Spin-label studies of the lipid and protein components of erythrocyte membranes. A comparison of electron paramagnetic resonance and saturation transfer electron paramagnetic resonance methods.

We have used both a protein spin label and a lipid spin probe to study some of the slow motions of proteins and of lipids, respectively, in intact erythrocyte membranes. Three electron paramagnetic resonance (EPR) methods, conventional (V1) EPR, second harmonic out-of-phase absorption saturation transfer (ST) EPR (V'2), and first harmonic out-of-phase dispersion ST EPR (U'1) were used to compare the experimental methods and spectral sensitivities with different kinds of molecular motions in human erythrocyte membranes under different experimental conditions. The results show that the V'2 display is relatively more sensitive to the protein motion, while the U'1 display appears more sensitive to the lipid motions, and the V'2 display is substantially more convenient to obtain than the U'1 display.     

The Lipid Peroxidation Product, 4-Hydroxy-2-trans-Nonenal, Alters the Conformation of Cortical Synaptosomal Membrane Proteins

Abstract: Alzheimer's disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid β-peptide (Aβ). Aβ-associated free radicals cause lipid peroxidation, a major product of which is 4-hydroxy-2- trans -nonenal (HNE). We determined whether HNE would alter the conformation of synaptosomal membrane proteins, which might be related to the known neurotoxicity of Aβ and HNE. Electron paramagnetic resonance spectroscopy, using a protein-specific spin label, MAL-6(2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl), was used to probe conformational changes in gerbil cortical synaptosomal membrane proteins, and a lipid-specific stearic acid label, 5-nitroxide stearate, was used to probe for HNE-induced alterations in the fluidity of the bilayer domain of these membranes. Synaptosomal membranes, incubated with low concentrations of HNE, exhibited changes in protein conformation and bilayer order and motion (fluidity). The changes in protein conformation were found to be concentration- and time-dependent. Significant protein conformational changes were observed at physiologically relevant concentrations of 1–10 µ M HNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque- and Aβ-rich AD hippocampal and inferior parietal brain regions. HNE-induced modifications in the physical state of gerbil synaptosomal membrane proteins were prevented completely by using excess glutathione ethyl ester, known to protect neurons from HNE-caused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 µ M ). The results obtained are discussed with relevance to the hypothesis of Aβ-induced free radical-mediated lipid peroxidation, leading to subsequent HNE-induced alterations in the structure and function of key membrane proteins with consequent neurotoxicity in AD brain.     

Temperature-induced protein conformational changes in barley root plasma membrane-enriched microsomes: I. Effect of temperature on membrane protein and lipid mobility

A protein spin label and lipid spin probes were used to study the temperature-dependent motion of protein and lipid, respectively, in barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes. Using membranes from seedlings grown at 20°C, the temperature-dependence of the relative motion of membrane surface spin probes and a spin label covalently attached to membrane proteins suggested abrupt changes in the lipid and protein mobilities at about 12°C. Spin probe spin-spin exchange broadening and fluorescent probe eximer formation indicated apparent temperature-induced alterations in probe lateral diffusion within the membrane at about 12 to 14°C. The results suggest the presence of temperature-induced quasicrystalline lipid clusters which may influence the activity of membrane-bound enzymes.     

Rotational dynamics of phospholamban determined by multifrequency electron paramagnetic resonance

We have used multifrequency electron paramagnetic resonance to define the multistate structural dynamics of an integral membrane protein, phospholamban (PLB), in a lipid bilayer. PLB is a key regulator of cardiac calcium transport, and its function requires transitions between distinct states of intramolecular dynamics. Monomeric PLB was synthesized with the TOAC spin label at positions 11 (in the cytoplasmic domain) and 46 (in the transmembrane domain) and reconstituted into lipid bilayers. Unlike other protein spin labels, TOAC reports directly the motion of the peptide backbone, so quantitative analysis of its dynamics is worthwhile. Electron paramagnetic resonance spectra at 9.4 GHz (X-band) and 94 GHz (W-band) were analyzed in terms of anisotropic rotational diffusion of the two domains. Motion of the transmembrane domain is highly restricted, while the cytoplasmic domain exhibits two distinct conformations, a major one with moderately restricted nanosecond dynamics (T) and another with nearly unrestricted subnanosecond motion (R). The global analysis of spectra at two frequencies yielded values for the rotational correlation times and order parameters that were much more precisely determined than at either frequency alone. Multifrequency EPR is a powerful approach for analysis of complex rotational dynamics of proteins.     

