Unusual properties of plastocyanin from the fern Dryopteris crassirhizoma.

The effect of pH on the (1)H NMR spectrum, reduction potential, and self-exchange rate constant of the novel plastocyanin (PCu) from the fern plant Dryopteris crassirhizoma has been studied. The results are compared with those for the higher-plant PCu from parsley. In the (1)H NMR spectrum of D. crassirhizoma PCu(I), there is no sign that either of the His ligands is protonated at pH* down to 5.4. The reduction potentials of D. crassirhizoma and parsley PCu are 382 and 379 mV, respectively, at pH 7.4. When the pH value is decreased, the reduction potential of parsley PCu is seen to increase quite dramatically, consistent with protonation at His87 in PCu(I). A pK(a) of 5.8 is obtained from the electrochemistry data, consistent with a value of 5.6 determined by NMR. The reduction potential of D. crassirhizoma PCu exhibits a much less pronounced dependence on pH. The self-exchange rate constant of D. crassirhizomaPCu(I) is 3.4 x 10(3) M(-1) s(-1) at pH* 7.9. This is the smallest self-exchange rate constant reported to date for a PCu and can be rationalized by considering the altered distribution of charged residues on the surface of the D. crassirhizoma protein compared to the charge distributions of other higher-plant PCus. The self-exchange rate constant increases to 9 x 10(3) M(-1) s(-1) at pH* 5.4, consistent with enhanced protein-protein association at lower pH*, and the absence of His87 protonation in D. crassirhizoma PCu(I) in the accessible pH range.     

Characterization of the Site‐Specific Acid‐Base Equilibria of 3‐Nitrotyrosine

The complete macro‐ and microequilibrium analyses of 3‐nitrotyrosine, a biomarker of oxidative stress damage, are presented for the first time. The protonation macroconstants were determined by ¹H‐NMR‐pH titration, while microconstants were elucidated by a combination of deductive and NMR methods, in which properties of the methyl ester derivative as an auxiliary compound were also studied. Combination of the NMR‐pH characterization of the title and auxiliary compounds and the pair‐interactivity parameters of 3‐iodotyrosine provided the sufficient system to evaluate all the microconstants. NMR‐pH profiles, macroscopic and microscopic protonation schemes, and species‐specific distribution diagrams are included. The phenolate basicity of 3‐nitrotyrosine is 500 times below that of tyrosine, and it is even lower than that of 3‐iodotyrosine. This phenomenon can be explained by the stronger electron withdrawing and the negative mesomeric effect of the nitro group. Based on our results, 89 % of the phenolic OH groups are deprotonated in 3‐NT molecules at the pH of the blood plasma.     

Electrostatic interactions in the acid denaturation of alpha-lactalbumin determined by NMR.

alpha-Lactalbumin (alpha-LA) undergoes a pH-dependent unfolding from the native state to a partially unfolded state (the molten globule state). To understand the role of electrostatic interactions in protein denaturation, NMR and CD pH titration experiments are performed on guinea pig alpha-LA. Variation of pH over the range of 7.0 to 2.0 simultaneously leads to the acid denaturation of the protein and the titration of individual ionizable groups. The pH titrations are interpreted in the context of these coupled events, and indicate that acid denaturation in alpha-LA is a cooperative event that is triggered by the protonation of two ionizable residues. Our NMR results suggest that the critical electrostatic interactions that contribute to the denaturation of alpha-LA are concentrated in the calcium binding region of the protein.     

NMR studies and semi-empirical energy calculations for cyclic ADP-ribose

A possible pH-dependent conformational switch was investigated for cyclic ADP-ribose. NMR signals for the exchangeable protons were observed in H2O at low temperature, but there was no direct evidence for the protonation of N-3 at neutral pH that has previously been postulated. MNDO calculations indicated that pH dependent 31P chemical shift changes are attributable to protonation of the phosphate adjacent to the N-1 of adenine, and not due to trans-annular hydrogen bonding with a protonated N-3.     

