31P nuclear magnetic resonance studies of the association of basic proteins with multilayers of diacyl phosphatidylserine

Lysozyme, cytochrome c, poly(l-lysine), myelin basic protein and ribonuclease were used to form multilayer dispersions containing about 50% protein (by weight) with bovine brain diacyl phosphatidylserine (PS). ³¹P nuclear magnetic resonance shift anisotropies, spin-spin (T₂) and spin-lattice (T₁) relaxation times for the lipid headgroup phosphorus were measured at 36.44 MHz. At pH 7.5, lysozyme, cytochrome c, poly(l-lysine) and ribonuclease were shown to increase the chemical shift anisotropy of PS by between 12–20%. Myelin basic protein altered the shape of the phosphate resonance, suggesting the presence of two lipid components, one of which had a modified headgroup conformation. The presence of cytochrome c led to the formation of a narrow spike at the isotropic shift position of the spectrum. Of the various proteins or peptides we have studied, only poly(l-lysine) and cytochrome c had any effect on the T₁ of PS (1050 ms). Both caused a 20–30% decrease in T₁ of the lamellar-phase phosphate peak. The narrow peak in the presence of cytochrome c had a very short T₁ of 156 ms. The possibility is considered that the cytochrome Fe³⁺ contributes to the phosphate relaxation in this case. The effect of all proteins on the T₂ of the phosphorus resonance was to cause an increase from the value for pure PS (1.6 ms) to between 2 and 5 ms. The results obtained with proteins are compared with the effects of small ions and intrinsic membrane proteins on the order and motion of the headgroups of lipids in bilayers.     

Dynamics in a protein-lipid complex: nuclear magnetic resonance measurements on the headgroup of cardiolipin when bound to cytochrome c.

Deuterium and phosphorus nuclear magnetic resonance (NMR) has been used to investigate the dynamics of slow motional processes induced in bilayer cardiolipin upon binding with cytochrome c. 31P NMR line shapes suggest that protein binding induces less restricted, isotropic-like motions in the lipid phosphates within the ms time scale of this measurement. However, these motions impart rapid transverse relaxation to methylene deuterons adjacent to the phosphate in the lipid headgroup and so did not feature strongly in the NMR line shapes recorded from these nuclei by using the quadrupolar echo. Nonetheless, motional characteristics of the headgroup deuterons were accessible to a dynamic NMR approach using the Carr-Purcell-Meiboom-Gill multiple-pulse experiment. Compared to the well-studied case of deuterons in fatty acyl chains of bilayer phosphatidylcholine, the motions determining the 2H spin transverse relaxation in the headgroup of bilayer cardiolipin were much faster, having a lower limit in the 5-10 kHz range. On binding with cytochrome c, the T2 effecting motions in the cardiolipin headgroup became faster still, with rates comparable to the residual quadrupolar coupling frequency of the headgroup deuterons (approximately 25 kHz) and so coincided with the time scale for recording the quadrupolar echo (approximately 40 microseconds). It is concluded that the headgroup of cardiolipin does not exclusively report localized dynamic information but is particularly sensitive to collective motions occurring throughout the bilayer molecules. Although the rates of collective modes of motion may be dependent on the lipid type in pure lipid bilayers, these low-frequency fluctuations appear to occupy a similar dynamic range in a variety of lipid-protein systems, including the natural membranes.     

Reversible unfolding of cytochrome c upon interaction with cardiolipin bilayers. 2. Evidence from phosphorus-31 NMR measurements.

