Fast hydride transfer in proton-translocating transhydrogenase revealed in a rapid mixing continuous flow device.

Transhydrogenase couples the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. Coupling is achieved through changes in protein conformation. Upon mixing, the isolated nucleotide-binding components of transhydrogenase (dI, which binds NAD(H), and dIII, which binds NADP(H)) form a catalytic dI(2).dIII(1) complex, the structure of which was recently solved by x-ray crystallography. The fluorescence from an engineered Trp in dIII changes when bound NADP(+) is reduced. Using a continuous flow device, we have measured the Trp fluorescence change when dI(2).dIII(1) complexes catalyze reduction of NADP(+) by NADH on a sub-millisecond scale. At elevated NADH concentrations, the first-order rate constant of the reaction approaches 21,200 s(-1), which is larger than that measured for redox reactions of nicotinamide nucleotides in other, soluble enzymes. Rather high concentrations of NADH are required to saturate the reaction. The deuterium isotope effect is small. Comparison with the rate of the reverse reaction (oxidation of NADPH by NAD(+)) reveals that the equilibrium constant for the redox reaction on the complex is >36. This high value might be important in ensuring high turnover rates in the intact enzyme.

Direct evidence for the cooperative unfolding of cytochrome c in lipid membranes from H-(2)H exchange kinetics.

The interaction of cytochrome c (cyt c) with anionic lipid membranes is known to disrupt the tightly packed native structure of the protein. This process leads to a lipid-inserted denatured state, which retains a native-like alpha-helical structure but lacks any specific tertiary interactions. The structural and dynamic properties of cyt c bound to vesicles containing an anionic phospholipid (DOPS) were investigated by amide H-(2)H exchange using two-dimensional NMR spectroscopy and electrospray ionisation mass spectrometry. The H-(2)H exchange kinetics of the core amide protons in cyt c, which in the native protein undergo exchange via an uncorrelated EX2 mechanism, exchange in the lipid vesicles via a highly concerted global transition that exposes these protected amide groups to solvent. The lack of pH dependence and the observation of distinct populations of deuterated and protonated species by mass spectrometry confirms that exchange occurs via an EX1 mechanism with a common rate of 1(+/-0.5) h(-1), which reflects the rate of transition from the lipid-inserted state, H(l), to an unprotected conformation, D(i), associated with the lipid interface.

Unfolding and refolding of cytochrome c driven by the interaction with lipid micelles.

Binding of native cyt c to L-PG micelles leads to a partially unfolded conformation of cyt c. This micelle-bound state has no stable tertiary structure, but remains as alpha-helical as native cyt c in solution. In contrast, binding of the acid-unfolded cyt c to L-PG micelles induces folding of the polypeptide, resulting in a similar helical state to that originated from the binding of native cyt c to L-PG micelles. Far-ultraviolet (UV) circular dichroism (CD) spectra showed that this common micelle-associated helical state (HL) has a native-like alpha-helix content, but is highly expanded without a tightly packed hydrophobic core, as revealed by tryptophan fluorescence, near-UV, and Soret CD spectroscopy. The kinetics of the interaction of native and acid-unfolded cyt c was investigated by stopped-flow tryptophan fluorescence. Formation of H(L) from the native state requires the disruption of the tightly packed hydrophobic core in the native protein. This micelle-induced unfolding of cyt c occurs at a rate approximately 0.1 s(-1), which is remarkably faster in the lipid environment compared with the expected rate of unfolding in solution. Refolding of acid-unfolded cyt c with L-PG micelles involves an early highly helical collapsed state formed during the burst phase (<3 ms), and the observed main kinetic event reports on the opening of this early compact intermediate prior to insertion into the lipid micelle.

Structure and dynamics of lipid-associated states of apocytochrome c.

Apocytochrome c (apocyt c), which in aqueous solution is largely unstructured, acquires an alpha-helical conformation upon association with lipid membranes. The extent of alpha-helix induced in apocyt c is lipid-dependent and this folding process is driven by both electrostatic and hydrophobic lipid-protein interactions. The structural and dynamic properties of apocyt c in lipid membranes were investigated by attenuated total reflection Fourier transform infrared spectroscopy combined with amide H-D exchange kinetics. Apocyt c acquires a higher content of alpha-helical structure with negatively charged membranes than with zwitterionic ones. For all membranes studied here, the helices of these partially folded states of apocyt c have a preferential orientation perpendicular to the plane of the lipid membrane. The H-D exchange revealed that a small fraction of amide protons of apocyt c, possibly associated with a stable folded domain protected by the lipid, remained protected from exchange over 20 min. However, a large fraction of amide protons exchanged in less than 20 min, indicating that the helical states of apocyt c in lipid membranes are very dynamic.

