Iso-halorhodopsin: a stable, 9-cis retinal containing photoproduct of halorhodopsin.

Dark-adapted halorhodopsin is a mixture of 13-cis and all-trans retinal chromophoric species. It is known that illumination with blue light increases the all-trans content, and this is reversed partially by brief red illumination. We now find that extended red-light illumination produces a third spectroscopic form. Analysis of composite absorption spectra recorded during various illumination regimes yielded the spectrum for the new species, whose absorption is shifted approximately 100 nm to the blue. The isomeric composition of retinal extracted from the illuminated pigment indicates that this form contains 9-cis retinal. This species, which we name iso-halorhodopsin, is stable in the dark at room temperature for at least a day, but can be quantitatively reconverted into a mixture of all-trans and 13-cis halorhodopsin by blue-light illumination. A kinetic scheme for the isomeric interconversions was drawn up, where iso-halorhodopsin is produced from either all-trans halorhodopsin only, or both 13-cis and all-trans forms. This kind of scheme is supported by the finding that red illumination of halo-opsin reconstituted with 13-trans-locked retinal will generate iso-halorhodopsin. A similar experiment with 13-cis-locked retinal could not be done because reconstitution with this retinal analogue was not possible. The photoreaction that leads to iso-halorhodopsin can be readily demonstrated in detergent-solubilized halorhodopsin or in halorhodopsin in liposomes made from phosphatidylcholine plus phosphatidyl-ethanolamine, but only to much reduced extent in cell envelope vesicles and in halorhodopsin incorporated into liposomes made from halobacterial polar lipids.     

Crystal structure of a thermally stable rhodopsin mutant

We determined the structure of the rhodopsin mutant N2C/D282C expressed in mammalian cells; the first structure of a recombinantly produced G protein-coupled receptor (GPCR). The mutant was designed to form a disulfide bond between the N terminus and loop E3, which allows handling of opsin in detergent solution and increases thermal stability of rhodopsin by 10 deg.C. It allowed us to crystallize a fully deglycosylated rhodopsin (N2C/N15D/D282C). N15 mutations are normally misfolding and cause retinitis pigmentosa in humans. Microcrystallographic techniques and a 5 microm X-ray beam were used to collect data along a single needle measuring 5 microm x 5 microm x 90 microm. The disulfide introduces only minor changes but fixes the N-terminal cap over the beta-sheet lid covering the ligand-binding site, a likely explanation for the increased stability. This work allows structural investigation of rhodopsin mutants and shows the problems encountered during structure determination of GPCRs and other mammalian membrane proteins.     

Two-photon absorption properties of fluorescent proteins

Two-photon excitation of fluorescent proteins is an attractive approach for imaging living systems. Today researchers are eager to know which proteins are the brightest and what the best excitation wavelengths are. Here we review the two-photon absorption properties of a wide variety of fluorescent proteins, including new far-red variants, to produce a comprehensive guide to choosing the right fluorescent protein and excitation wavelength for two-photon applications.     

Chromophore motion during the bacteriorhodopsin photocycle: polarized absorption spectroscopy of bacteriorhodopsin and its M-state in bacteriorhodopsin crystals.

The three-dimensional crystallization of bacteriorhodopsin was systematically investigated and the needle-shaped crystal form analysed. In these crystals the M-intermediate forms 10 times faster and decays 15 times more slowly than in purple membranes. Polarized absorption spectra of the crystals were measured in the dark and light adapted states. A slight decrease in the angle between the transition moment and the membrane plane was detected during dark adaptation. The crystallization of a mutated bacteriorhodopsin, in which the aspartic acid at residue 96 was replaced by asparagine, provided crystals with a long lived M-intermediate. This allowed polarized absorption measurements of the M-chromophore. The change in the polarization ratio upon formation of the M-intermediate indicates an increase in the angle between the main transition dipole and the membrane plane by 2.2 degrees +/- 0.5, corresponding to a 0.5 A displacement of one end of the chromophore out of the membrane plane of the bacteriorhodopsin molecule.     

