Protein domain library generation by overlap extension (PDLGO): a tool for enzyme engineering

We introduce an upgraded version of the error-prone polymerase chain reaction (epPCR) comprising three DNA polymerase-catalyzed steps. It improves the common epPCR strategy such that random mutations can be confined exactly to a distinct, but freely selectable, sequence region within a gene without the need for flanking restriction endonuclease sites. The new method is called protein domain library generation by overlap extension (PDLGO). To validate PDLGO, we generated a random library of EstE, a multidomain esterase from Xanthomonas vesicatoria. It was demonstrated that random mutations appear exclusively within the catalytic domains as intended. The domains of EstE flanking the catalytic domains are required for transport of EstE to the cell envelope and remain unaltered. Microplates with integrated pH sensors, providing a substrate-independent high-throughput screening tool, were used to analyze whole cells of E. coli expressing the variants of the EstE library. A variant (P286H) with substantially increased catalytic activity was identified. Our results indicate that combining PDLGO with microplates containing integrated pH sensors provides a simple and rapid toolbox for directed evolution of esterases.     

Chirality of peptide bond-forming condensation domains in nonribosomal peptide synthetases: the C5 domain of tyrocidine synthetase is a (D)C(L) catalyst

Nonribosomal peptides (NRP) such as the antibiotic tyrocidine have D-amino acids, introduced by epimerase (E) domains embedded within modules of the enzymatic assembly lines. We predict that the peptide bond-forming condensation (C) domains immediately downstream of E domains are D-specific for the peptidyl donor and L-specific for the aminoacyl acceptor ((D)C(L)). To validate this prediction and establish that the C(5) domain of tyrocidine synthetase is indeed (D)C(L), the apoT (thiolation) forms of module 4 (TycB(3) AT(4)E) and module 5 (TycC(1) C(5)AT(5)) were expressed. T(5) was posttranslationally primed with CoASH to introduce the HS-pantetheinyl group and autoaminoacylated with radiolabeled L-Asn* or L-Asp*. Alternate donor substrates were introduced by priming apo AT(4)E with synthetically prepared tetrapeptidyl-CoA's differing in the chirality of Phe-4, D-Phe-L-Pro-L-Phe-L-Phe-CoA, and D-Phe-L-Pro-L-Phe-D-Phe-CoA. The tetrapeptidyl-S-T(4) and L-Asp-S-T(5) were studied for peptide bond formation and chain translocation by C(5) to yield pentapeptidyl-S-T(5), whose chirality (D-L-L-D-L- vs D-L-L-L-L-) was assayed by thioester cleavage and chiral chromatography of the released pentapeptides. Only the D-Phe-4 pentapeptidyl-S-T(5) was generated, implying that only D-L-L-D-S-T(4) was utilized, proving C(5) is indeed a (D)C(L) catalyst. Furthermore, a mutant with an inactive E domain transferred tetrapeptide only when loaded with D-Phe-4 tetrapeptidyl donor, not L-Phe-4, confirming that in the wild-type assembly line C(5) only transfers D-L-L-L-tetrapeptidyl-S-T(4) after in situ epimerization by the E domain. These results contrast the observation that C(5) can make both L-Phe-L-Asn and D-Phe-L-Asn when assayed with Phe as the donor substrate. Hence, utilizing an aminoacyl-S-T(4) versus the natural peptidyl-S-T(4) donor produced misleading information regarding the specificity of the condensation domain.     

