Model phospholipid membranes affect the tertiary structure of holomyoglobin: Conformational changes at pH 6.2

The influence of model negatively charged membranes on the structure of sperm whale holomyoglobin at pH 6.2 has been investigated using a variety of techniques (far-UV and near-UV circular dichroism, tryptophan fluorescence, absorption spectroscopy, differential scanning microcalorimetry, and fast-performance liquid chromatography). It has been shown that, similarly to apomyoglobin, holomyoglobin in the presence of phospholipid vesicles undergoes a conformational transition from the native to the intermediate state, which is characterized by loss of the rigid tertiary structure and the native heme environment; at the same time, the content of -helical secondary structures remains virtually unchanged. The molar phospholipid/protein ratio required for this transition is higher than in the case of apomyoglobin. The properties of holomyoglobin in the presence of negatively charged membranes are largely similar to those of the molten globule state of its apo form in aqueous solution. A possible functional role of this novel non-native state of myoglobin in the cell is discussed.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 120–128.Original Russian Text Copyright © 2005 by Basova, Tiktopulo, Bychkova.     

The Conformational State of Apomyoglobin in the Presence of Phospholipid Vesicles at Neutral pH

The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near and far UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed.     

Conformational status of apomyoglobin in the presence of phospholipid vesicles at neutral pH

The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near- and far-UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed.     

Effect of phospholipids on conformational structure of bovine pancreatic trypsin inhibitor (BPTI) and its thermolabile mutants

The effects of negatively charged phosphatidylserine-prepared membranes (PS) and neutral phosphatidylcholine-prepared membranes (PC) on the structure of wild-type and mutant bovine pancreatic trypsin inhibitor (BPTI) at neutral pH were investigated. The presence of PC did not have any effect on the protein structure while PS induced a non-native structure in three mutant BPTI proteins. However, the negatively charged membrane did not have any effect on wild-type BPTI. The findings revealed that (i) elimination of some disulphide bonds results in dramatic change in protein structure, and, (ii) that this biochemical interaction is surface-driven and electrostatic interactions may play a very strong role in influencing the fore-stated changes in protein structure. Of further interest were the results obtained from investigating the possible role of PS fluidity and concentration in altering mutant. When the value of Gibbs free-energy change of unfolding (DeltaG(U)) was positive, various non-native structures were formed in a concentration-dependent manner. However, when the value of DeltaG(U) was negative, only two types of non-native structures were formed: one with high beta structure content at low PS fluidity state, and the other with a high alpha-helical content at high PS fluidity state. Our study reveals how particular combinations of phospholipid:protein interactions can induce a protein conformation transition from a native to a non-native one at neutral pH, especially when the native structure is predestabilized by amino acid substitutions. This revelation may open up opportunities to explore alternative ways in which phospholipids may play a role in protein mis-folding and the related pathologies.     

Lipids in the structure,folding, and function of the KcsA K+ channel

Lipid molecules surround an ion channel in its native environment of cellular membranes. The importance of the lipid bilayer and the role of lipid protein interactions in ion channel structure and function are not well understood. Here we demonstrate that the bacterial potassium channel KcsA binds a negatively charged lipid molecule. We have defined the potential binding site of the lipid molecule on KcsA by X-ray crystallographic analysis of a complex of KcsA with a monoclonal antibody Fab fragment. We also demonstrate that lipids are required for the in vitro refolding of the KcsA tetramer from the unfolded monomeric state. The correct refolding of the KcsA tetramer requires lipids, but it is not dependent on negatively charged lipids as refolding takes place in the absence of such lipids. We confirm that the presence of negatively charged lipids is required for ion conduction through the KcsA potassium channel, suggesting that the lipid bound to KcsA is important for ion channel function.     

