Water permeability of chloroplast envelope membranes. In vivo measurement by saturation-transfer NMR

In tulip tree (Liriodendron tulipifera) leaves, the proton NMR signal from chloroplast water is resolved from that of water in other leaf compartments. We used the saturation-transfer NMR method to measure the mean water molecule residence time within a chloroplast, (88 +/- 17) ms at 20 degrees C. From the measured chloroplast dimensions, we calculate an effective permeability coefficient of (9 +/- 2) X 10(-4) cm/s for the chloroplast envelope membrane. This is the first in vivo measurement of chloroplast water permeability.     

Thermal damage to chloroplast envelope membranes

Nuclear magnetic resonance was used to detect thermal injury to chloroplasts in vivo. A lesion occurs in the chloroplast envelope membrane at temperatures between 53°C and 57°C, depending on species, leaf condition, and heating rate. The injury is associated with a sudden loss of water from the chloroplast.     

Water is allocated differently to chloroplasts in sun and shade leaves

Hydrogen-1 nuclear magnetic resonance spectroscopy was used to study water allocation in cell compartments of sun and shade leaves. NMR spectra of Acer platanoides were resolved into two peaks that were assigned to chloroplast and nonchloroplast water. Sun leaves contained 1.7 times more water per unit area of surface than shade leaves, and the water was allocated differently. Chloroplasts in sun leaves contained 17% of the total leaf water versus 47% in shade leaves. Comparing equal leaf surface areas, the chloroplasts in shade leaves contained 60% more water than those in sun leaves.     

Two-dimensional NMR studies of Kazal proteinase inhibitors. 2. Sequence-specific assignments and secondary structure of reactive site modified turkey ovomucoid third domain

The solution structure of modified turkey ovomucoid third domain (OMTKY3*) was investigated by high-resolution proton NMR techniques. OMTKY3* was obtained by enzymatic hydrolysis of the scissile reactive site peptide bond (Leu18-Glu19) in turkey ovomucoid third domain (OMTKY3). All of the backbone proton resonances were assigned to sequence-specific residues except the NH's of Leu1 and Glu19, which were not observed. Over 80% of the side-chain protons also were assigned. The secondary structure of OMTKY3*, as determined from assigned NOESY cross-peaks and identification of slowly exchanging amide protons, contains antiparallel beta-sheet consisting of three strands (residues 21-25, 28-32, and 49-54), one alpha-helix (residues 33-44), and one reverse turn (residues 26-28). This secondary structure closely resembles that of OMTKY3 in solution [Robertson, A. D., Westler, W. M., & Markley, J. L. (1988) Biochemistry (preceding paper in this issue)]. On the other hand, changes in the tertiary structure of the protein near to and remote from the cleavage site are indicated by differences in the chemical shifts of numerous backbone protons of OMTKY3 and OMTKY3*.     

Functional properties of palmaris longus muscles of rhesus monkeys transplanted as index finger flexors

This experiment with skeletal muscle autografts in monkeys was designed to retest previous findings that transplanted skeletal muscle can regenerate to a functional degree in primates without predenervation and to test a new hypothesis that increased functional demands on regenerated muscle grafts in monkeys may result in improved functional capacity of the grafts. Rhesus monkey index flexors were replaced with free palmaris longus muscle autografts with microneural anastomoses between the graft motor nerve and the severed profundus motor nerve. One monkey was taught selective index flexion before grafting and continued with this program after grafting to test the effect of training on the graft. Mature grafts were evaluated for in vivo contractile properties and by histology and histochemistry and were compared with a group of normal Rhesus palmaris longus muscles. The results reconfirm the capacity of nonpredenervated monkey skeletal muscle grafts to regenerate and to achieve some contractile ability and suggest that training of free muscle grafts may enhance recovery of their functional and structural properties.     

