Laser Raman studies of lipid disordering by the B-protein of fd phage.

Complexes of the B-protein of fd phage with the model lipid dipalmitoyl phosphatidylcholine (DPPC) were made by sonication of the fd phage in the presence of dipalmitoyl phosphatidylcholine. Both laser Raman spectra and circular dichroism show the protein in the membrane to be almost entirely in the beta-sheet conformation. This beta-sheet conformation is found to be independent of the temperature between 10 degrees C and 50 degrees C. On the other hand, the protein has a very dramatic effect on the organization of the lipid bilayer. An aqueous dispersion of 1 : 1 lipid/protein mixture gives a broad conformational transition of DPPC which occurs between 10 degrees C and 30 degrees C. This contrasts markedly with simple aqueous DPPC dispersions which show a sharp transition at 41 degrees C. This appears to be the first reported example of the lowering of the conformational transition of a membrane bilayer by an intrinsic membrane protein.     

A stable disulfide-free gene-3-protein of phage fd generated by in vitro evolution

Disulfide bonds provide major contributions to the conformational stability of proteins, and their cleavage often leads to unfolding. The gene-3-protein of the filamentous phage fd contains two disulfides in its N1 domain and one in its N2 domain, and these three disulfide bonds are essential for the stability of this protein. Here, we employed in vitro evolution to generate a disulfide-free variant of the N1-N2 protein with a high conformational stability. The gene-3-protein is essential for the phage infectivity, and we exploited this requirement for a proteolytic selection of stabilized protein variants from phage libraries. First, optimal replacements for individual disulfide bonds were identified in libraries, in which the corresponding cysteine codons were randomized. Then stabilizing amino acid replacements at non-cysteine positions were selected from libraries that were created by error-prone PCR. This stepwise procedure led to variants of N1-N2 that are devoid of all three disulfide bonds but stable and functional. The best variant without disulfide bonds showed a much higher conformational stability than the disulfide-containing wild-type form of the gene-3-protein. Despite the loss of all three disulfide bonds, the midpoints of the thermal transitions were increased from 48.5 degrees C to 67.0 degrees C for the N2 domain and from 60.0 degrees C to 78.7 degrees C for the N1 domain. The major loss in conformational stability caused by the removal of the disulfides was thus over-compensated by strongly improved non-covalent interactions. The stabilized variants were less infectious than the wild-type protein, probably because the domain mobility was reduced. Only a small fraction of the sequence space could be accessed by using libraries created by error-prone PCR, but still many strongly stabilized variants could be identified. This is encouraging and indicates that proteins can be stabilized by mutations in many different ways.     

Interaction of melittin with dimyristoyl phosphatidylcholine liposomes. Evidence for boundary lipid by raman spectroscopy

The interaction of melittin, a polypeptide consisting of 26 amino acid residues, with dimyristoyl phosphatidylcholine bilayers was investigated by vibrational Raman spectroscopy. Spectral peak height intensity ratios, involving vibrational transitions in both the 3000 cm^?1 acyl chain methylene carbon-hydrogen stretching mode region and the 1100 cm^?1 acyl chain carbon-carbon skeletal stretching mode interval, served as temperature profile indices for monitoring the bilayer order-disorder processes. For a lipid : melittin molar ratio of 14 : 1 two order-disorder transitions were observed. In comparison to a gel to liquid crystalline phase transition of 22.5°C for the pure lipid, the lower transition, exhibiting a 2°C width, is centered at 17°C and is associated with a depression of the main lipid phase transition of dimyristoyl phosphatidylcholine. The second thermal transition, displaying a 7°C interval, occurs at approx. 29°C and is associated with the melting behavior of approximately seven immobilized boundary lipids which surround the inserted hydrophobic segment of the polypeptide. For a lipid : melittin molar ratio of 10 : 1 two thermal transitions are also observed at 11 and 30°C. As before, they represent, respectively, the main gel to liquid crystalline phase transition and the melting behavior of approximately four boundary lipids attached to melittin. From these data alternative schemes are suggested for disposing the immobilized lipids around the hydrophobic portion of the polypeptide within the bilayer.     

