The effect of metal cation binding on the protein, lipid and retinal isomeric ratio in regenerated bacteriorhodopsin of purple membrane

The effect of metal cation binding on bacteriorhodopsin (bR) in purple membrane has been examined using in situ attenuated total reflection-Fourier transform infrared difference spectroscopy in aqueous media. It is known that adding metal cations to deionized bR regenerates the purple state from its blue state and recovers the proton pump function. During this process, infrared spectral changes in the frequency region of 1800-1000 cm-1 are monitored. The results reveal that metal cation binding affects the protein conformation, the retinal isomeric composition as well as lipid head groups. It is also observed that metal cation binding induces conformational changes in the alpha 1-helix region of bR, converting the portion of its alpha 1-helical domain into beta-turn or disordered coil. In addition, the influence of Ho3+ binding on the protein and lipid is observed to be larger than that of Ca2+. These results suggest that some of the metal cation binding sites are on the membrane lipid domain, while others could be on the intrahelical domain or interhelical loops where the Asp and Glu are located (binding with their COO- groups). Our results also suggest that the removal of the C-terminal of bR increase the accessibility of the binding site of metal cations, which affects protein conformational structure. All these observations are discussed in terms of the two proposals given in the literature regarding the metal cation binding sites.     

TIME-RESOLVED RESONANCE RAMAN SPECTRA OF THE PHOTOCYCLE INTERMEDIATES OF ACID AND DEIONIZED BACTERIORHODOPSIN

Abstract— The photocycle of bacteriorhodopsin (bR) and its perturbed forms are investigated by a time-resolved resonance Raman study. These experiments were performed in the C=C stretching and in the fingerprint spectral regions for the acid blue, acid purple and deionized forms of bR.
The main observations are as follows: (1) isomerization of the retinal, from all- trans to 13- cis , occurs in native bR and in all of the acid and deionized perturbed bR species; (2) formation of the early intermediates (the K₆₁₀ and L₅₅₀ analogues) also occur in native bR and in all of the perturbed species; and (3) deprotonation of the protonated Schiff base (PSB), to give the M₄₁₂ type intermediate, occurs in native bR, but is inhibited in all of the perturbed bR species on the time-scale of the native bR photocycle.
The results show that isomerization alone is not a prerequisite for the PSB deprotonation process. The observed photocycle, initiated with retinal isomerization, is found to occur from all- trans to 13- cis in all of the perturbed forms of bR. In addition, the results imply that removal of the cations, of an increase in the hydrogen ion concentration, prevent only the PSB deprotonation process and not the formation of earlier cycle intermediates. Some attention is focused on the two blue forms of bR (acid and deionized) due to the fact that their ground-state absorption maximum, unphotolyzed Raman spectra, and Raman spectra changes during the photocycle are all very similar. The similarities between the acid blue and deionized blue forms in the fingerprint region support previous suggestions that both blue species have nearly the same retinal active site.     

Picosecond multidimensional fluorescence spectroscopy: a tool to measure real-time protein dynamics during function

Advanced multidimensional time-correlated single photon counting (mdTCSPC) and picosecond time-resolved fluorescence in combination with site-directed fluorescence labeling are valuable tools to study the properties of membrane protein surface segments on the pico- to nanoseconds time scale. Time-resolved fluorescence anisotropy changes of protein bound fluorescent probes reveal changes in protein dynamics and steric restriction. In addition, the change in fluorescence lifetime and intensity of the covalently bound fluorescent dye is indicative of environmental changes at the protein surface. In this study, we have measured the changes in fluorescence lifetime traces of the fluorescent dye fluorescein covalently bound to the first cytoplasmic loop of bacteriorhodopsin (bR) after light activation of protein function. The fluorescence is excited by a picosecond laser pulse. The retinylidene chromophore of bR is light-activated by a 10 ns laser pulse, which in turn triggers recording of a sequence of fluorescence lifetime traces in the mdTCSPC-module. The fluorescence decay changes upon protein function occur predominantly in the 100 ps time range. The kinetics of these changes shows two transitions between three intermediate states in the second part of the bR photocycle. Correlation with photocycle kinetics allows for the determination of reaction intermediates at the proteins surface which are coupled to changes in the retinal binding pocket.     