One- and two-dimensional NMR spectral analysis of the consequences of single amino acid replacements in proteins

The traditional approach of using homologous sequences to elucidate the role of specific amino acid residues in protein structure and function becomes more meaningful as the number of differences is minimized, with the limit being alteration of a single residue. For small proteins in solution, NMR spectroscopy offers a means of obtaining detailed information about each residue and its response to a given change in the protein sequence. Extraction of this information has been aided by recent progress in spectrometer technology (higher magnetic fields, more sensitive signal detection, more sophisticated computers) and experimental strategies (new NMR pulse sequences including multiple-quantum and two-dimensional NMR methods). The set of avian ovomucoid third domains, which consists of the third domain proper plus a short leader (connecting peptide) and has a maximum of 56 amino acid residues, offers an attractive system for developing experimental methods for investigating sequence-structure and structure-function relationships in proteins. Our NMR results provide examples of sequence effects on pKa' values, average conformation, and internal motion of amino acid side chains.     

Imino proton assignments in the proton nuclear magnetic resonance spectrum of the lambda phage OR3 deoxyribonucleic acid fragment

The 17 base pair duplex d(TATCACCGCAAGGGATAp) . d(TATCCCTTGCGGTGATAp) corresponding to the OR3 operator site of lambda phage has been synthesized and studied by 1H nuclear magnetic resonance spectroscopy at 470 MHz. The 13 imino proton resonances observed at 20 degrees C have been assigned to specific base pairs at positions 3-15 on the basis of nuclear Overhauser effect measurements and studies of the temperature dependence of peak intensities. Resonances from the A-T base pairs at positions 1, 2, 16, and 17 are assumed to be absent from the spectrum because of terminal fraying. Resonance from many of the base pairs suggested by Ohlendorf et al. [Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, Y., & Matthews, B. W. (1982) Nature (London) 298, 718-723] to be involved in specific binding of the lambda phage cro repressor are well resolved.     

14,15N, 13C, 57Fe, and 1,2H Q-band ENDOR study of Fe-S proteins with clusters that have endogenous sulfur ligands.

The benefits of performing ENDOR experiments at higher microwave frequency are demonstrated in a Q-band (35 GHz) ENDOR investigation of a number of proteins with [nFe-mS] clusters, n = 2, 3, 4. Each protein displays several resonances in the frequency range of 0-20 MHz. In all instances, features are seen near v approximately 13 and 8 MHz that can be assigned, respectively, to "distant ENDOR" from 13C in natural-abundance (1.1%) and from 14N (the delta m1 = +/- 2 transitions); the nuclei involved in this phenomenon are remote from and have negligible hyperfine couplings to the cluster. In addition, a number of proteins show local 13C ENDOR signals with resolved hyperfine interactions; these are assigned to the beta carbons of cysteines bound to the cluster [A(13C) approximately 1.0 MHz]. Five proteins show resolved, local delta m1 = +/- 2 ENDOR signals from 14N with an isotropic hyperfine coupling, 0.4 less than or equal to A(14N) less than or equal to 1.0, similar to those seen in ESEEM studies; these most likely are associated with N-H...S hydrogen bonds to the cluster. Anabaena ferredoxin further shows a signal corresponding to A(14N) approximately 4 MHz. Quadrupole coupling constants are derived for both local and distant 14N signals. The interpretation of the data is supported by studies on 15N- and 13C-enriched ferredoxin (Fd) from Anabaena 7120, where the 15N signals can be clearly correlated with the corresponding 14N signals and where the 13C signals are strongly enhanced. Thus, the observation of 14N delta m1 = +/- 2 signals at Q-band provides a new technique for examining weak interactions with a cluster.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of DON-related trichothecenes byFusarium graminearum Schw from Krasnodarski krai of the USSR

34Fusarium graminearum Schw isolates produced 4-deoxynivalenol to form significant amounts of 4, 7 — dideoxynivalenol and lesser amounts of 4 — deoxynivalenol monoacetates on grain substratesin vitro. This is the first report on the capability a large group of naturally occurring isolates to produce 4,7-dideoxynivalenol. The average levels of 4,7-dideoxynivalenol on rice, corn, barley, and wheat as a substrate were respectively 26.8, 14.0, 12.8, and 10.5% of the level of 4-deoxynivalenol. 4, 7 — dideoxynivalenol was present in all examined naturally contaminated wheat kernel samples at levels of 1.7 to 7.9% of the level of 4-deoxynivalenol. These findings suggest that more attention should be given to the occurrence of 4,7-dideoxynivalenol in cereals.