Evolutionary stabilization of the gene-3-protein of phage fd reveals the principles that govern the thermodynamic stability of two-domain proteins

The gene-3-protein (G3P) of filamentous phage is essential for their propagation. It consists of three domains. The CT domain anchors G3P in the phage coat, the N2 domain binds to the F pilus of Escherichia coli and thus initiates infection, and the N1 domain continues by interacting with the TolA receptor. Phage are thus only infective when the three domains of G3P are tightly linked, and this requirement is exploited by Proside, an in vitro selection method for proteins with increased stability. In Proside, a repertoire of variants of the protein to be stabilized is inserted between the N2 and the CT domains of G3P. Stabilized variants can be selected because they resist cleavage by a protease and thus maintain the essential linkage between the domains. The method is limited by the proteolytic stability of G3P itself. We improved the stability of G3P by subjecting the phage without a guest protein to rounds of random in vivo mutagenesis and proteolytic Proside selections. Variants of G3P with one to four mutations were selected, and the temperature at which the corresponding phage became accessible for a protease increased in a stepwise manner from 40 degrees C to almost 60 degrees C. The N1-N2 fragments of wild-type gene-3-protein and of the four selected variants were purified and their stabilities towards thermal and denaturant-induced unfolding were determined. In the biphasic transitions of these proteins domain dissociation and unfolding of N2 occur in a concerted reaction in the first step, followed by the independent unfolding of domain N1 in the second step. N2 is thus less stable than N1, and it unfolds when the interactions with N1 are broken. The strongest stabilizations were caused by mutations in domain N2, in particular in its hinge subdomain, which provides many stabilizing interactions between the N1 and N2 domains. These results reveal how the individual domains and their assembly contribute to the overall stability of two-domain proteins and how mutations are optimally placed to improve the stability of such proteins.     

A phage display‐selected peptide inhibitor of Agrobacterium vitis polygalacturonase

Agrobacterium vitis, the causal agent of crown gall of grapevine, is a threat to viticulture worldwide. A major virulence factor of this pathogen is polygalacturonase, an enzyme that degrades pectin components of the xylem cell wall. A single gene encodes for the polygalacturonase gene. Disruption of the polygalacturonase gene results in a mutant that is less pathogenic and produces significantly fewer root lesions on grapevines. Thus, the identification of peptides or proteins that could inhibit the activity of polygalacturonase could be part of a strategy for the protection of plants against this pathogen. A phage‐displayed combinatorial peptide library was used to isolate peptides with a high binding affinity to A. vitis polygalacturonase. These peptides showed sequence similarity to regions of Oryza sativa (EMS66324, Japonica) and Triticum urartu (NP_001054402, wild wheat) polygalacturonase‐inhibiting proteins (PGIPs). Furthermore, these panning experiments identified a peptide, SVTIHHLGGGS, which was able to reduce A. vitis polygalacturonase activity by 35% in vitro. Truncation studies showed that the IHHL motif alone is sufficient to inhibit A. vitis polygalacturonase activity.     

Resonance raman spectroscopy of chemically modified and isotopically labelled purple membranes. II. Kinetic studies

Kinetic resonance Raman spectra of native and isotopically labelled purple membranes are compared. Using these data and the assignments of the previous paper in this sequence, we have confirmed that the Schiff base is deprotonated at times that are short in comparison to M₄₁₂ evolution. In addition, by monitoring the kinetic resonance Raman spectra in ²H₂O with 488.0 nm excitation we have been able to characterize in more detail the vibrational features associated with this unprotonated intermediate that precedes M₄₁₂. Furthermore, the kinetic spectra of fully deuterated purple membranes in H₂O have allowed us to assign the 1465 cm⁻¹ band in these spectra to the C=C stretching frequency of BR₅₇₀ and the 1512 cm⁻¹ band to the C=C stretching frequency of M₄₁₂. These spectra have also provided an indication of a Raman spectral feature associated with O₆₄₀ and, finally, our kinetic spectra have provided evidence that there is a significant alteration in the rate constants for the evolution of the various intermediates when the non-exchangeable protons on the membrane are replaced by deuterons.     

A conformational unfolding reaction activates phage fd for the infection of Escherichia coli

Unfolding usually leads to the loss of the biological function of a protein. Here, we show that an unfolding reaction activates the gene-3-protein of the filamentous phage fd for its function during the infection of Escherichia coli. Before infection, the gene-3-protein is in a fully folded locked form, in which the binding site for the phage receptor TolA is buried at the domain interface. To expose this binding site, the gene-3-protein must be activated, and previously we identified the cis-to-trans isomerization at Pro213 in the hinge region between the two domains as a key step of activation. We now report that Pro213 isomerization destabilizes the protein and leads to a loss of folded structure, presumably in the hinge region. The partially unfolded form of the gene-3-protein is metastable, and trans-Pro213 arrests the protein in this activated form for an extended time, long enough to find the receptor TolA. The partial unfolding and its timing by prolyl isomerization are essential for the biological function.     