31P NMR measurements were conducted to determine the structural and chemical environment of beef heart cardiolipin when bound to cytochrome c. 31P NMR line shapes infer that the majority of lipid remains in the bilayer state and that the average conformation of the lipid phosphate is not greatly affected by binding to the protein. An analysis of the spin-lattice (T1) relaxation times of hydrated cardiolipin as a function of temperature describes a T1 minimum at around 25 degrees C which leads to a correlation time for the phosphates in the lipid headgroup of 0.71 ns. The relaxation behavior of the protein-lipid complex was markedly different, showing a pronounced enhancement in the phosphorus spin-lattice relaxation rate. This effect of the protein increased progressively with increasing temperature, giving no indication of a minimum in T1 up to 75 degrees C. The enhancement in lipid phosphorus T1 relaxation was observed with protein in both oxidation states, being somewhat less marked for the reduced form. The characteristics of the T1 effects and the influence of the protein on other relaxation processes determined for the lipid phosphorus (spin-spin relaxation and longitudinal relaxation in the rotating frame) point to a strong paramagnetic interaction from the protein. A comparison with the relaxation behavior of samples spinning at the "magic angle" was also consistent with this mechanism. The results suggest that cytochrome c reversibly denatures on complexation with cardiolipin bilayers, such that the electronic ground state prevailing in the native structure of both oxidized and reduced protein can convert to high-spin states with greater magnetic susceptibility.     

Association of ferri- and ferro-cytochrome c with lipid multilayers: a 31P solid-state NMR study

The 31P nuclear magnetic resonance anisotropies of dispersions of diacylphosphatidic acid and diacylphosphatidylserine were slightly increased in the presence of cytochrome c: no increase was observed with cardiolipin. However, the 31P spin-lattice relaxation times (T1) for all of these lipids were reduced markedly by the protein. As similar effects were observed with ferri-cytochrome c and with the reduced protein, which is diamagnetic, we suggest that the changes in T1 reflect a reduction in the spectral density of fast motions for the lipid headgroups attendant on binding of protein, rather than paramagnetic relaxation of the phosphorus nuclear spin.     

Electrostatic interaction of poly(L-lysine) with dipalmitoylphosphatidic acid studied by X-ray diffraction

Structure of dipalmitoylphosphatidic acid (DPPA) bilayers in the presence of poly(L-lysine) is proposed from the results of X-ray diffraction obtained by a storage phosphor detector with a high resolution called an imaging plate. The small-angle X-ray diffraction pattern exhibits that DPPA/poly(L-lysine) complex forms a highly ordered multilamellar structure. The electron density profile of the DPPA/poly(L-lysine) complex draws that only one poly(L-lysine) layer is intercalated between the neighboring DPPA bilayers. The wide-angle X-ray diffraction pattern suggests that the presence of poly(L-lysine) hardly affects the nature of hydrocarbon chain packing in the DPPA bilayers. The X-ray reflection from the DPPA/poly(L-lysine) complex indicates that the poly(L-lysine) molecules adopt a beta-sheet conformation on the surface of the DPPA bilayers. The both surface areas occupied by a headgroup of the DPPA and by a lysine residue in poly(L-lysine) are estimated from the observed spacings. The number ratio of lysine residues to DPPA headgroups per unit area is greater than unity. Therefore, one DPPA headgroup interacts with more than one lysine residue electrostatically, i.e., the electric charge distributions in both the surface of a DPPA bilayer and the poly(L-lysine) beta-sheet are incommensurate.     

Selective Release of Proteins from Spirillum itersonii by Tris(hydroxymethyl)aminomethane and Ethylenediaminetetraacetate

