Secondary structure of a truncated form of lecithin retinol acyltransferase in solution and evidence for its binding and hydrolytic action in monolayers

Lecithin retinol acyltransferase (LRAT) is a 230 amino acids membrane-associated protein which catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester. The enzymatic activity of a truncated form of LRAT (tLRAT) which contains the residues required for catalysis but which is lacking N- and C-terminal hydrophobic segments has been shown to depend on the detergent used for its solubilization. Moreover, it is unknown whether tLRAT can bind membranes in the absence of these hydrophobic segments. The present study has allowed to measure the membrane binding and hydrolytic action of tLRAT in lipid monolayers by use of polarization modulation infrared reflection absorption spectroscopy and Brewster angle microscopy. Moreover, the proportion of the secondary structure components of tLRAT was determined in three different detergents by infrared absorption spectroscopy, vibrational circular dichroism and electronic circular dichroism which allowed to explain its detergent dependent activity. In addition, the secondary structure of tLRAT in the absence of detergent was very similar to that in Triton X-100 thus suggesting that, compared to the other detergents assayed, the secondary structure of this protein is very little perturbed by this detergent.     

Enzymatic activity of Lecithin:retinol acyltransferase: A thermostable and highly active enzyme with a likely mode of interfacial activation

Lecithin:retinol acyltransferase (LRAT) plays a major role in the vertebrate visual cycle. Indeed, it is responsible for the esterification of all-trans retinol into all-trans retinyl esters, which can then be stored in microsomes or further metabolized to produce the chromophore of rhodopsin. In the present study, a detailed characterization of the enzymatic properties of truncated LRAT (tLRAT) has been achieved using in vitro assay conditions. A much larger tLRAT activity has been obtained compared to previous reports and to an enzyme with a similar activity. In addition, tLRAT is able to hydrolyze phospholipids bearing different chain lengths with a preference for micellar aggregated substrates. It therefore presents an interfacial activation property, which is typical of classical phospholipases. Furthermore, given that stability is a very important quality of an enzyme, the influence of different parameters on the activity and stability of tLRAT has thus been studied in detail. For example, storage buffer has a strong effect on tLRAT activity and high enzyme stability has been observed at room temperature. The thermostability of tLRAT has also been investigated using circular dichroism and infrared spectroscopy. A decrease in the activity of tLRAT was observed beyond 70 °C, accompanied by a modification of its secondary structure, i.e. a decrease of its α-helical content and the appearance of unordered structures and aggregated β-sheets. Nevertheless, residual activity could still be observed after heating tLRAT up to 100 °C. The results of this study highly improved our understanding of this enzyme.     

Azobenzene-containing poly(l-glutamates). Photochromism and conformation in solution

Photochromic polypeptides having various contents of azobenzene chromophores attached to th side chains have been prepared by condensing poly(l-glutamic acid) with p-amino-azobenzene. The photochemical and thermal cis-trans isomerizations of the azo chromophores have been investigated by absorption and circular dichroism spectroscopy, and the photochromic behaviour has been related to the conformation in solution. The azopolypeptides exhibit the α-helix c.d. pattern, in trimethylphosphate. The α-helix content markedly depends on the azo content, but it is not affected by the cis or trans geometric forms of the azo side chains. Strong solvent effects by H₂O or trifluoroethanol on the extrinsic azo c.d. bands, suggest the existence, in trimethylphosphate solution, of a super-ordered secondary structure involving a regular arrangement of the side chains, on th periphery of the helical peptide backbone.     

Unfolding of C-phycocyanin followed by loss of non-covalent chromophore-protein interactions: 1. Equilibrium experiments

Optical spectroscopic properties of the covalently linked chromophores of biliproteins are profoundly influenced by the state of the protein. This has been used to monitor the urea-induced denaturation of C-phycocyanin (CPC) from Mastigocladus laminosus and its subunits. Under equilibrium conditions, absorption, fluorescence and circular dichroism of the chromophores were monitored, as well as the circular dichroism of the polypeptide. Treatment of CPC trimers (αβ)₃ resulted first in monomerization (αβ), which was followed by a complex unfolding process of the protein. Loss of chromophore fluorescence is the next process at increasing urea concentrations; it indicates increased flexibility of the chromophore while maintaining the native, extended conformation, and a less compact but still native-like packing of the protein in the regions sampled by the chromophores. This was followed by relaxation of the chromophores from the energetically unfavorable extended to a cyclic-helical conformation, as reported by absorption and CD in the visible range, indicating local loss of protein structure. Only then is the protein secondary structure lost, as reported by the far-UV CD. Sequential processes were also seen in the subunits, where again the chromophore-protein interactions were reduced before the unfolding of the protein. It is concluded that the bilin chromophores are intrinsic probes suitable to differentiate among different processes involved in protein denaturation.     