Inframolecular studies of the protonation of adenophostin A: comparison with 1-D-myo-inositol 1,4,5-trisphosphate

Adenophostin A is a glyconucleotide natural product with the highest known potency for the D-myo-inositol 1,4,5-trisphosphate receptor. Using synthetic adenophostin A we have investigated the macroscopic and microscopic protonation process of this compound by performing (31)P NMR, (1)H NMR, and potentiometric titration experiments. The logarithms of the first to the fourth stepwise protonation constants are, respectively, log K(1) = 8.48, log K(2) = 6.20, log K(3) = 4.96, and log K(4) = 3.80. The latter constant refers to the protonation equilibrium involving the N1 adenine nitrogen. From the microconstants the protonation fractions of each individual phosphate group can be calculated. Remarkably, the ionization state of the phosphates of adenophostin A at near physiological pH is very similar to those of inositol 1,4,5-trisphosphate, indicating that differences in phosphate charge cannot account for the high potency of this molecule. The analysis of the (1)H chemical shifts vs pH provided complementary conformational information. In particular, a slight "wrongway shift" of H1" can be related to the protonation of P2, thus indicating a short H1"-P2 distance. Our results are in line with a recently published model in which, however, a certain degree of constraint would keep the ribose 2'-phosphate moiety close to the glucose ring phosphates.     

Asymmetric protonation of EmrE

The small multidrug resistance transporter EmrE is a homodimer that uses energy provided by the proton motive force to drive the efflux of drug substrates. The pKa values of its “active-site” residues—glutamate 14 (Glu14) from each subunit—must be poised around physiological pH values to efficiently couple proton import to drug export in vivo. To assess the protonation of EmrE, pH titrations were conducted with ¹H-¹⁵N TROSY-HSQC nuclear magnetic resonance (NMR) spectra. Analysis of these spectra indicates that the Glu14 residues have asymmetric pKa values of 7.0 ± 0.1 and 8.2 ± 0.3 at 45°C and 6.8 ± 0.1 and 8.5 ± 0.2 at 25°C. These pKa values are substantially increased compared with typical pKa values for solvent-exposed glutamates but are within the range of published Glu14 pKa values inferred from the pH dependence of substrate binding and transport assays. The active-site mutant, E14D-EmrE, has pKa values below the physiological pH range, consistent with its impaired transport activity. The NMR spectra demonstrate that the protonation states of the active-site Glu14 residues determine both the global structure and the rate of conformational exchange between inward- and outward-facing EmrE. Thus, the pKa values of the asymmetric active-site Glu14 residues are key for proper coupling of proton import to multidrug efflux. However, the results raise new questions regarding the coupling mechanism because they show that EmrE exists in a mixture of protonation states near neutral pH and can interconvert between inward- and outward-facing forms in multiple different protonation states.     

The pH-dependent structural variation of complementarity-determining region H3 in the crystal structures of the Fv fragment from an anti-dansyl monoclonal antibody.

The Fv fragment from an anti-dansyl antibody was optimally crystallized into two crystal forms having slightly different lattice dimensions at pH 5.25 and 6.75. The two crystal structures were determined and refined at high resolution at 112 K (at 1.45 A for the crystal at pH 5.25 and at 1.55 A for that at pH 6.75). In the two crystal structures, marked differences were identified in the first half of CDRH3 s having an amino acid sequence of Ile95H-Tyr96H-Tyr97H-His98H-Tyr99H-Pro1 00H-Trp100aH-Phe100bH-Ala101H- Tyr102H. NMR pH titration experiments revealed the p Kavalues of four histidine residues (His27dL, His93L, His55H and His98H) exposed to solvent. Only His98H (p Ka=6.3) completely changed its protonation state between the two crystallization conditions. In addition, the environmental structures including hydration water molecules around the four histidine residues were carefully compared. While the hydration structures around His27dL, His93L and His55H were almost invariant between the two crystal structures, those around His98Hs showed great difference in spite of the small conformational difference of His98H between the two crystal structures. These spectroscopic and crystallographic findings suggested that the change in the protonation state in His98H was responsible for the structural differences between pH 5.25 and 6.75. In addition, the most plausible binding site of the dansyl group was mapped into the present structural models with our previous NMR experimental results. The complementarity-determining regions H1, H3 and the N-terminal region in the VH domain formed the site. The side-chain of Tyr96H occupied the site and interacted with Phe27H of H1, giving a clue for the binding mode of the dansyl group in the site.     

Silybin, a new iron-chelating agent.