Treatment of Spirillum itersonii with tris(hydroxymethyl)aminomethane (Tris)-ethylenediaminetetraacetate (EDTA) results in the quantitative release of alkaline phosphatase and ribonuclease into the surrounding medium. At the same time, about 90% of the total cellular soluble cytochrome c is liberated. This process occurs within 1 min of treatment at both 24 and 4 C. Release of these proteins by Tris-EDTA treatment is highly selective, since only 9% of the total cell protein is liberated, concomitantly with less than 5% ribonucleic acid, deoxyribonucleic acid, and malate dehydrogenase. Different sigmoidal curves are obtained for release of proteins as a function of EDTA concentration. The order of liberation with increasing EDTA is as follows: alkaline phosphatase, protein, soluble cytochrome c, and ribonuclease. Treatment of cells with Tris-EDTA under conditions which cause extensive loss of alkaline phosphatase, soluble cytochrome c, and ribonuclease results in cell death, with cessation of protein and ribonucleic acid synthesis. Cells treated with EDTA in phosphate buffer (in the absence of Tris) liberate a large portion of their soluble cytochrome c, but negligible amounts of alkaline phosphatase and ribonuclease. Addition of Tris to cells pretreated with phosphate-buffered EDTA releases high levels of alkaline phosphatase, but not ribonuclease. These results suggest that a common surface alteration is not solely responsible for release of periplasmic proteins. More likely, each protein of the periplasm is bound in an independent and specific manner.     

A 13C NMR spin-lattice relaxation study of the interaction of myelin proteins with lipid vesicles

Natural abundance 13C nuclear magnetic spin-lattice relaxation times have been measured for bovine brain phosphatidylserine vesicles with and without bound proteins. The relaxation times were lower than published values for the corresponding nuclei in egg phosphatidylcholine, but showed the same trend, with relaxation times increasing along the acyl chains away from the polar headgroup. These times were inversely related to the degree of saturation of the lipid. Cytochrome c caused insignificant changes in the lipid acyl chain relaxation rates but reduced the resonance intensities, in agreement with Brown and Wüthrich (Biochem. Biophys. Acta 468 (1977) 389). In contrast, the basic protein from bovine myelin did not affect the intensities but reduced the relaxation times for 13C nuclei near the bilayer centre, and for nuclei near carbon-carbon double bonds. These proteins also dramatically broadened the serine headgroup carboxyl resonance. It appears, in accord with other recent evidence, that the basic protein does penetrate the hydrophobic region of the bilayer (possibly to the centre), producing quantitatively similar changes in the relaxation rates to proteolipid protein, an integral membrane protein.     

Phosphorus-31 NMR studies of E. coli ribosomes.

Phosphorus-31 nuclear magnetic resonance spectra, relaxation times and nuclear Overhauser (NOE) enhancement have been measured for E. coli ribosomes, subunits and rRNA. NOE and T1 experiments reveal that the phosphorus relaxation in this organelle is largely dipolar in origin. Moreover these results imply the presence of internal motion within the RNA chain with a correlation time of about 3-5 x 10(-9) sec. In all cases the predominant resonance is centered at about -1.5 ppm (relative to 85% H3PO4) as expected for a phosphodiester linkage where there is a large degree of double helix. The linewidth narrows by about a factor of four when the ribosomal proteins are removed indicating a substantial immobilization of the RNA when it is assembled into the ribosome. In addition to the phosphodiester resonance, ribosomes also reveal one or two narrower resonances shifted to low field by 1-4 ppm. Based on the observation that these resonances show a pH dependent chemical shift, we assign them to phosphate monoesters i.e. terminal 3' or 5' phosphate groups. These terminal phosphates are due to short oligomers of RNA derived from the terminus of the chain.     

A 31P-NMR spin-lattice relaxation and 31P[1H] nuclear Overhauser effect study of sonicated small unilamellar phosphatidylcholine vesicles.