Surface properties of membrane systems. Transport of staphylococcal delta-toxin from aqueous to membrane phase

Hemolytic delta-toxin from Staphylococcus aureus was soluble in either water, methanol or chloroform/methanol (2 : 1, v/v). The toxin spread readily from distilled water into films with pressures (pi) of 10 dynes/cm on water and 30 dynes/cm on 6 M urea; from chloroform/methanol it produced 40 dynes/cm pressure on distilled water. The toxin adsorbed barely from water (pi = 1 dyne/ cm) but it did rapidly from 6 M urea (pi = 35 dynes/cm). The protein films had unusually high surface potentials, which increased with the film pressure and decreased with increasing both pH and urea concentration in the aqueous phase. The fluorescence of 1-aniline 8-naphthalene sulfonate with delta-toxin was much greater than that with RNAase and dipalmitoyl phosphatidylcholine itself, indicating probably a marked lipid-binding character of the toxin. By circular dichroism the alpha-helix content of delta-toxin was 42% in water, 45% in methanol, 24% in 6 M urea. Infrared spectroscopy showed predominant alpha-helix in both 2H2O and deuterated chloroform/methanol as well as in films spread from either solvent on 2H2O. In spreading from 6 M [2H]urea, in which the major infrared absorption was that of [2H]urea with peaks at 1600 and 1480 cm(-1), the delta-toxin film showed prevalently non-alpha-helix structures with major peak intensities at 1633 cm(-1) > 1680 cm(-1), indicating the appearance of new beta-aggregated and beta-antiparallel pleated sheet structures in the film. The data prove that (1) high pressure protein films can consist of alpha-helix as well as non-alpha-helix structures and, differently from another cytolytic protein, melittin, delta-toxin does not resume the alpha-helix conformation in going into the film phase from the extended chain in 6 M urea; (2) conformational changes are important in the transport of proteins from aqueous to lipid or membrane phase; (3) delta-toxin is by far more versatile in structural dynamics and more surface active than alpha-toxin.     

Influence of the disulfide bond configuration on the dynamics of the spin label attached to cytochrome c

A series of multi-nanosecond molecular dynamics (MD) simulations of wild-type cytochrome c and its spin-labeled variants with the methanethiosulfonate moiety attached at position C102 were performed (1) to elucidate the effect of the spin probe presence on the protein structure and (2) to describe the structure and dynamics of the spin-label moiety. Comparisons with the reference crystal structure of cytochrome c (PDB entry: 1YCC) indicate that the protein secondary structure is well preserved during simulations of the wild-type cytochrome c but slightly changed in simulations of the cytochrome c labeled at position C102. At the time scale covered in our simulations, the spin label exhibits highly dynamical behavior. The number of observed distinct conformations of the spin label moiety is between 3 and 13. The spin probe was found to form short-lived hydrogen bonds with the protein. Temporary hydrophobic interactions between the probe and the protein were also detected. The MD simulations directly show that the disulfide bond in the tether linking a spin probe with a protein strongly influence the behavior of the nitroxide group. The conformational flexibility and interaction with the protein are different for each of the two low energy conformations of the disulfide bond.     

Conformational properties of streptokinase--secondary structure and localization of aromatic amino acids.

The conformational properties of streptokinase (Sk) have been assessed by several spectroscopic techniques. A solvent accessibility of about 70% of the 22 Tyr residues was found by u.v. perturbation spectroscopy. Fluorescence spectroscopy indicates also the surface localization of the single Trp 6 residue. Circular dichroism (c.d.), infrared (i.r.), and Raman spectra were analysed in order to estimate the contents of secondary structure elements of Sk. Values in the range of 14-23% alpha-helices, 38-46% beta-structures, 10-30% turns and 12-23% residual structures were found. The characteristics of the c.d. spectrum support the classification of Sk as an alpha + beta protein. Effects of temperature, pH, and denaturants were studied by c.d. spectroscopy, and on spin-labelled Sk, by e.p.r. spectroscopy. Structural effects were induced at temperatures above 40 degrees C, pH values below 3.0 and urea concentrations above 2 M. At temperatures above 70 degrees C, at pH 2.1, and at urea and Gu.HCl concentrations of 7 M and 5 M, respectively, no further structural changes are revealed in the spectra. At temperatures around 50 degrees C, at pH 3.0, and denaturant concentrations of about 1 M Gu.HCl and 1 M to 2 M urea, c.d. effects were observed in the near-u.v. region indicating an increase in the asymmetry for aromatic amino acids in comparison with the structure of Sk in low ionic strength buffers at neutral pH, 20 degrees C and in the absence of denaturants. These effects were most pronounced for the temperature dependence of the c.d. spectra. E.p.r. spectroscopy has shown that loosening of the protein surrounding of the spin label already begins at 1 M urea and that the mobility of the spin label points to a structural change in Sk at 46 degrees C.     