Optimal design of thermally stable proteins

MOTIVATION: For many biotechnological purposes, it is desirable to redesign proteins to be more structurally and functionally stable at higher temperatures. For example, chemical reactions are intrinsically faster at higher temperatures, so using enzymes that are stable at higher temperatures would lead to more efficient industrial processes. We describe an innovative and computationally efficient method called Improved Configurational Entropy (ICE), which can be used to redesign a protein to be more thermally stable (i.e. stable at high temperatures). This can be accomplished by systematically modifying the amino acid sequence via local structural entropy (LSE) minimization. The minimization problem is modeled as a shortest path problem in an acyclic graph with nonnegative weights and is solved efficiently using Dijkstra's method.     

Characterization of a thermally stable and organic solvent-adaptative NAD+ -dependent formate dehydrogenase from Bacillus sp. F1

Aims: To characterize a robust NAD⁺‐dependent formate dehydrogenase firstly obtained from a nonmethylotroph, Bacillus sp. F1. Methods and Results: The Bacillus sp. F1 NAD⁺‐dependent formate dehydrogenase (BacFDH) gene was cloned by TAIL‐PCR and heterologous expressed in Escherichia coli. BacFDH was stable at temperatures below 55°C, and the half‐life at 60°C was determined as 52·9 min. This enzyme also showed a broad pH stability and retained more than 80% of the activities after incubating in buffers with different pH ranging from 4·5 to 10·5 for 1 h. The activity of BacFDH was significantly enhanced by some metal ions. Moreover, BacFDH exhibited high tolerance to 20% dimethyl sulfoxide, 60% acetone, 10% methanol, 20% ethanol, 60% isopropanol and 20% n‐hexane. Like other FDHs, BacFDH displayed strict substrate specificity for formate. Conclusion: We isolated a robust formate dehydrogenase, designated as BacFDH, which showed excellent thermal stability, organic solvent stability and a broad pH stability. Significance and Impact of the Study: The multi‐aspect stability makes BacFDH a competitive candidate for coenzyme regeneration in practical applications of chiral chemicals and pharmaceuticals synthesis with a relatively low cost, especially for the catalysis performed in extreme pH conditions and organic solvents.     

A bacterial TrwC relaxase domain contains a thermally stable alpha-helical core

The TrwC protein is the relaxase-helicase responsible for the initiation and termination reactions of DNA processing during plasmid R388 conjugation. The TrwC-N275 fragment comprises the 275-amino-acid N-terminal domain of the protein that contains the DNA cleavage and strand transfer activities (the relaxase domain). It can be easily purified by keeping a cell lysate at 90 degrees C for 10 min. Infrared spectroscopy shows that this domain has a predominantly alpha/beta structure with some amount of unordered structure. Fast heating and cooling does not change the secondary structure, whereas slow heating produces two bands in the infrared spectrum characteristic of protein aggregation. The denaturation temperature is increased in the protein after the fast-heating thermal shock. Two-dimensional infrared correlation spectroscopy shows that thermal unfolding is a very cooperative two-state process without any appreciable steps prior to aggregation. After aggregation, the alpha-helix percentage is not altered and alpha-helix signal does not show in the correlation maps, meaning that the helices are not affected by heating. The results indicate that the domain has an alpha-helix core resistant to temperature and responsible for folding after fast heating and an outer layer of beta-sheet and unordered structure that aggregates under slow heating. The combination of a compact core and a flexible outer layer could be related to the structural requirements of DNA-protein binding.     

Electron cryomicroscopy of bacteriorhodopsin vesicles: mechanism of vesicle formation.

