Negative staining of myosin molecules

A reproducible method has been developed for the negative staining of myosin molecules. The dimensions of stained molecules are in close agreement with those obtained by metal shadowing. Sharp bends in the tail, indicative of hinge regions, were observed at two positions 44 nm and 76 nm from the head-tail junction. The tail was often ill-defined at the position of the first (44 nm) bend. The bend positions may be sites of proteolytic cleavage that result in the production of long and short myosin subfragment S2. About half the molecules exhibited bending to various degrees at one or both of these positions, but cases where the tail folded back on itself in a 180 degrees bend were comparatively rare (approximately equal to 10%). However, in the absence of EGTA, a large fraction of the molecules (approximately equal to 80%) exhibited 180 degrees bends. A small region, approximately 20 nm long, at the tip of the tail often appears to be significantly different from the rest. The heads are about 19 nm long and roughly pear-shaped. Although sometimes straight, more often they show a pronounced curvature. Both senses of curvature were observed, but those curved in a clockwise manner were the most common, indicating preferential binding of one side of the head to the carbon substrate. An analysis of the different combinations of head shapes in individual molecules indicates that each head can rotate independently around its long axis. No preferred angle of orientation between the two heads in a molecule, or between either head and the tail could be found. Substructure has been observed within the heads.     

A new model for the surface arrangement of myosin molecules in tarantula thick filaments

Three-dimensional reconstructions of the negatively stained thick filaments of tarantula muscle with a resolution of 50 A have previously suggested that the helical tracks of myosin heads are zigzagged, short diagonal ridges being connected by nearly axial links. However, surface views of lower contour levels reveal an additional J-shaped feature approximately the size and shape of a myosin head.We have modelled the surface array of myosin heads on the filaments using as a building block a model of a two-headed regulated myosin molecule in which the regulatory light chains of the two heads together form a compact head-tail junction. Four parameters defining the radius, orientation and rotation of each myosin molecule were varied. In addition, the heads were allowed independently to bend in a plane perpendicular to the coiled-coil tail at three sites, and to tilt with respect to the tail and to twist at one of these sites. After low-pass filtering, models were aligned with the reconstruction, scored by cross-correlation and refined by simulated annealing.Comparison of the geometry of the reconstruction and the distance between domains in the myosin molecule narrowed the choice of models to two main classes. A good match to the reconstruction was obtained with a model in which each ridge is formed from the motor domain of a head pointing to the bare zone together with the head-tail junction of a neighbouring molecule. The heads pointing to the Z-disc intermittently occupy the J-position. Each motor domain interacts with the essential and regulatory light chains of the neighbouring heads. A near-radial spoke in the reconstruction connecting the backbone to one end of the ridge can be identified as the start of the coiled-coil tail.     

Morphological aspects of thiophosphorylated and dephosphorylated myosin molecules from the plasmodium of Physarum polycephalum

Electron microscopically, the myosin molecule from the plasmodium of Physarum polycephalum has a long tail of 173 nm, having a flexible region over the range of 80 to 120 nm from the head-tail junction. In 0.6 M ammonium acetate, this region of the dephosphorylated myosin molecules is more flexible than that of the thiophosphorylated ones. In 50 mM ammonium acetate, the dephosphorylated myosin molecules exist in monomeric and oligomeric forms, independently of ATP and Mg2+, whereas the thiophosphorylated myosin molecules form dense aggregates of thick filaments. The tails of the monomeric dephosphorylated myosin molecules bend sharply at the flexible region at angles of more than 120 degrees. In oligomers of the dephosphorylated myosin molecules, the molecules are all associated side-to-side with straight tails and are oriented in the same direction. Based on these results, the regulation mechanism of cell motility of the plasmodium is discussed.     