Ceramide selectively displaces cholesterol from ordered lipid domains (rafts): implications for lipid raft structure and function

Ceramide is a membrane lipid involved in a number of crucial biological processes. Recent evidence suggests that ceramide is likely to reside and function within lipid rafts; ordered sphingolipid and cholesterol-rich lipid domains believed to exist within many eukaryotic cell membranes. Using lipid vesicles containing co-existing raft domains and disordered fluid domains, we find that natural and saturated synthetic ceramides displace sterols from rafts. Other raft lipids remain raft-associated in the presence of ceramide, showing displacement is relatively specific for sterols. Like cholesterol-containing rafts, ceramide-rich "rafts" remain in a highly ordered state. Comparison of the sterol-displacing abilities of natural ceramides with those of saturated diglycerides and an unsaturated ceramide demonstrates that tight lipid packing is critical for sterol displacement by ceramide. Based on these results, and the fact that cholesterol and ceramides both have small polar headgroups, we propose that ceramides and cholesterol compete for association with rafts because of a limited capacity of raft lipids with large headgroups to accommodate small headgroup lipids in a manner that prevents unfavorable contact between the hydrocarbon groups of the small headgroup lipids and the surrounding aqueous environment. Minimizing the exposure of cholesterol and ceramide to water may be a strong driving force for the association of other molecules with rafts. Furthermore, displacement of sterol from rafts by ceramide is very likely to have marked effects upon raft structure and function, altering liquid ordered properties as well as molecular composition. In this regard, certain previously observed physiological processes may be a result of displacement. In particular, a direct connection to the previously observed sphingomyelinase-induced displacement of cholesterol from plasma membranes in cells is proposed.     

A conformation change in the carboxyl terminus of Alzheimer's Abeta (1-40) accompanies the transition from dimer to fibril as revealed by fluorescence quenching analysis

Alzheimer's disease is characterized by the presence of insoluble, fibrous deposits composed principally of amyloid beta (Abeta) peptide. A number of studies have provided information on the fibril structure and on the factors affecting fiber formation, but the details of the fibril structure are not known. We used fluorescence quenching to investigate the solvent accessibility and surface charge of the soluble Abeta(1-40) dimer and amyloid fibrils. Analogs of Abeta(1-40) containing a single tryptophan were synthesized by substituting residues at positions 4, 10, 34, and 40 with tryptophan. Quenching measurements in the dimeric state indicate that the amino-terminal analogs (AbetaF4W and AbetaY10W) are accessible to polar quenchers, and the more carboxyl-terminal analog AbetaV34W is less accessible. AbetaV40W, on the other hand, exhibits a low degree of quenching, indicating that this residue is highly shielded from the solvent in the dimeric state. Correcting for the effect of reduced translational and rotational diffusion, fibril formation was associated with a selective increase in solvent exposure of residues 34 and 40, suggesting that a conformation change may take place in the carboxyl-terminal region coincident with the dimer to fibril transition.     

Identification of a peptide fragment from the carboxyl-terminal extension region (E-domain) of rat proinsulin-like growth factor-II

A fragment of the carboxyl-terminal extension region (E-peptide) of rat proinsulin-like growth factor-II has been purified from medium conditioned by cultured BRL-3A rat liver cells. The fragment, identified by microsequence analysis, was discovered in a biologically active fraction of insulin-like growth factor II (IGF-II). The fragment begins at position 117 in pro-IGF-II, two amino acids downstream from an Arg-Arg potential prohormone processing site. A synthetic analogue of the E-peptide at high concentrations stimulates [3H]thymidine incorporation in NIL8 hamster cells, raising the possibility that the E-peptide might bind with low affinity to a mitogen receptor. Peptides from the E-regions of pro-IGF-I and pro-IGF-II should be useful for development of radioimmunoassays for measurement of the somatic production of IGF-I and IGF-II, analogous to the radioimmunoassay for the insulin C-peptide.     

