Effects of thermal denaturation on protein glycation

Protein denaturation occurs at sites of inflammation. We hypothesized that denatured protein may provide a more susceptible target for glycation, which is a known mediator of inflammation. We examined the effects of thermal denaturation on the susceptibility of protein glycation using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and aspartate aminotransferase (AAT) as our target proteins. GAPDH and AAT are ubiquitous proteins that exhibited very different thermal stabilities. Glycating agents, methylglyoxal (MG) and glyceraldehyde (Glyc), caused an increase in the formation of advanced glycation endproducts (AGEs) in native and denatured GAPDH and AAT. The effects of the glycating agents were more pronounced with the denatured proteins. In addition to nitroblue tetrazolium (NBT)- reactivity, our measured endpoints were absorbance (lambda = 365 nm) and fluorescence (lambda(ex) = 370 nm; lambda(em) = 470 nm) properties that are typically associated with protein glycation. We also looked at carnosine's ability to prevent glycation of native and denatured protein. Carnosine, an endogenous histidine dipeptide, exhibits anti-inflammatory activity presumably due to its anti-oxidant and anti-glycation properties. Carnosine prevented Glyc-induced AGE formation in both native and denatured AAT suggesting that carnosine's anti-inflammatory activity may be due in part to carnosine's ability to prevent glycation of denatured protein.     

13C-NMR studies of membrane lipid-protein interactions upon protein heat denaturation.

Spinach chloroplast membranes were studied by natural abundance carbon-13 nuclear magnetic resonance (13C-NMR) spectroscopy in their normal state and after heat denaturation of membrane proteins. The membrane proteins were denatured by raising the temperature of the sample to 67 degrees C for 5 minutes [YashRoy, R.C. (1991) J. Biochem. Biophys. Methods 22, 55-59]. Line-broadening of 13C-NMR resonances arising from the 1st (carbonyl), 7th, 9th and 12th carbon atom of fatty-acyl chains with reference to the carbonyl (C-1) group shows increased immobilization of lipid fatty-acyl chains at these locations, obviously caused by changes in interactions between membrane lipids and proteins upon heat denaturation of membrane proteins.     

Lipids and topological rules of membrane protein assembly: balance between long and short range lipid-protein interactions

The N-terminal six-transmembrane domain (TM) bundle of lactose permease of Escherichia coli is uniformly inverted when assembled in membranes lacking phosphatidylethanolamine (PE). Inversion is dependent on the net charge of cytoplasmically exposed protein domains containing positive and negative residues, net charge of the membrane surface, and low hydrophobicity of TM VII acting as a molecular hinge between the two halves of lactose permease (Bogdanov, M., Xie, J., Heacock, P., and Dowhan, W. (2008) J. Cell Biol. 182, 925-935). Net neutral lipids suppress the membrane translocation potential of negatively charged amino acids, thus increasing the cytoplasmic retention potential of positively charged amino acids. Herein, TM organization of sucrose permease (CscB) and phenylalanine permease (PheP) as a function of membrane lipid composition was investigated to extend these principles to other proteins. For CscB, topological dependence on PE only becomes evident after a significant increase in the net negative charge of the cytoplasmic surface of the N-terminal TM bundle. High negative charge is required to overcome the thermodynamic block to inversion due to the high hydrophobicity of TM VII. Increasing the positive charge of the cytoplasmic surface of the N-terminal TM hairpin of PheP, which is misoriented in PE-lacking cells, favors native orientation in the absence of PE. PheP and CscB also display co-existing dual topologies dependent on changes in the charge balance between protein domains and the membrane lipids. Therefore, the topology of both permeases is dependent on PE. However, CscB topology is governed by thermodynamic balance between opposing lipid-dependent electrostatic and hydrophobic interactions.     