Conformation-Dependent Interaction of α-Lactalbumin with Model and Biological Membranes: A Spin-Label ESR Study

Alpha-lactalbumin (alpha-LA) is biosynthesized and stored at the smooth endoplasmic reticulum (ER), then transferred to the Golgi lumen when prolactin stimulation of lactose biosynthesis and secretion takes place. Because both environments are composed of membranes, it was of interest to examine the interactions of alpha-LA with relevant model and biological membranes. Using the ESR spin-labeled fatty acid analog 5-doxyl stearic acid, we found evidence reflecting the insertion of "acid-shocked" molten globule (MG) alpha-LA into lecithin or phosphatidylserine (PS) multi-lamellar vesicles. An additional approximately 3 G immobilization was observed in the alpha-LA-lecithin sample versus the lipid alone. With PS, the increased immobilization was almost 6 G, reflecting an enhanced effect caused by strong electrostatic interactions between the positively charged protein with the negatively charged headgroup at pH 2.4. This was also reflected in the broadening of the PS:alpha-LA phase transition. Additionally, we have demonstrated that alpha-LA in its apo-form also shows similar insertion characteristics with both model and natural lipid membranes. Upon addition of calcium, the apo-form is released from the membrane as the Ca(2+)-bound protein.     

Conformational exchange of fatty acid binding protein induced by protein-nanodisc interactions

Fatty acid binding proteins (FABPs) can facilitate the transfer of long-chain fatty acids between intracellular membranes across considerable distances. The transfer process involves fatty acids, their donor membrane and acceptor membrane, and FABPs, implying that potential protein-membrane interactions exist. Despite intensive studies on FABP-membrane interactions, the interaction mode remains elusive, and the protein-membrane association and dissociation rates are inconsistent. In this study, we used nanodiscs (NDs) as mimetic membranes to investigate FABP-membrane interactions. Our NMR experiments showed that human intestinal FABP interacts weakly with both negatively charged and neutral membranes, but it prefers the negatively charged one. Through simultaneous analysis of NMR relaxation in the rotating-frame (R_1ρ), relaxation dispersion, chemical exchange saturation transfer, and dark-state exchange saturation transfer data, we estimated the affinity of the protein to negatively charged NDs, the dissociation rate, and apparent association rate. We further showed that the protein in the ND-bound state adopts a conformation different from the native structure and the second helix is very likely involved in interactions with NDs. We also found a membrane-induced FABP conformational state that exists only in the presence of NDs. This state is native-like, different from other conformational states in structure, unbound to NDs, and in dynamic equilibrium with the ND-bound state.     

Kinetics of interactions between apomyoglobin and phospholipid membrane

The interaction of apomyoglobin and its mutant forms with phospholipid membranes was studied using tryptophan fluorescence and circular dichroism in the far UV region. It is shown that a negatively charged phospholipid membrane can have a dual effect on the structure of protein molecule upon their interaction. On the one hand, the membrane induces denaturation of the protein native structure to its intermediate state, acting as a moderate denaturing agent. On the other hand, it can stabilize the structure of unfolded protein to the same intermediate state, acting as a moderate structuring agent. The kinetics of interaction between apomyoglobin and its mutant forms and the phospholipid membrane depends on the membrane surface charge. Here the interaction rate depends on the concentration of phospholipids vesicles and stability of protein molecule, which increase with a decrease in the latter. The roles of these factors in the folding of membrane proteins and the choice of the targeted delivery pathways for protein drugs are discussed.     

Conformational states of the water-soluble fragment of cytochrome b5. I. pH-induced denaturation

Cytochrome b5 is a membrane protein that comprises two fragments: one is water-soluble and heme-containing, and the other is hydrophobic and membrane-embedded. The function of electron transfer is performed by the former whose crystal structure is known; however, its conformational states when in the membrane field and interacting with other proteins are still to be studied. Previously, we proposed water-alcohol mixtures for modeling the effect of membrane surface on proteins, and used this approach to study the conformational behavior of positively charged cytochrome c as well as relatively neutral retinol-binding protein also functioning in the field of negatively charged membrane. The current study describes the conformational behavior of the negatively charged water-soluble fragment of cytochrome b5 as dependent on pH. Decreasing pH was shown to transform the fragment state from native to intermediate, similar to the molten globule reported earlier for other proteins in aqueous solutions: at pH 3.0, the fragment preserved a pronounced secondary structure and compactness but lost its rigid tertiary structure. A possible role of this intermediate state in cytochrome b5 functioning is discussed.     