The motional properties of the inner and outer monolayer headgroups of egg phosphatidylcholine (PC) small unilamellar vesicles (SUV) were investigated by 31P-NMR temperature-dependent spin-lattice relaxation time constant (T1) and 31P[1H] nuclear Overhauser effect (NOE) analyses. Three different aspects of the dynamics of PC headgroups were investigated using the T1 analysis. First, differences in the dynamics of the headgroup region of both surfaces of the SUV were measured after application of a chemical shift reagent, PrCl3, to either the extra- or intravesicular volumes. Second, the ability of the T1 experiment to resolve the different motional states was evaluated in the absence of shift reagent. Third, comparison between correlation times obtained from a resonance frequency dependent 31P[1H] NOE analysis allowed a determination of the applicability of a simplified motional model to describe phosphorus dipolar relaxation. Temperature-dependent 31P-NMR T1 values obtained for the individual monolayers at 81.0 and 162.0 MHz were modelled assuming that phosphorus undergoes both a dipolar and an anisotropic chemical shielding relaxation mechanism, each being described by the same correlation time, tau. At 162.0 MHz, the position of the T1 minimum for the inner monolayer was 9 degrees higher than that of the outer region, indicating a higher level of motional restriction for the inner leaflet, in agreement with 31P[1H] NOE measurements. The 162.0 MHz T1 profile of the combined SUV monolayers exhibited a smooth minimum located at the midpoint of the monolayer minima positions, effectively masking the presence of the individual surfaces. 31P[1H] NOE results obtained at 32.3, 81.0 and 162.0 MHz did not agree with those predicted from a simple dipolar relaxation model. These results suggest a T1-temperature method can neither discriminate two or more closely related motional time scales in a heterogeneous environment (such as incorporation of protein into lipid bilayers) nor allow accurate determination of the correlation time at the position of the minimum when the dipolar relaxation rate makes a significant contribution to the overall rate.     

Cytochrome c interactions with cardiolipin in bilayers: a multinuclear magic-angle spinning NMR study.

The influence of cytochrome c binding to cardiolipin bilayers on the motional characteristics of each component has been analyzed by magic-angle spinning (MAS) NMR. Observations were made by NMR of natural abundance 31P, 13C, and 1H nuclei in the lipid as well as sites enriched with 13C in the protein. Analysis of methyl carbons enriched in ([epsilon-13CH3]methionine)cytochrome c at residues 65 and 80 reveal quite different behavior for these sites when the protein was bound at a 1:15 molar ratio with hydrated cardiolipin. Cross-polarization (CP) shows a single broad resonance downfield in the methyl region which corresponds to the spectral characteristics of methionine 65 in the solution protein when subjected to moderate thermal perturbations. These observations suggest that although methionine 65 remains motionally restricted when the protein binds to the lipid bilayers, this residue becomes less shielded and exposed to more chemically distinct environments than in the native state of the protein. In contrast to its behavior in native oxidized protein, the methionine 80 methyl could be detected following direct pi/2 pulse excitation, and this residue is assumed to be released from the axial ligand site on the heme iron to become more exposed and highly mobile in the protein-lipid complex. An analysis of the CP response for natural abundance 13C nuclei in the lipid reveals a general increase in motions with slower rates (tens of kilohertz) on binding with cytochrome c, except for sites within the region of fatty acyl chain unsaturation which appear to be selectively mobilized in the complex with protein. It is concluded that, aside from effects on the unsaturated segments, the bound protein induces new modes of slow motions in the lipid assemblies rather than restricting the overall reorientation freedom of the lipid. The strong paramagnetic effects observed previously on the relaxation of phosphorus in protein-bound lipid [Spooner, P.J.R., & Watts, A. (1991) Biochemistry 30, 3880-3885] were not extended to any carbon and proton sites observable by MAS NMR in the lipid, and this infers a specific interaction of lipid phosphate groups with the heme. However, when protein was bound to cardiolipin mixed at a 1:4 mole ratio with dioleoylphosphatidylcholine in bilayers, no direct interaction with the heme was apparent from the phosphorus NMR relaxation behavior in this component, resolved by MAS. Instead, the spectral anisotropy of cardiolipin phosphorus was determined to be reduced, indicating that, on binding with cytochrome c, the headgroup organization was perturbed in this component.(ABSTRACT TRUNCATED AT 400 WORDS)     

Poly(2-methylthio-7-deazainosinic acid)--hydrophobic stabilization of polynucleotide secondary structure by the 2-methylthio group.