Spectroscopic exploration and thermodynamic characterization of desvenlafaxine interacting with fluorescent bovine serum albumin

The mechanism of the interaction between bovine serum albumin (BSA) and desvenlafaxine was studied using fluorescence, ultraviolet absorption, 3‐dimensional fluorescence spectroscopy, circular dichroism, synchronous fluorescence spectroscopy, cyclic voltametry, differential scanning calorimetry, and attenuated total reflection–Fourier transform infrared spectroscopic techniques under physiological condition at pH 7.4. Stern‐Volmer calculations authenticate the fluorescence of BSA that was quenched by desvenlafaxine in a collision quenching mode. The fluorescence quenching method was used to evaluate number of binding sites “n” and binding constant K _A that were measured, and various thermodynamic parameters were evaluated at different temperatures by using the van't Hoff equation and differential scanning calorimetry technique, which indicated a spontaneous and hydrophobic interaction between BSA and desvenlafaxine. According to the Förster theory we calculate the distance between the donor, BSA and acceptor, desvenlafaxine molecules. Furthermore, circular dichroism and attenuated total reflection–Fourier transform infrared spectroscopy indicate nominal changes in the secondary structure of the protein.     

Structure analysis of the membrane protein TatCd from the Tat system of B. subtilis by circular dichroism

The twin arginine translocation (Tat) system can transport fully folded proteins, including their cofactors, across bacterial and thylakoid membranes. The Tat system of Bacillus subtilis that serves to export the phosphodiesterase (PhoD) consists of only two membrane proteins, TatA_d and TatC_d. The larger component TatC_d has a molecular weight of 28 kDa and several membrane-spanning segments. This protein has been expressed in Escherichia coli and purified in sufficient amounts for structure analysis by circular dichroism (CD) and NMR spectroscopy. TatC_d was reconstituted in detergent micelles and in lipid bilayers for CD analysis in solution and in macroscopically oriented samples, to examine the stability of the protein. Suitable protocols and model membrane systems have been established, by which TatC_d maintains the level of helicity close to theoretically predicted, and its transmembrane alignment could been verified.     

Conformation of membrane‐bound proteins revealed by vacuum‐ultraviolet circular‐dichroism and linear‐dichroism spectroscopy

Knowledge of the conformations of a water‐soluble protein bound to a membrane is important for understanding the membrane‐interaction mechanisms and the membrane‐mediated functions of the protein. In this study we applied vacuum‐ultraviolet circular‐dichroism (VUVCD) and linear‐dichroism (LD) spectroscopy to analyze the conformations of α‐lactalbumin (LA), thioredoxin (Trx), and β‐lactoglobulin (LG) bound to phosphatidylglycerol liposomes. The VUVCD analysis coupled with a neural‐network analysis showed that these three proteins have characteristic helix‐rich conformations involving several helical segments, of which two amphiphilic or hydrophobic segments take part in interactions with the liposome. The LD analysis predicted the average orientations of these helix segments on the liposome: two amphiphilic helices parallel to the liposome surface for LA, two hydrophobic helices perpendicular to the liposome surface for Trx, and a hydrophobic helix perpendicular to and an amphiphilic helix parallel to the liposome surface for LG. This sequence‐level information about the secondary structures and orientations was used to formulate interaction models of the three proteins at the membrane surface. This study demonstrates the validity of a combination of VUVCD and LD spectroscopy in conformational analyses of membrane‐binding proteins, which are difficult targets for X‐ray crystallography and nuclear magnetic resonance spectroscopy. Proteins 2016; 84:349–359. © 2016 Wiley Periodicals, Inc.     