We obtained vesicles from purple membrane of Halobacterium halobium at different suspension compositions (pH, electrolytes, buffers), following the procedure of Kouyama et al. (1994) (J. Mol. Biol. 236:990-994). The vesicles contained bacteriorhodopsin (bR) and halolipid, and spontaneously formed during incubation of purple membrane suspension in the presence of detergent octylthioglucoside (OTG) if the protein:OTG ratio was 2:1 by weight. The size distribution of the vesicles was precisely determined by electron cryomicroscopy and was found to be almost independent on the incubation conditions (mean radius 17.9-19 nm). The size distribution in a given sample was close to the normal one, with a standard deviation of approximately +/- 1 nm. During dialysis for removal of the detergent, the vesicles diminished their radius by 2-2.5 nm. The results allow us to conclude that the driving force for the formation of bR vesicles is the preferential incorporation of OTG molecules in the cytoplasmic side of the membrane (with possible preferential delipidation of the extracellular side), which creates spontaneous curvature of the purple membrane. From the size distribution of the vesicles, we calculated the elasticity bending constant, K(B) approximately 9 x 10(-20) J, of the vesicle wall. The results provide some insight into the possible formation mechanisms of spherical assembles in living organisms. The conditions for vesicle formation and the mechanical properties of the vesicles could also be of interest with respect to the potential technological application of the bR vesicles as light energy converters.     

Light-driven proton transport of bacteriorhodopsin incorporated into long-term stable liposomes of a polymerizable sulfolipid

The chromoprotein bacteriorhodopsin from Halobacterium halobium has been incorporated into liposomes made of a fully synthetic, polymerizable lipid. Bacteriorhodopsin is found to be active in these polymer liposomes. The advantage in the use of such polymer systems concerning long-term stability in comparison with liposomes made of natural lipid is demonstrated.     

Complete stereospecific repair of a synthetic dinucleotide spore photoproduct by spore photoproduct lyase

Spore photoproduct lyase (SP lyase), a member of the radical S-adenosylmethionine superfamily of enzymes, catalyzes the repair of 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)], a type of UV-induced DNA damage unique to bacterial spores. The anaerobic purification and characterization of Clostridium acetobutylicum SP lyase heterologously expressed in Escherichia coli, and its catalytic activity in repairing stereochemically defined synthetic dinucleotide SPs was investigated. The purified enzyme contains between 2.3 and 3.1 iron atoms per protein. Electron paramagnetic resonance (EPR) spectroscopy reveals an isotropic signal centered at g = 1.99, characteristic of a [3Fe–4S]⁺ cluster accounting for 3–4% of the iron in the sample. Upon reduction, a nearly axial signal (g = 2.03, 1.93 and 1.92) characteristic of a [4Fe–4S]⁺ cluster is observed that accounts for 34–45% of total iron. Addition of S-adenosylmethionine to the reduced enzyme produces a rhombic signal (g = 2.02, 1.93, 1.82) unique to the S-adenosyl-l-methionine complex while decreasing the overall EPR intensity. This reduced enzyme is shown to rapidly and completely repair the 5R diastereomer of a synthetic dinucleotide SP with a specific activity of 7.1 ± 0.6 nmol min⁻¹ mg⁻¹, whereas no repair was observed for the 5S diastereomer.     

Regeneration of bacteriorhodopsin from thermally unfolded bacterio-opsin and all-trans retinal at high temperatures

The temperature dependence of regeneration of bacteriorhodopsin (bR) from its apoprotein, bacterio-opsin (bO), and all-trans retinal was investigated using two different procedures to probe the structural properties of bO at high temperatures. Regeneration experiments performed at 25 degrees C after incubation of bO within the temperature range of 35-75 degrees C indicate that irreversible thermal unfolding begins at 50 degrees C. When bO is incubated for one hour and mixed with retinal at the same elevated temperatures, however, a greater extent of regeneration to bR occurs, even at temperatures ranging from 50 to 65 degrees C. These experimental results indicate that regeneration of bR occurs from thermally unfolded bO and suggest dynamic structural fluctuation of bO in the unfolded state.     

The presence of a thermally stable domain in the spiral part of a collagen molecule

It is shown that in 0,5 M NaCl 8 M CH3COOH heat absorption and the second structure alteration in a heated solution proceed between two stages following one another, and besides, salts not only decrease the macromolecule denaturation temperature in total, but produce different destabilization effect on different regions. The presence of the thermostable domain in the macromolecule helical part permits investigation of the folding mechanism of the triple collagen helix under partial denaturation. The localization and biological role of the stable domain in the triple helix formation are suggested.     

