The effect of chloride on the thermal inactivation of oxygen evolution.

The effect of Cl depletion on the sensitivity of the oxygen-evolving complex of Photosystem II (PS II) to heat treatment was examined by a parallel study of the Hill activity (H2O→2,6-dichlorophenolindophenol), Cl(-) binding (by (35)Cl-NMR) and Mn release (by EPR). The extent of thermal inactivation in spinach thylakoids was found to depend on the degree of Cl(-) depletion in the sample. In partially Cl(-)-depleted thylakoids, mild heating (38°C, 3 min) was found to eliminate inflections in plots of both Hill activity versus [Cl(-)] (at low light intensity) and excess (35)Cl-NMR linewidth versus [Cl(-)] (in the dark). In PS II membranes, the same treatment reduced the differences between the linewidth maxima and minima, particularly in the region of 0.3 mM and 7.0 mM Cl(-), as compared to unheated membranes. These results indicate that mild heating affects the Cl(-)-binding domains within the oxygen-evolving complex, OEC, EPR measurements of the temperature dependence of Mn release from heated thylakoids show that Mn release begins to correlate with the loss of Hill activity only at higher temperatures, where the OEC is already substantially inactivated. We conclude from these studies that the Cl(-)-binding domains of the OEC constitute a principal site of damage by heat treatment.

Origin and behavior of deuteron spin echoes in selectively labeled amino acids, myoglobin microcrystals, and purple membranes.

We have obtained deuterium NMR spin-echo spectra of crystalline DL-[gamma-2H6]valine, [S-methyl-2H3]-methionine, cyanoferrimyoglobin from sperm whale (Physeter catodon), containing deuteriomethyl groups at methionine-55 and methionine-131, and [gamma-2H6]valine-labeled bacteriorhodopsin in the purple membrane of Halobacterium halobium R1. By using 90-tau-beta 90 degrees (XY) and 90-tau-beta 0 degrees (XX) pulse sequences and observing the dependence of the spin-echo amplitude upon the interpulse spacing tau, we have determined that the so-called "quadrupole echoes" obtained in these typical selectively deuterated condensed-phase biological systems are in fact strongly modulated by proton-deuteron and deuteron-deuteron dipolar interactions. The two amino acids and the protein crystals behaved as typical organic solids, with no evidence of "liquid-like" behavior, even in the presence of excess water (in the case of the ferrimyoglobin crystals). However, with the valine-labeled bacteriorhodopsin, the tau-dependence of XY echoes as a function of temperature emphasized the "solid-like" behavior of the membrane "matrix", while the basic nature of the spin-echo response for the narrow central component of the spectrum clearly indicated the very "fluid" or "mobile" nature of a series of residues that are shown elsewhere [Keniry, M., Gutowsky, H. S., & Oldfield, E. (1984) Nature (London) 307, 383-386] to arise from the membrane surface. Our results thus suggest that such NMR methods may yield useful information on side-chain dynamics complementary to that of line-shape and spin-lattice relaxation time analyses.

NMR study of chloride ion interactions with thylakoid membranes.

The role of Cl(-) in photosynthetic O(2) evolution has been investigated by observing the (35)Cl NMR linewidth under a variety of conditions in aqueous suspensions of chloroplasts, primarily for the halophytes Avicennia germinans, Avicennia marina, and Aster tripolium but also for spinach. The line broadening shows there is weak, ionic binding of Cl(-) to thylakoids, the bound Cl(-) exchanging rapidly (>>10(4) sec(-1)) with free Cl(-) in solution. The binding is necessary for O(2) evolution to occur. Michaelis-Menten constants obtained from the Cl(-) dependence of the O(2) evolution rate are approximately 15-70 mM for the halophytes compared with 0.6 mM for spinach (0.5 mM with Br(-)). There appear to be two types of Cl(-) binding sites in halophytes, of which the stronger is the activator, at lower [Cl(-)], of O(2) evolution. The (35)Cl line broadening includes a nonspecific interaction, which becomes apparent at high Cl(-) concentrations (>/=0.5 M).

Nuclear magnetic resonance studies of amino acids and proteins. Deuterium nuclear magnetic resonance relaxation of deuteriomethyl-labeled amino acids in crystals and in Halobacterium halobium and Escherichia coli cell membranes.