Silybin, a natural occurring flavolignan isolated from the fruits of Silibum marianum, has been reported to exert antioxidant and free radical scavenging abilities. It was suggested to act also as an iron chelator. The complexation and protonation equilibria of the ferric complex of this compound have been studied by potentiometric, spectrophotometric and electrochemical techniques. The formation of the complex silybin-Ga(III) in anhydrous DMSO-d6 has been studied by 1H NMR spectroscopy. Mass spectrometry and infrared spectroscopy on silybin-Fe(III) complex confirm all data obtained by 1H NMR spectroscopy. The experimental results show that silybin binds Fe(III) even at acidic pH. Different ternary complexes were observed at increasing methoxide ion concentration and their stability constants have been calculated. The results show the possible role of silybin in relation to the chelation therapy of chronic iron overload, as occurs in the treatment of Cooley's anemia.     

Structure and dynamics of the influenza A M2 Channel: a comparison of three structures

The M2 protein is an essential component of the Influenza virus’ infectivity cycle. It is a homo-tetrameric bundle forming a pH-gated H⁺ channel. The structure of M2 was solved by three different groups, using different techniques, protein sequences and pH environment. For example, solid-state NMR spectroscopy was used on a protein in lipid bilayers, while X-ray crystallography and solution NMR spectroscopy were applied on a protein in detergent micelles. The resulting structures from the above efforts are rather distinct. Herein, we examine the different structures under uniform conditions such as a lipid bilayer and specified protonation state. We employ extensive molecular dynamics simulations, in several protonation states, representing both closed and open forms of the channel. Exploring the properties of each of these structures has shown that the X-ray structure is more stable than the other structures according to various criteria, although its water conductance and water-wire formation do not correlate to the protonation state of the channel.     

Bayesian estimation of the number of protonation sites for urinary metabolites from NMR spectroscopic data

Introduction

To aid the development of better algorithms for \(^1\)H NMR data analysis, such as alignment or peak-fitting, it is important to characterise and model chemical shift changes caused by variation in pH. The number of protonation sites, a key parameter in the theoretical relationship between pH and chemical shift, is traditionally estimated from the molecular structure, which is often unknown in untargeted metabolomics applications.

Objective

We aim to use observed NMR chemical shift titration data to estimate the number of protonation sites for a range of urinary metabolites.

Methods

A pool of urine from healthy subjects was titrated in the range pH 2–12, standard \(^1\)H NMR spectra were acquired and positions of 51 peaks (corresponding to 32 identified metabolites) were recorded. A theoretical model of chemical shift was fit to the data using a Bayesian statistical framework, using model selection procedures in a Markov Chain Monte Carlo algorithm to estimate the number of protonation sites for each molecule.

Results

The estimated number of protonation sites was found to be correct for 41 out of 51 peaks. In some cases, the number of sites was incorrectly estimated, due to very close pKa values or a limited amount of data in the required pH range.

Conclusions

Given appropriate data, it is possible to estimate the number of protonation sites for many metabolites typically observed in \(^1\)H NMR metabolomics without knowledge of the molecular structure. This approach may be a valuable resource for the development of future automated metabolite alignment, annotation and peak fitting algorithms.

     

Membrane fusion activity of succinylated melittin is triggered by protonation of its carboxyl groups

The membrane fusion activity of melittin and its succinylated derivative was studied as a function of pH by the transfer of spin-labeled phosphatidylcholine as well as by internal content mixing and electron microscopy. The protonation process of the carboxyl groups introduced into melittin was studied by 13C NMR spectroscopy using derivative prepared with [1,4(-13)C]succinic anhydride. Melittin causes fusion of sonicated phosphatidylcholine vesicles in a wide range of pH. In marked contrast, melittin with all four amino groups succinylated induces fusion only at acidic pH lower than 5.2, with the maximum at pH 5.1. The fusion reactions are very rapid, reaching a saturation level within 1 min. The fusion efficiency depends on the peptide-to-phospholipid ratio in the reaction mixture. Trypsinized succinylated melittin, which has lost the four positively charged C-terminal residues, causes aggregation of vesicles at acidic pH but cannot induce fusion. The 13C NMR peaks for the carboxyl and carbonyl groups of succinylated melittin shifted to higher field as the pH was lowered. The pKa value of the four carboxyl groups was obtained as 5.19 and 4.83 in the presence and absence of vesicles, respectively. The pKa value in the presence of vesicles agrees quite well with the half-maximal pH for fusion of 5.15, indicating that the fusion activity is triggered by protonation of the carboxyl groups in the hydrophobic segment of the peptide. The higher shift of pKa value in the presence of vesicles can be due to stabilization of the protonated form by entrance into lipid bilayer hydrocarbon layer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spermidine oxidase in human pregnancy serum. Probable identity with diamine oxidase.