Purification and characterization of a transmembrane domain-deleted form of lecithin retinol acyltransferase

Lecithin retinol acyltransferase (LRAT) catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester, an essential reaction in the vertebrate visual cycle. Since all-trans-retinyl esters are the substrates for the isomerization reaction that generates 11-cis-retinoids, this esterification reaction is essential in the operation of the visual cycle. In addition, LRAT is the founder member of a series of proteins, which are of novel sequence and have unknown functions. Native LRAT is an integral membrane protein and has never been purified. To obtain a pure LRAT, the N- and C-transmembrane termini were deleted and replaced with a poly His tag for the purpose of purification. This truncated form of LRAT, referred to as tLRAT, has been expressed in bacteria and fully purified. tLRAT is catalytically active and processes all-trans-retinol at least 10-fold more efficiently than 11-cis-retinol, the precursor to the visual chromophore. While tLRAT can be robustly expressed in bacteria, it requires detergent for extraction, as the enzyme still contains hydrophobic domains, which may interact. Indeed, tLRAT can oligomerize and forms dimers. Native LRAT also forms functional homodimers. These studies pave the way for the preparation of large-scale amounts of pure tLRAT for further mechanistic and structural studies.     

Importance of trimer-trimer interactions for the native state of the plant light-harvesting complex II

Aggregates and solubilized trimers of LHCII were characterized by circular dichroism (CD), linear dichroism and time-resolved fluorescence spectroscopy and compared with thylakoid membranes in order to evaluate the native state of LHCII in vivo. It was found that the CD spectra of lamellar aggregates closely resemble those of unstacked thylakoid membranes whereas the spectra of trimers solubilized in n-dodecyl-β,d-maltoside, n-octyl-β,d-glucopyranoside, or Triton X-100 were drastically different in the Soret region. Thylakoid membranes or LHCII aggregates solubilized with detergent exhibited CD spectra similar to the isolated trimers. Solubilization of LHCII was accompanied by profound changes in the linear dichroism and increase in fluorescence lifetime. These data support the notion that lamellar aggregates of LHCII retain the native organization of LHCII in the thylakoid membranes. The results indicate that the supramolecular organization of LHCII, most likely due to specific trimer-trimer contacts, has significant impact on the pigment interactions in the complexes.     

The Full-Length Mu-Opioid Receptor: A Conformational Study by Circular Dichroism in Trifluoroethanol and Membrane-Mimetic Environments

The secondary structure content of the recombinant human mu-opioid receptor (HuMOR) solubilized in trifluoroethanol (TFE) and in detergent micelles was investigated by circular dichroism. In both conditions, this G protein-coupled receptor adopts a characteristic alpha-helical structure, with minima at 208 and 222 nm as observed in the circular dichroism spectra. After deconvolution of spectra, the alpha-helix contents were estimated to be in the range of 50% in TFE and in sodium dodecyl sulfate at pH 6. These values are in accordance with the predicted secondary structure content determined for the mu-opioid receptor. A pH-dependent effect was observed on the secondary structure of the receptor solubilized in detergents, which demonstrates the essential role of ionic and hydrophobic interactions on the secondary structure. Circular dichroism spectra of EGFP-HuMOR, a fusion protein between the enhanced green fluorescent protein (EGFP) and the mu-opioid receptor, and EGFP solubilized in TFE were also analyzed as part of this study.     

Circular dichroism of double-helical oligoribonucleotides

The ultraviolet circular dichroism and absorption of 15 double-stranded helical oligoribonucleotides have been measured. These molecules of chain-length 6 to 12 contain all 10 possible nearest neighbors of Watson-Crick base pairs. They are thus good models for short double-stranded regions in RNA molecules. The contribution to the circular dichroism of each of the nearest neighbor base pairs has been obtained. The circular dichroism is found to be very sequence-dependent and may be useful in distinguishing possible secondary structures. However, the nearest neighbor approximation for circular dichroism fails to give a quantitative measure of the circular dichroism of double-strand regions.     