The physical state of water in living cells and model systems. XII. The influence of the conformation of a protein on the solubility of Na+ (sulfate), sucrose, glycine and urea in the water in which the protein is also dissolved

In this report, we describe the result of an extensive investigation of the effects of the conformations of proteins on the solvency of the bulk-phase water in which the proteins are dissolved. The concentrations of the proteins used were usually between 20 to 40%; the temperature was 25 degrees +/- 1 degree C. To probe the solvency of the water, the apparent equilibrium distribution coefficients (or p-values) of 4 solutes were studied: Na+ (sulfate), glycine, sucrose, and urea. From 8 to 14 isolated proteins in three types of conformations were investigated: native; denatured by agents that unravel the secondary structure (e.g., alpha-helix, beta-pleated sheet) of the protein (i.e., 9 M urea, 3 M guanidine HCl); denatured by agents that only disrupt the tertiary structure but leave the secondary structure intact or even strengthened (i.e., 0.1 M sodium dodecylsulfate or SDS, 2 M n-propanol). The results are as follows: (1) as a rule, native proteins have no or weak effect on the solvency of the water for all 4 probes; (2) exposure to 0.1 M SDS and to 2 M n-propanol, as a rule, does not significantly decrease the p-value of all 4 probes; (3) exposure to 9 M urea and to 3 M guanidine HCl consistently lowers the p-values of sucrose, glycine and Na+ (sulfate) and equally consistently produces no effect on the p-value of urea. Sucrose, glycine, and Na+ are found in low concentrations in cell water while urea is not. These experiments were designed and carried out primarily to test two subsidiary theories of the AI hypotheses: the polarized multilayer (PM) theory of cell water; and the theory of size-dependent solute exclusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and use of new spin labeled derivatives of phosphorylcholine in a comparative study of human, dogfish, and Limulus C-reactive proteins

New spin labeled derivatives of phosphorylcholine have been synthesized. The compounds cause reversible inhibition of the precipitation reactions between pneumococcal C-polysaccharide and the C-reactive proteins from humans, dogfish sharks (Mustelus canis), and horseshoe crabs (Limulus polyphemus). The spin labeled phosphorylcholine derivatives also rival phosphorylcholine as a ligand for the human, dogfish, and Limulus C-reactive proteins. The interactions of the purified C-reactive proteins with the spin labeled derivatives of phosphorylcholine have been studied using electron spin resonance spectrometry. The dramatic decrease in the ESR signal of some of the spin labels is due to immobilization of the label. Only the well known phosphate spin label, 4-phosphate-2,2,6,6-tetramethylpiperidine-1-oxyl could be used for binding studies on human and Limulus C-reactive proteins. Thus, by Scatchard analysis, the human C-reactive protein bound 1 mol of phosphate spin label per mol of protein with a Ka = 3.91 X 10(3) M-1, whereas the Limulus C-reactive protein bound only 0.5 mol of phosphate spin label per mol of protein with an overall Ka = 1.95 X 10(3) M-1. Inhibition studies using purified C-polysaccharide-induced inhibition of the phosphate spin label-human C-reactive protein interaction showed competitive inhibition with a KI of 4.78 X 10(-5) M at 18 degrees C. The phosphate spin label did not bind to dogfish C-reactive protein. However, one new phosphorylcholine spin label did bind and was used for Scatchard and Hill plot analyses. The dogfish C-reactive protein, which exists as a Mr = 50,000 dimer, bound 2 mol of the phosphorylcholine spin label per mol of protein, and this binding exhibited negative cooperativity as indicated by a Hill coefficient of 0.75.     