Folding of apocytochrome c in lipid micelles: formation of alpha-helix precedes membrane insertion.

Apocytochrome c, which in aqueous solution is largely unstructured, acquires a highly alpha-helical structure upon interaction with lipid. The alpha-helix content induced in apocytochrome c depends on the lipid system, and this folding process is driven by both electrostatic and hydrophobic lipid-protein interactions. The folding kinetic mechanism of apocytochrome c induced by zwitterionic micelles of lysophosphatidylcholine (L-PC), predominantly driven by hydrophobic lipid-protein interactions, was investigated by fluorescence stopped-flow measurements of Trp 59 and fluorescein-phosphatidylethanolamine-(FPE) labeled micelles, in combination with stopped-flow far-UV circular dichroism. It was found that formation of the alpha-helical structure of apocytochrome c precedes membrane insertion. The unfolded state in solution (U(W)) binds to the micelle surface in a helical conformation (I(S)) and is followed by insertion into the lipid micelle, i.e., formation of the final helical state H(L). Binding of apocytochrome c to the lipid micelle (U(W) --> I(S)) is concurrent with formation of a large fraction (75-100%, depending on lipid concentration) of the alpha-helical structure of the final lipid-inserted state H(L). The highly helical intermediate I(S) is formed on the time scale of 3-12 ms, depending on lipid concentration, and inserts into the lipid micelle (I(S) --> H(L)) in the time range of approximately 200 ms to >1 s, depending on lipid-to-protein ratio. The final lipid-inserted helical state H(L) in L-PC micelles has an alpha-helix content approximately 65% of that of cytochrome c in solution and has no compact stable tertiary structure as revealed by circular dichroism results.

Folding of apocytochrome c induced by the interaction with negatively charged lipid micelles proceeds via a collapsed intermediate state.

Unfolded apocytochrome c acquires an alpha-helical conformation upon interaction with lipid. Folding kinetic results below and above the lipid's CMC, together with energy transfer measurements of lipid bound states, and salt-induced compact states in solution, show that the folding transition of apocytochrome c from the unfolded state in solution to a lipid-inserted helical conformation proceeds via a collapsed intermediate state (I(C)). This initial compact state is driven by a hydrophobic collapse of the polypeptide chain in the absence of the heme group and may represent a heme-free analogue of an early compact intermediate detected on the folding pathway of cytochrome c in solution. Insertion into the lipid phase occurs via an unfolding step of I(C) through a more extended state associated with the membrane surface (I(S)). While I(C) appears to be as compact as salt-induced compact states in solution with substantial alpha-helix content, the final lipid-inserted state (Hmic) is as compact as the unfolded state in solution at pH 5 and has an alpha-helix content which resembles that of native cytochrome c.

Electrostatic and hydrophobic contributions to the folding mechanism of apocytochrome c driven by the interaction with lipid.

In aqueous solution, while cytochrome c is a stably folded protein with a tightly packed structure at the secondary and tertiary levels, its heme-free precursor, apocytochrome c, shows all features of a structureless random coil. However, upon interaction with phospholipid vesicles or lysophospholipid micelles, apocytochrome c undergoes a conformational transition from its random coil in solution to an alpha-helical structure on association with lipid. The driving forces of this lipid-induced folding process of apocytochrome c were investigated for the interaction with various phospholipids and lysophospholipids. Binding of apocytochrome c to negatively charged phospholipid vesicles induced a partially folded state with approximately 85% of the alpha-helical structure of cytochrome c in solution. In contrast, in the presence of zwitterionic phospholipid vesicles, apocytochrome c remains a random coil, suggesting that negatively charged phospholipid headgroups play an important role in the mechanism of lipid-induced folding of apocytochrome c. However, negatively charged lysophospholipid micelles induce a higher content of alpha-helical structure than equivalent negatively charged diacylphospholipids in bilayers, reaching 100% of the alpha-helix content of cytochrome c in solution. Furthermore, micelles of lysolipids with the same zwitterionic headgroup of phospholipid bilayer vesicles induce approximately 60% of the alpha-helix content of cytochrome c in solution. On the basis of these results, we propose a mechanism for the folding of apocytochrome c induced by the interaction with lipid, which accounts for both electrostatic and hydrophobic contributions. Electrostatic lipid-protein interactions appear to direct the polypeptide to the micelle or vesicle surface and to induce an early partially folded state on the membrane surface. Hydrophobic interactions between nonpolar residues in the protein and the hydrophobic core of the lipid bilayer stabilize and extend the secondary structure upon membrane insertion.

