected. Furthermore, the computational method provides insights about FRET data that are important for assessing the stability of the alpha-helix separating the SH1 and SH2 reactive thiols in skeletal myosin.

Domain motion between the regulatory light chain and the nucleotide site in skeletal myosin.

Resonance energy transfer probes were attached to skeletal myosin's nucleotide site and regulatory light chain (RLC) to examine nucleotide analog-induced structural transitions. A novel chemical modification of the RLC was developed for specific labeling of the basic N-terminus without affecting myosin ATPase activity. The modification allows attachment of a terbium chelate to rabbit skeletal RLC and was mapped by tryptic digestion to an amino group on the six N-terminal RLC residues. The use of terbium as a resonance energy transfer donor allowed the determination of the efficiency of energy transfer by sensitized emission lifetime measurements that practically eliminate background from unlabeled donor and acceptor sites as well as potential orientation factor artifacts in the calculation of the critical transfer distance. The nucleotide site was labeled with a functional CY3-labeled nucleotide as an energy transfer acceptor. Of the nucleotide states examined, ADP, ADP. vanadate, ADP. A1F4, and ADP. BeFx, the difference between the ADP and ADP. vanadate states was greatest (0.4-nm change), but was not considered to be statistically significant. The binding of actin to ADP-myosin also failed to produce a statistically significant change (0.3-nm change). These results are not consistent with a number of versions of the swinging lever arm hypothesis.

In situ molecular association of dystrophin with actin revealed by sensitized emission immuno-resonance energy transfer.

A novel method was developed to detect molecular associations of dystrophin with actin in cryostat muscle tissue sections by combining resonance energy transfer technology with immunohistochemical techniques. This method takes advantage of the long phosphorescent lifetime of terbium chelates, a property that enables the accurate determination of energy transfer in biological tissues by lifetime measurements of sensitized emission. After a brief excitation pulse, terbium chelates emit for milliseconds after the intrinsically high autofluorescence of biological specimens has decayed to negligible levels. Rat skeletal muscle tissue sections were labeled with both anti-dystrophin monoclonal antibody conjugated to a terbium-based resonance energy transfer donor and anti-actin tetramethylrhodamine phalloidin as an acceptor. Resonance energy transfer between the two probes indicated that the distance separating the probes is within 10 nm (about the size of an IgG2b antibody molecule). The fraction of antibodies that participated in resonance energy transfer was estimated to be 80-90% because of the close agreement between the quenching of donor phosphorescence and the efficiency of resonance energy transfer revealed by lifetime measurements of sensitized emission by tetramethyl-rhodamine phalloidin. Sensitized emission was detectable only when both anti-dystrophin antibody and tetramethyl-rhodamine phalloidin were present. These results indicate that actin and dystrophin are closely associated within the cell. This method is potentially applicable to the investigation of many types of intracellular associations.

Calmodulin-sensitive interaction of human nebulin fragments with actin and myosin.

Nebulin is a giant protein ruler of the actin filament of skeletal muscle. This modular protein primarily consists of a repeating sequence (module) of 35 residues and a superrepeat of seven modules. These modules are thought to be actin binding domains along the length of nebulin. Cloned human nebulin fragments of 7-8 modules bind with high affinity to calmodulin, actin, myosin, and myosin head. The stoichiometry of high-affinity binding is approximately one actin monomer bound per nebulin fragment and one myosin bound per nebulin fragment, as determined by cosedimentation binding assays. These observations raise the intriguing possibility that nebulin might have regulatory functions on actomyosin interactions. Nebulin fragments from the N-terminal half situated in the actomyosin overlap region of the sarcomere inhibit actomyosin ATPase activity and the sliding velocity of actin over myosin in motility assays, while a nebulin fragment near the C-terminus, which is localized to the Z-line, does not prevent actin sliding. Calmodulin reverses the inhibition of ATPase and accelerates actin sliding in calcium-dependent manner. Calmodulin with calcium greatly reduces the binding of nebulin fragments to both actin and myosin. The data suggest that the nebulin can interact with both actin and myosin in a calcium/calmodulin-dependent manner and might have regulatory functions in skeletal muscle contraction in a fashion analogous to caldesmon in smooth muscle.