Use of covariance analysis for the prediction of structural domain boundaries from multiple protein sequence alignments

Current methods for identification of domains within protein sequences require either structural information or the identification of homologous domain sequences in different sequence contexts. Knowledge of structural domain boundaries is important for fold recognition experiments and structural determination by X-ray crystallography or nuclear magnetic resonance spectroscopy using the divide-and-conquer approach. Here, a new and conceptually simple method for the identification of structural domain boundaries in multiple protein sequence alignments is presented. Analysis of covariance at positions within the alignment is first used to predict 3D contacts. By the nature of the domain as an independent folding unit, inter-domain predicted contacts are fewer than intra-domain predicted contacts. By analysing all possible domain boundaries and constructing a smoothed profile of predicted contact density (PCD), true structural domain boundaries are predicted as local profile minima associated with low PCD. A training data set is constructed from 52 non-homologous two-domain protein sequences of known 3D structure and used to determine optimal parameters for the profile analysis. The alignments in the training data set contained 48 +/- 17 (mean +/- SD) sequences and lengths of 257 +/- 121 residues. Of the 47 alignments yielding predictions, 35% of true domain boundaries are predicted to within 15 amino acids by the local profile minimum with the lowest profile value. Including predictions from the second- and third-lowest local minima increases the correct domain boundary coverage to 60%, whereas the lowest five local minima cover 79% of correct domain boundaries. Through further profile analysis, criteria are presented which reliably identify subsets of more accurate predictions. Retrospective analysis of CASP3 targets shows predictions of sufficient accuracy to enable dramatically improved fold recognition results. Finally, a prediction is made for geminivirus AL1 protein which is in full agreement with biochemical data, yielding a plausible, novel threading result.     

Binding of cytochrome c to liposomes as revealed by the quenching of fluorescence from pyrene-labeled phospholipids

Resonance energy transfer from pyrene-fatty acid containing phospholipid derivatives to the heme of cytochrome c (cyt c) was used to observe the binding of this protein to liposomal membranes. Liposomes were formed of egg yolk phosphatidic acid (PA) and either egg yolk phosphatidylcholine or dipalmitoylphosphatidylcholine with 1 mol % of the fluorescent lipid. Binding of cyt c to liposomes was monitored by measuring the decrease either in the fluorescence intensity or in the lifetime of pyrene emission. The requirement for the presence of the acidic phospholipid in the membrane for the binding of cyt c could be reconfirmed. Below 5 mol % of phosphatidic acid in the membrane, no significant attachment of cyt c to liquid-crystalline bilayers was evident whereas upon increasing the concentration of PA further the association of cyt c progressively increased until a saturation was reached at about 30 mol % of phosphatidic acid. Addition of NaCl caused the fluorescence intensity and lifetimes to return to values observed in the absence of cyt c, thus revealing the dissociation of the protein from the membrane. The pyrene-labeled phosphatidic acid derivatives PPHPA and PPDPA were quenched more effectively than the corresponding phosphatidylcholines, apparently due to the direct involvement of the acidic head group in binding cyt c. When dipalmitoylphosphatidylcholine (DPPC) with 5 mol % of phosphatidic acid was used, no binding of cyt c to the liposomes above the phase transition temperature of the former lipid could be demonstrated whereas below the transition temperature (Tm) binding did take place.(ABSTRACT TRUNCATED AT 250 WORDS)     

Permeability of lipid bilayers to methylmercuric chloride: Quantification by fluorescence quenching of a carbazole-labeled phospholipid

We investigated the permeabilities of lipid bilayers to the neurotoxin methylmercuric chloride (MMC). This mercurial is an efficient collisional quencher of the fluorescence of N-alkyl carbazole derivatives. Quenching of the fluorescence of β-(3-(9-carbazole)-propionyl-L--phosphatidylcholine (CPA-PC) in vesicles of dimyristoyl phosphatidylcholine and of dioleoyl phosphatidylcholine reveal rapid diffusion of MMC in the alkyl side chain regions of these bilayers. By a combination of (1) the lipid concentration dependence of the apparent quenching constants, (2) the solubility of MMC in concentrated lipid dispersions and (3) the 270 MHz proton magnetic resonance of methylmercury in the presence of lipid bilayers we conclude that the lipid-water partition coefficient of this mercurial is less than or equal to two. Using the fluorescence quenching and the partitioning data we estimate the diffusion coefficient of MMC in these bilayers to range from 0.13 to 0.31 × 10⁻⁵ cm²/sec, or 20–47% of its diffusion coefficient in ethanol. These data indicate that lipid bilayers do not pose a significant permeability barrier to the diffusional transport of MMC.     