Structural and kinetic description of cytochrome c unfolding induced by the interaction with lipid vesicles.

The interaction of cytochrome c with anionic lipid vesicles of DOPS induces an extensive disruption of the native structure of the protein. The kinetics of this lipid-induced unfolding process were investigated in a series of fluorescence- and absorbance-detected stopped-flow measurements. The results show that the tightly packed native structure of cytochrome c is disrupted at a rate of approximately 1.5 s-1 (independent of protein and lipid concentration), leading to the formation of a lipid-inserted denatured state (DL). Comparison with the expected rate of unfolding in solution (approximately 2 x 10(-3) s-1 at pH 5.0 in the absence of denaturant) suggests that the lipid environment dramatically accelerates the structural unfolding process of cytochrome c. We propose that this acceleration is in part due to the low effective pH in the vicinity of the lipid headgroups. This hypothesis was tested by comparative kinetic measurements of acid unfolding of cytochrome c in solution. Our absorbance and fluorescence kinetic data, combined with a well-characterized mechanism for folding/unfolding of cytochrome c in solution, allow us to propose a kinetic mechanism for cytochrome c unfolding at the membrane surface. Binding of native cytochrome c in water (NW) to DOPS vesicles is driven by the electrostatic interaction between positively charged residues in the protein and the negatively charged lipid headgroups on the membrane surface. This binding step occurs within the dead time of the stopped-flow experiments (<2 ms), where a membrane-associated native state (NS) is formed. Unfolding of NS driven by the acidic environment at the membrane surface is proposed to occur via a native-like intermediate lacking Met 80 ligation (MS), as previously observed during unfolding in solution. The overall unfolding process (NS --> DL) is limited by the rate of disruption of the hydrophobic core in MS. Equilibrium spectroscopic measurements by near-IR and Soret absorbance, fluorescence, and circular dichroism showed that DL has native-like helical secondary structure, but shows no evidence for specific tertiary interactions. This lipid-denatured equilibrium state (DL) is clearly more extensively unfolded than the A-state in solution, but is distinct from the unfolded protein in water (UW), which has no stable secondary structure.

Phospholipid headgroup dynamics in DOPG-d5-cytochrome c complexes as revealed by 2H and 31P NMR: the effects of a peripheral protein on collective lipid fluctuations.

The dynamics of the glycerol headgroup of dioleoylphosphatidylglycerol (DOPG) in hydrated bilayers were studied by 2H and 31P NMR spectroscopy, and the effects of binding a peripheral protein, cytochrome c, were evaluated. The fast headgroup segmental motions (tau c, 10(-10)-(-13) s) of DOPG in fully hydrated bilayers were not affected upon binding of cytochrome c, as evaluated by the spin-lattice (T1) relaxation of deuterons in the DOPG glycerol headgroup. In contrast, the spin-spin (T2e) relaxation is strongly affected, indicating that slow cooperative bilayer motions (tau c, 10(-3)-10(-6) s) are enhanced upon the interaction with cytochrome c, 2H and 31P NMR spectral lineshape analysis reveal details of the nature of these motions. The importance of these effects are discussed in terms of a possible mechanism for modulating membrane-associated processes.

Spin label and 2H-NMR studies on the interaction of melanotropic peptides with lipid bilayers.

The interaction of the cationic tridecapeptide alpha-melanocyte stimulating hormone (alpha-MSH) and the biologically more active analog [Nle4, DPhe7]-alpha-MSH with lipid membranes was investigated by means of ESR of spin probes incorporated in the bilayer, and NMR of deuterated lipids. All spin labels used here, stearic acid and phospholipid derivatives labeled at the 5th and 12th position of the hydrocarbon chain, and the cholestane label, incorporated into anionic vesicles of DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) in the liquid-crystalline phase, indicated that both peptides decrease the motional freedom of the acyl chains. No peptide effect was detected with neutral lipid bilayers. Changes in the alpha-deuteron quadrupolar splittings and spin lattice relaxation time of DMPG deuterated at the glycerol headgroup paralleled the results obtained with ESR, showing that the peptides cause a better packing both at the headgroup and at the acyl chain bilayer regions. The stronger effect caused by the more potent analog in the membrane structure, when compared to the native hormone, is discussed in terms of its larger lipid association constant and/or its deeper penetration into the bilayer.

Phospholipid headgroup-headgroup electrostatic interactions in mixed bilayers of cardiolipin with phosphatidylcholines studied by 2H NMR.