Tyrosine protonation changes in bacteriorhodopsin. A Fourier transform infrared study of BR548 and its primary photoproduct

The structural alterations which occur in bacteriorhodopsin (bR) during dark adaptation (BR570----BR548) and the primary phototransition of the dark photocycle (BR548----KD610) have been investigated by Fourier transform infrared and UV difference spectroscopy. Possible contributions of tyrosine to the Fourier transform infrared difference spectra of these transitions were assigned by incorporating ring per-deuterated tyrosine into bR. Based on these data and UV difference measurements, we conclude that a stable tyrosinate exists in BR570 at physiological temperature and that it protonates during formation of BR548. A tyrosinate protonation has also been observed at low temperature during the primary phototransition of BR570 to the red-shifted photoproduct K630 (1). However, we now find that no tyrosine protonation change occurs during the primary phototransition of BR548 to the red-shifted intermediate KD610. Through analysis of bR containing isotopically labeled retinals, it was also determined that the chromophore of KD610 exits in a 13-trans, 15-cis configuration. On the basis of this evidence and previous studies on the structure of the chromophore in BR570, BR548, and K630, it appears that only the 13-trans,15-trans configuration of the protonated chromophore leads to a stable tyrosinate group. It is proposed that a tyrosinate residue is stabilized due to its interaction with the Schiff base positive charge in the BR570 chromophore. Isomerization of the chromophore about either the C13 = C14 or C = N bond disrupts this interaction causing a protonation of the tyrosinate.     

Fourier transform infrared spectroscopic evidence for the existence of two conformations of the bacteriorhodopsin primary photoproduct at low temperature

Fourier transform infrared difference spectroscopy of bacteriorhodopsin at low temperature reveals at least two stable forms of bacteriorhodopsin570 and the K photoproduct. In the case of bacteriorhodopsin570, warming from 81 to 135 K causes a reduction in absorption of several chromophore vibrations, but not the C = N stretching mode. These changes are consistent with a reorientation of the chromophore which leaves the angle of the C = N bond unchanged relative to the membrane plane. In the case of the K intermediate, two different forms can be isolated at 135 K on the basis of wavelength-dependent photoalteration. One form is identical to the low temperature K630 species, whereas a second blue-shifted form is present only above 135 K. This new form exhibits a 985 cm-1 peak in the hydrogen-out-of-plane bending region, which is similar to a reported room-temperature resonance Raman spectrum of K. Temperature-dependent changes in the conformation of the protein involving possible alterations in peptide hydrogen bonding are also detected.     

The structure of a thermally stable 3-phosphoglycerate kinase and a comparison with its mesophilic equivalent

The structure of the phosphoglycerate kinase (PGK) from Bacillus stearothermophilus, a moderate thermophile, has been determined and compared with that of its mesophilic equivalent from yeast. The Bacillus enzyme structure was solved by molecular replacement and improved using constrained rigid-body, molecular dynamics and conventional refinement procedures. The refinement residual, calculated using all the measured data between 8 and 1.65 Å, is 0.18(1). The stereo chemical deviations of the final model from ideality are 0.01 Å for both bonds and planes. The mid-point temperatures of the Bacillus and yeast enzymes are 67 and 53°C, respectively. Differential scanning calorimetry indicates that the energy difference (ΔΔG) between the mesophilic and thermophilic enzymes is of the order of 5 kcal mol^?1 at room temperature. The structure comparison indicates that the features most likely to be responsible for the increased thermal stability of the Bacillus enzyme are the increased internal hydrophobicity, additional ion pairs, and better α-helix stability resulting from the removal of helix destablising residues and extra helix–dipole/helix side chain ionic interactions. © 1993 Wiley-Liss, Inc.     