Lipid-protein interactions in membranes

The interactions of lipids with integral and peripheral proteins can be studied in reconstituted and natural membranes using spin label electron spin resonance (ESR) spectroscopy. The ESR spectra reveal a reduction in mobility of the spin-labelled lipid species, and in certain cases evidence is obtained for a partial penetration of the peripheral proteins into the membrane. The latter may be relevant to the import mechanism of apocytochrome c into mitochondria. Integral proteins induce a more direct motional restriction of the spin-labelled lipid chains, allowing the stoichiometry and specificity of the interaction, and the lipid exchange rate at the protein interface to be determined from the ESR spectra. In this way, a population of very slowly exchanging cardiolipin associated with the mitochondrial ADP-ATP carrier has been identified. The residues involved in the specificity for charged lipids of the myelin proteolipid protein have been localized to the deletion in the DM-20 mutant, and the difference in lipid-protein interactions with the beta-sheet and alpha-helical conformations of the M-13 coat protein, has been characterized.     

Solvent interactions and protein dynamics in spin-labeled T4 lysozyme

Aspects of T4 lysozyme dynamics and solvent interaction are investigated using atomically detailed Molecular Dynamics (MD) simulations. Two spin-labeled mutants of T4 lysozyme are analyzed (T4L-N40C and T4L-K48C), which have been found from electronic paramagnetic resonance (EPR) experiments to exhibit different mobilities at the site of spin probe attachment (N- and C-terminus of helix B, respectively). Similarities and differences in solvent distribution and diffusion around the spin label, as well as around exposed and buried residues within the protein, are discussed. The purpose is to capture possible strong interactions between the spin label (ring) and solvent molecules, which may affect EPR lineshapes. The effect of backbone motions on the water density profiles is also investigated. The focus is on the domain closure associated with the T4 lysozyme hinge-bending motion, which is analyzed by Essential Dynamics (ED). The N-terminus of helix B is found to be a "hinge" residue, which explains the high degree of flexibility and motional freedom at this site.     

Electron spin-labelling of the EutC subunit in B12-dependent ethanolamine ammonia-lyase reveals dynamics and a two-state conformational equilibrium in the N-terminal,signal-sequence-associated domain

Abstract

The B₁₂ (adenosylcobalamin)-dependent ethanolamine ammonia-lyase (EAL) is a product of the ethanolamine utilisation (eut) gene cluster, that is involved in human gut microbiome homeostasis and in disease conditions caused by pathogenic strains of Salmonella and Escherichia coli. Toward elucidation of the molecular basis of EAL catalysis, and its intracellular trafficking and targeting to the Eut biomicrocompartment (BMC), we have applied electron spin-labelling and electron paramagnetic resonance spectroscopy to wild-type (wt) EAL from Salmonella typhimurium, by using the sulphydryl-specific, 4-maleimido-TEMPO (4MT) spin label. One cysteine residue per active site displays exceptional reactivity with 4MT. This site is identified as βC37 on the EutC subunit, by using 4MT-labeling of site-specific cysteine-to-alanine mutants, enzyme kinetics, and accessible surface area calculations. Electron paramagnetic resonance (EPR) spectra of 4MT-labelled wt EAL are collected over 200–265 K in frozen, polycrystalline water-only, and 1% v/v DMSO solvents. EPR simulations reveal two mobility components for each condition. Detectable spin probe reorientational motion of the two components occurs at 215 and 225 K with 1% v/v DMSO, relative to the water-only condition, consistent with formation of an aqueous-DMSO solvent mesodomain around EAL. Parallel trends in fast- and slow-reorientational correlation times and interconversion of the two populations with increasing temperature, indicate 4MT labelling of a single site (βC37). A two-state model is proposed, in which the fast and slow motional populations represent EAL-bound and free conformations of the EutC N-terminal domain. The approximately equal proportion of each state may represent a balance between EutC and EAL protein stability and efficient targeting to the BMC.     

Electron paramagnetic resonance reveals a large-scale conformational change in the cytoplasmic domain of phospholamban upon binding to the sarcoplasmic reticulum Ca-ATPase

We used EPR spectroscopy to probe directly the interaction between phospholamban (PLB) and its regulatory target, the sarcoplasmic reticulum Ca-ATPase (SERCA). Synthetic monomeric PLB was prepared with a single cytoplasmic cysteine at residue 11, which was then spin labeled. PLB was reconstituted into membranes in the presence or absence of SERCA, and spin label mobility and accessibility were measured. The spin label was quite rotationally mobile in the absence of SERCA, but became more restricted in the presence of SERCA. SERCA also decreased the dependence of spin label mobility on PLB concentration in the membrane, indicating that SERCA reduces PLB-PLB interactions. The spin label MTSSL, attached to Cys11 on PLB by a disulfide bond, was stable at position 11 in the absence of SERCA. In the presence of SERCA, the spin label was released and a covalent bond was formed between PLB and SERCA, indicating direct interaction of one or more SERCA cysteine residues with Cys11 on PLB. The accessibility of the PLB-bound spin label IPSL to paramagnetic agents, localized in different phases of the membrane, indicates that SERCA greatly reduces the level of interaction of the spin label with the membrane surface. We propose that the cytoplasmic domain of PLB associates with the lipid surface, and that association with SERCA induces a major conformational change in PLB in which the cytoplasmic domain is drawn away from the lipid surface by SERCA.     