We have obtained deuterium (2H) Fourier transform nuclear magnetic resonance (NMR) spectra of zwitterionic L-[beta-2H3]alanine, DL-[gamma-2H6]valine, DL-[beta, gamma-2H4]threonine, L-[delta-2H3]leucine, and L-[alpha, beta, gamma, gamma', delta-2H10]isoleucine in the crystalline solid state and have determined the deuteriomethyl group spin-lattice relaxation rates as a function of temperature. The results yield the Arrhenius activation energies (delta E) for methyl rotation, and through use of a suitable mathematical model, rotational correlation times, tau c. For alanine, valine, threonine, leucine, and isoleucine at 37 degrees C, tau c and delta E values are 780, 100, 40, 38, and 18 ps and 22, 14.0, 17.6, 15.5, and 8.6 kJ, respectively. For L-[beta-2H3]alanine in the zwitterionic lattice, a spin-lattice relaxation time (T1) minimum of 2.1 +/- 0.3 ms is observed (at 0 degree C), in excellent agreement with the 1.92-ms prediction of the mathematical model. Similar tau c and delta E measurements are reported for bacteriorhodopsin in the purple membrane of Halobacterium halobium R1 and for Escherichia coli cell membranes. Overall, our results demonstrate a great similarity between the dynamics in amino acid crystals and in membrane proteins. However, threonine exhibits a nonlinear Arrhenius behavior in bacteriorhodopsin, and in the valine-, leucine-, and isoleucine-labeled membrane samples at higher temperatures (approximately greater than 37 degrees C), there is evidence of an additional slow side-chain motion. The lipid phase state in E. coli does not appear to influence, on the average, the dynamics of the valine side chains. These results indicate that the sensitivity of the deuterium NMR technique is now adequate to study in moderate detail the dynamics of most types of amino acids in a membrane protein and that adequate sensitivity, in some instances, should be available for the study of individual amino acids in suitably labeled membrane proteins.

Surface dynamics of the integral membrane protein bacteriorhodopsin.

Recently, there has been great interest in determining the three-dimensional structures of membrane proteins, particularly bacteriorhodopsin, for which a variety of possible folding arrangements have been suggested. In this paper we present nuclear magnetic resonance (NMR) spectra of deuterated bacteriorhodopsin, and use the data to help interpret the various suggested bacteriorhodopsin folding patterns. The results strongly indicate that (1) a membrane surface (+/- 1 residue) may be defined by NMR in bacteriorhodopsin; (2) all amino acids inside the surface are essentially crystalline; (3) all amino acids outside the surface (surface residues) in bacteriorhodopsin are highly mobile on the time scale of the 2H NMR experiments; (4) NMR data may be used to help evaluate the various structural models that have been proposed; (5) aggregation of purple membrane sheets may lead to an immobilization of the surface residues.

Nuclear magnetic resonance studies of amino acids and proteins. Side-chain mobility of methionine in the crystalline amino acid and in crystalline sperm whale (Physeter catodon) myoglobin.

We have obtained deuterium (2H) nuclear magnetic resonance (NMR) spectra and spin-lattice relaxation times (T1) of L-[epsilon-2H3]methionine, L-[epsilon-2H3]methionine in a D,L lattice, and [S-methyl-2H3]methionine in the crystalline solid state, as a function of temperature, in addition to obtaining 2H T1 and line-width results as a function of temperature on [epsilon-2H3]methionine-labeled sperm whale (Physeter catodon) myoglobins by using the method of magnetic ordering [Rothgeb, T. M., & Oldfield, E. (1981) J. Biol. Chem. 256, 1432-1446]. The results indicate that in the L-amino acid, methyl rotation having an activation energy (delta E) of 8.3 +/- 1 kJ dominates T1 at low temperatures (less than or equal to 10 degrees C), while at higher temperatures an additional large-amplitude side-chain motion occurs which causes changes in the 2H NMR line shape and T1. This motion is inhibited in the D,L lattice, indicating that lattice effects may have a strong effect on the mobility of anhydrous amino acids in the solid state. Further substitution at S delta to form the sulfonium salt [S-methyl-2H3]-methionine causes a large increase in delta E, to 15.9 +/- 2 kJ, a value comparable to the 14-16 kJ found in valine and leucine, which contain the structurally similar isopropyl moiety. These results suggest that the very low barriers to methyl rotation in the methionine side chain are due to long C-S bond lengths and the presence of only two substituents on sulfur, while the anomalous high-temperature behavior is due to a lattice-packing effect. 2H T1 results with methionine-labeled myoglobin are complex, reflecting the presence of fast large-amplitude side-chain motions, in addition to rapid methyl rotation. Our data indicate that Met-55 and Met-131 are motionally inequivalent in crystalline cyanoferrimyoglobin, in contrast to solution NMR results. We have also recorded 13C cross-polarization "magic-angle" sample-spinning NMR spectra of [epsilon-13C]methionine-labeled crystalline cyanoferrimyoglobin (at 37.7 MHz, corresponding to a magnetic field strength of 3.52 T) and of the same protein in aqueous solution. Cross-polarization transfer rates and proton rotating-frame relaxation time results again indicate that Met-55 and Met-131 are motionally inequivalent in the solid state, and the TCH data indicate that Met-55 is more solidlike. However, we find that 13C chemical shifts in solution and those in the crystalline solid state are in very close agreement, suggesting that the average solution and crystal conformations are the same, in the area of Met-55 and Met-131.