Diamine oxidase was previously measured in human pregnancy serum with putrescine or histamine as substrate. We have now documented the presence of spermidine oxidase activity in pregnancy serum by means of a specific radioactive assay with [14C]spermidine as substrate and Dowex 50 cation-exchange chromatography to separate products from substrate. The apparent Km of a partially purified preparation of this enzyme for spermidine was 10.9 microM and the Ki for aminoguanidine was 0.8 microM. The pH optimum (pH 9.0) and temperature optimum (55 degrees C) were identical with those for diamine oxidase. Spermidine oxidase activity and diamine oxidase activity eluted in a concerted fashion when pregnancy serum was subjected to cadaverine-Sepharose chromatography, gel filtration and ion-exchange chromatography. Spermidine oxidase became detectable in serum during pregnancy in the human approx. 8 weeks after the last menstrual period and increased with gestational age in concert with the increase in diamine oxidase activity, reaching a plateau at 20 weeks of gestation. Foetal-cord serum displayed virtually no activity of either enzyme. A 400-fold-purified preparation of diamine oxidase retained the same diamine oxidase/spermidine oxidase ratio as exhibited by crude pregnancy serum. These data suggest that in pregnancy serum, unlike foetal bovine serum, spermidine oxidase and diamine oxidase activity may be a single enzyme protein.     

Proton nuclear magnetic resonance study of the effect of pH on tRNA structure.

The low-field 220-MHz proton nuclear magnetic resonance (NMR) spectra of four tRNA molecules, Escherichia coli tRNAPhe, tRNA1Val, and tRNAfMet1, and yeast tRNAPhe, at neutral and mildly acidic pH are compared. We find a net increase in the number of resonances contributing to the -9.9-ppm peak (downfield from sodium 4,4-dimethyl-4-silapentanesulfonate) in three of these tRNAs at pH 6, while tRNAfMet1 does not clearly exhibit this behavior. The increase in intensity at this resonance position is half-completed at pH 6.2 in the case of yeast tRNAPhe. An alteration at the 5'-phosphate terminus is not involved, since removal of the terminal phosphate does not affect the gain in intensity at -9.9 ppm. Based on a survey of the tertiary interactions in the four molecules, assuming that they possess tertiary structures like that of yeast tRNAPhe at neutral pH, we tentatively attribute this altered resonance in E. coli and yeast tRNAPhe to the protonation of the N3 of the adenine residue at position 9 which results in the stabilization of the tertiary triple A23-U12-A9. This intepretation is supported by model studies on the lowfield proton NMR spectrum of AN oligomers at acid pH, which reveal an exchanging proton resonance at -9.4 ppm if the chain length N greater than or equal to 6.     

Protein mobility and self-association by deuterium nuclear magnetic resonance.

Hen egg white lysozyme has been prepared in which the C epsilon position of the single histidine residue is substituted by a deuterium atom as a nondisturbing stable isotope probe. The deuterium nuclear magnetic resonance (2H NMR) spectrum in H2O shows a broad resonance (500--1000 Hz) due to the histidine deuteron and a sharp signal from residual HOD. The line width of the deuterium signal increases with pH, reflecting the self-association of lysozyme which is known to involve this histidine [shindo, H., Cohen, J.S., & Rupley, J. A. (1977) Biochemistry 16, 3879]. Correlation times calculated from spin-spin relaxation times (T2) derived from the 2H widths indicate that His-15 is restricted in motion and that lysozyme is predominantly dimerized at pH 7.5. Controls carried out with [epsilon-2H]imidazole showed a small pH dependence of the spin-lattice relaxation time (T1), which parallels the 2H chemical shift change upon ionization of the imidazole. Similar results cannot generally be observed by proton nuclear magnetic resonance (1H NMR) because of paramagnetic relaxation due to trace metal ion impurities. The pH dependence of the 2H T1 values indicates a change in the 2H quadrupole coupling constant upon protonation of the imidazole ring.     