Poly(2-methylthio-7-deazainosinic acid) [poly(ms2c7I)] was enzymatically synthesized by polymerization of 2-methylthio-7-deazainosine 5'-diphosphate with polynucleotide phosphorylase from Micrococcus luteus in high yield. The homopolymer shows much higher thermal stability than its parent polynucleotides poly(7-deazainosinic acid) [poly(c7I)] and poly(I). Its sigmoidal melting curve and pronounced hypochromicity imply a rigid, ordered structure. Poly(ms2c7I), like poly(2-methylthio-inosinic acid) [poly(ms2I)], does not form a complex with poly(C) because of the bulky 2-methylthio substituent. On the other hand, two poly(ms2c7I) strands form very rigid triple strands with poly(A). Different from poly(I) and poly(c7I) the homopolymer poly(ms2c7I) is very stable against cleavage by nuclease S1 and ribonuclease T2 as expected from its rigid secondary structure.     

Amphiphilic poly(L-lactide)-b-dendritic poly(L-lysine)s synthesized with a metal-free catalyst and new dendron initiators: chemical preparation and characterization

This study presents investigations on new approaches to novel biodegradable amphiphilic poly(L-lactide)-b-dendritic poly(L-lysine)s bearing well-defined structures. First, two new Boc-protected poly(L-lysine) dendron initiators G(2)OH 4 (generation = 2) and G(3)OH 6 (generation = 3) with hydroxyl end functional groups were efficiently derived from corresponding precursors 3 and 5 via methyl ester substitution with ethanolamine. Subsequently, two series of new diblock copolymers of poly(L-lactide)-b-dendritic Boc-protected poly(L-lysine)s (S1-S2, S3-S4) were prepared in chloroform through ring-opening copolymerization of poly(L-lactide)s with a metal-free catalyst of organic 4-(dimethylamino) pyridine (DMAP) in the presence of a corresponding new poly(L-lysine) dendron initiator. Further, molecular structures of the prepared new dendron initiators as well as those of poly(L-lactide)-b-dendritic Boc-protected poly(L-lysine)s bearing different dendron blocks and PLLA lengths were examined by means of nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography (GPC), mass spectrometry (ESI-MS, MALDI-FTMS), and thermal gravimetric analysis (TGA). The results demonstrated successful formation of the synthetic precursors, functional dendron initiators, and new diblock copolymers. In addition, the very narrow molecular weight distributions (PDI = 1.10-1.14) of these poly(L-lactide)-b-dendritic Boc-protected poly(L-lysine)s further indicated their well-defined molecular structures. After the efficient Boc-deprotection for the dendron amino groups with TFA/CH(2)Cl(2), new diblock poly(L-lactide)-b-dendritic poly(L-lysine)s bearing lipophilic PLLA and hydrophilic dendritic PLL were finally prepared. It was noteworthy that the MALDI-FTMS result showed that no appreciable intermolecular chain transesterification happened during the ROP of L-lactide catalyzed by the DMAP. Moreover, self-assembly of these new biodegradable amphiphilic copolymers in diverse solvents were also preliminarily studied.     

Interaction between cytochrome c and cytochrome c peroxidase: excited-state reactions of zinc- and tin-substituted derivatives