Stoichiometry of lipid-protein interaction and integral membrane protein structure

The stoichiometries of lipid-protein interaction obtained from spin label electron spin resonance experiments with integral membrane proteins are compared with simple geometric models for the intramembranous perimeter that are based on the predicted numbers of transmembrane helices. Deviations from the predicted values provide evidence for oligomerization of the protein in the membrane and/or more complex arrangements of the transmembrane segments. Received: 16 January 1997 / Accepted: 5 March 1997     

Effects of potassium on lipid-protein interactions in light sarcoplasmic reticulum

This study shows the effect of K+ on phospholipid-protein interactions in light sarcoplasmic reticulum (LSR) as measured by 31P NMR. In the presence of 110 mM K+, a substantial effect of the membrane protein on the behavior of the phospholipids was detected. Subtracting the spectrum of the LSR lipid extract from the spectrum of the intact LSR membrane produced a difference spectrum of much greater breadth than the normal phospholipid bilayer powder pattern. This powder pattern is indicative of a phospholipid domain considerably more motionally restricted than the phospholipids in a normal phospholipid bilayer. The apparent axially symmetric powder pattern is consistent with axial diffusion. In a reconstituted membrane containing the calcium pump protein at a lipid/protein ratio much less than in the light sarcoplasmic reticulum, the broad component was more prominent. The relative resonance intensity of the broad component appeared to be proportional to the lipid/protein ratio of the membrane. In 10 mM K+, no broad powder pattern is observed in the corresponding difference spectrum. Thus, in the absence of potassium, the membrane protein has much less influence on the phospholipid of the membrane, as measured by 31P NMR. In addition to the effects of K+ on the membrane structure of the sarcoplasmic reticulum, K+ modulated the function of the calcium pump. The rate of calcium-dependent ATP hydrolysis increased in light sarcoplasmic reticulum when [K+] increased from 10 to 110 mM. The rate of calcium transport was also stimulated by an increase in K+.     

Effects of melittin on lipid-protein interactions in sarcoplasmic reticulum membranes.

To investigate the physical mechanism by which melittin inhibits Ca-adenosine triphosphatase (ATPase) activity in sarcoplasmic reticulum (SR) membranes, we have used electron paramagnetic resonance spectroscopy to probe the effect of melittin on lipid-protein interactions in SR. Previous studies have shown that melittin substantially restricts the rotational mobility of the Ca-ATPase but only slightly decreases the average lipid hydrocarbon chain fluidity in SR. Therefore, in the present study, we ask whether melittin has a preferential effect on Ca-ATPase boundary lipids, i.e., the annular shell of motionally restricted lipid that surrounds the protein. Paramagnetic derivatives of stearic acid and phosphatidylcholine, spin-labeled at C-14, were incorporated into SR membranes. The electronic paramagnetic resonance spectra of these probes contained two components, corresponding to motionally restricted and motionally fluid lipids, that were analyzed by spectral subtraction. The addition of increasing amounts of melittin, to the level of 10 mol melittin/mol Ca-ATPase, progressively increased the fraction of restricted lipids and increased the hyperfine splitting of both components in the composite spectra, indicating that melittin decreases the hydrocarbon chain rotational mobility for both the fluid and restricted populations of lipids. No further effects were observed above a level of 10 mol melittin/mol Ca-ATPase. In the spectra from control and melittin-containing samples, the fraction of restricted lipids decreased significantly with increasing temperature. The effect of melittin was similar to that of decreased temperature, i.e., each spectrum obtained in the presence of melittin (10:1) was nearly identical to the spectrum obtained without melittin at a temperature approximately 5 degrees C lower. The results suggest that the principal effect of melittin on SR membranes is to induce protein aggregation and this in turn, augmented by direct binding of melittin to the lipid, is responsible for the observed decreases in lipid mobility. Protein aggregation is concluded to be the main cause of inactivation of the Ca-ATPase by melittin, with possible modulation also by the decrease in mobility of the boundary layer lipids.     