The headgroup-headgroup interactions in binary mixed bilayers of diacylphosphatidylcholines (PC) and cardiolipin were analyzed by 2H NMR. Specific changes in the quadrupole splittings of the choline headgroup deuterated PC at alpha,beta-methylenes, and gamma-methyls are observed upon the insertion of the negatively charged tetraacylphospholipid, cardiolipin. The effects are consistent with an electrostatic interaction between PC and cardiolipin headgroups, in which a concerted conformational reorientation of the entire phosphocholine moiety toward the membrane surface is involved. On the basis of the "choline-tilt" model by Macdonald and co-workers (1991) the variations in the quadrupole splittings are consistent with a change in orientation of the choline P-N vector up to 23 degrees for the highest cardiolipin concentrations. Additional information on headgroup conformational changes was obtained through the analysis of the dependence on temperature of the quadrupole splittings for the various deuterium-labeled segments. Evaluation of the deuterium spin-lattice (T1) relaxation times for the deuterons in the various positions of the choline headgroup in mixed bilayers of PC and cardiolipin showed that the internal fast segmental motions were not affected on addition of cardiolipin to PC membranes.

Lipid specificity in the interaction of cytochrome c with anionic phospholipid bilayers revealed by solid-state 31P NMR.

Phosphorus-31 NMR has been used to investigate the interaction of cytochrome c with bilayers of the anionic lipids dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylserine (DOPS), and diacylphosphatidylinositol (diacylPI). All 31P NMR spectra revealed the typical line shapes characteristics of phospholipids in liquid-crystalline bilayers. The effects on the 31P chemical shift anisotropy (CSA) for each system reflect particular modes of phospholipid headgroup interaction with cytochrome c. A distinct increase in the CSA for DOPS bilayers was observed upon binding of cytochrome c, which is likely to arise from a partial restriction of the amplitude of motion on this phospholipid headgroup. 31P NMR spin-lattice (T1) relaxation times of the various phospholipid-cytochrome c complexes show that conformational changes occur in the protein on binding to anionic phospholipids. These protein conformational changes are observed through paramagnetic enhancement of the measured 31P spin-lattice relaxation times for lipid phosphates. However, the 31P T1 values for the various complexes with cytochrome c show a different temperature dependence for each lipid, revealing different modes of protein interaction for each of the different lipid headgroups. The phosphate of DOPS was most efficiently relaxed by cytochrome c, while the relaxation of the phosphate in the PI headgroup was not affected. The relaxation profile for DOPG-bound cytochrome c shows a more complex behavior, where the lipid phosphorus relaxation is strongly enhanced above 15 degrees C, but not significantly affected at lower temperatures. It was found that the enhancement of lipid phosphorus relaxation is a result of the conformational changes in the protein, in which the heme becomes accessible to lipid phosphate upon binding to charged bilayer surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Resolution of individual lipids in mixed phospholipid membranes and specific lipid-cytochrome c interactions by magic-angle spinning solid-state phosphorus-31 NMR.

A model of the inner mitochondrial membrane was constructed with dioleoyphosphatidylcholine (PC), dioleoylphosphatidylethanolamine (PE), and cardiolipin (CL) at a PC:PE:CL molar ratio of 2:2:1, and the interaction of the peripheral membrane protein cytochrome c with this mixed membrane has been investigated by static and magic-angle spinning (MAS) solid-state 31P NMR. The static 31P NMR spectrum of the three-component membrane is a typical broad powder pattern for phospholipids in a bilayer structure, and is a result of three overlapping spectra of each individual phospholipid component in the mixed membrane, with an average effective chemical shift anisotropy of approximately 41 ppm. Using magic-angle spinning NMR methods, three resolved resonances are observed in the narrowed MAS 31P NMR spectrum, each of which has been assigned to each lipid component in the mixed membrane. This allows the investigation of individual phospholipid-protein interactions in multicomponent lipid bilayers. The interaction of cytochrome c with each lipid in a model mitochondrial membrane could now be evaluated. Phosphorus-31 spin-lattice (T1) relaxation times for each lipid phosphate were measured as a function of temperature, in the absence and presence of bound cytochrome c. T1 was not affected for any lipid upon binding of cytochrome c over the temperature range analyzed. However, averaging of the phosphorus-31 chemical shift anisotropy for the cardiolipin component in mixed PC/PE/CL bilayers at lower temperatures ceases to be axially symmetric on binding of cytochrome c, while for PC and PE components the axial symmetry is retained over the temperature interval studied here.(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction of horse heart cytochrome c with phospholipid bilayers. Structural and dynamic effects.