Pressure-induced isomerization of retinal on bacteriorhodopsin as disclosed by fast magic angle spinning NMR

Bacteriorhodopsin (bR) is a retinal protein in purple membrane of Halobacterium salinarum, which functions as a light-driven proton pump. We have detected pressure-induced isomerization of retinal in bR by analyzing 15N cross polarization-magic angle spinning (CP-MAS) NMR spectra of [zeta-15N]Lys-labeled bR. In the 15N-NMR spectra, both all-trans and 13-cis retinal configurations have been observed in the Lys N(zeta) in protonated Schiff base at 148.0 and 155.0 ppm, respectively, at the MAS frequency of 4 kHz in the dark. When the MAS frequency was increased up to 12 kHz corresponding to the sample pressure of 63 bar, the 15N-NMR signals of [zeta-15N]Lys in Schiff base of retinal were broadened. On the other hand, other [zeta-15N]Lys did not show broadening. Subsequently, the increased signal intensity of [zeta-15N]Lys in Schiff base of 13-cis retinal at 155.0 ppm was observed when the MAS frequency was decreased from 12 to 4 kHz. These results showed that the equilibrium constant of [all-trans-bR]/[13-cis-bR] in retinal decreased by the pressure of 63 bar. It was also revealed that the structural changes induced by the pressure occurred in the vicinity of retinal. Therefore, microscopically, hydrogen-bond network around retinal would be disrupted or distorted by a constantly applied pressure. It is, therefore, clearly demonstrated that increased pressure induced by fast MAS frequencies generated isomerization of retinal from all-trans to 13-cis state in the membrane protein bR.     

Dynamics of Bacteriorhodopsin in the Dark-Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy

To achieve efficient proton pumping in the light-driven proton pump bacteriorhodopsin (bR), the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark-adapted state, and were found to be mostly well ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10 % residual proton pumping activity, are less well ordered, suggesting a link between side-chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground-state bR.     

INTERACTION OF DIBUCAINEHCl LOCAL ANESTHETICS WITH BACTERIORHODOPSIN IN PURPLE MEMBRANE: A SPECTROSCOPIC STUDY

Several spectroscopic techniques (absorption, emission, transient absorption and differential scanning calorimetry--DSC) were used to investigate the deprotonation of dibucaine.HCl in a hydrophobic environment, and the interaction sites and mechanisms of the local anesthetic dibucaine.HCl on bacteriorhodopsin (bR) in purple membrane. The important results are summarized as follows: (1) the visible absorption features of native (lambda max = 568 nm) and deionized (lambda max = 608 nm) bR are sensitive to the amount of dibucaine.HCl added; (2) the emission spectrum of dibucaine.HCl embedded in the retinal-free mutant bR is similar to that of dibucaine free base in Triton X-100 micellar solutions; (3) the phosphorescence emission of dibucaine at 77 K is completely quenched by bR and the fluorescence quenching rate for the incorporated dibucaine.HCl in bR was determined as kq = 4.09 x 10(13) M-1 s-1; (4) the incorporation of dibucaine.HCl in bR inhibits the slow component rate of formation of M412 and decreases the amount of M412 formation in the photochemical cycle of bR; and (5) the thermal stability of native bR was measured by DSC in the presence and absence of dibucaine and yielded an endothermic transition at 95.9 +/- 1.0 degrees C with 13.6 J/g (3.25 +/- 0.12 cal/g) of enthalpy changes. All observations suggest that the action site of the local anesthetic, dibucaine.HCl, is near or at the chromophore, i.e. the retinal Schiff base of bR. The anesthetic action on bR purple membrane is probably via a specific site binding, but not a conformational mechanism.     

Characterization and photochemistry of 13-desmethyl bacteriorhodopsin

The photochemistry of the 13-desmethyl (DM) analogue of bacteriorhodopsin (BR) is examined by using spectroscopy, molecular orbital theory, and chromophore extraction followed by conformational analysis. The removal of the 13-methyl group permits the direct photochemical formation of a thermally stable, photochemically reversible state, P1(DM) (lambda(max) = 525 nm), which can be generated efficiently by exciting the resting state, bR(DM) with yellow or red light (lambda > 590 nm). Chromophore extraction analysis reveals that the retinal configuration in P1(DM) is 9-cis, identical to that of the retinal configuration in the native BR P1 state. Fourier transform infrared and Raman experiments on P1(DM) indicate an anti configuration around the C15=N bond, as would be expected of an O-state photoproduct. However, low-temperature spectroscopy and ambient, time-resolved studies indicate that the P1(DM) state forms primarily via thermal relaxation from the L(D)(DM) state. Theoretical studies on the BR binding site show that 13-dm retinal is capable of isomerizing into a 9-cis configuration with minimal steric hindrance from surrounding residues, in contrast to the native chromophore in which surrounding residues significantly obstruct the corresponding motion. Analysis of the photokinetic experiments indicates that the Arrhenius activation energy of the bR(DM) --> P1(DM) transition in 13-dm-BR is less than 0.6 kcal/mol (vs 22 +/-5 kcal/mol measured for the bR --> P (P1 and P2) reaction in 85:15 glycerol:water suspensions of wild type). Consequently, the P1(DM) state in 13-dm-BR can form directly from all-trans, 15-anti intermediates (bR(DM) and O(DM)) or all-trans, 15-syn (K(D)(DM)/L(D)(DM)) intermediates. This study demonstrates that the 13-methyl group, and its interactions with nearby binding site residues, is primarily responsible for channeling one-photon photochemical and thermal reactions and is limited to the all-trans and 13-cis species interconversions in the native protein.     