Cryo-atomic force microscopy of unphosphorylated and thiophosphorylated single smooth muscle myosin molecules

The purpose of this study was to determine whether steric blockage of one head by the second head of native two-headed myosin was responsible for the inactivity of nonphosphorylated two-headed myosin compared with the high activity of single-headed myosin, as suggested on the basis of electron microscopy of two-dimensional crystals of heavy meromyosin (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385-1390; and Wendt, T., Taylor, D., Trybus, K. M., and Taylor, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4361-4366). Our earlier cryo-atomic force microscopy (cryo-AFM) (Zhang, Y., Shao, Z., Somlyo, A. P., and Somlyo, A. V. (1997) Biophys. J. 72, 1308-1318) indicates that thiophosphorylation of the regulatory light chain increases the separation of the two heads of a single myosin molecule, but the thermodynamic probability of steric hindrance by strong binding between the two heads was not determined. We now report this probability determined by cryo-AFM of single whole myosin molecules shown to have normal low ATPase activity (0.007 s-1). We found that the thermodynamic probability of the relative head positions of nonphosphorylated myosin was approximately equal between separated heads as compared with closely apposed heads (energy difference of 0.24 kT (where k is a Boltzman constant and T is the absolute temperature)), and thiophosphorylation increased the number of molecules having separated heads (energy advantage of -1.2 kT (where k is a Boltzman constant and I is the absolute temperature)). Our results do not support the suggestion that strong binding of one head to the other stabilizes the blocked conformation against thermal fluctuations resulting in steric blockage that can account for the low activity of nonphosphorylated two-headed myosin.     

Skip residues correlate with bends in the myosin tail

Sharp bends have previously been observed in the tail of the skeletal myosin molecule at well-defined positions 44, 75 and 135 nm from the head-tail junction, and in vertebrate smooth myosin at two positions about 45 and 96 nm from this junction. The amino acid sequence of the heavy chain does not straightforwardly account for such bending on the original model of the tail in which an invariant proline residue is present at the head-tail junction and the repeating seven amino acid pattern of hydrophobic residues lies entirely in the tail. Recently, a revised model has been proposed by Rimm et al. in which the first seven to eight heptads lie in the heads. It is shown here that with this model the observed bends in the tail of skeletal myosin coincide with three of the four additional (skip) residues that interrupt the heptad repeat. It is concluded that the skip residues, by causing localized instability of the coiled-coil, are responsible for the bends. Smooth myosin lacks the second of these skip residues explaining the absence of a bend at 75 nm.     

Kinetics of smooth muscle heavy meromyosin with one thiophosphorylated head

Actin-activated MgATPase of smooth muscle heavy meromyosin is activated by thiophosphorylation of two regulatory light chains, one on each head domain. To understand cooperativity between heads, we examined the kinetics of heavy meromyosin (HMM) with one thiophosphorylated head. Proteolytic gizzard heavy meromyosin regulatory light chains were partially exchanged with recombinant thiophosphorylated His-tagged light chains, and HMM with one thiophosphorylated head was isolated by nickel-affinity chromatography. In vitro motility was observed. By steady-state kinetic analysis, one-head thiophosphorylated heavy meromyosin had a similar K(m) value for actin but a V(max) value of approximately 50% of the fully thiophosphorylated molecule. However, single turnover analysis, which is not sensitive to small amounts of active heads, showed that one-head thiophosphorylated heavy meromyosin was 46-120 times more active than unphosphorylated HMM but only 7-19% as active as the fully thiophosphorylated molecule. Discrepancy between the single turnover and steady-state values could be explained by a small fraction of rigor heads. These rigor heads would have a large effect on the steady-state kinetics of one-head thiophosphorylated HMM. In summary, thiophosphorylation of one head leads to a molecule with unique intermediate kinetics suggesting that thiophosphorylation of one head cooperatively alters the kinetics of the partner head and vice versa.     

Two-headed binding of the unphosphorylated nonmuscle heavy meromyosin.ADP complex to actin

The enzymatic and motor function of smooth muscle and nonmuscle myosin II is activated by phosphorylation of the regulatory light chains located in the head portion of myosin. Dimerization of the heads, which is brought about by the coiled-coil tail region, is essential for regulation since single-headed fragments are active regardless of the state of phosphorylation. Utilizing the fluorescence signal on binding of myosin to pyrene-labeled actin filaments, we investigated the interplay of actin and nucleotide binding to thiophosphorylated and unphosphorylated recombinant nonmuscle IIA heavy meromyosin constructs. We show that both heads of either thiophosphorylated or unphosphorylated heavy meromyosin bind very strongly to actin (K(d) < 10 nM) in the presence or absence of ADP. The heads have high and indistinguishable affinities for ADP (K(d) around 1 microM) when bound to actin. These findings are in line with the previously observed unusually loose coupling between nucleotide and actin binding to nonmuscle myosin IIA subfragment-1 (Kovács et al. (2003) J. Biol. Chem. 278, 38132.). Furthermore, they imply that the structure of the two heads in the ternary actomyosin-ADP complex is symmetrical and that the asymmetrical structure observed in the presence of ATP and the absence of actin in previous investigations (Wendt et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 4361) is likely to represent an ATPase intermediate that precedes the actomyosin-ADP state.     