Conformational analysis by CD and NMR spectroscopy of a peptide encompassing the amphipathic domain of YopD from Yersinia.

To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III secretion machine that permits the translocation of several anti-host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre-secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N-terminus and in a predicted amphipathic domain located near the C-terminus. This latter domain is also critical for Yersinia virulence. In this study, we designed synthetic peptides encompassing the C-terminal amphipathic domain of YopD. A solution structure of YopD278-300, a peptide that strongly interacted with LcrH, was obtained by NMR methods. The structure is composed of a well-defined amphipathic alpha helix ranging from Phe280 to Tyr291, followed by a type I beta turn between residues Val292 and His295. The C-terminal truncated peptides, YopD278-292 and YopD271-292, lacked helical structure, implicating the beta turn in helix stability. An interaction between YopD278-300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the peptide-LcrH complex. These effects were not observed for the unstructured peptide, YopD278-292, which confirms that the alpha helical structure of the YopD amphipathic domain is a critical binding region of LcrH.     

Investigation of the plastoquinone pool size and fluorescence quenching in thylakoid membranes and Photosystem II (PS II) membrane fragments

The efficiency of oxidized endogenous plastoquinone-9 (PQ-9) as a non-photochemical quencher of chlorophyll fluorescence has been analyzed in spinach thylakoids and PS II membrane fragments isolated by Triton X-100 fractionation of grana stacks. The following results were obtained: (a) After subjection of PS II membrane fragments to ultrasonic treatment in the presence of PQ-9, the area over the induction curve of chlorophyll fluorescence owing to actinic cw light increases linearly with the PQ-9/PS II ratio in the reconstitution assay medium; (b) the difference of the maximum fluorescence levels, F_max, of the induction curves, measured in the absence and presence of DCMU, is much more pronounced in PS II membrane fragments than in thylakoids; (c) the ratio F_max(-DCMU)/F_max(+DCMU) increases linearly with the content of oxidized PQ-9 that is varied in the thylakoids by reoxidation of the pool after preillumination and in PS II membrane fragments by the PQ-9/PS II ratio in the reconstitution assay; (d) the reconstitution procedure leads to tight binding of PQ-9 to PS II membrane fragments, and PQ-9 cannot be replaced by other quinones; (e) the fluorescence quenching by oxidized PQ-9 persists at low temperatures, and (f) oxidized PQ-9 preferentially affects the F695 of the fluorescence emission spectrum at 77 K. Based on the results of this study the oxidized PQ-9 is inferred to act as a non-photochemical quencher via a static mechanism. Possible implications for the nature of the quenching complex are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photosystem II chlorophyll a fluorescence lifetimes and intensity are independent of the antenna size differences between barley wild-type and chlorina mutants: Photochemical quenching and xanthophyll cycle-dependent nonphotochemical quenching of fluorescence