Assessing the acid-base and conformational properties of histidine residues in human prion protein (125-228) by means of pK(a) calculations and molecular dynamics simulations

A thorough study of the acid-base behavior of the four histidines and the other titratable residues of the structured domain of human prion protein (125-228) is presented. By using multi-tautomer electrostatic calculations, average titration curves have been built for all titratable residues, using the whole bundles of NMR structures determined at pH 4.5 and 7.0. According to our results, (1) only histidine residues are likely to be involved in the first steps of the pH-driven conformational transition of prion protein; (2) the pK(a)'s of His140 and His177 are approximately 7.0, whereas those of His155 and His187 are < 5.5. 10-ns long molecular dynamics simulations have been performed on five different models, corresponding to the most significant combinations of histidine protonation states. A critical comparison between the available NMR structures and our computational results (1) confirms that His155 and His187 are the residues whose protonation is involved in the conformational rearrangement of huPrP in mildly acidic condition, and (2) shows how their protonation leads to the destructuration of the C-terminal part of HB and to the loss of the last turn of HA that represent the crucial microscopic steps of the rearrangement.     

Coupling of drug protonation to the specific binding of aminoglycosides to the A site of 16 S rRNA: elucidation of the number of drug amino groups involved and their identities

2-Deoxystreptamine (2-DOS) aminoglycoside antibiotics bind specifically to the central region of the 16S rRNA A site and interfere with protein synthesis. Recently, we have shown that the binding of 2-DOS aminoglycosides to an A site model RNA oligonucleotide is linked to the protonation of drug amino groups. Here, we extend these studies to define the number of amino groups involved as well as their identities. Specifically, we use pH-dependent 15N NMR spectroscopy to determine the pK(a) values of the amino groups in neomycin B, paromomycin I, and lividomycin A sulfate, with the resulting pK(a) values ranging from 6.92 to 9.51. For each drug, the 3-amino group was associated with the lowest pK(a), with this value being 6.92 in neomycin B, 7.07 in paromomycin I, and 7.24 in lividomycin A. In addition, we use buffer-dependent isothermal titration calorimetry (ITC) to determine the number of protons linked to the complexation of the three drugs with the A site model RNA oligomer at pH 5.5, 8.8, or 9.0. At pH 5.5, the binding of the three drugs to the host RNA is independent of drug protonation effects. By contrast, at pH 9.0, the RNA binding of paromomycin I and neomycin B is coupled to the uptake of 3.25 and 3.80 protons, respectively, with the RNA binding of lividomycin A at pH 8.8 being coupled to the uptake of 3.25 protons. A comparison of these values with the protonation states of the drugs predicted by our NMR-derived pK(a) values allows us to identify the specific drug amino groups whose protonation is linked to complexation with the host RNA. These determinations reveal that the binding of lividomycin A to the host RNA is coupled to the protonation of all five of its amino groups, with the RNA binding of paromomycin I and neomycin B being linked to the protonation of four and at least five amino groups, respectively. For paromomycin I, the protonation reactions involve the 1-, 3-, 2'-, and 2"'-amino groups, while, for neomycin B, the binding-linked protonation reactions involve at least the 1-, 3-, 2', 6'-, and 2"'-amino groups. Our results clearly identify drug protonation reactions as important thermodynamic participants in the specific binding of 2-DOS aminoglycosides to the A site of 16S rRNA.     

Effect of histidine 6 protonation on the active site structure and electron-transfer capabilities of pseudoazurin from Achromobacter cycloclastes

The paramagnetic (1)H NMR spectrum of Cu(II) pseudoazurin [PACu(II)] contains eight directly observed hyperfine-shifted resonances which we have assigned using saturation transfer experiments on a 1:1 mixture of PACu(I) and PACu(II). The spectrum exhibits a number of similarities to those of other cupredoxins, but differences are found concerning the Cu-S(Met) interaction. The spectrum is dependent on pH* in the range 8.5-4.5 (pK(a)* 6.4), and a conformational change involving movement of the copper ion away from the Met toward the equatorial ligands, as a consequence of protonation of the surface His6 residue, is identified. Corresponding changes are also seen in the UV/vis spectrum. The protonation/deprotonation equilibrium of His6 influences the reduction potential of the protein in the same pH range. The self-exchange rate constant of PACu at pH* 6.0 (25 degrees C) is considerably smaller (1.1 x 10(3) M(-1) s(-1)) than the value obtained at pH* 7.6 (3.7 x 10(3) M(-1) s(-1)). The effect on the self-exchange reactivity is mainly due to an alteration in the reorganization energy of the copper site brought about by the structural change resulting from His6 protonation.     