Protein-cation interactions: structural and thermodynamic aspects

Cations are specifically recognized by numerous proteins. Cations may play a structural role, as cofactors stabilizing their binding partners, or a functional role, as cofactors activating their binding partners or being themselves involved in enzymatic reactions. Despite their small size, their charge density and their specific interaction with highly charged residues allow them to induce significant conformational changes on their binding proteins. The protein conformational change induced by cation binding may be as large as to account for the complete folding of a protein (as evidenced in Hepatitis C NS3 protease, or human rhinovirus 2A protease), and they may also trigger oligomerization (as in calcium-binding protein 1). Especially intriguing is the ability of cation-binding proteins of discriminating between very similar cations. In particular, calcium and magnesium are recognized by proteins with markedly different binding affinities and cause significantly different conformational changes and stabilization effects in the binding proteins (as in the fifth ligand binding repeat of the LDL receptor binding domain, calcium-binding protein 1, or parvalbumin). This article summarizes recent findings on the structural and energetic impact of cation binding to different proteins. A general framework can be envisaged in which cations can be considered as a special type of allosteric effectors able to modulate the functional properties of proteins, in particular the ability to interact with biological targets, by altering their conformational equilibrium.     

Photoreduction of bacteriorhodopsin Schiff base at low humidity. A study with C13=C14 nonisomerizable artificial pigments

The retinal protonated Schiff base of bacteriorhodopsin is photoreactive to reducing agents such as NaBH4. In the present work we have studied the effect of different protein hydration levels on the photoreductive reaction, as well as the consequences of preventing isomerization around the critical C13=C14 retinal double bond. It was revealed that the rate of light-induced NaBH4 reaction can be fitted to three phases, between 100 and 87%, from 87 to 35% and below 35% relative humidities (r.h.). The three phases are attributed to three protein regions characterized by different water affinities. Furthermore, it is shown that the PSB reduction reaction is light catalyzed even in artificial pigments derived from retinal analogs, in which isomerization around the C13=C14 double bond is prevented. It is suggested that the protein experiences light-induced conformational alterations that are not associated with C13=C14 double bond isomerization. In the 13-cis locked pigment the rate of reduction reaction is affected by r.h. levels only below 35%. The relatively low r.h. required for withdrawing water from the protein is attributed to the increased protein-water affinity in this specific pigment.     

Time-resolved thermodynamic changes photoinduced in 5,12-trans-locked bacteriorhodopsin. Evidence that retinal isomerization is required for protein activation

Structural volume changes upon excitation of isomerization-blocked 5,12-trans-locked bacteriorhodopsin (bR) (bacterio-opsin + 5-12-trans-locked retinal) were studied using photothermal methods. The very small prompt expansion detected using laser-induced optoacoustics (0.3 mL/mol of absorbed photons) is assigned to a charge reorganization in the chromophore protein pocket concomitant with the formation of the intermediate T5.12. The subsequent contraction associated with a 300 ns lifetime is assigned to protein movements required to reach the entire chromoprotein free energy minimum, after the 17 ps optical decay of T5.12. The volume changes comprise the entropy of medium rearrangement during T5.12 formation and decay. The slow changes detected in previous studies by atomic force microscopy might be explained by the slowing down of movements in films containing 5,12-trans-locked bR. Photothermal beam deflection data with the 5,12-trans-locked bR suspensions indicate no further changes in microseconds to hundreds of milliseconds. Thus, all the absorbed energy is either released to the solution as heat or used for entropy changes within the first 300 ns after the pulse, supporting the paradigm that isomerization is required for signal transduction in retinal proteins. Bacterio-opsin assembled with all-trans-retinal afforded (similar to data reported with wild-type bR) an expansion of 2.6 mL/mol (assigned to the production of KE) followed by a further expansion of 0.8 mL/mol (KE-->KL; KE, KL, early and late K's) involving no heat loss. For KL decay to L, a contraction of 6 mL/mol of phototransformed reconstituted all-trans bR was determined.     