Phosphorylation regulates the ADP-induced rotation of the light chain domain of smooth muscle myosin.

We have observed the effects of MgADP and thiophosphorylation on the conformational state of the light chain domain of myosin in skinned smooth muscle. Electron paramagnetic resonance (EPR) spectroscopy was used to monitor the orientation of spin probes attached to the myosin regulatory light chain (RLC). Two spectral states were seen, termed here "intermediate" and "final", that are distinguished by a approximately 24 degrees axial rotation of spin probes attached to the RLC. The two observed conformations are similar to those found previously for smooth muscle myosin S1; the final state corresponds to the major conformation of S1 in the absence of ADP, while the intermediate state corresponds to the conformation of S1 with ADP bound. Light chain domain orientation was observed as a function of the MgADP concentration and the extent of RLC thiophosphorylation. In rigor (no MgADP), LC domains were distributed equally between the intermediate state and the final state; upon addition of saturating (3.5 mM) MgADP, about one-third of the LC domains in the final state rotated approximately 20 degrees axially to the intermediate state. The progression of the change in populations was fit to a simple binding equation, yielding an apparent dissociation constant of approximately 110 microM for skinned smooth muscle fibers and approximately 730 microM for thiophosphorylated, skinned smooth muscle fibers. These observations suggest a model that explains the behavior of "latch bridges" in smooth muscle.     

Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown state

Remodelling the contractile apparatus within smooth muscle cells allows effective contractile activity over a wide range of cell lengths. Thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. Using negative stain electron microscopy of individual folded myosin molecules from turkey gizzard smooth muscle, we show that they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin, with features identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2D crystals on lipid monolayers. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2D crystal. The tail of whole myosin is bent sharply and consistently close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. Tail segments 2 and 3 associate only with the blocked head, such that the second bend is near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young's modulus of about 0.5 GPa. The folded tail of the whole myosin is less flexible, indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule.     

Atomic force microscopy of the myosin molecule.

Atomic force microscopy (AFM) has been used to study the structure of rabbit skeletal muscle myosin deposited onto a mica substrate from glycerol solution. Images of the myosin molecule have been obtained using contact mode AFM with the sample immersed in propanol. The molecules have two heads at one end of a long tail and have an appearance similar to those prepared by glycerol deposition techniques for electron microscopy, except that the separation of the two heads is not so well defined. The average length of the tail (155 +/- 5 nm) agrees well with previous studies. Bends in the myosin tail have been observed at locations similar to those observed in the electron microscope. By raising the applied force, it has been possible locally to separate the two strands of the alpha-helical coiled-coil tail. We conclude that the glycerol-mica technique is a useful tool for the preparation of fibrous proteins for examination by scanning probe microscopy.     

Coiled-coil unwinding at the smooth muscle myosin head-rod junction is required for optimal mechanical performance

Myosin II has two heads that are joined together by an alpha-helical coiled-coil rod, which can separate in the region adjacent to the head-rod junction (Trybus, K. M. 1994. J. Biol. Chem. 269:20819-20822). To test whether this flexibility at the head-rod junction is important for the mechanical performance of myosin, we used the optical trap to measure the unitary displacements of heavy meromyosin constructs in which a stable coiled-coil sequence derived from the leucine zipper was introduced into the myosin rod. The zipper was positioned either immediately after the heads (0-hep zip) or following 15 heptads of native sequence (15-hep zip). The unitary displacement (d) decreased from d = 9.7 +/- 0.6 nm for wild-type heavy meromyosin (WT HMM) to d = 0.1 +/- 0.3 nm for the 0-hep zip construct (mean +/- SE). Native values were restored in the 15-hep zip construct (d = 7.5 +/- 0.7 nm). We conclude that flexibility at the myosin head-rod junction, which is provided by an unstable coiled-coil region, is essential for optimal mechanical performance.     