Photosystem II (PS II) chlorophyll (Chl) a fluorescence lifetimes were measured in thylakoids and leaves of barley wild-type and chlorina f104 and f2 mutants to determine the effects of the PS II Chl a+b antenna size on the deexcitation of absorbed light energy. These barley chlorina mutants have drastically reduced levels of PS II light-harvesting Chls and pigment-proteins when compared to wild-type plants. However, the mutant and wild-type PS II Chl a fluorescence lifetimes and intensity parameters were remarkably similar and thus independent of the PS II light-harvesting antenna size for both maximal (at minimum Chl fluorescence level, F_o) and minimal rates of PS II photochemistry (at maximum Chl fluorescence level, F_m). Further, the fluorescence lifetimes and intensity parameters, as affected by the trans-thylakoid membrane pH gradient (pH) and the carotenoid pigments of the xanthophyll cycle, were also similar and independent of the antenna size differences. In the presence of a pH, the xanthophyll cycle-dependent processes increased the fractional intensity of a Chl a fluorescence lifetime distribution centered around 0.4–0.5 ns, at the expense of a 1.6 ns lifetime distribution (see Gilmore et al. (1995) Proc Natl Acad Sci USA 92: 2273–2277). When the zeaxanthin and antheraxanthin concentrations were measured relative to the number of PS II reaction center units, the ratios of fluorescence quenching to [xanthophyll] were similar between the wild-type and chlorina f104. However, the chlorina f104, compared to the wild-type, required around 2.5 times higher concentrations of these xanthophylls relative to Chl a+b to obtain the same levels of xanthophyll cycle-dependent fluorescence quenching. We thus suggest that, at a constant pH, the fraction of the short lifetime distribution is determined by the concentration and thus binding frequency of the xanthophylls in the PS II inner antenna. The pH also affected both the widths and centers of the lifetime distributions independent of the xanthophyll cycle. We suggest that the combined effects of the xanthophyll cycle and pH cause major conformational changes in the pigment-protein complexes of the PS II inner or core antennae that switch a normal PS II unit to an increased rate constant of heat dissipation. We discuss a model of the PS II photochemical apparatus where PS II photochemistry and xanthophyll cycle-dependent energy dissipation are independent of the Peripheral antenna size.Abbreviations Ax antheraxanthin - BSA bovine serum albumin - c_x lifetime center of fluorescence decay component x - CP chlorophyll binding protein of PS II inner antenna - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DTT dithiothreitol - f_x fractional intensity of fluorescence lifetime component x - F_m, F_m maximal PS II Chl a fluorescence intensity with all Q_A reduced in the absence, presence of thylakoid membrane energization - F_o minimal PS II Chl a fluorescence intensity with all Q_A oxidized - F_v=F_m–F_o variable level of PS II Chl a fluorescence - HPLC high performance liquid chromatography - k_A rate constant of all combined energy dissipation pathways in PS II except photochemistry and fluorescence - k_F rate constant of PS II Chl a fluorescence - LHCIIb main light harvesting pigment-protein complex (of PS II) - N_pig mols Chl a+b per PS II - NPQ=(F_m/F_m–1) nonphotochemical quenching of PS II Chl a fluorescence - PAM pulse-amplitude modulation fluorometer - PFD photon-flux density, mols photons m^–2 s^–1 - PS II Photosystem II - P680 special-pair Chls of PS II reaction center - Q_A primary quinone electron acceptor of PS II - V_x violaxanthin - w_x width at half maximum of Lorentzian fluorescence lifetime distribution x - Zx zeaxanthin - pH trans-thylakoid proton gradient - % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% GabaqaaiGacaGaamqadaabaeaafiaakeaacqGH8aapcqaHepaDcqGH% +aGpdaWgaaWcbaGaamOraiaad2gaaeqaaaaa!4989!\[< \tau > _{Fm}\],% MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% GabaqaaiGacaGaamqadaabaeaafiaakeaacqGH8aapcqaHepaDcqGH% +aGpdaWgaaWcbaGaamOraiaad+gaaeqaaOGaeyypa0Zaaabqaeaaca% WGMbWaaSbaaSqaaiaadIhaaeqaaOGaam4yamaaBaaaleaacaWG4baa% beaaaeqabeqdcqGHris5aaaa!50D3!\[< \tau > _{Fo} = \sum {f_x c_x }\] average lifetime of Chl a fluorescence calculated from a multi-exponential model under F_m, F_o conditions     

Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain

Conjugation to cationic cell penetrating peptides (such as Tat, Penetratin, or oligo arginines) efficiently improves the cellular uptake of large hydrophilic molecules such as oligonucleotides and peptide nucleic acids, but the cellular uptake is predominantly via an unproductive endosomal pathway and therefore mechanisms that promote endosomal escape (or avoid the endosomal route) are required for improving bioavailability. A variety of auxiliary agents (chloroquine, calcium ions, or lipophilic photosensitizers) has this effect, but improved, unaided delivery would be highly advantageous in particular for future in vivo applications. We find that simply conjugating a lipid domain (fatty acid) to the cationic peptide (a CatLip conjugate) increases the biological effect of the corresponding PNA (CatLip) conjugates in a luciferase cellular antisense assay up to 2 orders of magnitude. The effect increases with increasing length of the fatty acid (C8-C16) but in parallel also results in increased cellular toxicity, with decanoic acid being optimal. Furthermore, the relative enhancement is significantly higher for Tat peptide compared to oligoarginine. Confocal microscopy and chloroquine enhancement indicates that the lipophilic domain increases the endosomal uptake as well as promoting significantly endosomal escape. These results provide a novel route for improving the (cellular) bioavailability of larger hydrophilic molecules.     

A four-to-one association between peptide motifs: four C-terminal domains from cholinesterase assemble with one proline-rich attachment domain (PRAD) in the secretory pathway.

The major type of acetylcholinesterase in vertebrates (AChET) is characterized by the presence of a short C-terminal domain of 40 residues, the 'tryptophan amphiphilic tetramerization' (WAT) domain. The presence of this domain is not necessary for catalytic activity but is responsible for hydrophobic interactions and for the capacity of AChET subunits to form quaternary associations with anchoring proteins, thereby conditioning their functional localization. In the collagen tail of asymmetric forms, we characterized a small conserved region that is sufficient for binding an AChET tetramer, the proline-rich attachment domain (PRAD). We show that the WAT domain alone is sufficient for association with the PRAD, and that it can attach foreign proteins (alkaline phosphatase, GFP) to a PRAD-containing construct with a glycophosphatidylinositol anchor (GPI), and thus anchor them to the cell surface. Furthermore, we show that isolated WAT domains, or proteins containing a WAT domain, can replace individual AChET subunits in PRAD-linked tetramers. This suggests that the four WAT domains interact with the PRAD in a similar manner. These quaternary interactions can form without intercatenary disulfide bonds. The common catalytic domains of AChE are not necessary for tetrameric assembly, although they may contribute to the stability of the tetramer.     

hsp70 binds specifically to a peptide derived from the highly conserved domain (I) region of p53.

Products of a number of mutant p53 genes bind with high affinity to members of the hsp70 family of chaperonin proteins, whereas wild type p53 lacks this type of association. Examination of the sequences of p53 genes from five different species enabled us to predict domains on p53 which may be involved in the association with hsp70 family members. A synthetic polypeptide (Pro-17-Gly) corresponding to the candidate hsp70 binding domain bound to in vitro translated hsp70 as determined by affinity chromatography and nondenaturing gel mobility shift assays. In addition, the Pro-17-Gly peptide competitively inhibited association between hsp70 and p53, an activity which was determined by immunoprecipitation with anti-p53 monoclonal antibody PAb240. The data indicate that p53 contains a hsp70 binding domain, which is located in a highly conserved region at the amino terminus of the protein, and may participate in the cellular function of wild-type p53 or in the transforming capacity of p53 mutants.     

Prediction of the substrate for nonribosomal peptide synthetase (NRPS) adenylation domains by virtual screening