Role of hydrogen-bond network in energy storage of bacteriorhodopsin's light-driven proton pump revealed by ab initio normal-mode analysis

Vibrational modes of the hydrogen-bond network in the binding site of bacteriorhodopsin (bR), a protein in halobacteria functioning as a light-driven proton pump, were investigated by an ab initio quantum mechanical/molecular mechanical (QM/MM) method. Normal-mode analysis calculations for O-D and N-D stretching modes of internal water molecules and the Schiff base of the retinal chromophore in the early intermediate state, K, reproduced well experimentally observed vibrational spectra. Supported by agreement with observed spectra, the QM/MM calculation suggests that weakened hydrogen bonds upon photoisomerization of the chromophore are an important means of energy storage in bR.     

Participation of the BC Loop in the Correct Folding of Bacteriorhodopsin as Revealed by Solid‐state NMR†

Structural changes in bacteriorhodopsin (bR) in two different processes of retinal reconstitutions were investigated by observing the ¹³C and ¹⁵N solid‐state NMR spectra of [1‐¹³C]Val‐ and [¹⁵N]Pro‐labeled bR. We found that NMR signals of the BC loop were sensitive to changes in protein structure and dynamics, from wild‐type (WT) bR to bacterio‐opsin (bO), regenerated bR and E1001 bR. Regenerated bR was prepared following the addition of retinal into bO obtained from photobleached WT‐bR. E1001 bR was cultured from a retinal‐deficient strain termed E1001 following the addition of retinal to growing cells. ¹⁵N NMR signal at Pro70 in the BC loop in WT‐bR was observed at 122.4 p.p.m., whereas signals were not apparent or partly suppressed in bO and regenerated bR, respectively. Similarly, the ¹³C NMR signal at Val69 in the BC loop at 172.0 p.p.m. that was observed in WT‐bR was significantly decreased in both regenerated bR and bO. These results suggest that the dynamic structure of the BC loop in bO was substantially altered following the removal of retinal. As a consequence, the correct protein structure failed to be recovered via the regenerating process of retinal to bO. On the other hand, ¹³C and ¹⁵N NMR signals at the BC loop in E1001 bR appeared at positions identical to those of WT‐bR. The results of the current study indicate that the BC loop may not always fold correctly in the regenerated bR, which leads to different properties in the regenerated bR compared to that of WT‐bR.     

ISOMER COMPOSITION and SPECTRA OF THE DARK and LIGHT ADAPTED FORMS OF ARTIFICIAL BACTERIORHODOPSINS*

Abstract– The isomer composition and spectral properties of 15 artificial bacteriorhodopsin (bR) pigments, based on a series of retinal analogs with polyene residue modified below C₉ are determined for both dark-adapted (DA) and light-adapted (LA) forms. Similarly to native bR, in all cases only two isomers, C₁₃=C₁₄cis (13-cis) and M-trans, are observed. However, the artificial DA pigments have a lower 13-d.s content than native DA bR (? 66%) while the corresponding LA pigments have a much higher 13-cis content (11-69%) than native LA bR (<2%). Thus, in variance with the native pigment, in all of the artificial systems light also induced the reversed all-trans13-cis process. The data are accounted for in terms of specific steric interactions between the polyene and the protein binding site which allow a (C₁₅-anti)(C_ls-syn) isomerization during the photocycle of the artificial pigments, but not in the case of native bR. This accounts for the high proton pumping efficiency of the natural pigment. The nature of a highly red shifted light-adapted form of two of the artificial pigments is investigated and discussed. It is also shown that, in variance with native bR, several artificial pigments exhibit identical absorption spectra for their 13-cis and all-trans isomers. It is concluded that the spectral data for the above species of artificial pigments do not lead to a clear molecular model for the origin of the spectral shift between 13-cis and all-trans bR.     