Location of the head-tail junction of myosin

The tails of double-headed myosin molecules consist of an alpha-helical/coiled-coil structure composed of two identical polypeptides with a heptad repeat of hydrophobic amino acids that starts immediately after a conserved proline near position 847. Both muscle and nonmuscle myosins have this heptad repeat and it has been assumed that proline 847 is physically located at the head-tail junction. We present two lines of evidence that this assumption is incorrect. First, we localized the binding sites of several monoclonal antibodies on Acanthamoeba myosin-II both physically, by electron microscopy, and chemically, with a series of truncated myosin-II peptides produced in bacteria. These data indicate that the head-tail junction is located near residue 900. Second, we compared the lengths of two truncated recombinant myosin-II tails with native myosin-II. The distances from the NH2 termini to the tips of these short tails confirms the rise per residue (0.148 nm/residue) and establishes that the 86-nm tail of myosin-II must start near residue 900. We propose that the first 53 residues of heptad repeat of Acanthamoeba myosin-II and other myosins are located in the heads and the proteolytic separation of S-1 from rod occurs within the heads.     

Time-resolved measurements of phosphate release by cycling cross-bridges in portal vein smooth muscle.

The rate of release of inorganic phosphate (Pi) from cycling cross-bridges in rabbit portal-anterior mesenteric vein smooth muscle was determined by following the fluorescence of the Pi-reporter, MDCC-PBP (Brune, M., J. L. Hunter, S. A. Howell, S. R. Martin, T. L. Hazlett, J. E. T. Corrie, and M. R. Webb. 1998. Biochemistry. 37:10370-10380). Cross-bridge cycling was initiated by photolytic release of ATP from caged-ATP in Triton-permeabilized smooth muscles in rigor. When the regulatory myosin light chains (MLC20) had been thiophosphorylated, the rate of Pi release was biphasic with an initial rate of 80 microM s-1 and amplitude 108 microM, decreasing to 13.7 microM s-1. These rates correspond to fast and slow turnovers of 1.8 s-1 and 0.3 s-1, assuming 84% thiophosphorylation of 52 microM myosin heads. Activation by Ca2+-dependent phosphorylation subsequent to ATP release resulted in slower Pi release, paralleling the rate of contraction that was also slower than after thiophosphorylation, and was also biphasic: 51 microM s-1 and 13.2 microM s-1. These rates suggest that the activity of myosin light chain kinase and phosphatase ("pseudo-ATPase") contributes <20% of the ATP usage during cross-bridge cycling. The extracellular "ecto-nucleotidase" activity was reduced eightfold by permeabilization, conditions in which the ecto-ADPase was 17% of the ecto-ATPase. Nevertheless, the remaining ecto-ATPase activity reduced the precision of the estimate of cross-bridge ATPase. We conclude that the transition from fast to slow ATPase rates reflects the properties and forces directly acting on cross-bridges, rather than the result of a time-dependent decrease in activation (MLC20 phosphorylation) occurring in intact smooth muscle. The mechanisms of slowing may include the effect of positive strain on cross-bridges, inhibition of the cycling rate by high affinity Mg-ADP binding, and associated state hydrolysis.     

Identification of functional regions on the tail of Acanthamoeba myosin- II using recombinant fusion proteins. II. Assembly properties of tails with NH2- and COOH-terminal deletions

We used purified fusion proteins containing parts of the Acanthamoeba myosin-II tail to localize those regions of the tail responsible for each of the three steps in the successive dimerization mechanism (Sinard, J. H., W. F. Stafford, and T. D. Pollard. 1989. J. Cell Biol. 107:1537-1547) for Acanthamoeba myosin-II minifiliment assembly. Fusion proteins containing the terminal approximately 90% of the myosin-II tail assemble normally, but deletions within the last 100 amino acids of the tail sequence alter or prevent assembly. The first step in minifilament assembly, formation of antiparallel dimers, requires the COOH-terminal approximately 30 amino acids that are thought to form a nonhelical domain at the end of the coiled-coil. The second step, formation of antiparallel tetramers, requires the last approximately 40 residues in the coiled-coil. The final step, the association of two antiparallel tetramers to form the completed octameric minifilament, requires residues approximately 40-70 from the end of the coiled-coil. A region of the tail near the junction with the heads is important for tight packing of the tails in the minifilaments. Divalent cations induce the lateral aggregation of minifilaments formed from native myosin-II or fusion proteins containing a nonmyosin "head," but under the same conditions fusion proteins composed essentially only of myosin tail sequences with very little nonmyosin sequences form paracrystals. The region of the tail necessary for this paracrystal formation lies NH2-terminal to amino acid residue 1,468 in the native myosin-II sequence.     