Efficient identification of phosphatidylserine-binding proteins by ORF phage display

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in ∼300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressed for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.     

A comparative study of dynamic structures between phage 434 Cro and repressor proteins by normal mode analysis

Two DNA binding proteins, Cro and the amino-terminal domain of the repressor of bacteriophage 434 (434 Cro and 434 repressor) that regulate gene expression and contain a helix-turn-helix (HTH) motif responsible for their site-specific DNA recognition adopt very similar three-dimensional structures when compared to each other. To reveal structural differences between these two similar proteins, their dynamic structures, as examined by normal mode analysis, are compared in this paper. Two kinds of structural data, one for the monomer and the other for a complex with DNA, for each protein, are used in the analyses. From a comparison between the monomers it is found that the interactions of Ala-24 in 434 Cro or Val-24 in 434 repressor, both located in the HTH motif, with residues 44, 47, 48, and 51 located in the domain facing the motif, and the interactions between residues 17, 18, 28, and 32, located in the HTH motif, cause significant differences in the correlative motions of these residues. From the comparison between the monomer and the complex with DNA for each protein, it was found that the first helix in the HTH motif is distorted in the complex form. While the residues in the HTH motif in 434 Cro have relatively larger positive correlation coefficients of motions with other residues within the HTH motif, such correlations are not large in the HTH motif of 434 repressor. It is suggestive to their specificity because the 434 repressor is less specific than 434 Cro. Although a structural comparison of proteins has been performed mainly from a static or geometrical point of view, this study demonstrates that the comparison from a dynamic point of view, using the normal mode analysis, is useful and convenient to explore a difference that is difficult to find only from a geometrical point of view, especially for proteins very similar in structure. © 1996 Wiley-Liss, Inc.     

Structure of the dna binding cleft of the gene 5 protein from bacteriophage fd

The structure of the gene 5 DNA unwinding protein from bacteriophage fd has been solved to 2.3-Å resolution by X-ray diffraction techniques. The molecule contains an extensive cleft region that we have identified as the DNA binding site on the basis of the residues that comprise its surface. The interior of the groove has a rather large number of basic amino acid residues that serve to draw the polynucleotide backbone into the cleft. Arrayed along the external edges of the groove are a number of aromatic amino acid side groups that are in position to stack upon the bases of the DNA and fix it in place. The structure and binding mechanism as we visualize it appear to be fully consistent with evidence provided by physical-chemical studies of the protein in solution.     

Aminoacyl-tRNA synthetase-interacting multi-functional protein, p43, is imported to endothelial cells via lipid rafts

An aminoacyl-tRNA synthetase subunit, p43, was previously demonstrated to be released from mammalian cells, and to function as an extracellular regulator of both angiogenesis and inflammatory responses (Ko et al., [2001] J Biol Chem, 276; 23028; Park et al.[2002], J Biol Chem 277; 45243). Here, we report that p43 is internalized to the endothelial cells via lipid rafts. Exogenous p43 was co-localized on bovine aorta endothelial cells with cholera toxin B (CTB), which binds to cholesterol-enriched lipid rafts. The p43 was rapidly internalized to the cells, as early as 5 min after binding to the surfaces of the cells. p43 bound to the isolated lipid rafts, and its interaction with the lipid rafts, was prevented by high salt content, but not by detergent. This suggests that ionic bonds are involved in the molecular association of p43 with the lipid rafts. Taken together, we conclude that p43 binds to the endothelial cell surface via lipid rafts.     

A phage display system for studying the sequence determinants of protein folding.

We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein.     

The structural analysis of three‐dimensional fibrous collagen hydrogels by raman microspectroscopy