Behaviour of bovine phosphatidylethanolamine-binding protein with model membranes. Evidence of affinity for negatively charged membranes.

The ability of phosphatidylethanolamine-binding protein (PEBP) to bind membranes was tested by using small and large unilamellar vesicles and monolayers composed of l-alpha-1,2-dimyristoylphosphatidylcholine, l-alpha-1,2-dimyristoylphosphatidylglycerol and l-alpha-1,2-dimyristoylphosphatidylethanolamine. PEBP only bound to model membranes containing l-alpha-1,2-dimyristoylphosphatidylglycerol; the interaction was primarily due to electrostatic forces between the basic protein and the acidic phospholipids. Further experiments indicated that the interaction was not dependent on the length and unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane. PEBP affinity for negatively charged membranes is puzzling considering the previous identification of the protein as a phosphatidylethanolamine-binding protein, and suggests that the association of PEBP with phospholipid membranes is driven by a mechanism other than its binding to solubilized phosphatidylethanolamine. An explanation was suggested by its three-dimensional structure: a small cavity at the protein surface has been reported to be the binding site of the polar head of phosphatidylethanolamine, while the N-terminal and C-terminal parts of PEBP, exposed at the protein surface, appear to be involved in the interaction with membranes. To test this hypothesis, we synthesized the two PEBP terminal regions and tested them with model membranes in parallel with the whole protein. Both peptides displayed the same behaviour as whole PEBP, indicating that they could participate in the binding of PEBP to membranes. Our results strongly suggest that PEBP directly interacts with negatively charged membrane microdomains in living cells.     

Molten globule-like state of cytochrome c induced by polyanion poly(vinylsulfate) in slightly acidic pH.

The effect of polyanion, poly(vinylsulfate), used as a model of negatively charged surface, on ferric cytochrome c (ferricyt c) structure in acidic pH has been studied by absorbance spectroscopy, circular dichroism (CD), tryptophan (Trp) fluorescence and microcalorimetry. The polyanion induced only small changes in the native structure of the protein at neutral pH, but it profoundly shifted the acid induced high spin state of the heme in the active center of cyt c to a more neutral pH region. Cooperativity of the acidic transition of ferricyt c in the presence of the polyanion was disturbed, in comparison with uncomplexed protein, as followed from different apparent pK(a) values observed in a distinct regions of the ferricyt c electronic absorbance spectrum (4.55+/-0.08 in the 620 nm band region and 5.47+/-0.15 in the Soret region). The ferricyt c structure in the complex with the polyanion at acidic pH (below pH 5.0) has properties of a molten globule-like state. Its tertiary structure is strongly disturbed according to CD and microcalorimetry measurements; however, its secondary structure, from CD, is still native-like and ferricyt c is in a compact state as evidenced by quenched Trp fluorescence. These findings are discussed in the context of the molten globule state of proteins induced on a negatively charged membrane surface under physiological conditions.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b5 has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b5-vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b5 upon the interaction with its redox partners is discussed.     

The interaction of the Bax C-terminal domain with negatively charged lipids modifies the secondary structure and changes its way of insertion into membranes

Fourier transform infrared spectroscopy (FTIR) was used to study the secondary structure of peptides which imitate the amino acid sequences of the C-terminal domain of the pro-apoptotic protein Bax (Bax-C) when incorporated into different lipid vesicles with or without negatively charged phospholipids. The infrared spectroscopy results showed that while the beta-sheet components are predominant in the membrane-free Bax-C secondary structure as well as in the presence of phosphatidylcholine vesicles, the peptide changes its secondary structure in the presence of negatively charged membranes, including phospholipids such as phosphatidylglycerol or phosphatidylinositol, depending on both the lipid composition and their molar ratio. The negative charges in the model membrane surface caused a marked change from beta-sheet to alpha-helix structure. Moreover, using attenuated total reflection infrared spectroscopy (ATR-FTIR), we investigated the orientation of Bax-C alpha-helical structures with respect to the normal to the internal reflection element. The orientation of Bax-C in membranes was also affected by negatively charged lipids, the presence of phosphatidylglycerol reduced the angle it forms with the normal to the germanium plate from 45 degrees in phosphatidylcholine to 27 degrees in phosphatidylglycerol vesicles. These results highlight the importance of lipid-protein interaction for the correct folding of membrane proteins and they suggest that the C-terminal domain of Bax will only span membranes with a net negative charge in their surface.     