Selectivity of lipid-protein interactions

The spin label ESR and intrinsic fluorescence quenching methods of determining the selectivity of interactions of lipids with integral membrane proteins are summarized. The selectivity patterns of phospholipids, fatty acids, and steroids are reviewed for a variety of integral proteins. Where appropriate, correlations are established with biochemical assays of the effects of specific lipids on enzymatic activity and transport function.     

The role of lipid-protein interactions in amyloid-type protein fibril formation

Structural transition of polypeptide chains into the beta-sheet state followed by amyloid fibril formation is the key characteristic of a number of the so-called conformational diseases. The multistep process of protein fibrillization can be modulated by a variety of factors, in particular by lipid-protein interactions. A wealth of experimental evidence provides support to the notion that amyloid fibril assembly and the toxicity of pre-fibrillar aggregates are closely related and are both intimately membrane associated phenomena. The present review summarizes the principal factors responsible for the enhancement of fibril formation in a membrane environment, viz. (i) structural transformation of polypeptide chain into a partially folded conformation, (ii) increase of the local concentration of a protein upon its membrane binding, (iii) aggregation-favoring orientation of the bound protein, and (iv) variation in the depth of bilayer penetration affecting the nucleation propensity of the membrane associated protein. The molecular mechanisms of membrane-mediated protein fibrillization are discussed. Importantly, the toxicity of lipid-induced pre-fibrillar aggregates is likely to have presented a very strong negative selection pressure in the evolution of amino acid sequences.     

Effects of unsaturated fatty acids and triacylglycerols on phosphatidylethanolamine membrane structure

Lipid intake in diet regulates the membrane lipid composition, which in turn controls activities of membrane proteins. There is evidence that fatty acids (FAs) and triacylglycerols (TGs) can alter the phospholipid (PL) mesomorphism. However, the molecular mechanisms involved are not fully understood. This study focuses on the effect of the unsaturation degree of the C-18 FAs, oleic acid (OA), linoleic acid and linolenic acid, and their TGs, triolein (TO), trilinolein, and trilinolenin, on the structural properties of phosphoethanolamine PLs. By means of X-ray diffraction and 31P-NMR spectroscopy, it is shown that both types of molecules stabilize the HII phase in 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) liposomes. Several structural factors are considered to explain the correlation between the FA unsaturation degree and the onset temperature of the HII phase. It is proposed that TGs could act as lateral spacers between polar DEPE groups, providing an increase in the effective surface area per lipid molecule that would account for the structural parameters of the HII phase. Fluorescence polarization data indicated a fluidification effect of OA on the lamellar phase. TO increased the viscosity of the hydrophobic core with a high effect on the HII phase.     

To flip or not to flip: lipid-protein charge interactions are a determinant of final membrane protein topology

The molecular details of how lipids influence final topological organization of membrane proteins are not well understood. Here, we present evidence that final topology is influenced by lipid–protein interactions most likely outside of the translocon. The N-terminal half of Escherichia coli lactose permease (LacY) is inverted with respect to the C-terminal half and the membrane bilayer when assembled in mutants lacking phosphatidylethanolamine and containing only negatively charged phospholipids. We demonstrate that inversion is dependent on interactions between the net charge of the cytoplasmic surface of the N-terminal bundle and the negative charge density of the membrane bilayer surface. A transmembrane domain, acting as a molecular hinge between the two halves of the protein, must also exit from the membrane for inversion to occur. Phosphatidylethanolamine dampens the translocation potential of negative residues in favor of the cytoplasmic retention potential of positive residues, thus explaining the dominance of positive over negative amino acids as co- or post-translational topological determinants.     