A flexible domain is essential for the large step size and processivity of myosin VI

Myosin VI moves processively along actin with a larger step size than expected from the size of the motor. Here, we show that the proximal tail (the approximately 80-residue segment following the IQ domain) is not a rigid structure but, rather, a flexible domain that permits the heads to separate. With a GCN4 coiled coil inserted in the proximal tail, the heads are closer together in electron microscopy (EM) images, and the motor takes shorter processive steps. Single-headed myosin VI S1 constructs take nonprocessive 12 nm steps, suggesting that most of the processive step is covered by a diffusive search for an actin binding site. Based on these results, we present a mechanical model that describes stepping under an applied load.     

Phosphorylation-dependent structural changes in the regulatory light chain domain of smooth muscle heavy meromyosin.

Smooth muscle heavy meromyosin, a double-headed proteolytic fragment of myosin lacking the COOH-terminal two-thirds of the tail, has been shown previously to be regulated by phosphorylation. To examine phosphorylation-dependent structural changes near the head-tail junction, we prepared five well regulated heavy meromyosins containing single-cysteine mutants of the human smooth muscle regulatory light chain labeled with the photocross-linking reagent, benzophenone-iodoacetamide. For those mutants that generated cross-links, only one type of cross-linked species was observed, a regulatory light chain dimer. Irradiated mutants fell into two classes. First, for Q15C, A23C, and wild type (Cys-108), a regulatory light chain dimer was formed for dephosphorylated but not thiophosphorylated heavy meromyosin. These data provide direct chemical evidence that in the dephosphorylated state, Gln-15, Ala-23, and Cys-108 on one head are positioned near (within 8.9 A) the regulatory light chain of the partner head and that thiophosphorylation abolishes proximity. This behavior was also observed for the Q15C mutant on a truncated heavy meromyosin lacking both catalytic domains. For the actin-heavy meromyosin complex, cross-links were formed in both de- and thiophosphorylated states. S59C and T134C mutants were in a second mutant class, where regulatory light chain dimers were not detected in dephosphorylated or thiophosphorylated heavy meromyosin, suggesting positions outside the region of interaction of the regulatory light chains.     

The relationship between ATPase activity, isometric force, and myosin light-chain phosphorylation and thiophosphorylation in skinned smooth muscle fiber bundles from chicken gizzard

Isometric force developed by skinned gizzard muscle fiber bundles and levels of phosphorylation and thiophosphorylation of the 20,000-dalton myosin light chain were determined. These data showed a highly non-linear relationship between isometric force and myosin light-chain phosphorylation. Maximum force was developed at approximately 0.2 mol of phosphate/mol of light chain as reported previously (Hoar, P. E., Kerrick, W. G. L., and Cassidy, P. S. (1979) Science 204, 503-506). In contrast, the relationship between isometric force and myosin light-chain thiophosphorylation was linear, with maximum force occurring at 1.0 mol of thiophosphate/mol of myosin light chain. These observations are consistent with the latch-bridge hypothesis for conditions of varying myosin light-chain phosphatase/myosin light-chain kinase activity ratios as discussed by Hai and Murphy [1988) Am. J. Physiol. 254, C99-C106). To further test the latch-bridge hypothesis, ATPase activity was also measured during isometric force development in these fiber bundles. The relationship between isometric force and ATPase activity was linear whether the myosin light chains were phosphorylated or thiophosphorylated. Thus the number of cycling myosin cross-bridges, as measured by ATPase activity, was directly proportional to the force the muscle developed, not to the level of myosin light-chain phosphorylation. This finding that high levels of tension generated at low levels of light-chain phosphorylation are associated with high levels of ATPase activity is inconsistent with the latch-bridge model (Hai and Murphy, 1988).     