Stabilization of acetylcholine receptor secondary structure by cholesterol and negatively charged phospholipids in membranes

Fourier-transform infrared (FTIR) spectroscopy was used to study the secondary structure of purified Torpedo californica nicotinic acetylcholine receptor (AChR) in reconstituted membranes. Functional studies have previously demonstrated that the ion channel activity requires the presence of both sterol and negatively charged phospholipids in membranes. The present studies are designed to test the hypothesis that the alpha-helical structure of AChR may be stabilized by specific lipid molecules (sterol and/or negatively charged phospholipids) and that these alpha-helices may be responsible for the formation of a potential ion channel. FTIR data show statistically significant (p less than 0.005) spectral changes due to cholesterol and negatively charged phospholipids, respectively. On the basis of standard curves describing the relationship between the spectral properties and the secondary structural contents of water-soluble proteins, the observed spectral change at 931 cm-1 can be interpreted as an apparent change in the alpha-helix content from about 17% in the absence of sterols to about 20% in the presence of sterols, suggesting that protein-sterol interactions increase the helical structure of the AChR molecule. Similarly, the spectral change at 988 cm-1 can be interpreted as an apparent increase of beta-sheet content in the AChR molecule from about 20% to about 24% due to the presence of negatively charged phospholipids. Functional AChR in membranes thus appears to be correlated with higher alpha-helical and beta-sheet contents. It is concluded that one role of specific interactions between membrane protein and lipid molecules may be to maintain specific secondary structures necessary to support the ion channel function of AChR.     

Latent membrane perturbation activity of a mitochondrial precursor protein is exposed by unfolding.

We have purified milligram amounts of an importable mitochondrial precursor protein [the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase (DHFR)]. This has made it possible, for the first time, to perform detailed studies on the conformation of a precursor protein and its interaction with lipid membranes. The precursor protein closely resembled authentic mouse DHFR with respect to secondary structure (measured by CD spectra) and stability towards urea (measured by tryptophan fluorescence and enzyme activity). With this precursor protein, the presequence thus does not significantly alter the folding of the attached 'passenger protein'. In contrast to the corresponding presequence peptide, the native precursor exhibited only weak ability to disrupt vesicles with a low mol% of negatively charged lipids, suggesting that the passenger protein masks the amphiphilic properties of the presequence. The membrane-perturbing properties of the precursor were greatly enhanced by increasing the vesicles' content of negatively charged lipid or by denaturing the precursor in 5 M urea. Interaction with vesicles rich in acidic phospholipid was accompanied by partial unfolding of the precursor, suggesting that such a conformational change may also be involved in the interaction of the precursor with the mitochondrial membranes.     

Conformational States of the Water-Soluble Fragment of Cytochrome b5. I. pH-Induced Denaturation

Cytochrome b ₅ is a membrane protein that comprises two fragments: one is water-soluble and heme-containing, and the other is hydrophobic and membrane-embedded. The function of electron transfer is performed by the former whose crystal structure is known; however, its conformational states when in the membrane field and interacting with other proteins are still to be studied. Previously, we proposed water–alcohol mixtures for modeling the effect of membrane surface on proteins, and used this approach to study the conformational behavior of positively charged cytochrome c as well as relatively neutral retinol-binding protein also functioning in the field of a negatively charged membrane. The current study describes the conformational behavior of the negatively charged water-soluble fragment of cytochrome b ₅ as dependent on pH. Decreasing pH was shown to transform the fragment state from native to intermediate, similar to the molten globule reported earlier for other proteins in aqueous solutions: at pH 3.0, the fragment preserved a pronounced secondary structure and compactness but lost its rigid tertiary structure. A possible role of this intermediate state in cytochrome b ₅ functioning is discussed.     