Hydrophobic membrane thickness and lipid-protein interactions of the leucine transport system of Lactococcus lactis

The effect of the phospholipid acyl chain carbon number on the activity of the branched-chain amino acid transport system of Lactococcus lactis has been investigated. Major fatty acids identified in a total lipid extract of L. lactis membranes are palmitic acid (16:0), oleic acid (18:1) and the cyclopropane-ring containing lactobacillic acid (19 delta). L. lactis membrane vesicles were fused with liposomes prepared from equimolar mixtures of synthetic phosphatidylethanolamine (PE) and phosphatidylcholine (PC) with cis mono-unsaturated acyl chains. The activity of the branched-chain amino acid carrier is determined by the bulk properties of the membrane (Driessen, A.J.M., Zheng, T., In 't Veld, G., Op den Kamp, J.A.F. and Konings, W.N. (1988) Biochemistry 27, 865-872). PE acts as an activator and PC is ineffective. Counterflow and protonmotive-force driven transport of leucine is sensitive to changes in the acyl chain carbon number of both phospholipids and maximal with dioleoyl-PE/dioleoyl-PC. Above the gel to liquid-crystalline phase transition temperature of the lipid species, membrane fluidity decreased with increasing acyl chain carbon number. Our data suggest that the carbon number of the acyl chains of PE and PC determine to a large extent the activity of the transport system. This might be relevant for the interaction of PE with the transport protein. Variations in the acyl chain composition of PC exert a more general effect on transport activity. The acyl chain composition of phospholipids determines the membrane thickness (Lewis, B.A. and Engelman, D.M. (1983) J. Mol. Biol. 166, 211-217). We therefore propose that the degree of matching between the lipid-bilayer and the hydrophobic thickness of the branched-chain amino acid carrier is an important parameter in lipid-protein interactions.     

Bacterial osmosensing: roles of membrane structure and electrostatics in lipid-protein and protein-protein interactions

Bacteria act to maintain their hydration when the osmotic pressure of their environment changes. When the external osmolality decreases (osmotic downshift), mechanosensitive channels are activated to release low molecular weight osmolytes (and hence water) from the cytoplasm. Upon osmotic upshift, osmoregulatory transporters are activated to import osmolytes (and hence water). Osmoregulatory channels and transporters sense and respond to osmotic stress via different mechanisms. Mechanosensitive channel MscL senses the increasing tension in the membrane and appears to gate when the lateral pressure in the acyl chain region of the lipids drops below a threshold value. Transporters OpuA, BetP and ProP are activated when increasing external osmolality causes threshold ionic concentrations in excess of about 0.05 M to be reached in the proteoliposome lumen. The threshold activation concentrations for the OpuA transporter are strongly dependent on the fraction of anionic lipids that surround the cytoplasmic face of the protein. The higher the fraction of anionic lipids, the higher the threshold ionic concentrations. A similar trend is observed for the BetP transporter. The lipid dependence of osmotic activation of OpuA and BetP suggests that osmotic signals are transmitted to the protein via interactions between charged osmosensor domains and the ionic headgroups of the lipids in the membrane. The charged, C-terminal domains of BetP and ProP are important for osmosensing. The C-terminal domain of ProP participates in homodimeric coiled-coil formation and it may interact with the membrane lipids and soluble protein ProQ. The activation of ProP by lumenal, macromolecular solutes at constant ionic strength indicates that its structure and activity may also respond to macromolecular crowding. This excluded volume effect may restrict the range over which the osmosensing domain can electrostatically interact. A simplified version of the dissociative double layer theory is used to explain the activation of the transporters by showing how changes in ion concentration could modulate interactions between charged osmosensor domains and charged lipid or protein surfaces. Importantly, the relatively high ionic concentrations at which osmosensors become activated at different surface charge densities compare well with the predicted dependence of 'critical' ion concentrations on surface charge density. The critical ion concentrations represent transitions in Maxwellian ionic distributions at which the surface potential reaches 25.7 mV for monovalent ions. The osmosensing mechanism is qualitatively described as an "ON/OFF switch" representing thermally relaxed and electrostatically locked protein conformations.     

