Predictive energy landscapes for folding membrane protein assemblies.

We study the energy landscapes for membrane protein oligomerization using the Associative memory, Water mediated, Structure and Energy Model with an implicit membrane potential (AWSEM-membrane), a coarse-grained molecular dynamics model previously optimized under the assumption that the energy landscapes for folding α-helical membrane protein monomers are funneled once their native topology within the membrane is established. In this study we show that the AWSEM-membrane force field is able to sample near native binding interfaces of several oligomeric systems. By predicting candidate structures using simulated annealing, we further show that degeneracies in predicting structures of membrane protein monomers are generally resolved in the folding of the higher order assemblies as is the case in the assemblies of both nicotinic acetylcholine receptor and V-type Na(+)-ATPase dimers. The physics of the phenomenon resembles domain swapping, which is consistent with the landscape following the principle of minimal frustration. We revisit also the classic Khorana study of the reconstitution of bacteriorhodopsin from its fragments, which is the close analogue of the early Anfinsen experiment on globular proteins. Here, we show the retinal cofactor likely plays a major role in selecting the final functional assembly.

Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes.

The expression of membrane proteins for functional and structural studies or medicinal applications is still not very well established. Membrane-spanning proteins that mediate the information flow of the extracellular side with the interior of the cell are prime targets for drug development methods that would allow screening techniques or high throughput formats are of particular interest. Here we describe a systematic approach to the liposome-assisted cell-free synthesis of functional membrane proteins. We demonstrate the synthesis of bacteriorhodopsin (bR(cf)) in presence of small unilamellar liposomes. The yield of bR(cf) per volume cell culture is comparable to that of bacteriorhodopsin in its native host. The functional analysis of bR(cf) was performed directly using the cell-free reaction mixture. Photocycle measurements reveal kinetic data similar to that determined for bR in Halobacterium salinarum cell-envelope vesicles. The liposomes can be attached directly to black lipid membranes (BLM), which allows measuring light activated photocurrents in situ. The results reveal a functional proton pump with properties identical to those established for the native protein.

Synthesis of N-heteroaryl retinals and their artificial bacteriorhodopsins.

N-Heteroaryl retinals derived from indole, 1-indolizine and 3-indolizine (10 a-c) have been synthesized after their UV/Vis red-shifted absorption properties had been predicted by time-dependent density functional theory (TD-DFT) computations. The three new analogues form artificial pigments upon recombination with bacterioopsin: indolyl retinal 10 a undergoes fast and efficient reconstitution to form a species with a UV/Vis absorbance maximum similar to that of wild-type bacteriorhodopsin, whilst the indolizinyl retinals 10 b and 10 c also reconstitute in significant proportion to give noticeably red-shifted, although unstable, pigments. Significant changes in the pK(a) values of these artificial bacteriorhodopsins are interpreted as arising from nonoptimal binding-site occupancy by the chromophore due to steric constraints.

A study on the fabrication of chemically modified bacteriorhodopsin film and its photochemical properties.

Chemically modified bacteriorhodopsin (BR) films embedded in gelatin matrix were fabricated. It was found that chemically modified BR/gelatin film remained the original conformation of BR and possessed homogeneity. Then, the photochemical conversion from all-trans O state to 9-cis P state in the chemically modified BR film/gelatin was investigated. For comparison purpose, the same conversion in the wild-type BR (BRwt) film was also presented here. To our know, it is the first report to show that the absorption changes of the P state in the chemically modified BR film were larger than in the BRwt film upon illumination with red light at the low actinic power density.

Probing the folding and unfolding of wild-type and mutant forms of bacteriorhodopsin in micellar solutions: evaluation of reversible unfolding conditions.

Wild-type and mutant forms of bacteriorhodopsin (sbR) from Halobacterium salinarium, produced by Escherichia coli overexpression of a synthetic gene, were reversibly unfolded in 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxyl-1-propane (CHAPSO), and sodium dodecyl sulfate (SDS) mixed micelles. To study the effect on protein stability by substitutions on the hydrophobic surface with polar residues, the unfolding behavior of a G113Q, G116Q mutant [sbR(Q2)] was compared to the wild-type sbR [sbR(WT)]. sbR(Q2) was more sensitive to SDS-induced unfolding than sbR(WT) under equilibrium conditions, and kinetic experiments showed that sbR(Q2) was more sensitive to acid-induced denaturation and thermal unfolding than sbR(WT). Since the mutations in sbR(Q2) were on the detergent-embedded hydrophobic surface of sbR, protein destabilization by these mutations supports the concept that the membrane-embedded segments are important for the stability of sbR. Our experiments provide the basis for studying the thermodynamic stability of sbR by evaluating reversible folding and unfolding conditions in DMPC/CHAPSO/SDS mixed micelles.

13-Demethylbacteriorhodopsin: formation and some properties.

Incorporation of 9-cis-13-demethylretinal into bacterioopsin was shown to form the covalent purple complex. This result was predicted by our hypothesis about the structure of the BR chromophore cavity (Mol. Biologiya 29:1398-1407 (1995) (in Russian)). It supports the hypothesis and eliminates the main objection known from the literature.