Efficient calculation of two-dimensional adiabatic and free energy maps: Application to the isomerization of the C13C14 and C15N16 bonds in the retinal of bacteriorhodopsin

Accurate calculation of potential energy and free-energy profiles along reaction coordinates of biological processes such as enzymatic reactions or conformational changes is fundamental to the obtention of theoretical insight into protein function. We describe here the practical implementation of the Automatic Map Refinement Procedure (AMRP) and two-dimensional Weighted Histogram Analysis Method (WHAM) for efficient computation of adiabatic potential energy and free-energy maps, respectively. Methods for efficiently sampling configuration space with high-energy barriers and for removing hysteresis in the case of periodic reaction coordinates are presented. The application of these techniques to the isomerization of the C13C14 and C15N16 bonds in the retinal of bacteriorhodopsin is described. In dark-adapted bacteriorhodopsin (bR), the retinal moiety exists in two conformers, all-trans and (13,15)cis, with the latter making ≃67% of the population. This experimental free energy difference is reproduced here to within k_BT. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 1644–1658, 1999     

Monitoring intracellular proteins using fluorescence techniques: from protein synthesis and localization to activity

The recent breakthroughs in genomics and proteomics and improvements of optical methods have made it possible to obtain localized, real-time information on intracellular proteins dynamics, through dynamic three-dimensional (3D) maps of the living cell with nanometric resolution of individual molecules. On one side, brighter variants of the Green Fluorescence Protein (GFP) have been engineered that have different excitation and/or emission spectra that better match available light sources. Like their parent molecule, these variants retain their fluorescence when fused to heterologous proteins on the N- and C- terminals, and this binding generally does not affect the functionality of the tagged protein leading the way to their use as an intracellular reporter. On the other side, optical methods have been improved to allow reaching the level of single-molecule detection inside living cells. Nevertheless some limitations exist for the use of GFP variants for probing 3D conformational changes of proteins. First, these variants are fused to the N and/or C terminals of the studied protein, which are generally not the best location to detect conformational changes resulting from the binding to other proteins or enzyme substrates. Then their own relatively large size makes them unusable for tagging small proteins. These limitations suggest that new tagging processes, permitting the location of the right fluorescent markers at the right places, must be found to built up inter- and/or intra-molecular rulers allowing one to monitor conformational changes resulting from intracellular protein-protein, protein-membrane, and enzyme-substrate binding. These specific locations can be obtained from in vitro studies of 3D conformational changes that occur during protein docking.     

Combined QM/MM study of the opsin shift in bacteriorhodopsin

Combined quantum mechanical and molecular mechanical (QM/MM) calculations and molecular dynamics simulations of bacteriorhodopsin (bR) in the membrane matrix have been carried out to determine the factors that make significant contributions to the opsin shift. We found that both solvation and interactions with the protein significantly shifts the absorption maximum of the retinal protonated Schiff base, but the effects are much more pronounced in polar solvents such as methanol, acetonitrile, and water than in the protein environment. The differential solvatochromic shifts of PSB in methanol and in bR leads to a bathochromic shift of about 1800 cm(-1). Because the combined QM/MM configuration interaction calculation is essentially a point charge model, this contribution is attributed to the extended point-charge model of Honig and Nakanishi. The incorporation of retinal in bR is accompanied by a change in retinal conformation from the 6-s-cis form in solution to the 6-s-trans configuration in bR. The extension of the pi-conjugated system further increases the red-shift by 2400 cm(-1). The remaining factors are due to the change in dispersion interactions. Using an estimate of about 1000 cm(-1) in the dispersion contribution by Houjou et al., we obtained a theoretical opsin shift of 5200 cm(-1) in bR, which is in excellent agreement with the experimental value of 5100 cm(-1). Structural analysis of the PSB binding site revealed the specific interactions that make contributions to the observed opsin shift. The combined QM/MM method used in the present study provides an opportunity to accurately model the photoisomerization and proton transfer reactions in bR.     

Second-harmonic generation for studying structural motion of biological molecules in real time and space

SHG and sum-frequency generation (SFG) are surface-selective, nonlinear optical techniques whose ability to measure the average tilt angle of molecules on surfaces is well known in non-biological systems. By labeling molecules with a second-harmonic-active dye probe, SHG detection is extended to any biological molecule. The method has been used in previous work to detect biomolecules at an interface and their ligand-induced conformational changes. Here I demonstrate that SHG can be used to study structural motion quantitatively using a probe placed at a specific site (Cys-77) in adenylate kinase, a protein. The protein is also labeled non-site-specifically via amines. Labeled protein is absorbed to a surface and a baseline SH signal is measured. Upon introducing ATP, AMP or a specific inhibitor, AP(5)A, the baseline signal changes depending on the ligand and the labeling site. In particular, a substantial change in SH intensity is produced upon binding ATP to the amine-labeled protein, consistent with the X-ray crystal structures. In contrast, SHG polarization measurements are used to quantitatively determine that no rotation occurs at site Cys-77, in agreement with the lack of motion observed at this site in the X-ray crystal structures. A method for building a global map of conformational change in real time and space is proposed using a set of probes placed at different sites in a biomolecule. For this purpose, SH-active unnatural amino acids are attractive complements to exogenous labels.     