FtsY binds to the Escherichia coli inner membrane via interactions with phosphatidylethanolamine and membrane proteins

Targeting of many polytopic proteins to the inner membrane of prokaryotes occurs via an essential signal recognition particle-like pathway. FtsY, the Escherichia coli homolog of the eukaryotic signal recognition particle receptor alpha-subunit, binds to membranes via its amino-terminal AN domain. We demonstrate that FtsY assembles on membranes via interactions with phosphatidylethanolamine and with a trypsin-sensitive component. Both interactions are mediated by the AN domain of FtsY. In the absence of phosphatidylethanolamine, the trypsin-sensitive component is sufficient for binding and function of FtsY in the targeting of membrane proteins. We propose a two-step mechanism for the assembly of FtsY on the membrane similar to that of SecA on the E. coli inner membrane.     

Protein heat denaturation and study of membrane lipid-protein interactions by spin label ESR.

Spinach chloroplast membranes labelled with stearic acid-spin probe-bearing nitroxyl (label) moiety at 5th, 9th, 12th, 13th, 14th or 16th carbon locations with respect to the carboxylic group of stearic acid were studied (in the dark) by electron spin resonance (ESR) spectroscopy. Spectra were recorded at sample temperatures of 5, 30 and 67 degrees C. After heat denaturation of the membrane proteins for 5 min at 67 degrees C, the spectra were re-recorded at 30 and 5 degrees C for comparison. The results unequivocally show that membrane lipid fatty-acyl chains become substantially more rigid after protein heat-denaturation. The data throw light on the degree of lipid-protein interactions at various microlocations along the length of fatty-acyl chains of the membrane lipid matrix.     

Divalent cation and lipid-protein interactions of biomembranes

Divalent cations play an important role in the functions of biomembranes. This review deals with three topics: (1) Mg²⁺-mediated change in physical state of phospholipid induces conformation and activity change of reconstituted mitochondrial H⁺-ATPase, (2) a proper transmembrane Ca²⁺ gradient is essential for the higher enzymatic activity of adenylate cyclase, and (3) role of transmembrane Ca²⁺ gradient in the modulation of reconstituted sarcoplasmic reticulm Ca²⁺-ATPase activity.     

Effects of solute-solute interactions on protein stability studied using various counterions and dendrimers

Much work has been performed on understanding the effects of additives on protein thermodynamics and degradation kinetics, in particular addressing the Hofmeister series and other broad empirical phenomena. Little attention, however, has been paid to the effect of additive-additive interactions on proteins. Our group and others have recently shown that such interactions can actually govern protein events, such as aggregation. Here we use dendrimers, which have the advantage that both size and surface chemical groups can be changed and therein studied independently. Dendrimers are a relatively new and broad class of materials which have been demonstrated useful in biological and therapeutic applications, such as drug delivery, perturbing amyloid formation, etc. Guanidinium modified dendrimers pose an interesting case given that guanidinium can form multiple attractive hydrogen bonds with either a protein surface or other components in solution, such as hydrogen bond accepting counterions. Here we present a study which shows that the behavior of such macromolecule species (modified PAMAM dendrimers) is governed by intra-solvent interactions. Attractive guanidinium-anion interactions seem to cause clustering in solution, which inhibits cooperative binding to the protein surface but at the same time, significantly suppresses nonnative aggregation.     

Thermal stability of outer membrane protein porin from Paracoccus denitrificans: FT-IR as a spectroscopic tool to study lipid-protein interaction

Lipid protein interactions play a key role in the stability and function of various membrane proteins. Earlier we have reported the extreme thermal stability of porin from Paracoccus denitrificans reconstituted into liposomes. Here, we used Fourier transform infrared spectroscopy for a label free analysis of the global secondary structural changes and local changes in the tyrosine microenvironment. Our results show that a mixed lipid system (non-uniform bilayer) optimizes the thermal stability of porin as compared to the porin in pure lipids (uniform bilayer) or detergent micelles. This is in line with the fact that the bacterial outer membrane is a dynamic system made up of lipids of varying chain lengths, head groups and the barrel wall height contacting the membrane is uneven.