Kinetic silver staining and quantification of proteins adsorbed to microtiter plates.

A silver stain was used to detect and quantitate proteins adsorbed to microtiter plate wells. The kinetics of the development of the silver stain were analyzed with an automated microtiter plate reader. The lag time for stain development was found to be a consistent indicator of the amount of protein adsorbed to a microtiter plate well. Protein which was not preadsorbed to the microtiter plate was not effectively stained by silver. Complete adsorption of protein applied to the microtiter plate was possible by drying small amounts of protein in very dilute buffers. Variations in sensitivity for different proteins were less than 30% for the panel of proteins examined. Determinations from kinetic silver staining agreed with those from copper staining for bovine albumin adsorbed to microtiter plates. The precision of kinetic silver staining assay was optimal in the range of 40 to 200 ng per microtiter plate well. In this range, the standard deviations averaged less than 5%. Even smaller amounts of protein can be detected and interpolated down to approximately 10 ng per well. The kinetic silver staining method can be used on standard microtiter plate readers without special filters and is readily adaptable to automated systems.

Silver-enhanced copper staining of protein blots.

Copper staining of protein blots has previously been shown to detect proteins rapidly, quantitatively, and sensitively. The sensitivity of this assay can now be enhanced nearly 10-fold with an additional step of silver staining. The silver-enhanced assay retains many of the virtues of copper staining, including a quantitative optical response with the amount of adsorbed protein, low variation between proteins, and reversibility. In addition, both copper staining and silver-enhanced copper staining do not detect nucleic acid, thus allowing for selective measurement of nucleic acid binding proteins or protein contamination in nucleic acid preparations. Silver-enhancement requires only a 5-min incubation with a single reagent. Since copper staining is performed first, it can be used to determine if the greater sensitivity provided by silver enhancement is desirable. For quantitation, an inexpensive reflectance scanning densitometer utilizing optical scanners and NIH Image is described.

Cooperativity of thiol-modified myosin filaments. ATPase and motility assays of myosin function.

The effects of chemical modifications of myosin's reactive cysteines on actomyosin adenosine triphosphatase (ATPase) activities and sliding velocities in the in vitro motility assays were examined in this work. The three types of modifications studied were 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3- diazole labeling of SH2 (based on Ajtai and Burghart. 1989. Biochemistry. 28:2204-2210.), phenylmaleimide labeling of SH1, and phenylmaleimide labeling of myosin in myofibrils under rigor conditions. Each type of modified myosin inhibited the sliding of actin in motility assays. The sliding velocities of actin over copolymers of modified and unmodified myosins in the motility assay were slowest with rigor-modified myosin and most rapid with SH2-labeled myosin. The actin-activated ATPase activities of similarly copolymerized myosins were lowest with SH2-labeled myosin and highest with rigor-modified myosin. The actin-activated ATPase activities of myosin subfragment-1 obtained from these modified myosins decreased in the same linear manner with the fraction of modified heads. These results are interpreted using a model in which the sliding of actin filaments over myosin filaments decreases the probability of myosin activation by actin. The sliding velocity of actin over monomeric rigor-modified myosin exceeded that over the filamentous form, which suggests for this myosin that filament structure is important for the inhibition of actin sliding in motility assays. The fact that all cysteine modifications examined inhibited the actomyosin ATPase activities and sliding velocities of actin over myosin poses questions concerning the information about the activated crossbridge obtained from probes attached to SH1 or SH2 on myosin.

The accessibility of etheno-nucleotides to collisional quenchers and the nucleotide cleft in G- and F-actin.

