For a long time, NMR chemical shifts have been used to identify protein secondary structures. Currently, this is accomplished through comparing the observed (1)H(alpha), (13)C(alpha), (13)C(beta), or (13)C' chemical shifts with the random coil values. Here, we present a new protocol, which is based on the joint probability of each of the three secondary structural types (beta-strand, alpha-helix, and random coil) derived from chemical-shift data, to identify the secondary structure. In combination with empirical smooth filters/functions, this protocol shows significant improvements in the accuracy and the confidence of identification. Updated chemical-shift statistics are reported, on the basis of which the reliability of using chemical shift to identify protein secondary structure is evaluated for each nucleus. The reliability varies greatly among the 20 amino acids, but, on average, is in the order of: (13)C(alpha)>(13)C'>(1)H(alpha)>(13)C(beta)>(15)N>(1)H(N) to distinguish an alpha-helix from a random coil; and (1)H(alpha)>(13)C(beta) >(1)H(N) approximately (13)C(alpha) approximately (13)C' approximately (15)N for a beta-strand from a random coil. Amide (15)N and (1)H(N) chemical shifts, which are generally excluded from the application, in fact, were found to be helpful in distinguishing a beta-strand from a random coil. In addition, the chemical-shift statistical data are compared with those reported previously, and the results are discussed. A JAVA User Interface program has been developed to make the entire procedure fully automated and is available via http://ccsr3150-p3.stanford.edu.
We show that a transient lactic fermentation provides the signal triggering ethanol production in hypoxic maize root tips. The signal is cytoplasmic pH. This interaction between lactic and ethanolic fermentation permits tight cytoplasmic pH regulation during hypoxia-cytoplasmic pH remaining near neutrality for several hours. Mutant roots unable to synthesize ethanol can neither regulate cytoplasmic pH nor maintain ATP levels during extended periods of hypoxia and, like vertebrate tissues, are less tolerant of hypoxia than normal maize. This indicates that cytoplasmic pH regulation is an important factor in survival under hypoxia.Certain higher plant tissues, such as maize roots, although requiring oxygen for normal functioning, can survive long periods (>18 hr) of anaerobiosis (1), with glycolysis continuing during most of this period (2,3). Most vertebrate tissues, on the other hand, can survive only short periods of hypoxia (<2 hr), after which glycolysis is greatly inhibited, and in most cases irreversible cell damage occurs (4,5). This difference between plants and animals cannot be due simply to the lower metabolic rates often seen in plants: the maize root tips (at 250C) used in this study respire five times the rate of a resting man (at 370C) and about half the rate of a resting mouse (at 370C) (6). It is possible, however, that the ability of higher plants to undergo a mainly ethanolic fermentation (2, 3, 7-9), rather than the exclusively lactic fermentation seen in higher animals, is to some degree responsible for their ability to withstand long periods of hypoxia. We considered this view after showing that the cytoplasmic pH of maize root tips falls to a stable value -0.5 pH unit below aerobic values within ""20 min after transfer to an anaerobic environment (10). This behavior is in complete contrast to active, hypoxic vertebrate tissues, where cytoplasmic pH falls throughout hypoxia because of continuous lactic acid accumulation (4, 11, 12) until glycolysis ceases. Thus, in hypoxic vertebrate tissues, energy production (glycolysis) leads to cytoplasmic acidosis, which eventually inhibits continued energy production; in plants, continued energy production does not involve generation of acid (other than carbon dioxide), and so no inhibition of glycolysis due to acidosis is observed.We describe here the mechanism by which cytoplasmic pH is regulated, and ethanol production induced, in hypoxic maize root tips. The mechanism is consistent with in vitro data (13,14). We also show that root tips of mutants that lack the ability to make ethanol during hypoxia-and have decreased viability-cannot regulate cytoplasmic pH but instead, like vertebrate tissues, undergo cytoplasmic acidification throughout hypoxia. MATERIALS AND METHODSExperiments were performed with -=1.5-g samples of 2-mm hybrid maize (WW x Br38) (obtained from Customaize Research, Decateur, IL) root tips excised from 2-day-old seedlings, perfused as described in the figure legends (10). NADH fluorescence (15) was measured in a Perkin-Elm...