Nonribosomal peptide synthetases (NRPSs) synthesize a diverse array of bioactive small peptides, many of which are used in medicine. There is considerable interest in predicting NRPS substrate specificity in order to facilitate investigation of the many “cryptic” NRPS genes that have not been linked to any known product. However, the current sequence similarity‐based methods are unable to produce reliable predictions when there is a lack of prior specificity data, which is a particular problem for fungal NRPSs. We conducted virtual screening on the specificity‐determining domain of NRPSs, the adenylation domain, and found that virtual screening using experimentally determined structures results in good enrichment of the cognate substrate. Our results indicate that the conformation of the adenylation domain and in particular the conformation of a key conserved aromatic residue is important in determining the success of the virtual screening. When homology models of NRPS adenylation domains of known specificity, rather than experimentally determined structures, were built and used for virtual screening, good enrichment of the cognate substrate was also achieved in many cases. However, the accuracy of the models was key to the reliability of the predictions and there was a large variation in the results when different models of the same domain were used. This virtual screening approach is promising and is able to produce enrichment of the cognate substrates in many cases, but improvements in building and assessing homology models are required before the approach can be reliably applied to these models. Proteins 2015; 83:2052–2066. © 2015 Wiley Periodicals, Inc.     

Effect of the structure of lipids favoring disordered domain formation on the stability of cholesterol-containing ordered domains (lipid rafts): identification of multiple raft-stabilization mechanisms

Despite the importance of lipid rafts, commonly defined as liquid-ordered domains rich in cholesterol and in lipids with high gel-to-fluid melting temperatures (T_m), the rules for raft formation in membranes are not completely understood. Here, a fluorescence-quenching strategy was used to define how lipids with low T_m, which tend to form disordered fluid domains at physiological temperatures, can stabilize ordered domain formation by cholesterol and high-T_m lipids (either sphingomyelin or dipalmitoylphosphatidylcholine). In bilayers containing mixtures of low-T_m phosphatidylcholines, cholesterol, and high-T_m lipid, the thermal stability of ordered domains decreased with the acyl-chain structure of low-T_m lipids in the following order: diarachadonyl > diphytanoyl > 1-palmitoyl 2-docosahexenoyl = 1,2 dioleoyl = dimyristoleoyl = 1-palmitoyl, 2-oleoyl (PO). This shows that low-T_m lipids with two acyl chains having very poor tight-packing propensities can stabilize ordered domain formation by high-T_m lipids and cholesterol. The effect of headgroup structure was also studied. We found that even in the absence of high-T_m lipids, mixtures of cholesterol with PO phosphatidylethanolamine (POPE) and PO phosphatidylserine (POPS) or with brain PE and brain PS showed a (borderline) tendency to form ordered domains. Because these lipids are abundant in the inner (cytofacial) leaflet of mammalian membranes, this raises the possibility that PE and PS could participate in inner-leaflet raft formation or stabilization. In bilayers containing ternary mixtures of PO lipids, cholesterol, and high-T_m lipids, the thermal stability of ordered domains decreased with the polar headgroup structure of PO lipids in the order PE > PS > phosphatidylcholine (PC). Analogous experiments using diphytanoyl acyl chain lipids in place of PO acyl chain lipids showed that the stabilization of ordered lipid domains by acyl chain and headgroup structure was not additive. This implies that it is likely that there are two largely mutually exclusive mechanisms by which low-T_m lipids can stabilize ordered domain formation by high-T_m lipids and cholesterol: 1), by having structures resulting in immiscibility of low-T_m and high-T_m lipids, and 2), by having structures allowing them to pack tightly within ordered domains to a significant degree.     

Insolubility of lipids in triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts)

The insolubility of lipids in detergents is a useful method for probing the structure of biological membranes. Insolubility in detergents like Triton X-100 is observed in lipid bilayers that exist in physical states in which lipid packing is tight. The Triton X-100-insoluble lipid fraction obtained after detergent extraction of eukaryotic cells is composed of detergent-insoluble membranes rich in sphingolipids and cholesterol. These insoluble membranes appear to arise from sphingolipid- and cholesterol-rich membrane domains (rafts) in the tightly packed liquid ordered state. Because the degree of lipid insolubility depends on the stability of lipid-lipid interactions relative to lipid-detergent interactions, the quantitative relationship between rafts and detergent-insoluble membranes is complex, and can depend on lipid composition, detergent and temperature. Nevertheless, when used conservatively detergent insolubility is an invaluable tool for studying cellular rafts and characterizing their composition.