To investigate molecular effects of 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC), EDC/N‐hydroxysuccinimide (NHS), glyceraldehyde cross‐linking as well as polymerization temperature and concentration on the three‐dimensional (3D) collagen hydrogels, we analyzed the structures in situ by Raman microspectroscopy. The increased intensity of the 814 and 936 cm^?1 Raman bands corresponding to the C—C stretch of a protein backbone and a shift in the amide III bands from 1241 cm^?1/1268 cm^?1 in controls to 1247 cm^?1/1283 cm^?1 in glyceraldehyde‐treated gels indicated changes to the alignment of the collagen molecules, fibrils/fibers and/or changes to the secondary structure on glyceraldehyde treatment. The increased intensity of 1450 cm^?1 band and the appearance of a strong peak at 1468 cm^?1 reflected a change in the motion of lysine/arginine CH₂ groups. For the EDC‐treated collagen hydrogels, the increased intensity of 823 cm^?1 peak corresponding to the C—C stretch of the protein backbone indicated that EDC also changed the packing of collagen molecules. The 23% decrease in the ratio of 1238 cm^?1 to 1271 cm^?1 amide III band intensities in the EDC‐modified samples compared with the controls indicated changes to the alignment of the collagen molecules/fibrils and/or the secondary structure. A change in the motion of lysine/arginine CH₂ groups was detected as well. The addition of NHS did not induce additional Raman shifts compared to the effect of EDC alone with the exception of a 1416 cm^?1 band corresponding to a COO^? stretch. Overall, the Raman spectra suggest that glyceraldehyde affects the collagen states within 3D hydrogels to a greater extent compared to EDC and the effects of temperature and concentration are minimal and/or not detectable. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 349–356, 2013.     

Structure of Shigella sonnei lipid A

The detailed chemical structure of lipid A of Shigella sonnei phase II was elucidated. The lipid A backbone consists of a β-1,6-linked glucosamine disaccharide substituted with (mono) phosphates both at C-1 and C-4′. This was shown by selective degradation followed by ³¹P-NMR studies. C-4 and C-6′ were found to contain unsubstituted hydroxyl groups, the latter being the point of attachment of KDO as reported for other enterobacterial lipids A.The amino groups of the glucosamine disaccharide are substituted by 3-hydroxy fatty acids: 3-O-(14:0) 14:0 at the non-reducing glucosamine and 3-O-(12:0) 14:0 at the reducing glucosamine. In contrast to earlier reports, no ethanolamine or phosphodiester linkages were found in lipid A.     

NMR studies of glycerol permeability in lipid vesicles, erythrocytes and the alga Dunaliella

Glycerol diffusional permeabilities through the cytoplasmic cell membrane of Dunaliella salina, the cell envelope of pig erythrocyte and egg phosphattidylcholine vesicles were measured by NMR spectroscopy employing the spin-echo method and nuclear T₁ relaxation. The following permeability coefficients (P) and corresponding enthalpies of activation (ΔH^‡) were determined for glycerol at 25°C: for phosphatidylcholine vesicles 5·10⁻⁶ cm/s and 11±2 kcal/mol; for pig erythrocytes 7·10⁻⁸ cm/s and 18±3 kcal/mol, respectively; for the cytoplasmic membrane of D. salina the permeability at 17°C was found to be exceptionally low and only a lower limit (P<5·10⁻¹¹cm/s) could be calculated. At temperatures above 50°C a change in membrane permeability occurred leading to rapid leakage of glycerol accompanied by cell death. The data reinforce the notion that the cytoplasmic membrane of Dunaliella represents a genuine anomaly in its exceptional low permeability to glycerol.     

Conformational order of the hydrocarbon chains in lipid bilayers. A raman spectroscopic study

Raman spectra of dihexadecylphosphatidic acid (DHPA) and of dimyristoylphosphatidylcholine (DMPC) and its longer chain homologues have been obtained as a function of temperature in order to study the conformational order of the hydrocarbon chains in lipid bilayers. The frequency of the longitudinal acoustical (LA) vibration band is evaluated in terms of the length of all-trans chain segments. In the ordered phase, the chains are found to be overwhelmingly in the all-trans conformation. In the fluid phase, definite all-trans segments occur predominantly, the length of which coincides with the extension of the order parameter plateau known from deuterium magnetic resonance (DMR). The frequency of the skeletal optical (SO) trans vibration band leads to the same result, if evaluated under the assumption of vibrational decoupling by gauche bands in the fluid phase, thus lending support to this assumption. The intensity of this band determined from the band area increases linearly with chain length in the ordered phase and is independent of chain length in the fluid phase. Evaluating the intensity for the length of all-trans segments, the same result for the chain conformation is obtained as derived from the frequencies, with the additional information that the length of the all-trans segments in the fluid phase does not vary with chain length.     