A lens intercellular junction protein, MP26, is a phosphoprotein

The major protein present in the plasma membrane of the bovine lens fiber cell (MP26), thought to be a component of intercellular junctions, was phosphorylated in an in vivo labeling procedure. After fragments of decapsulated fetal bovine lenses were incubated with [32P]orthophosphate, membranes were isolated and analyzed by SDS PAGE and autoradiography. A number of lens membrane proteins were routinely phosphorylated under these conditions. These proteins included species at Mr 17,000 and 26,000 as well as a series at both 34,000 and 55,000. The label at Mr 26,000 appeared to be associated with MP26, since (a) boiling the membrane sample in SDS led to both an aggregation of MP26 and a loss of label at Mr 26,000, (b) the label at 26,000 was resistant to both urea and nonionic detergents, and (c) two-dimensional gels showed that a phosphorylated Mr 24,000 fragment was derived from MP26 with V8 protease. Studies with proteases also provided for a localization of most label within approximately 20 to 40 residues from the COOH-terminus of MP26. Published work indicates that the phosphorylated portion of MP26 resides on the cytoplasmic side of the membrane, and that this region of MP26 contains a number of serine residues. The same region of MP26 was labeled when isolated lens membranes were reacted with a cAMP-dependent protein kinase prepared from the bovine lens. After the in vivo labeling of lens fragments, phosphoamino acid analysis of MP26 demonstrated primarily labeled serines, with 5-10% threonines and no tyrosines. Treatments that lowered the intracellular calcium levels in the in vivo system led to a selective reduction of MP26 phosphorylation. In addition, forskolin and cAMP stimulated the phosphorylation of MP26 and other proteins in concentrated lens homogenates. These findings are of interest because MP26 appears to serve as a protein of cell-to-cell channels in the lens, perhaps as a lens gap junction protein.     

Solvent Interactions and Protein Dynamics in Spin-labeled T4 Lysozyme

Abstract

Aspects of T4 lysozyme dynamics and solvent interaction are investigated using atomically detailed Molecular Dynamics (MD) simulations. Two spin-labeled mutants of T4 lysozyme are analyzed (T4L-N40C and T4L-K48C), which have been found from electronic paramagnetic resonance (EPR) experiments to exhibit different mobilities at the site of spin probe attachment (N- and C-terminus of helix B, respectively). Similarities and differences in solvent distribution and diffusion around the spin label, as well as around exposed and buried residues within the protein, are discussed. The purpose is to capture possible strong interactions between the spin label (ring) and solvent molecules, which may affect EPR lineshapes. The effect of backbone motions on the water density profiles is also investigated. The focus is on the domain closure associated with the T4 lysozyme hinge-bending motion, which is analyzed by Essential Dynamics (ED). The N-terminus of helix B is found to be a “hinge” residue, which explains the high degree of flexibility and motional freedom at this site.     

Tryptic digestion of bovine secretory IgA at elevated temperature and in urea. Isolation of SC domain 1 which is covalently bound to IgA dimer and binds non-covalently to IgM

1. Tryptic cleavage sites in bovine secretory component (SC) which become inaccessible when SC is bound to IgA dimer remained inaccessible at 60 degrees C and in 4 M urea at 37 degrees C. 2. This suggests the presence of strong interactions compatible with published affinity constants of ca 10(8) M-1. 3. In 5 M urea at 37 degrees C further cleavage of bound SC did occur to produce a fragment consisting of domain 1 which was disulphide bridged to the IgA dimer. 4. Binding studies on the isolated fragment showed that domain 1 did not account for all the binding by SC. 5. Cleavage of the isolated fragment with iodosobenzoic produced a smaller fragment consisting of the n-terminal third of domain 1 (residues 1-35). This N-terminal fragment showed significant binding.     