Two processes lead to a stable all-trans and 13-cis isomer equilibrium in dark-adapted bacteriorhodopsin; effect of high pressure on bacteriorhodopsin,bacteriorhodopsin mutant D96N and fluoro-bacteriorhodopsin analogues

The combination of absorption spectroscopy and extraction techniques was applied to study the effect of high pressure on the dark-adapted state of bacteriorhodopsin, 14-(12-,10-)fluoro-bacteriorhodopsin, a D96N bacteriorhodopsin mutant, and 14-(12-,10-)fluoro-D96N. Evidence is presented that, at high pressure, the isomers' equilibrium is shifted from all- trans isomers towards the 13-cis isomers. Two groups of values for calculated molar volume changes indicate that there are at least two different processes leading to a stable all-trans and 13-cis isomers' equilibrium called the dark-adapted bacteriorhodopsin. The first process may be attributed to changes in the distances and rearrangement of functionally important residues and a retinal Schiff base. It is suggested that the moved residues (probably Asp-212 with the contribution of Tyr-185 and/or Asp-85) closer to the chromophore could catalyse its trans-cis isomerization. These changes require smaller pressure changes and induce larger volume changes (large-volume-change process). The second process may be attributed to the formation of the three hydrogen bonds that additionally decrease the volume and strengthen further stabilization of the 13-cis isomer. To induce these changes, larger changes of pressure are required and the final molar volume changes are smaller (small-volume-change process). The total molar volume change between all-trans bacteriorhodopsin and 13-cis bacteriorhodopsin in the dark-adapted state of native bacteriorhodopsin was found to be about -28 mL/mol, which is much higher than the value of about -7 mL/mol obtained previously (Tsuda and Ebrey 1980, Schulte and Bradley 1995). The data provide a novel insight into factors leading to stable isomer equilibrium in dark-adapted bacteriorhodopsin.     

Luffa cylindrica sponges as a thermally and chemically stable support for Aspergillus niger lipase

The use of biopolymer compounds as matrices for enzyme immobilization is currently a focus of increasing interest. In the present work we propose the use of Luffa cylindrica vegetable sponges as a support for the lipase extracted from Aspergillus niger. Effectiveness of immobilization was analyzed using Fourier transform infrared spectroscopy, elemental analysis and the Bradford method. An initial enzyme solution concentration of 1.0 mg/mL and an immobilization time of 12 h were selected as the parameters that produce a system retaining the highest hydrolytic activity (84% of free enzyme). The resulting biocatalyst system also exhibited high thermal and chemical stability, reusability and storage stability, which makes it a candidate for use in a wide range of applications. Kinetic parameters for the native and immobilized lipase were also calculated. The value of the Michaelis–Menten constant for the immobilized lipase (0.47 mM) is higher than for the free enzyme (0.21 mM), which indicates that the adsorbed enzyme exhibits a lower affinity to the substrate than native lipase. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:657–665, 2016     

Synthesis of a red-shifted fluorescence polarization probe for Hsp90

The synthesis of a red-shifted cy3B-GM ligand and its evaluation as a fluorescence polarization probe for Hsp90 is presented.     

Substitution of amino acids in helix F of bacteriorhodopsin: effects on the photochemical cycle

The effects of amino acid substitutions in helix F of bacteriorhodopsin on the photocycle of this light-driven proton pump were studied. The photocycles of Ser-183----Ala and Glu-194----Gln mutants were qualitatively similar to that of wild-type bacteriorhodopsin produced in Escherichia coli and bacteriorhodopsin from Halobacterium halobium. The substitution of a Phe for either Trp-182 or Trp-189 significantly reduced the fraction of photocycling bacteriorhodopsin. The amino acid substitutions Tyr-185----Phe and Ser-193----Ala substantially increased the lifetime of the photocycle without substantially increasing the lifetime of the M photocycle intermediate. Similar results were also obtained with the Pro-186----Gly substitution. In contrast, replacing Pro-186 with the larger residue Leu inhibited the formation of the M photocycle intermediate. These results are consistent with a structural model of the retinal-binding pocket suggested by low-temperature UV/visible and Fourier transform infrared difference spectroscopies that has Trp-182, Tyr-185, Pro-186, and Trp-189 forming part of the binding pocket.