Synthesis, protonation behavior, conformational analysis, and regioselective enzymatic acylation of the novel diamino analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU)

(E)-3',5'-Diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. The protonation behavior of 5 has been studied by means of pH-metric measurements and NMR spectroscopy. This study allows the determination of the basicity constants and the stepwise protonation sites. Thus, the main species at physiological pH is the monoprotonated form. The conformational analysis of this nucleoside analogue was also carried out through 1H NMR spectroscopy. In addition, a convenient synthesis of N-3' and N-5' acylated derivatives was developed by regioselective enzymatic acylation. Thus, Candida antarctica lipase B (CAL-B) selectively acylated the 5'-amino group, thus furnishing nucleosides 8. On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) exhibited the opposite selectivity, conferring acylation at the 3'-amino group, thus affording derivatives 9.     

The interactions between nucleic acids and polyamines: I. High resolution carbon-13 and hydrogen-1 nuclear magnetic resonance studies of spermidine and 5'-AMP

In 0.5 M solution at pH 7.6, interaction of spermidine and 5'-AMP is demonstrated by downfield proton NMR shifts. Shifts of ribose and adenine protons support a model in which triprotonated spermidine engages the phosphate, anion with the C-3 diamine segment in a conformation to maximize interaction and the C-4 ammo segment extended to interact with adenine N-7 (base anti, 2'endo, g'g' and gg nucleoside conformation). Changes in carbon-13 chemical shifts for ribose C-5' (downfield), C-2' C-3', and C-4' (upfield) and for adenine C-6 and C-8 (upfield) support this model. In 0.006 M solution no significant changes in proton shifts and therefore no evidence for interaction was found. Spermidine and 5'-UMP (0.006 M) showed interaction at pH 10.5 (small upfield shifts in the proton nmr) interpreted as changing conformation by solvent interaction. In 0.006 M 3'-UMP at pH 10.5 small downfield proton shifts induced by spennidine are attributed to interactions with phosphate anion.     

Acid- and calcium-induced structural changes in phosphatidylethanolamine membranes stabilized by cholesteryl hemisuccinate

The membrane stabilization effect of cholesteryl hemisuccinate (CHEMS) and the sensitivity of the CHEMS-phosphatidylethanolamine membranes to protons and calcium ions were studied by differential scanning calorimetry, freeze-fracture electron microscopy, and 31P NMR. (1) At neutral pH, the addition of 8 mol % CHEMS to transesterified egg phosphatidylethanolamine (TPE) raised the lamellar-hexagonal transition temperature of TPE by 11 degrees C. Stable bilayer vesicles were formed when the incorporated CHEMS exceeded 20 mol %. (2) At a pH below 5.5, the protonation of CHEMS enhanced the formation of the hexagonal phase (HII) of TPE. At 25 mol % CHEMS the bilayer-hexagonal transition temperature was lowered by 30 degrees C at pH 4.5. (3) The endothermic acid-induced hexagonal hexagonal transition of TPE-CHEMS was suppressed at 35 mol % CHEMS. However, 31P NMR and electron microscopy indicated that a lamellar-hexagonal transition still occurred at this composition. (4) The main transition of TPE was not affected by the protonation of the incorporated CHEMS, indicating that no macroscopic phase separation occurred in TPE-CHEMS mixtures at low pH. (5) In contrast to the HII-promoting effect of H+, the neutralization of the negative charge on TPE-CHEMS by Ca2+ resulted in aggregates that remained in the lamellar structure even at the hexagonal transition temperature of TPE. It is suggested that calcium might form a complex between CHEMS in apposed bilayers. These results are related to the possible biological function of acidic cholesterol esters in biomembranes.