Rotational dynamics of protein and boundary lipid in sarcoplasmic reticulum membrane

We have used spin labels and electron paramagnetic resonance (EPR) to study the correlation between the rotational dynamics of protein and lipid in sarcoplasmic reticulum (SR) membranes. A short-chain maleimide spin label was used to monitor the submillisecond rotational mobility of the Ca-ATPase enzyme (using saturation transfer EPR); a free fatty acid spin label was used to monitor the submicrosecond rotational mobility of the bulk lipid hydrocarbon chains (using conventional EPR); and a fatty acid spin label derivative (long-chain maleimide) attached to the enzyme was used to monitor the mobility of hydrocarbon chains adjacent to the protein (i.e., boundary lipid). In the native SR membranes, the protein was highly mobile (effective correlation time 50 microseconds). The spectra of the hydrocarbon probes both contained at least two components. For the unattached probe, the major component indicated nearly as much mobility as in the absence of protein (effective rotational correlation time 3 ns), while a minor component, corresponding to 25-30% of the total signal, indicated strong immobilization (effective correlation time greater than or equal to 10 ns). For the attached hydrocarbon probe, the major component (approximately 70% of the total) was strongly immobilized, and the mobile component was less mobile than that of the unattached probe. When the lipid-to-protein ratio was reduced 55% by treatment with deoxycholate, protein mobility decreased considerably, suggesting protein aggregation. A concomitant increase was observed in the fraction of immobilized spin labels for both the free and attached hydrocarbon probes. The observed hydrocarbon immobilization probably arises in part from immobilization at the protein-lipid boundary, but protein-protein interactions that trap hydrocarbon chains may also contribute. When protein aggregation was induced by glutaraldehyde crosslinking, submillisecond protein mobility was eliminated, but there was no effect on either hydrocarbon probe. Thus protein aggregation does not necessarily cause hydrocarbon chain immobilization.     

Dependence of reaction rate of 5,5′-dithiobis-(2-nitrobenzoic acid) to free sulfhydryl groups of bovine serum albumin and ovalbumin on the protein conformations

The effect of protein conformations on the reaction rate of Ellman's reagent, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) with sulfhydryl (SH) groups of proteins was examined. The stopped-flow method was applied to follow the reaction of DTNB with SH group of two proteins, bovine serum albumin (BSA) and ovalbumin (OVA), at various concentrations of guanidine hydrochloride and urea. The rates for both the proteins were faster in guanidine than in urea. The rate sharply depended on the protein conformations, which were monitored by changes of helix contents on the basis of the circular dichroism measurements. The reaction rate of DTNB with SH groups of BSA was maximal around 2 M guanidine and 5 M urea. On the other hand, the reaction rate of DTNB with OVA was maximal at 3.5 M guanidine, while it gradually increased with an increase in the urea concentration. The amount of reactive SH group participating in the reaction with DTNB was also estimated by the absorbance change at 412 nm. The magnitudes of absorbance change for the reaction with free SH groups of OVA at low concentrations of the denaturants were appreciably smaller than those for BSA with one free SH group. Most of the four SH groups of OVA might react with DTNB above 5 M guanidine, although only a part of them did even at 9 M urea.     

Asymmetric dipping of bacteriophage M13 coat protein with increasing lipid bilayer thickness

Knowledge about the vertical movement of a protein with respect to the lipid bilayer plane is important to understand protein functionality in the biological membrane. In this work, the vertical displacement of bacteriophage M13 major coat protein in a lipid bilayer is used as a model system to study the molecular details of its anchoring mechanism in a homologue series of lipids with the same polar head group but different hydrophobic chain length. The major coat proteins were reconstituted into 14:1PC, 16:1PC, 18:1PC, 20:1PC, and 22:1PC bilayers, and the fluorescence spectra were measured of the intrinsic tryptophan at position 26 and BADAN attached to an introduced cysteine at position 46, located at the opposite ends of the transmembrane helix. The fluorescence maximum of tryptophan shifted for 700 cm^-1 on going from 14:1PC to 22:1PC, the corresponding shift of the fluorescence maximum of BADAN at position 46 was approximately 10 times less (∼ 70 cm^-1). Quenching of fluorescence with the spin label CAT 1 indicates that the tryptophan is becoming progressively inaccessible for the quencher with increasing bilayer thickness, whereas quenching of BADAN attached to the T46C mutant remained approximately unchanged. This supports the idea that the BADAN probe at position 46 remains at the same depth in the bilayer irrespective of its thickness and clearly indicates an asymmetrical nature of the protein dipping in the lipid bilayer. The anchoring strength at the C-terminal domain of the protein (provided by two phenylalanine residues together with four lysine residues) was estimated to be roughly 5 times larger than the anchoring strength of the N-terminal domain.