Brigitte Schobert

Unusual solution properties of proline and its interaction with proteins

Proline in aqueous solution shows several properties which are unusual for low molecular weight substances. Investigations of solubility, density and viscosity revealed behaviour which is characteristic for hydrophilic colloids. 1H-NMR studies indicated a strong hydrogen bonding of water in proline solutions, especially at high concentrations of the solute. From these results it was concluded that proline forms aggregates by stepwise stacking and hydrophobic interaction of the pyrrolidine ring. Thus, the proposed multimer contans a hydrophobic backbone and hydrophilic groups on the surface, exposed to water. Proline solutions are able to increase the solubility of sparingly soluble proteins. The enhancement effect depends on the nature of the protein and on the proline concentration. It is postulated that by a hydrophobic interaction of proline with hydrophobic surface residues of proteins their hydrophilic area is increased. The presence of proline in solutions of the well soluble protein bovine albumin reduces the precipitation of this protein by ethanol and (NH4)2SO4, presumably by an increased water-binding capacity of the proline-protein solution.

Crystallographic structure of the K intermediate of bacteriorhodopsin: conservation of free energy after photoisomerization of the retinal.

The K state, an early intermediate of the bacteriorhodopsin photocycle, contains the excess free energy used for light-driven proton transport. The energy gain must reside in or near the photoisomerized retinal, but in what form has long been an open question. We produced the K intermediate in bacteriorhodopsin crystals in a photostationary state at 100K, with 40% yield, and determined its X-ray diffraction structure to 1.43 A resolution. In independent refinements of data from four crystals, the changes are confined mainly to the photoisomerized retinal. The retinal is 13-cis,15-anti, as known from vibrational spectroscopy. The C13=C14 bond is rotated nearly fully to cis from the initial trans configuration, but the C14-C15 and C15=NZ bonds are partially counter-rotated. This strained geometry keeps the direction of the Schiff base N-H bond vector roughly in the extracellular direction, but the angle of its hydrogen bond with water 402, that connects it to the anionic Asp85 and Asp212, is not optimal. Weakening of this hydrogen bond may account for many of the reported features of the infrared spectrum of K, and for its photoelectric signal, as well as the deprotonation of the Schiff base later in the cycle. Importantly, although 13-cis, the retinal does not assume the expected bent shape of this configuration. Comparison of the calculated energy of the increased angle of C12-C13=C14, that allows this distortion, with the earlier reported calorimetric measurement of the enthalpy gain of the K state indicates that a significant part of the excess energy is conserved in the bond strain at C13.

Crystallographic structure of xanthorhodopsin, the light-driven proton pump with a dual chromophore.

Homologous to bacteriorhodopsin and even more to proteorhodopsin, xanthorhodopsin is a light-driven proton pump that, in addition to retinal, contains a noncovalently bound carotenoid with a function of a light-harvesting antenna. We determined the structure of this eubacterial membrane protein–carotenoid complex by X-ray diffraction, to 1.9-Å resolution. Although it contains 7 transmembrane helices like bacteriorhodopsin and archaerhodopsin, the structure of xanthorhodopsin is considerably different from the 2 archaeal proteins. The crystallographic model for this rhodopsin introduces structural motifs for proton transfer during the reaction cycle, particularly for proton release, that are dramatically different from those in other retinal-based transmembrane pumps. Further, it contains a histidine–aspartate complex for regulating the pKa of the primary proton acceptor not present in archaeal pumps but apparently conserved in eubacterial pumps. In addition to aiding elucidation of a more general proton transfer mechanism for light-driven energy transducers, the structure defines also the geometry of the carotenoid and the retinal. The close approach of the 2 polyenes at their ring ends explains why the efficiency of the excited-state energy transfer is as high as ≈45%, and the 46° angle between them suggests that the chromophore location is a compromise between optimal capture of light of all polarization angles and excited-state energy transfer.

Mechanism of Proton Transport in Bacteriorhodopsin from Crystallographic Structures of the K, L, M1, M2, and M2′ Intermediates of the Photocycle

We produced the L intermediate of the photocycle in a bacteriorhodopsin crystal in photo-stationary state at 170 K with red laser illumination at 60% occupancy, and determined its structure to 1.62 A resolution. With this model, high-resolution structural information is available for the initial bacteriorhodopsin, as well as the first five states in the transport cycle. These states involve photo-isomerization of the retinal and its initial configurational changes, deprotonation of the retinal Schiff base and the coupled release of a proton to the extracellular membrane surface, and the switch event that allows reprotonation of the Schiff base from the cytoplasmic side. The six structural models describe the transformations of the retinal and its interaction with water 402, Asp85, and Asp212 in atomic detail, as well as the displacements of functional residues farther from the Schiff base. The changes provide rationales for how relaxation of the distorted retinal causes movements of water and protein atoms that result in vectorial proton transfers to and from the Schiff base.