Recent publication of the atomic structure of G-actin (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., & Holmes, K. C., 1990, Nature 347, 37-44) raises questions about how the conformation of actin changes upon its polymerization. In this work, the effects of various quenchers of etheno-nucleotides bound to G- and F-actin were examined in order to assess polymerization-related changes in the nucleotide phosphate site. The Mg(2+)-induced polymerization of actin quenched the fluorescence of the etheno-nucleotides by approximately 20% simultaneously with the increase in light scattering by actin. A conformational change at the nucleotide binding site was also indicated by greater accessibility of F-actin than G-actin to positively, negatively, and neutrally charged collisional quenchers. The difference in accessibility between G- and F-actin was greatest for I-, indicating that the environment of the etheno group is more positively charged in the polymerized form of actin. Based on calculations of the change in electric potential of the environment of the etheno group, specific polymerization-related movements of charged residues in the atomic structure of G-actin are suggested. The binding of S-1 to epsilon-ATP-G-actin increased the accessibility of the etheno group to I- even over that in Mg(2+)-polymerized actin. The quenching of the etheno group by nitromethane was, however, unaffected by the binding of S-1 to actin. Thus, the binding of S-1 induces conformational changes in the cleft region of actin that are different from those caused by Mg2+ polymerization of actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalytic cooperativity induced by SH1 labeling of myosin filaments.

Modifications of SH1 groups on isolated myosin subfragment 1 (S-1) and myosin in muscle fibers affect differently the acto-S-1 ATPase and the fiber properties. Consistent with the findings of earlier work on fibers, the modification of SH1 groups in relaxed myofibrils with phenylmaleimide caused a loss of their shortening. This loss paralleled the decrease in the Vmax of extracted myosin but was not linear with the extent of SH1 labeling. Strikingly, the decrease in Vmax of S-1 prepared from the modified myofibrils was directly proportional to the extent of SH1 labeling. The specificity of SH1 labeling in myofibrils was verified by ATPase activities, thiol titrations, radiolabeling experiments, and comparisons to myosin labeled on SH1 in solution. To test for intermolecular interactions in the myosin filaments and their contribution to the differences between S-1 and myosin, the catalytic properties of copolymers of myosin were examined. Copolymers of myosin and rod minifilaments were formed in 5 mM citrate-Tris (pH 8.0) buffer, and their homogeneity was verified by sedimentation velocity analysis. The inhibition of actomyosin ATPase by rod particles was related to the decrease in the Km value. When rod particles were replaced in these minifilaments by SH1-modified myosin, the ATPase of the copolymers was increased over that of the combined ATPases of the individual filaments. The actomyosin ATP turnover rates on the unmodified heads were increased severalfold by the modified heads.(ABSTRACT TRUNCATED AT 250 WORDS)

Copper iodide staining and determination of proteins adsorbed to microtiter plates.

Copper iodide staining and determination of proteins adsorbed to polystyrene microtiter plates are described. The minimum amount of copper iodide-stained protein detected in densitometric measurements is approximately 20 pg/mm2. Enzyme immunoassay readers may also be used for the determination of copper iodide-stained proteins, but are less sensitive than densitometers. The densitometric readings of copper iodide-stained proteins vary linearly with the amount of protein present as verified by enzymatic and radioactive probes. Staining is complete in 2-3 min and may be removed by a 30-min treatment with EDTA without loss of adsorbed protein or immunoreactivity. The exact amount of protein adsorbed to microtiter plate wells can be measured by using protein bound and stained on nitrocellulose as a calibration curve. Copper iodide staining is a rapid, convenient, and inexpensive alternative to radioactive measurements of similar parameters.

Copper iodide staining of protein blots on nitrocellulose membranes.

Copper iodide staining which can detect protein levels as low as 100-150 pg/mm2 on nitrocellulose membranes is described. The staining is quantitative as measured by densitometry. Staining is complete within 5 min and may be removed by washing the membrane for 15 min without loss of immunoreactivity. The stain utilizes a reddish-brown precipitate of copper iodide in highly alkaline conditions. Because of its high sensitivity, convenience, and low cost, this stain may be more practical than amido black or gold- and silver-based stains for most laboratory purposes.