We present evidence that cytoplasmic acidosis is a cause of meristematic death in hypoxic root tips of maize and pea seedlings. Usually, leakage of acid from the vacuole is responsible for cytoplasmic acidosis. Leakage of acid, which occurs earlier during hypoxia in pea root tips than in maize root tips, appears to account for the lower tolerance of peas for hypoxia. Cytoplasmic acidosis is accelerated in maize root tips that are either (i) deficient in alcohol dehydrogenase, so that lactic acid production continues throughout hypoxia, or (ii) exposed to external CO2 during hypoxia, or (iii) perfused slowly so that escape of CO2 produced during ethanolic fermentation is retarded. All three conditions decrease the length of time maize root tips can tolerate hypoxia; more rapid cytoplasmic acidosis is associated with more rapid death under hypoxia. Possible mechanisms by which cytoplasmic acidosis leads to death are suggested; the mechanism does not involve inhibition of glycolysis by low pH. It is clear that even species described as "non-flood-tolerant" (9), such as peas, can survive extended periods (days) of flooding (10). Thus, there are no truly flood-intolerant plant species; all plants survive far longer under hypoxia than does any vertebrate (11). We recently presented evidence (11) that the ability of plants to carry out a mainly ethanolic fermentation, rather than the exclusively lactic fermentation seen in higher animals, was responsible for their ability to withstand long periods of hypoxia. In contrast to lactic fermentation, ethanolic fermentation does not result in severe cytoplasmic acidosis (12). Cytoplasmic acidosis appears to be responsible for loss of viability in excised animal organs (13,14). However, our previous experiments (11) did not explain why plant species, all of which apparently carry out a mainly ethanolic fermentation (5, 15-17), differ in their ability to withstand hypoxia. In the study described here we quantitated the ability of excised maize and pea root tips to tolerate extreme hypoxia and measured simultaneously the intracellular pH and the rate of energy production (i.e., rate of ethanolic fermentation) during hypoxia. We present evidence that cytoplasmic acidosis, due to leakage of acid from the vacuole, is a cause of meristematic death in hypoxic root tips. MATERIALS AND METHODSPlant Material. Experiments were carried out using 1-to 2-mm-long root tips from 2-day-old maize (hybrid Funk 4323, obtained from Germain's Seeds, Los Angeles, CA; or F3 progeny, described below) or pea (var. Alaska, from Burpee, Riverside, CA) seedlings. The F3 progeny used in the experiments shown in Fig. 1 were derived from a cross between the maize hybrid, Funk 4343, and plants homozygous for a mutation at the Adhl locus ] that make no active alcohol dehydrogenase-1 (ADH1). Both the normal and the mutant roots contain a low level of ADH2 enzymatic activity, which we previously showed to be insufficient to sustain normal rates of ethanol production and thus permit cytoplasmic pH regulat...
In this study, we report nearest neighbor residue effects statistically determined from a chemical shift database. For an amino acid sequence XYZ, we define two correction factors, Delta((X)Y)n,s and Delta(Y(Z))n,s, representing the effects on Y's chemical shifts from the preceding residue (X) and the following residue (Z), respectively, where X, Y, and Z are any of the 20 naturally occurring amino acids, n stands for (1)H(N), (15)N, (1)H(alpha), (13)C(alpha), (13)C(beta), and (13)C' nuclei, and s represents the three secondary structural types beta-strand, random coil, and alpha-helix. A total of approximately 14400 Delta((X)Y)n,s and Delta(Y(Z))n,s, representing nearly all combinations of X, Y, Z, n, and s, have been quantitatively determined. Our approach overcomes the limits of earlier experimental methods using short model peptides, and the resulting correction factors have important applications such as chemical shift prediction for the folded proteins. More importantly, we have found, for the first time, a linear correlation between the Delta((X)Y)n,s (n = (15)N) and the (13)C(alpha) chemical shifts of the preceding residue X. Since (13)C(alpha) chemical shifts of the 20 amino acids, which span a wide range of 40-70 ppm, are largely dominated by one property, the electron density of the side chain, the correlation indicates that the same property is responsible for the effect on the following residue. The influence of the secondary structure on both the chemical shifts and the nearest neighbor residue effect are also investigated.
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