Effects of genotype, season, and nitrogen nutrition on gene expression and protein accumulation in wheat grain.

Six commercial U.K. cultivars of winter wheat selected to represent different abilities to partition nitrogen into grain protein were grown in replicated field trials at five different sites over three seasons. The proportion of LMW glutenin subunits decreased and the proportion of gliadins increased during grain development and in response to N application. Differences were observed between the proportions of LMW glutenin subunits and gliadins in low- and high-protein grain, these two fractions being decreased and increased, respectively. There was little effect of grain protein content on the proportions of either the HMW glutenin subunits or large glutenin polymers, which are enriched in these subunits, with the latter increasing during development in all cultivars. The proportion of total protein present in polymers in the mature grain decreased with increasing N level. Correlations were also observed between the abundances of gliadin protein transcripts and the corresponding proteins.

The effects of phosphorylation of cardiac troponin-I on its interactions with actin and cardiac troponin-C.

It is known that the phosphorylation of two serine residues on the NH2-terminal extension specific to cardiac troponin-I (Tn-I) modulates the calcium-dependent activation of the myofilaments. The process by which this occurs remains an unsolved puzzle. We have applied a dissective approach to study the effect of this phosphorylation on the interactions between Tn-I and its partner proteins, actin and troponin-C (Tn-C). Using N-[14C]ethylmaleimide-labelled Tn-I in sedimentation assays with F-actin, we found that the dephosphorylated Tn-I binds to F-actin with pronounced positive cooperativity, both in the absence and the presence of tropomyosin. Phosphorylation of the protein slightly weakens the interaction in the absence of tropomyosin, but the cooperativity remained. In the presence of tropomyosin, phosphorylation of the Tn-I also appeared to slightly weaken the interaction as well but, more significantly, the cooperativity was eliminated. These data can only be explained simply by a cooperative interaction between the monomer units in the actin filament. The interactions between cardiac Tn-I and Tn-C were studied by labelling the Tn-C with the fluorescent probe dansyl aziridine. As expected, the binding of the dephosphorylated Tn-I to Tn-C was strengthened by over 20-fold upon the addition of calcium to the assay. Phosphorylation of the protein, however, had a dramatic effect on the interaction in that it appeared to desensitise the complex to the effect of calcium: the Ka values obtained for both interactions (+/- Ca2+) were almost identical. These results clearly indicate that the phosphorylation of Tn-I in the cardiac system has dramatic effects on the isolated inter-protein interactions. We also discuss the possible significance of such an effect on the interactions of the isolated proteins in their roles within the intact cardiac regulatory complex.

Dynamic quenching studies of fluorophore-labelled myosin subfragment 1 and its alkali light chain subunits.

The technique of fluorescence quenching by the non-ionic quenchers acrylamide and nicotinamide has been used to probe the accessibility of the environmentally sensitive N-(bromoacetyl)-N'-(1-sulpho-5-naphthyl) ethylenediamine (1,5-Br-AEDANS) fluorophore attached to either Cys-177 of the A1-light chain or the SH1 thiol (Cys-707) of the myosin subfragment (S1) heavy chain. Neither quencher caused any detrimental effects to the ATPase activities of S1 under the conditions of the experiments. It was found that the fluorophore on the isolated light chain was highly exposed to solvent and although this exposure was reduced on hybridization into S1(A1-AEDANS), the probe was still accessible to solvent. This exposure was unaltered by formation of binary complexes with either Mg.ATP or actin or by the formation of a weakly associated acto-S1 complex (in which the Cys-697 and Cys-707 residues of S1 were crosslinked with p-phenylenedimaleimide). The lack of corresponding change in lambda max of emission and quantum yield supported the quenching date and indicated that actin neither binds directly to this region nor induces any significant conformational changes in this locality despite the observation that the A1-Cys-707 moves some 3 nm closer to a point on actin in the weak-binding state (Trayer, H.R. and Trayer, I.P. (1988) Biochemistry, 27, 5718-5727). Parallel experiments with the fluorophore attached to the Cys-707 of the S1 indicated that this region was less accessible to solvent than the light chain thiol despite its ease of labelling. This exposure was not significantly altered by binary complex formation with actin and Mg.ATP, although spectral changes in the absence of quencher support the notion that some conformational change is occurring in this region.

1H-NMR study of mobility and conformational constraints within the proline-rich N-terminal of the LC1 alkali light chain of skeletal myosin. Correlation with similar segments in other protein systems.

Analysis by 1H-NMR spectroscopic techniques of the conformation of the N-terminal segment of the LC1 alkali light chain of rabbit skeletal muscle has shown that this portion of the molecule adopts a well-defined elongated configuration. This rod-like feature is a consequence of the Ala/Pro-rich composition and the functional aspects of such conformational preference in this and similar segments in other proteins are discussed.

Resonance energy transfer evidence for two attached states of the actomyosin complex.

Resonance energy transfer measurements were made between a donor fluorophore, N-(bromacetyl)-N'-(1-sulpho-5-naphthyl)ethylenediamine, located on the single cysteine of the Al light chain of myosin (S1(A1), and an acceptor fluorophore, 5-(iodoacetamido)fluorescein, sited on Cys-374 of actin. In the binary rigor complex a transfer efficiency of 24% was noted, representing a spatial separation of about 6 nm. When the same measurements were made using a stable analogue of S1 X ATP, in which the fast reacting SH1 thiol group is crosslinked to another thiol group in the 20 kDa domain of S1, the 2 fluorophores were found to have moved closer together by greater than or equal to 3 nm. This provides, for the first time, direct experimental evidence for a change in structure of the myosin crossbridge that could account for tension generation.