The effect of cytochrome c peroxidase (CCP) and apoCCP on the fluorescence and phosphorescence of Zn and Sn cytochrome c (cyt c) and the effect of cyt c on the fluorescence and phosphorescence of Zn CCP were examined. We found the following: The fluorescence yields of Zn and Sn cyt c were quenched by about 20% by CCP, consistent with energy transfer between the two chromophores with a separation of about 1.8 nm. The phosphorescence spectrum of Zn cyt c (but not Sn cyt c) shifts by 20 nm to the blue upon complexation with either CCP or apoCCP; at the same time the phosphorescence lifetime of Zn cyt c decreases from 12 +/- 2 to 6 ms with apoCCP addition. Zn CCP phosphorescence decay increases from 8.3 to 9.1 ms upon addition of poly(L-lysine) used to mimic cyt c. It is concluded from these results that binding of the redox partner or an analogue to Zn CCP and Zn cyt c results in a conformational change. The respective phosphorescence lifetimes of Zn and Sn cyt c were 13 and 3 ms in the absence of CCP and 1.6 and 1.1 ms in the presence of CCP; this corresponds to a quenching rate due to CCP of 519 and 570 s-1, for Zn and Sn cyt c, respectively. The phosphorescence of Zn CCP is also affected by native cyt c but is dramatically less than the complementary pair; the quenching rate constant is 17 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transient protein interactions studied by NMR spectroscopy: the case of cytochrome C and adrenodoxin

The interaction between yeast iso-1-cytochrome c (C102T) and two forms of bovine adrenodoxin, the wild type and a truncated form comprising residues 4-108, has been investigated using a combination of one- and two-dimensional heteronuclear NMR spectroscopy. Chemical shift perturbations and line broadening of amide resonances in the [(15)N,(1)H]HSQC spectrum for both (15)N-labeled cytochrome c and adrenodoxin in the presence of the unlabeled partner protein indicate the formation of a transient complex, with a K(a) of (4 +/- 1) x 10(4) M(-)(1) and a lifetime of <3 ms. The perturbed residues map over a large surface area for both proteins. For cytochrome c, the dominating effects are located around the exposed heme edge but with other areas also affected upon formation of the complex. In the case of adrenodoxin, effects are seen in both the recognition and core domains, with the largest perturbations in the recognition domain. These results indicate that the complex has a dynamic nature, with delocalized binding of cytochrome c on adrenodoxin. A comparison with other transient complexes of redox proteins places this complex between well-defined complexes such as the cytochrome c-cytochrome c peroxidase complex and entirely dynamic complexes such as the cytochrome b(5)-myoglobin complex.     

Cytochrome function in the cyclic electron transport pathway of chloroplasts.

Flash excitation of isolated intact chloroplasts promoted absorbance transients corresponding to the electrochromic effect (P-518) and the alpha-bands of cytochrome b6 and cytochrome f. Under conditions supporting coupled cyclic electron flow, the oxidation of cytochrome b6 and the reduction of cytochrome f had relaxation half-times of 15 and 17 ms, respectively. Optimal poising of cyclic electron flow, achieved by addition of 0.1 microM 3-(3,4-dichlorophenyl)-1,1-dimethylurea, increased phosphorylation of endogenous ADP and prolonged these relaxation times. The presence of NH4Cl, or monensin plus NaCl, decreased the half-times for cytochrome relaxation to approximately 2 ms. Uncouplers also revealed the presence of a slow rise component in the electrochromic absorption shift with formation half-time of about 2 ms. Ths inhibitors of cyclic phosphorylation antimycin and 2,5-dibromo-3-methyl-6-isoprophy-p-benzoquinone abolished the slow rise in the electrochromic shift and prolonged the uncoupled relaxation times of cytochromes b6 and f by factors of ten or more. These observations indicate that cytochrome b6, plastoquinone and cytochrome f participated in a coupled electron transport process responsible for cyclic phosphorylation in intact chloroplasts. Estimations of cyclic phosphorylation rates from 40 to 120 mumol ATP/mg chlorophyll per h suggest that this process can provide a substantial fraction of the ATP needed for CO2 fixation.     