乙型肝炎病毒表面抗原对细胞脂质合成作用的研究

乙型肝炎病毒(hepatitis B virus,HBV)合成的蛋白调节细胞脂质代谢的研究不断被报道,但乙型肝炎病毒表面抗原(hepatitis B virus surface antigen,HBsAg)与脂质代谢的相互调控研究较少,且机制尚不明确。本研究通过对细胞转录组学的分析,揭示HBsAg对脂质代谢的调控机制。选用稳定表达HBsAg的细胞系HepG2-S-G2与其对照细胞系HepG2-neo-F4进行转录组学分析。利用定量聚合酶链反应(polymerase chain reaction,PCR)、蛋白质印迹法(Western blot,WB)分别检测重要差异基因OXCT1和CYP4F3在mRNA水平和蛋白水平的表达差异。为验证HBsAg促进脂质合成上调的表型,对两种细胞系进行油红O染色并检测细胞脂肪酸、总胆固醇水平。进一步对稳定转染HBV的细胞系HepG2.2.15进行降脂处理,以观察细胞上清液中HBsAg与脂质合成之间是否存在相互调控。结果显示,参与脂质代谢的差异基因发生显著变化,提示HBsAg引起了宿主细胞脂质合成途径的上调和消耗途径下调。定量PCR结果显示,相对于HepG2-neo-F4细胞,HepG2-S-G2细胞的3-酮酸辅酶A转移酶1(3-oxoacid CoA-transferase 1,OXCT1)mRNA水平升高约9倍,与转录组测序结果基本一致;CYP4F3基因在HepG2-S-G2细胞中转录相对下调。 WB结果显示,OXCT1和CYP4F3蛋白表达均出现相应的显著上调或下调,并且趋势与转录组分析一致。油红O染色以及细胞脂肪酸、总胆固醇水平检测结果证实HepG2-S-G2细胞中脂滴更明显,且游离脂肪酸和总胆固醇均显著升高。降脂处理结果显示细胞上清液中HBsAg显著降低。上述结果表明,HBsAg可上调脂质代谢、促进脂质合成,提示降脂可能成为抑制HBsAg的潜在有效途径。     

Photoelectrospectrometry of bilayer lipid membranes

Three different bilayer lipid membrane systems were studied under visible and ultraviolet illumination. The first system consisted of a bilayer lipid membrane formed with a mixture of phospholipids and cholesterol, to one side of which purple membrane fragments from Halobacterium halobium were added. The second system consisted of a membrane formed from spinach chloroplast extract. When either of these membrane systems was illuminated with ultraviolet and visible radiation, photopotentials were observed and photoelectric action spectra were recorded (the technique is termed photoelectrospectrometry). Each spectrum had a definite structure which was characteristic of each of the modified membranes. The third system studied consisted of an otherwise photoinactive membrane formed with a mixture of phospholipids and cholesterol, to one side of which chymotrypsin was added. When the membrane was illuminated with visible light no photoresponse was observed. On the other hand, a photopotential which increased with incubation time was observed when the membrane was illuminated with ultraviolet light. Since, in our systems, the photoresponses have been observed to be due to certain species incorporated into the membrane, it appears that photoelectrospectrometry is a useful tool for studying lipid-protein interactions, constituent organization and energy transfer in membranes.     

Glutathione-dependent protection by rat liver microsomal protein against lipid peroxidation

GSH is an important cellular defense against oxidant injury. Its effect in the rat liver microsomal lipid peroxidation system has been examined. Incubation of fresh rat liver microsomes with ascorbic acid and ADP-chelated iron leads to the peroxidation of microsomal lipids (production of thiobarbituric acid-reactive substances and destruction of polyunsaturated fatty acids) following a 2 to 5 min lag. Addition of 0.1 mM GSH to the system lengthened the lag period by 5 to 15 min without affecting the rate or the extent of lipid peroxidation. GSH could not be replaced in prolonging the lag by cysteine, mercaptoethanol, dithiothreitol, propylthiouracil, or GSSG. The GSH effect on the lag was abolished by heating or trypsin digestion of the microsomes, indicating that microsomal protein is required for its expression. Progressively longer lags were observed as the GSH concentration was increased from 0.1 to 5 mM, but there was no evidence of GSH oxidation as a consequence of the protection against lipid peroxidation. GSH protected against heat inactivation of the microsomal protein responsible for the GSH effect. Experiments with an oxygen electrode revealed that the GSH protection did not alter the ratio of O₂ consumed to thiobarbituric acid-reactive substances produced. This implicated free radical scavenging as the mechanism of protection. These results indicate the existence of a GSH-dependent rat liver microsomal protein which scavenges free radical. This protein may be an important defense against free radical injury to the microsomal membrane.