[Spatial structure of bacterioopsin 87-136 fragment]. / Prostranstvennaya struktura fragmenta 87-136 bacterioopsina.

The [Nle18]-(87-136)-bacterioopsin, a fragment of bacterioopsin from Halobacterium salinarium synthesized by solid phase technique, was solubilized in a 1:1 chloroform-methanol mixture containing 0.1 M LiClO4 and studied by two-dimensional 1H NMR spectroscopy. The complete assignment of proton resonances was performed in the DQF-COSY, TOCSY, and NOESY spectra of this peptide, and its spatial structure was computed. As a result, two helical regions (92-100 and 108-130) were identified, which correspond to the C-terminal part of segment C and to segment D of bacteriorhodopsin, respectively. The 92-100 region forms a right-handed alpha-helix, and the 108-130 region can adopt right-handed alpha-helical, 3(10)-helical, and combined (from the two) conformations. A comparison of the structure computed with the bacteriorhodopsin model deduced from the electron cryomicroscopy data showed good agreement in the 91-100 region (the root-mean-square deviation of the backbone atoms was less than 0.51 A) and considerable differences in the 108-130 region (1.82 A). A dynamic model of the conformation of the D transmembrane segment was suggested, and the accordance of the model to the functional dynamics of bacteriorhodopsin was discussed.

Primary picosecond molecular events in the photoreaction of the BR5.12 artificial bacteriorhodopsin pigment.

The picosecond dynamics of the photoreaction of an artificial bacteriorhodopsin (BR) pigment containing a retinal in which a five-membered ring spans the C-12 to C-14 positions of the polyene chain (BR5.12) is examined by using time-resolved absorption and fluorescence and resonance Raman spectroscopy. The ring within the retinal chromophore of BR5.12 blocks the C-13 = C-14 isomerization proposed to be a primary step in the energy storage/transduction mechanism in the BR photocycle. Relative to the native BR pigment (BR-570), the absorption spectrum of BR5.12 is red-shifted by 8 nm. The fluorescence spectrum of BR5.12 closely resembles that of BR-570 although the relative fluorescence yield is higher (approximately 10-fold). Picosecond transient absorption (4-ps pulses, 568-662 nm) measurements reveal an intermediate absorbing to the red side of BR5.12. Kinetic fits show that the red-absorbing intermediate appears within < 3 ps and decays with a time constant of 17 +/- 1 ps to form only BR5.12. No emission in the 650- to 900-nm region can be attributed to the red-absorbing species. Since rotation around C-12 - C-13 and isomerization around C-13 = C-14 are prevented in BR5.12, these results demonstrate that motion in these regions of the retinal is (i) necessary to form the K-like intermediate observed in the native BR-570 photocycle and (ii) not necessary to form a red-absorbing intermediate that has spectral and kinetic properties analogous to those of J-625 in the native BR photocycle. Discussions of the excited and ground electronic state assignments for the intermediate observed in the BR5.12 photoreaction are presented.

Azidotetrafluorophenyl retinal analogue: synthesis and bacteriorhodopsin pigment formation.

The retinal derivative, all-trans-9-(4-azido-2,3,5,6-tetrafluorophenyl)-3,7- dimethyl-2,4,6,8-nonatetraenal, was synthesized by two routes as a potential photoactivatable cross-linking agent for studies in bacteriorhodopsin (BR) of the chromophore interaction with its apoprotein. The retinal analogue formed a stable, moderately functional BR pigment confirming that the ring cavity of the retinal binding site has a significant tolerance for derivatization on that portion of the molecule. Attempts to cross-link the azido chromophore to the protein by photoactivation were unsuccessful. The electron delocalization effect of the conjugated polyene side chain of the retinal appears to interfere with the formation or reactivity of the nitrene intermediate to the extent that photoactivated cross-linking is not achieved. These results demonstrate a limitation to the use of fluorinated aryl azides as photoaffinity reagents.

Bacteriorhodopsins with chromophores modified at the beta-ionone site. Formation and light-driven action of the proton pump.

The binding to bacterioopsin of the all-trans isomers of retinal analogues lacking the six-membered ring and differing in length of the conjugated chain, as well as the light-driven action of the proton pump of the resulting bacteriorhodopsin analogues, were studied. The 'opsin shifts' in these modified bacteriorhodopsins are all around 2700 cm-1 and do not depend on the number of double bonds in the chromophore. These experimental results suggest that the 4800 cm-1 'opsin shift' in unmodified bacteriorhodopsin consists of a contribution of about 2700 cm-1 due to the interaction of the protonated Schiff-base with the counterion. The extra 2100 cm-1 shift in bacteriorhodopsin is due to the specific interaction of the cyclohexene ring and the protein. Only the bacteriorhodopsin analogue with the same number of conjugated double bonds in the chromophore as bacteriorhodopsin itself shows light-driven proton pump action.