Microsecond rotational motions of eosin-labeled myosin measured by time-resolved anisotropy of absorption and phosphorescence

We have studied submicrosecond and microsecond rotational motions within the contractile protein myosin by observing the time-resolved anisotropy of both absorption and emission from the long-lived triplet state of eosin-5-iodoacetamide covalently bound to a specific site on the myosin head. These results, reporting anisotropy data up to 50 microseconds after excitation, extend by two orders of magnitude the time range of data on time-resolved site-specific probe motion in myosin. Optical and enzymatic analyses of the labeled myosin and its chymotryptic digests show that more than 95% of the probe is specifically attached to sulfhydryl-1 (SH1) on the myosin head. In a solution of labeled subfragment-1 (S-1) at 4 degrees C, absorption anisotropy at 0.1 microseconds after a laser pulse is about 0.27. This anisotropy decays exponentially with a rotational correlation time of 210 ns, in good agreement with the theoretical prediction for end-over-end tumbling of S-1, and with times determined previously by fluorescence and electron paramagnetic resonance. In aqueous glycerol solutions, this correlation time is proportional to viscosity/temperature in the microsecond time range. Furthermore, binding to actin greatly restricts probe motion. Thus the bound eosin is a reliable probe of myosin-head rotational motion in the submicrosecond and microsecond time ranges. Our submicrosecond data for myosin monomers (correlation time 400 ns) also agree with previous results using other techniques, but we also detect a previously unresolvable slower decay component (correlation time 2.6 microseconds), indicating that the faster motions are restricted in amplitude. This restriction is not consistent with the commonly accepted free-swivel model of S-1 attachment in myosin. In synthetic thick filaments of myosin, both fast (700 ns) and slow (5 microseconds) components of anisotropy decay are observed. In contrast to the data for monomers, the anisotropy of filaments has a substantial residual component (26% of the initial anisotropy) that does not decay to zero even at times as long as 50 microseconds, implying significant restriction in overall rotational amplitude. This result is consistent with motion restricted to a cone half-angle of about 50 degrees. The combined results are consistent with a model in which myosin has two principal sites of segmental flexibility, one giving rise to submicrosecond motions (possibly corresponding to the junction between S-1 and S-2) and the other giving rise to microsecond motions (possibly corresponding to the junction between S-2 and light meromyosin).(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure of the vanadate-induced crystals of sarcoplasmic reticulum Ca2+-ATPase

The projected structure of the vanadate-induced crystalline aggregates of Ca2+-ATPase molecules in isolated sarcoplasmic reticulum membranes has been determined. The molecules form tubular crystals with an oblique surface lattice having cell dimensions a = 65.9 A, b = 114.4 A and gamma = 77.9 degrees. The space group is P2. The crystalline tubules are formed through lateral aggregation of chains made up of dimers of Ca2+-ATPase molecules.     

Lipid/myelin basic protein multilayers. A model for the cytoplasmic space in central nervous system myelin

A multilayered complex forms when a solution of myelin basic protein is added to single-bilayer vesicles formed by sonicating myelin lipids. Vesicles and multilayers have been studied by electron microscopy, biochemical analysis, and X-ray diffraction. Freeze-fracture electron microscopy shows well-separated vesicles before myelin basic protein is added, but afterward there are aggregated, possibly multilayered, vesicles and extensive planar multilayers. The vesicles aggregate and fuse within seconds after the protein is added, and the multilayers form within minutes. No intra-bilayer particles are seen, with or without the protein. Some myelin basic protein, but no lipid, remains in the supernatant after the protein is added and the complex sedimented for X-ray diffraction. A rather variable proportion of the protein is bound. X-ray diffraction patterns show that the vesicles are stable in the absence of myelin basic protein, even under high g-forces. After the protein is added, however, lipid/myelin basic protein multilayers predominate over single-bilayer vesicles. The protein is in every space between lipid bilayers. Thus the vesicles are torn open by strong interaction with myelin basic protein. The inter-bilayer spaces in the multilayers are comparable to the cytoplasmic spaces in central nervous system myelins . The diffraction indicates the same lipid bilayer thickness in vesicles and multilayers, to within 1 A. By comparing electron-density profiles of vesicles and multilayers, most of the myelin basic protein is located in the inter-bilayer space while up to one-third may be inserted between lipid headgroups. When cytochrome c is added in place of myelin basic protein, multilayers also form. In this case the protein is located entirely outside the unchanged bilayer. Comparison of the various profiles emphasizes the close and extensive apposition of myelin basic protein to the lipid bilayer. Numerous bonds may form between myelin basic protein and lipids. Cholesterol may enhance binding by opening gaps between diacyl-lipid headgroups.     

