Factors affecting the collection and fitting of nuclear magnetic cross-relaxation spectroscopy data with application to waxy corn starch.

An examination of the methods for nuclear magnetic cross-relaxation spectroscopy (CRS) data collection and analysis was conducted using water and an aqueous waxy corn starch suspension to better perform and interpret the results obtained using CRS. The CRS data collection properties evaluated were the time to achieve steady state saturation, the direct saturation of liquid protons, generation of transverse magnetization, and dependence of the offset frequency and radio frequency (RF) field strength of longitudinal relaxation in the presence of RF saturation. Effects were evaluated for variations of input values of RF saturation field strength, apparent cross-relaxation rate, and solid longitudinal relaxation rate on the results for solid content and solid internal mobility from fitting NMR data to modified theoretical expressions. Discrepancies between fitted and stoichiometric values for the solid to liquid proton ratio were investigated. The fitting procedure used a Gaussian line shape for RF saturation of the solid-like spin system and a Lorentzian line shape for RF saturation of the liquid-spin system. Conditions under which acceptable results can be obtained with limited data sets are discussed.

Ionic effects on the rotational dynamics of cross-bridges in myosin filaments, measured by triplet absorption anisotropy.

We have measured the rotational motion of myosin heads in synthetic thick filaments at 4 degrees C in the time range from 10(-7) to 10(-4) seconds, by measuring transient absorption anisotropy of an eosin probe attached to a reactive sulfhydryl on the myosin head. Under conditions that result in monomeric myosin (500 mM ionic strength), the anisotropy decay is independent of pH in the range from 7.0 to 8.2 and [Mg2+] in the range from 0.1 to 10 mM; the anisotropy decays bi-exponentially with correlation times of 0.4 and 2 microseconds to a constant value of 0.016. Under more physiological conditions (115 mM ionic strength), resulting in filament formation, the anisotropy decay is sensitive to both pH and [Mg2+]. The anisotropy at pH 8.2 and 0.1 mM-Mg2+ decays with correlation times of 0.5 and 3.8 microseconds to a constant limiting anisotropy of 0.038. When the [Mg2+] is increased to 10 mM, the correlation times are 0.6 and 5.7 microseconds and the limiting anisotropy value is 0.055. Identical changes in the anisotropy decay are caused by an increase in [H+] to pH 7.0, in the presence of 0.1 mM-Mg2+. Increasing the total ionic strength to 187 mM decreases the amplitude of the cation effects. These results provide direct evidence that the rotational dynamics of myosin heads in thick filaments are influenced by physiological concentrations of cations. The results are qualitatively consistent with the proposal that these and other ionic conditions regulate transitions between "spread" and "compact" cross-bridge conformations, but the quantitative results indicate that cross-bridges undergo large-amplitude microsecond rotations even under conditions where the compact state should predominate.

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)

Backbone and side-chain motion in myosin, subfragment 1, and rod determined by natural abundance carbon-13 NMR.

We report natural abundance carbon-13 nuclear magnetic resonance studies of myosin, subfragment 1 (S-1), and myosin rod in solution and of myosin filaments. We measured solution spectra at 37.7 MHz, 20 degrees C with scalar proton decoupling. If the native proteins were rigid particles, only S-1 would have observable intensity. In fact, 30% of myosin, 70% of S-1, and 30% of rod carbons are observable. Observable carbons possess rotational correlation times less than 10(-7) s which effectively average 13C-1H dipolar coupling and anisotropic chemical shift interactions. Short alpha-carbon region spin-lattice relaxation times (T1) and nonminimal 13C-1H nuclear Overhauser enhancements suggest restricted nanosecond motions of backbone atoms. This is direct evidence for internal motion of backbone and side-chain carbons in myosin and its fragments. Double resonance experiments at 15.1 MHz and 5 degrees C with pelleted myosin filaments detected carbon atoms in rigid domains. While most (80%) aliphatic carbons are strongly 13C-1H dipolar coupled due to limited motion, they have short T1 values and large nuclear Overhauser enhancement values; this is evidence for high frequency restricted motion. Cross-polarization experiments show that the 13C carbonyl line width is motionally narrowed, suggesting broad backbone motions in the 100 mus range. Thus, motion in filaments is highly anisotropic.