The interaction of cytochrome c with phospholipid bilayers is reviewed. Special emphasis is given to the structural and dynamic perturbations induced, either in the membrane lipid component or protein itself, by the lipid-protein interaction. The lipid-induced perturbations in the structure of cytochrome c involve: i) conformational changes in and around the heme crevice, converting the heme iron to a high-spin state: and ii) a destabilisation/loosening of the overall tertiary and secondary structure. This highly mobile, partially unfolded intermediate of cytochrome c has a remarkable resemblance to partially folded membrane-bound intermediates of the precursor protein. The functional implications of lipid-protein intermediates for (apo) cytochrome c in (protein-translocation) electron-transfer are discussed.

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.

Physical studies on membrane lipids of Bacillus stearothermophilus temperature and calcium effects.

Bacillus stearothermophilus was grown at the optimal temperature range (center, 65 degrees C), below it (48 and 55 degrees C), and above it (68 degrees C), in a complex medium with or without 2.5 mM Ca2+. The Ca(2+)-supplement improves growth at sub- and supraoptimal temperatures and extends it to higher temperatures (Jurado et al. (1987) J. Gen. Microbiol. 133, 507-513). The phospholipid composition of cultures obtained in the different growth conditions was studied. Phosphatidylethanolamine was always the major phospholipid (40 to 50% of the total phospholipid). Diphosphatidylglycerol, phosphatidylglycerol, a phosphoglycolipid (pgl) and two minor phospholipids (not identified) were also found in the polar lipid extract. The pgl shows a threefold concentration increase as the growth temperature raises from 48 to 68 degrees C. The thermotropic behavior of membrane lipids was studied by differential scanning calorimetry (DSC) and by means of two fluorescent probes of fluidity, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1,3-di(2-pyrenyl)propane (2Py(3)2Py). The results reveal similar features and clearly show a shift of the temperature range of the phase transition to higher values and an increased structural order of the bilayer, as the growth temperature rises from 55 to 68 degrees C, but an opposite effect was observed from 48 to 55 degrees C. Although the Ca(2+)-supplement to the growth medium has no detectable effect, the addition of Ca2+ to the buffer of liposomes (Ca(2+)-liposomes) has a significant ordering effect at all growth temperatures. These liposomes show a shift of the transition range to higher temperatures and the fluorescent parameters (DPH polarization and intramolecular excimerization of the 2Py(3)2Py) detected an order increase of the probes environment, along and above the main phase transition. Spectra of 31P-NMR and polarized light microscopy clearly show that the lipid extracts exhibit, in all the conditions, typical lamellar phase geometry. We concluded that B. stearothermophilus controls the membrane lipid composition to compensate for the destabilizing effect of high temperatures on the membrane organization or to provide an appropriate packing of phospholipid molecules in a stable bilayer. At high temperatures, Ca(2+)-stimulatory effect on growth is presumably due to a direct Ca2+ interaction with the membrane phospholipids, inducing an increased structural order on the bilayer. The increase of the phase transition temperature in the total lipid extracts as compared with the respective polar lipid fractions probably indicates a stabilizing effect of neutral lipids on membrane bilayers.

A 31P-NMR study on multilamellar liposomes formed from the lipids of a thermophilic bacterium.

The membrane lipids of a thermophilic bacterium, Thermus SPS11, isolated from thermal springs in São Pedro do Sul, Portugal, were fractionated by chromatography on silica gel. The total lipid extract was found to contain one major phospholipid (PL), which accounts for about 90% of the total lipid phosphorous, and one major glycolipid (GL), which accounts for about 95% of the total carbohydrate in the non-phospholipid fraction. The membranes also contain about 11% by weight of a complex mixture of carotenoids (CA). Multilamellar liposomes, in excess water, formed from PL and mixtures of PL with GL and CA in proportions found in the natural membrane were investigated by proton-decoupled 31P-nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction. All mixtures examined were found to be in a lamellar phase with disordered hydrophobic chains with no evidence for "non-bilayer structures" between 23 degrees and 85 degrees C. Compared to bilayers formed from pure PL or mixtures of PL and CA, significantly larger values for the chemical shift anisotropy of the 31P-NMR powder patterns were obtained from bilayers formed from mixtures of PL and GL, at temperatures above 75 degrees C, and mixtures of PL, GL and CA at all temperatures examined. These differences are interpreted in terms of changes in the order of the bilayer and/or changes in the orientation of the phosphate moiety of PL. The significance of these results to the thermophily of the bacterium is discussed.