Putative interhelix ion pairs involved in the stability of myoglobin.

An earlier theoretical study predicted that specific ion pair interactions between neighboring helices should be important in stabilizing myoglobin. To measure these interactions in sperm whale myoglobin, single mutations were made to disrupt them. To obtain reliable DeltaG values, conditions were found in which the urea induced unfolding of holomyoglobin is reversible and two-state. The cyanomet form of myoglobin satisfies this condition at pH 5, 25 degrees C. The unfolding curves monitored by far-UV CD and Soret absorbance are superimposable and reversible. None of the putative ion pairs studied here makes a large contribution to the stability of native myoglobin. The protein stability does decrease somewhat between 0 and 0.1 M NaCl, however, indicating that electrostatic interactions contribute favorably to myoglobin stability at pH 5.0. A previous mutational study indicated that the net positive charge of the A[B]GH subdomain of myoglobin is an important factor affecting the stability of the pH 4 folding intermediate and potential ion pairs within the subdomain do not contribute significantly to its stability. One of the assumptions made in that study is tested here: replacement of either positively or negatively charged residues outside the A[B]GH subdomain has no significant effect on the stability of the pH 4 molten globule.     

Interaction of the lantibiotic nisin with mixed lipid bilayers: a 31P and 2H NMR study

Nisin is a positively charged antibacterial peptide which binds to the negatively charged membranes of Gram-positive bacteria. The initial interaction of the peptide with model membranes of neutral (phosphatidylcholine) and negatively charged (phosphatidylcholine/phosphatidylglycerol) model lipid membranes was studied using nonperturbing solid state magic angle spinning (MAS) (31)P NMR and (2)H wide-line NMR. In the presence of nisin, the coexistence of two bilayer lipid environments was observed both in charged and in neutral membranes. One lipid environment was found to be associated with lipid directly interacting with nisin and one with noninteracting lipid. Solid state (31)P MAS NMR results show that the acidic membrane lipid component partitions preferentially into the nisin-associated environment. Deuterium NMR ((2)H NMR) of the selectively headgroup-labeled acidic lipid provides further evidence of a strong interaction between the charged lipid component and the peptide. The segregation of acidic lipid into the nisin-bound environment was quantified from (2)H NMR measurements of selectively headgroup-deuterated neutral lipid. It is suggested that the observed lipid partitioning in the presence of nisin is driven, at least initially, by electrostatic interactions. (2)H NMR measurements from chain-perdeuterated neutral lipids indicate that nisin perturbs the hydrophobic region of both charged and neutral bilayers.     

Positive Charges on Lysine Residues of the Extrinsic 18 kDa Protein Are Important to Its Electrostatic Interaction with Spinach Photosystem Ⅱ Membranes

To determine the contribution of charged amino acids to binding with the photosystem II complex （PSII）, the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N- succinimidyl propionate （NSP） or glycine methyl ester （GME） in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.     

Binding of prion protein to lipid membranes and implications for prion conversion

The binding of the Syrian hamster prion protein, SHaPrP(90-231), to model lipid membranes was investigated by tryptophan fluorescence. Membranes composed of negatively charged or zwitterionic lipids, and raft-like membranes containing dipalmitoylphosphatidylcholine(1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol and sphingomyelin, were investigated. It was found that SHaPrP(90-231) binds to negatively charged lipid membranes and raft-like membranes. Binding of PrP to negatively charged lipid membranes involves both electrostatic and hydrophobic lipid-protein interactions and results in partial insertion of PrP into the lipid bilayer. This membrane-inserted conformation of PrP is richer in beta-sheet structure and has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents. In contrast, the binding of PrP to raft-like membranes is driven by hydrophobic lipid-protein interactions and induces the formation of alpha-helical structure. This conformation of PrP with a high content of alpha-helix is formed only at pH 7 and does not destabilize the lipid bilayer. Our findings support the view that an interaction of PrP with lipid membranes could play a role in PrP conversion.