Structure of tomato bushy stunt virus: V. Coat protein sequence determination and its structural implications

We report the chemically determined sequence of most of the polypeptide chain of the coat protein of tomato bushy stunt virus. Peptide locations have been determined by comparison with the high-resolution electron density map from X-ray crystallographic analysis as well as by conventional chemical overlaps. Three small gaps remain in the 387-residue sequence. Positively charged side-chains are concentrated in the N-terminal part of the polypeptide (the R domain) as well as on inward-facing surfaces of the S domain. There is homology of S-domain sequences with structurally corresponding residues in southern bean mosaic virus.     

Structural implications of primary sequences from a family of Balbiani ring-encoded proteins inChironomus

Summary DNA sequencing has revealed an internal, tandemly repetitive structure in the family of giant polypeptides encoded by three types of Balbiani ring (BR) genes, in three different species ofChironomus. Each major BR repeat can be subdivided into two halves: a region consisting of short subrepeats and a more constant region that lacks obvious subrepeats. Comparative predictions of secondary structure indicate that an -helical segment is consistently present in the amino-terminal half of the constant region in all known BR proteins. Comparative predictions, coupled with consideration of the known phosphorylation of serine and threonine residues in BR proteins, suggest that the -helical structure may also extend into the carboxy-terminal half of the constant region, possibly interrupted by -turn(s). However, it is also possible that the structure is variable, and that a -strand is present in that half in some cases. All of the constant regions conserve one methionine and one phenylalanine residue, as well as all four cysteines; these residues presumably play roles in the packing or cross-linking of aligned constant regions. The structure of the subrepeat region is not clear, but the prevalence of a tripeptide pattern (basic-proline-acidic) suggests some type of structural regularity, possibly an extended helix. The possible significance of these conserved molecular features is discussed in the context of how they may serve the elasticity, insolubility, and hydrophilicity of the fibrils and threads formed by the BR polypeptides.     

β-decay,Bremsstrahlen, and the origin of molecular chirality

Summary A brief review is presented of the Vester-Ulbricht -decay Bremsstrahlen hypothesis for the origin of optical activity, and of subsequent experiments designed to test it. Certain of our experiments along these lines, begun in 1974 and involving the irradiation of racemic and optically active amino acids in a 61.7 KCi⁹⁰Sr–⁹⁰Y Bremsstrahlen source, have now been completed and are described. After 10.89 years of irradiation with a total Bremsstrahlen dose of 2.5×10⁹ rads, crystallinedl-leucine, norleucine, and norvaline suffered 47.2, 33.6, and 27.4% radiolysis, respectively, but showed no evidence whatsoever of asymmetric degradation.d- andl-Leucine underwent about 48% radiolysis and showed 2.4–2.9% radioracemization. Other samples in solution were too severely degraded to analyze. Probable intrinsic reasons for the failure of the Vester-Ulbricht mechanism to afford asymmetric radiolysis in the present and related experiments involving -decay Bremsstrahlen are enumerated.A portion of this material was presented at the 7th International Conference on the Origins of Life, Mainz, FRG, July 10–15, 1983     

Interaction of coupled nonlinear oscillators having different intrinsic resting levels

The interaction among coupled oscillators is governed by oscillator properties (intrinsic frequency and amplitude) and coupling mechanisms. This study considers another oscillator property, the intrinsic resting level, and evaluates its role in governing oscillator interactions. The results of computer experiments on a chain of either three or five bidirectionally coupled nonlinear oscillators, suggest that an intrinsic resting level gradient, if present, is one of the factors governing the interaction between coupled oscillators. If there is no intrinsic frequency gradient, then an intrinsic resting level gradient is sufficient to produce many features of interaction among coupled oscillators. If both intrinsic frequency and intrinsic resting level gradients are present, then both of them determine the manner in which the coupled oscillators interact with each other.