Structure of the myosin projections on native thick filaments from vertebrate skeletal muscle

Rabbit psoas muscle filaments, isolated in relaxing buffer from non-glycerinated muscle, have been applied to hydrophilic carbon films and stained with uranyl acetate. Electron micrographs were obtained under low-dose conditions to minimize specimen damage. Surrounding the filament backbone, except in the bare zone, is a fringe of clearly identifiable myosin heads. Frequently, both heads of individual myosin molecules are seen, and sometimes a section of the tail can be seen connecting the heads to the backbone. About half the expected number of heads can be counted, and they are uniformly distributed along the filament. The majority of heads appear curved. The remainder could be curved heads viewed from another aspect. Three times as many heads curve in a clockwise sense than in an anticlockwise sense, suggesting a preferential binding of one side of the head to the carbon film. The two heads of myosin molecules exhibit all the possible combinations of clockwise, anticlockwise and straight heads, and analysis of their relative frequencies suggests that the heads rotate freely and independently. The heads also adopt a wide range of angles of attachment to the tail. The lengths of heads cover a range of 14 to 26 nm, with a peak at 19 nm. The average maximum width is 6.5 nm. Both measurements are in excellent agreement with values for shadowed molecules. Since our data are from heads adsorbed to the film in relaxing conditions and the shadowed molecules were free of nucleotide, gross shape changes are not likely to be produced by nucleotide binding. The length of the link between the heads and the backbone was found to vary between 10 nm and 52 nm, with a broad peak at about 25 nm. Thus, the hinge point detected in the tail of isolated molecules was not usually the point from which the crossbridges swung out from the filament surface. The angle made by the link to the filament axis was between 20 degrees and 80 degrees, with a broad maximum around 45 degrees. These lengths and angles concur with our observation of an average limit of the crossbridges from the filament surface of 30 nm. This is sufficient to enable heads in the myofibril lattice to reach out beyond the nearest thin filament and should allow considerable flexibility for stereospecific binding to actin in active muscle.     

Intermolecular versus intramolecular interactions of Dictyostelium myosin: possible regulation by heavy chain phosphorylation

Dictyostelium myosin has been examined under conditions that reveal intramolecular and intermolecular interactions that may be important in the process of assembly and its regulation. Rotary shadowed myosin molecules exhibit primarily two configurations under these conditions: straight parallel dimers and folded monomers. All of the monomers bend in a specific region of the 1860-A-long tail that is 1200 A from the head-tail junction. Molecules in parallel dimers are staggered by 140 A, which is a periodicity in the packing of myosin molecules originally observed in native thick filaments of muscle. The most common region for interaction in the dimers is a segment of the tail about 200-A-long, extending from 900 to 1100 A from the head-tail junction. Parallel dimers form tetramers by way of antiparallel interactions in their tail regions with overlaps in multiples of 140 A. The folded configuration of the myosin molecules is promoted by phosphorylation of the heavy chain by Dictyostelium myosin heavy chain kinase. It appears that the bent monomers are excluded from filaments formed upon addition of salt while the dimeric molecules assemble. These results may provide the structural basis for primary steps in myosin filament assembly and its regulation by heavy chain phosphorylation.     

Selective enrichment of thiophosphorylated polypeptides as a tool for the analysis of protein phosphorylation

A chemoselective alkylation method is described for the isolation and subsequent identification of thiophosphorylated peptides/proteins. The method involves thiophosphorylation of proteins using adenosine 5'-O-(thiotriphosphate) (ATPgammaS) followed by selective in situ alkylation of the newly thiophosphorylated proteins resulting in a stable covalent bond. The chemoselective alkylation exploits the relatively high nucleophilicity at low pH of the sulfur in thiophosphate residues, whereas the nucleophilicities of phosphates, amines, and other functionality of amino acids are negligible or significantly suppressed. Modified alkylation reagents linked to biotin or solid supports (e.g. glass or Sepharose beads) with or without a photocleavable linker facilitate the isolation of the thiophosphorylated peptide/proteins. This approach is demonstrated through the localization of phosphorylation sites on myosin regulatory light chain. We anticipate that this technique will be useful for isolation and subsequent identification of newly thiophosphorylated proteins, produced either in vivo or in vitro, thus facilitating the dissection of protein phosphorylation networks.