Phosphorylation/dephosphorylation of the beta light chain of clathrin from rat liver coated vesicles

The phosphorylation in vitro, on serine residues by endogenous casein kinase 2, of the clathrin beta light chain (33 kDa) of rat liver coated vesicles requires the presence of poly(L-lysine) which acts through binding to the beta light chain. The phosphorylation of other proteins is also increased in the presence of poly(L-lysine) and casein kinase 2. In contrast, the phosphorylation of the upper band of the 50-kDa protein doublet from rat liver coated vesicles is inhibited. Rat liver coated vesicles display a protein phosphatase activity which preferentially dephosphorylates clathrin beta light chain. This activity is different from the protein phosphatase which dephosphorylates the 50-kDa protein. This enzyme seems to be unrelated to the ATP/Mg-dependent protein phosphatase, or the polycation-stimulated protein phosphatases, which dephosphorylate the 50-kDa protein and beta light chain very efficiently, but with a different specificity. After dissociation of coated vesicles the beta-light-chain phosphatase activity is recovered in the membrane fraction. This phosphatase activity is inhibited by 50 microM orthovanadate and 5 mM p-nitrophenyl phosphate but not by 10 mM EDTA.     

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Relaxation behavior.

The aromatic regions in proton-decoupled natural abundance 13C Fourier transform nuclear magnetic resonance spectra (at 14.2 kG) of small native proteins contain broad methine carbon bands and narrow nonprotonated carbon resonances. Some factors that affect the use of natural abundance 13C Fourier transform NMR spectroscopy for monitoring individual nonprotonated aromatic carbon sites of native proteins in solution are discussed. The effect of protein size is evaluated by comparing the 13C NMR spectra of horse heart ferrocytochrome c, hen egg white lysozyme, horse carbon monoxide myoglobin, and human adult carbon monoxide hemoglobin. Numerous single carbon resonances are observed in the aromatic regions of 13C NMR spectra of cytochrome c, lysozyme, and myoglobin. The much larger hemoglobin yields few resolved individual carbon resonances. Theoretical and some experimental values are presented for the natural linewidths (W), spin-lattice relaxation times (T1), and nuclear Overhauser enhancements (NOE) of nonprotonated aromatic carbons and Czeta of arginine residues. In general, the 13C-1H dipolar mechanism dominates the relaxation of these carbons. 13C-14N dipolar relaxation contributes significantly to 1/T1 of C epsilon2 of tryptophan residues and Czeta of arginine residues of proteins in D2O. The NOE of each nonprotonated aromatic carbon is within experimental error of the calculated value of about 1.2. As a result, integrated intensities can be used for making a carbon count. Theoretical results are presented for the effect of internal rotation on W, T1, and the NOE. A comparison with the experimental T1 and NOE values indicates that if there is internal rotation of aromatic amino acid side chains, it is not fast relative to the over-all rotational motion of the protein.     

Conformational study of poly(L-lysine) interacting with acidic phospholipid vesicles

Circular dichroism measurements were carried out on poly(L-lysine) in the presence of vesicles of the negatively charged phospholipids, phosphatidylserine (PS; from bovine brain), phosphatidic acid (PA; prepared from egg yolk lecithin) and dimyristoylphosphatidylglycerol (DMPG). PS vesicles induced a conformational change in poly(L-lysine) from random coil to alpha-helix structure in 5 mM Tes (pH 7.0), whereas PA vesicles gave rise to beta-structure in the same buffer. The fraction of alpha-helix, F alpha (or beta-structure, F beta), increased with increasing PS (or PA) concentration, reaching a saturation value of about 0.7 (or about 1). Mixed vesicles comprising PS and dilauroylphosphatidylcholine (DLPC) also induced alpha-helix conformation, however, the saturation value of F alpha diminished with decreasing PS content in mixed vesicles. On the other hand, the spectral patterns for poly(L-lysine) in DMPG vesicle suspensions exhibited the coexistence of alpha-helix and beta-structure. Both F alpha and F beta increased with DMPG concentration and reached saturation values of about 0.5. Mixed vesicles composed of DMPG and dimyristoylphosphatidylcholine (DMPC) led to a reduction in F beta, while F alpha remained almost constant. The diversity in ordered structure induced by different phospholipid vesicles suggests the participation of lipid head groups in determining the secondary structure of poly(L-lysine) adsorbed on the vesicular surface.     