An NADH:Quinone oxidoreductase of the halotolerant bacterium Ba1 is specifically dependent on sodium ions☆

The rate of NADH oxidation by inverted membrane vesicles prepared from the halotolerant bacterium Ba1 of the Dead Sea is increased specifically by sodium ions, as observed earlier in whole cells. The site of this sodium effect is identified as the NADH: quinone oxidoreductase, similarly to the other such system known, Vibrio alginolyticus (H. Tokuda and T. Unemoto (1984) J. Biol. Chem. 259, 7785-7790). Sodium accelerates quinone reduction severalfold, but oxidation of the quinol, with oxygen as terminal electron acceptor, is unaffected. The sodium-dependent pathway of quinone reduction exhibits higher apparent affinity to extraneous quinone (Q-2) than the sodium-insensitive pathway, and is specifically inhibited by 2-heptyl-4-hydroxyquinoline N-oxide. ESR spectra of the membranes contain a feature at g = 1.98 which is tentatively identified as one originating from semiquinone. This feature is increased by NADH and decreased by addition of Na+, suggesting that, as proposed from different kinds of evidence for the V. alginolyticus system, sodium affects the semiquinone reduction step. As in the other system, the site of sodium stimulation in Ba1 probably corresponds to the site of sodium translocation, which was shown earlier (S. Ken-Dror, R. Shnaiderman, and Y. Avi-Dor (1984) Arch. Biochem. Biophys. 229, 640-649) to be linked directly to a redox reaction in the respiratory chain.

Spin label studies on osmotically-induced changes in the aqueous cytoplasm of Phaeodactylum tricornutum

The effects of hyperosmotic stress and adaption on the aqueous cytoplasm of Phaeodactylum tricornutum have been studied with spin labels using 0.2M external Ni2+ to obtain spectra solely from labels within the cells. From partitioning of the TEMPO spin label between the internal aqueous phase and the membrane it is found that the internal volume of the cells decreased by approx. 50-60% in media of high osmotic strength (1.9 osmol/l). During the accumulation of proline in the cells (8.8 mg/ml packed cells) on incubation in the medium of high osmolarity for 3 days, the recovery of the volume was 80%. Further addition of proline to the medium resulted in an increase in the proline concentration in the cells (12.2 mg/ml packed cells) and a recovery in volume of 90%. Cells incubated in the absence of any nitrogen source showed very little recovery and were in a stressed state even in the absence of an osmotic gradient. From the rotational correlation times of the TEMPONE spin label it was found that the effective microviscosity in the cytoplasm of normal cells (approx. 3-8 cP) was considerably higher than that of the external medium (1 cP) and increased 1.5-2-fold under high osmotic stress (1.9 osmol/l). Adaption during the accumulation of proline only decreased the effective microviscosity by approx. 50% of the stressed-induced increase, a considerably smaller recovery than that of the cell volume.

ESR studies of light-dependent volume changes in cell envelope vesicles from Halobacterium halobium

Abstract Volume changes in illuminated cell envelope vesicles, prepared from various Halobacterium halobium strains, were measured with an ESR method. We demonstrated light-dependent swelling of vesicles which contained halorhodopsin (an inward-directed light-driven chloride pump), and shrinking of vesicles which contained bacteriorhodopsin (an outward-directed light-driven proton pump coupled to a proton/sodium antiporter). The swelling of the halorhodopsin vesicles was not inhibited by uncouplers or gramicidin, but the shrinking of the bacteriorhodopsin-vesicles was abolished by these ionophores. These findings confirm earlier models for ion circulation in these systems. Vesicles from strains which contained both pigments showed relatively small net volume changes upon illumination. A scheme of ionic transport in H. halobium cells is suggested, in which the inward movement of K + exceeds the outward movement of Na + , and the difference equals the Cl − uptake, so as to provide the net gain of KCl necessary for volume increases during cell growth.

Effects of arginine modification on the photocycle of halorhodopsin

Exhaustive reaction with phenylglyoxal removed 9 of the 12 arginine and 1 of the 2 lysine residues in detergent-solubilized halorhodopsin, without affecting the chromophore. The consequences of this extensive removal of positive charges on various chloride-binding equilibria and the photochemistry were evaluated. No significant effects were seen on the affinity of Site I to chloride and on the increase in the pKa of Schiff-base deprotonation, which is caused by the chloride binding at this site. No significant effects were seen on the affinity of Site II to chloride, either. However, the photocycle of the pigment was affected. Kinetic modeling of the observed changes in flash-induced absorption changes suggests that the modification increases the affinity of the main halorhodopsin photointermediate to chloride by about fourfold. If chloride translocation involves release of chloride from this intermediate during the transport cycle, the result might explain the observed partial inhibitory effects on chloride transport. Plausible models of chloride translocation include reversible binding of the anion by positively charged groups, strategically arranged in the protein. The results indicate that two of the three spectroscopically observable chloride-dependent equilibria do not depend on a large number of positively charged residues in the protein. To the extent that the unaffected equilibria represent association and dissociation which occur during chloride translocation, at least part of the chloride translocation might be accomplished with the participation of only a few positively charged residues.

[38] Light-driven chloride transport in halorhodopsin-containing cell envelope vesicles

Publisher Summary Halorhodopsin is the second retinal protein discovered in the plasma membrane of Halobacterium halobium. Like bacteriorhodopsin, it functions as a light-driven ion pump in the cytoplasmic membrane of this organism, but its substrate is chloride instead of protons, and it transports this ion into the cells, rather than outward. For unambiguous results the halobacterial strain used in assays of halorhodopsin should be defective in bacteriorohodopsin, as is strain L-33, but such strains usually contain also slowly cycling rhodopsin, the third bacterial retinal protein. Cell envelope vesicles are prepared by the sonication method. This procedure results in a preparation with 85–90% right-side-out membrane orientation. The vesicle concentration can be measured by a protein determination, with serum albumin as standard. The functional assays of halorhodopsin are either direct—that is, measurement of the chloride transported, or more usually indirect—that is, consist of following a secondary consequence of the chloride transport, such as membrane potential, passive proton uptake, or volume change.