Federal funding of basic research: the red tape mill.

Federal regulations and concerns about accountability for public funds have added greatly to the administrative burden associated with federal support of research at universities. Much of the added burden is viewed as unnecessary and counterproductive by the scientists and administrators who must bear the load. They feel that funds and effort intended for research are being diverted and wasted. The various types of costs are reviewed, including some thoughts as to their origin and estimates of their magnitude. Topics covered include project versus programmatic support, the indirect cost game, accountability, federal regulations, and the bureaucratic syndrome. There are no simple solutions, but several promising initiatives have been taken and more should be forthcoming.

Deuterium nuclear magnetic resonance investigation of the effects of proteins and polypeptides on hydrocarbon chain order in model membrane systems.

Deuterium Fourier-transform nuclear magnetic resonance spectra have been obtained of 1-myristoyl 2-(14,14,14-trideutero)myristoyl phosphatidylcholine bilayers at 34.1 MHz by using the quadrupole echo pulse technique. Thereby, we have investigated the effects upon the deuterated dimyristoyl phosphatidylcholine bilayers of the following proteins and polypeptides: gramicidin A, bacteriophage f1 coat protein, beef brain myelin proteolipid apoprotein, cytochrome b(5), and cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). Above T(c), the transition temperature between the gel and liquid crystal phases, the quadrupole splitting of the deuterium-labeled methyl group is reduced or collapsed in the presence of protein or polypeptide. No evidence has been found for ordered "boundary lipid." Below T(c), the spectra show that the hydrocarbon chains are prevented from crystallizing by the protein (or polypeptide) incorporated in the membrane. Similar disordering effects above T(c) are also seen when an unsaturated lipid, 1-(16,16,16-trideutero)palmitoyl 2-palmitoleyl phosphatidylcholine is complexed with cytochrome oxidase.

Nuclear magnetic relaxation by the manganese in aqueous suspensions of chloroplasts.

Proton and oxygen-17 NMR relaxation rate (T1-1 and T2-1) data are presented for aqueous suspensions of dark-adapted chloroplasts. It is concluded from the dependence of the proton relaxation rates (PRR) upon Mn concentration that T1-1 and T2-1 are determined largely by the loosely bound Mn present in the chloroplast membranes. The frequency and temperature dependences of PRR are characteristic of Mn(II). The effects of oxidants (e.g., ferricyanide) and reductants (e.g., tetraphenylboron) on the PRR indicate that only about one-third to one-fourth of the loosely bound Mn is present in the dark-adapted chloroplasts as Mn(II), the remainder being in a higher oxidation state(s), probably Mn(III). The frequency dependence of the PRR for the chloroplast suspensions was fitted by a simplified form of the Solomon-Bloembergen-Morgan equations, and the following parameters were obtained: tauS = (1.1 +/- 0.1) X 10(-8) S; tauM = (2.2 +/- 0.2) X 10(-8) S; and B = (0.9 +/- 0.09) X 10(19). The oxygen-17 T1 and T2 data for suspensions before and after treatment with a detergent are consistent with the location of the manganese in the interior of the thylakoids. An analysis of the relaxation rates shows that the average lifetime of a water molecule inside a thylakoid is greater than 1 ms.

Proton relaxation and charge accumulation during oxygen evolution in photosynthesis.

The water proton spin-spin (transverse) relaxation rate of chloroplast suspensions has been measured after each of a series of 2.4 musec light flashes. The sequence of relaxation rates shows a damped oscillatory pattern with a period of four and peaks after the 3rd, 7th, 11th, and 15th flashes. This result indicates that water proton relaxation can be used to monitor the charge-accumulating states as postulated by Kok and coworkers for the oxygen-evolving mechanism in green plants [(1970) Photochem. Photobiol. 11, 457-475]. Other experiments [Wydrzynski et al. (1975) Biochim. Biophys. Acta 408, 349-354] have shown that the proton relaxation rate is strongly influenced by membrane-bound manganese in various oxidation states, suggesting that manganese participates in the charge accumulation process during oxygen evolution.