Subpicosecond protein backbone changes detected during the green-absorbing proteorhodopsin primary photoreaction

Recent studies demonstrate that photoactive proteins can react within several picoseconds to photon absorption by their chromophores. Faster subpicosecond protein responses have been suggested to occur in rhodopsin-like proteins where retinal photoisomerization may impulsively drive structural changes in nearby protein groups. Here, we test this possibility by investigating the earliest protein structural changes occurring in proteorhodopsin (PR) using ultrafast transient infrared (TIR) spectroscopy with approximately 200 fs time resolution combined with nonperturbing isotope labeling. PR is a recently discovered microbial rhodopsin similar to bacteriorhodopsin (BR) found in marine proteobacteria and functions as a proton pump. Vibrational bands in the retinal fingerprint (1175-1215 cm(-1)) and ethylenic stretching (1500-1570 cm(-1)) regions characteristic of all-trans to 13-cis chromophore isomerization and formation of a red-shifted photointermediate appear with a 500-700 fs time constant after photoexcitation. Bands characteristic of partial return to the ground state evolve with a 2.0-3.5 ps time constant. In addition, a negative band appears at 1548 cm(-1) with a time constant of 500-700 fs, which on the basis of total-15N and retinal C15D (retinal with a deuterium on carbon 15) isotope labeling is assigned to an amide II peptide backbone mode that shifts to near 1538 cm(-1) concomitantly with chromophore isomerization. Our results demonstrate that one or more peptide backbone groups in PR respond with a time constant of 500-700 fs, almost coincident with the light-driven retinylidene chromophore isomerization. The protein changes we observe on a subpicosecond time scale may be involved in storage of the absorbed photon energy subsequently utilized for proton transport.     

Chromophore interaction in xanthorhodopsin--retinal dependence of salinixanthin binding

Xanthorhodopsin is a light-driven proton pump in the extremely halophilic bacterium Salinibacter ruber. Its unique feature is that besides retinal it has a carotenoid, salinixanthin, with a light harvesting function. Tight and specific binding of the carotenoid antenna is controlled by binding of the retinal. Addition of all-trans retinal to xanthorhodopsin bleached with hydroxylamine restores not only the retinal chromophore absorption band, but causes sharpening of the salinixanthin bands reflecting its rigid binding by the protein. In this report we examine the correlation of the changes in the two chromophores during bleaching and reconstitution with native all-trans retinal, artificial retinal analogs and retinol. Bleaching and reconstitution both appear to be multistage processes. The carotenoid absorption changes during bleaching occurred not only upon hydrolysis of the Schiff base but continued while the retinal was leaving its binding site. In the case of reconstitution, the 13-desmethyl analog formed the protonated Schiff base slower than retinal, and provided the opportunity to observe changes in carotenoid binding at various stages. The characteristic sharpening of the carotenoid bands, indicative of its reduced conformational heterogeneity in the binding site, occurs when the retinal occupies the binding site but the covalent bond to Lys-240 via a Schiff base is not yet formed. This is confirmed by the results for retinol reconstitution, where the Schiff base does not form but the carotenoid exhibits its characteristic spectral change from the binding.     

Artificial Accelerators of the Molecular Chaperone Hsp90 Facilitate Rate‐Limiting Conformational Transitions

The molecular chaperone Hsp90 undergoes an ATP‐driven cycle of conformational changes in which large structural rearrangements precede ATP hydrolysis. Well‐established small‐molecule inhibitors of Hsp90 compete with ATP‐binding. We wondered whether compounds exist that can accelerate the conformational cycle. In a FRET‐based screen reporting on conformational rearrangements in Hsp90 we identified compounds. We elucidated their mode of action and showed that they can overcome the intrinsic inhibition in Hsp90 which prevents these rearrangements. The mode of action is similar to that of the co‐chaperone Aha1 which accelerates the Hsp90 ATPase. However, while the two identified compounds influence conformational changes, they target different aspects of the structural transitions. Also, the binding site determined by NMR spectroscopy is distinct. This study demonstrates that small molecules are capable of triggering specific rate‐limiting transitions in Hsp90 by mechanisms similar to those in protein cofactors.