Pressure dependence of activity and stability of dihydrofolate reductases of the deep-sea bacterium Moritella profunda and Escherichia coli

To understand the pressure-adaptation mechanism of deep-sea enzymes, we studied the effects of pressure on the enzyme activity and structural stability of dihydrofolate reductase (DHFR) of the deep-sea bacterium Moritella profunda (mpDHFR) in comparison with those of Escherichia coli (ecDHFR). mpDHFR exhibited optimal enzyme activity at 50 MPa whereas ecDHFR was monotonically inactivated by pressure, suggesting inherent pressure-adaptation mechanisms in mpDHFR. The secondary structure of apo-mpDHFR was stable up to 80 °C, as revealed by circular dichroism spectra. The free energy changes due to pressure and urea unfolding of apo-mpDHFR, determined by fluorescence spectroscopy, were smaller than those of ecDHFR, indicating the unstable structure of mpDHFR against pressure and urea despite the three-dimensional crystal structures of both DHFRs being almost the same. The respective volume changes due to pressure and urea unfolding were − 45 and − 53 ml/mol at 25 °C for mpDHFR, which were smaller (less negative) than the corresponding values of − 77 and − 85 ml/mol for ecDHFR. These volume changes can be ascribed to the difference in internal cavity and surface hydration of each DHFR. From these results, we assume that the native structure of mpDHFR is loosely packed and highly hydrated compared with that of ecDHFR in solution.     

VCD studies on cyclic peptides assembled from L‐α‐amino acids and a trans‐2‐aminocyclopentane‐ or trans‐2‐aminocyclohexane carboxylic acid

The increasing interest in peptidomimetics of biological relevance prompted us to synthesize a series of cyclic peptides comprising trans‐2‐aminocyclohexane carboxylic acid (Achc) or trans‐2‐aminocyclopentane carboxylic acid (Acpc). NMR experiments in combination with MD calculations were performed to investigate the three‐dimensional structure of the cyclic peptides. These data were compared to the conformational information obtained by electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectroscopy. Experimental VCD spectra were compared to theoretical VCD spectra computed quantum chemically at B3LYP/6‐31G(d) density functional theory (DFT) level. The good agreement between the structural features derived from the VCD spectra and the NMR‐based structures underlines the applicability of VCD in studying the conformation of small cyclic peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

A circular dichroism study of human erythrocyte ghost proteins during exposure to 2450 MHz microwave radiation

The effect of 2450 MHz microwave radiation on the proteins of human erythrocyte ghosts has been investigated using circular dichroism spectroscopy. A specially constructed waveguide inserted into the spectropolarimeter allowed the continuous recording of optical activity before, during and after microwave irradiation. The data indicate that high levels of microwave radiation (600 mW/g, specific absorption rate) induce decreases in α-helical conformation that may result from both thermal vibrations and increased strain on the intramolecular hydrogen bonds that maintain secondary structure. The latter effect may result from differential intramolecular interactions with the oscillating electric field. Spectrin (bands 1 and 2) isolated from the ghosts was more sensitive to microwave irradiation than intact ghosts, and spectrin-depleted vesicles were the least sensitive. The data, therefore, indicate that the α-helical conformation of spectrin is altered by high levels of microwave radiation.     

Conformation and orientation of penetratin in phospholipid membranes.

The binding, conformation and orientation of a hydrophilic vector peptide penetratin in lipid membranes and its state of self-association in solution were examined using circular dichroism (CD), analytical ultracentrifugation and fluorescence spectroscopy. In aqueous solution, penetratin exhibited a low helicity and sedimented as a monomer in the concentration range approximately 50-500 microM. The partitioning of penetratin into phospholipid vesicles was determined using tryptophan fluorescence anisotropy titrations. The apparent penetratin affinity for 20% phosphatidylserine/80% egg phosphatidylcholine vesicles was inversely related to the total peptide concentration implying repulsive peptide-peptide interactions on the lipid surface. The circular dichroism spectra of the peptide when bound to unaligned 20% phosphatidylserine/80% egg phosphatidylcholine vesicles and aligned hydrated phospholipid multilayers were attributed to the presence of both alpha-helical and beta-turn structures. The orientation of the secondary structural elements was determined using oriented circular dichroism spectroscopy. From the known circular dichroism tensor components of the alpha-helix, it can be concluded that the orientation of the helical structures is predominantly perpendicular to the membrane surface, while that of the beta-type carbonyls is parallel to the membrane surface. On the basis of our observations, we propose a novel model for penetratin translocation.     