Freezing of phosphocholine headgroup in fully hydrated sphingomyelin bilayers and its effect on the dynamics of nonfreezable water at subzero temperatures.

Differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) spectroscopy are applied to characterize the nonfreezable water molecules in fully hydrated D2O/sphingomyelin at temperatures below 0 degrees C. Upon cooling, DSC thermogram displays two thermal transitions peaked at -11 and -34 degrees C. The high-temperature exothermic transition corresponds to the freezing of the bulk D2O, and the low-temperature transition, which has not previously been reported, can be ascribed to the freezing of the phosphocholine headgroup in the lipid bilayer. The dynamics of nonfreezable water are also studied by 2H NMR T1 (spin-lattice relaxation time) and T2e (spin-spin relaxation time obtained by two pulse echo) measurements at 30.7 MHz and at temperatures down to -110 degrees C. The temperature dependence of the T1 relaxation time is characterized by a distinct minimum value of 2.1 +/- 0.1 ms at -30 degrees C. T2e is discontinuous at temperature around -70 degrees C, indicating another freezing-like event for the bound water at this temperature. Analysis of the relaxation data suggest that nonfreezable water undergoes both fast and slow motions at characteristic NMR time scales. The slow motions are affected when the lipid headgroup freezes.     

The mitochondrial precursor protein apocytochrome c strongly influences the order of the headgroup and acyl chains of phosphatidylserine dispersions. A 2H and 31P NMR study

Deuterium and phosphorus nuclear magnetic resonance techniques were used to study the interaction of the mitochondrial precursor protein apocytochrome c with headgroup-deuterated (dioleoylphosphatidyl-L-[2-2H1]serine) and acyl chain deuterated (1,2-[11,11-2H2]dioleoylphosphatidylserine) dispersions. Binding of the protein to dioleoylphosphatidylserine liposomes results in phosphorus nuclear magnetic resonance spectra typical of phospholipids undergoing fast axial rotation in extended liquid-crystalline bilayers with a reduced residual chemical shift anisotropy and an increased line width. 2H NMR spectra on headgroup-deuterated dioleoylphosphatidylserine dispersions showed a decrease in quadrupolar splitting and a broadening of the signal on interaction with apocytochrome c. Addition of increasing amounts of apocytochrome c to the acyl chain deuterated dioleoylphosphatidylserine dispersions results in the gradual appearance of a second component in the spectra with a 44% reduced quadrupolar splitting. Such large reduction of the quadrupolar splitting has never been observed for any protein studied yet. The lipid structures corresponding to these two components could be separated by sucrose gradient centrifugation, demonstrating the existence of two macroscopic phases. In mixtures of phosphatidylserine and phosphatidylcholine similar effects are observed. The induction of a new spectral component with a well-defined reduced quadrupolar splitting seems to be confined to the N-terminus since addition of a small hydrophilic amino-terminal peptide (residues 1-38) also induces a second component with a strongly reduced quadrupolar splitting. A chemically synthesized peptide corresponding to amino acid residues 2-17 of the presequence of the mitochondrial protein cytochrome oxidase subunit IV also has a large perturbing effect on the order of the acyl chains, indicating that the observed effects may be a property shared by many mitochondrial precursor proteins. In contrast, binding of the mature protein, cytochrome c, to acyl chain deuterated phosphatidylserine dispersions has no effect on the deuterium and phosphorus nuclear magnetic resonance spectra, thereby demonstrating precursor-specific perturbation of the phospholipid order. The inability of holocytochrome c to perturb the phospholipid order is due to folding of this protein, since unfolding of cytochrome c by heat or urea treatment results in similar effects on dioleoylphosphatidylserine bilayers, as observed for the unfolded precursor. Implications of these data for the import of apocytochrome c into mitochondria will be discussed.