Analysis of the Secondary Structure of the Cys-Less Yeast Mitochondrial Citrate Transport Protein and Four Single-Cys Variants by Circular Dichroism

Utilizing cysteine scanning mutagenesis, with functional Cys-less citrate transport protein (CTP) serving as the starting template, we previously demonstrated that four single-Cys mutants located in transmembrane domains III and IV, rendered the CTP nonfunctional. The present investigations assess and quantify the secondary structure of the Cys-less CTP and the four single-Cys mutants, both in the absence and presence of citrate, via circular dichroism (CD) spectroscopy. In detergent micelles, highly purified Cys-less CTP contained approximately 50% alpha-helix and approximately 20% beta-sheet. The CD spectra of the G119C, E122C, R181C, and R189C mutants in detergent micelles were virtually superimposable with that of the functional Cys-less CTP, thereby suggesting that the wild-type residues, rather than affecting structure, may assume important mechanistic roles. Exogenously added citrate caused a significant change in the CD spectra of all solubilized CTP samples. Analyses of the spectra of the Cys-less CTP indicated an approximately 10% increase in its alpha-helical content in the presence of citrate. The conformational changes effected by the addition of substrate were less pronounced with the single-Cys mutants. Studies of the Cys-less CTP reconstituted in liposomes indicated that while the CD spectra was red-shifted, the net secondary structure of the reconstituted carrier is approximately equivalent to that of the transporter in detergent micelles, and displayed a response to added citrate. In combination, the above studies indicate that purified Cys-less CTP in either sarkosyl micelles or in liposomes, and the four inactive single-Cys mutants in sarkosyl micelles, retain native-like structure, and thus represent ideal material for detailed structural characterization.     

The Retinol Dehydrogenase Rdh10 Localizes to Lipid Droplets during Acyl Ester Biosynthesis

Rdh10 catalyzes the first step of all-trans-retinoic acid biogenesis physiologically, conversion of retinol into retinal. We show that Rdh10 associates predominantly with mitochondria/mitochondrial-associated membrane (MAM) in the absence of lipid droplet biosynthesis, but also locates with lipid droplets during acyl ester biosynthesis. Targeting to lipid droplets requires the 32 N-terminal residues, which include a hydrophobic region followed by a net positive charge. Targeting to mitochondria/MAM and/or the stability of Rdh10 require both the N-terminal and the 48 C-terminal hydrophobic residues. Rdh10 behaves similarly to cellular retinol-binding protein, type 1, which also localizes to mitochondria/MAM before lipid droplet synthesis, and associates with lipid droplets during acyl ester synthesis (Jiang, W., and Napoli, J. L. (2012) Biochem. Biophys. Acta 1820, 859–8692). LRAT, an ER protein, also associates with lipid droplets upon acyl ester biosynthesis. Colocalization of Rdh10, Crbp1, and LRAT on lipid droplets suggests a metabolon that mediates retinol homeostasis.     

Interpretations of the effects of pH on the spectra of purple membrane.

The absorption and circular dichroism of the purple membrane in solution and the linear and circular dichroism of the purple membrane oriented in a film were used to detect changes in the membrane protein structure and membrane organization in the pH range of 2.4 to 12.6. Main findings are (a) the membrane protein structure is stable at every level of organization to pH changes over the range of 5.0 to 8.5. (b) Tertiary structural changes occur in the membrane protein structure in the pH range of 2.4 to 5.0 and 8.5 to 11.8 without any secondary structural involvement. (c) An irreversible change occurs in the membrane organization in the pH range of 11.8 to 12.6 involving large tertiary and secondary structural changes in the membrane protein. (d) The retinyl chromophore is influenced by a nearby ionizable group. (e) The membrane crystalline structure is highly stable to pH perturbation except at the high pH range of 11.0 to 11.8.     

Self-assembly of the hydrophobin SC3 proceeds via two structural intermediates

Hydrophobins self assemble into amphipathic films at hydrophobic-hydrophilic interfaces. These proteins are involved in a broad range of processes in fungal development. We have studied the conformational changes that accompany the self-assembly of the hydrophobin SC3 with polarization-modulation infrared reflection absorption spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, and circular dichroism, and related them to changes in morphology as observed by electron microcopy. Three states of SC3 have been spectroscopically identified previously as follows: the monomeric state, the alpha-helical state that is formed upon binding to a hydrophobic solid, and the beta-sheet state, which is formed at the air-water interface. Here, we show that the formation of the beta-sheet state of SC3 proceeds via two intermediates. The first intermediate has an infrared spectrum indistinguishable from that of the alpha-helical state of SC3. The second intermediate is rich in beta-sheet structure and has a featureless appearance under the electron microscope. The end state has the same secondary structure, but is characterized by the familiar 10-nm-wide rodlets.