Attenuated total reflection infrared spectroscopy of proteins and lipids in biological membranes.
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Animals↗
Biomedical subjects
E Goormaghtigh
Publications and source records attributed to E Goormaghtigh.
Fourier transform infrared spectroscopy study of the secondary and tertiary structure of the reconstituted Na+/Ca2+ exchanger 70-kDa polypeptide.
The secondary structure of the purified 70-kDa protein Na+/Ca2+ exchanger, functionally reconstituted into asolectin lipid vesicles, was examined by Fourier transform infrared attenuated total reflection spectroscopy. Fourier transform infrared attenuated total reflection spectroscopy provided evidence that the protein is composed of 44% alpha-helices, 25% beta-sheets, 16% beta-turns, and 15% random structures, notably the proportion of alpha-helices is greater than that corresponding to the transmembrane domains predicted by exchanger hydropathy profile. Polarized infrared spectroscopy showed that the orientation of helices is almost perpendicular to the membrane. Tertiary structure modifications, induced by addition of Ca2+, were evaluated by deuterium/hydrogen exchange kinetic measurements for the reconstituted exchanger. This approach was previously proven as a useful tool for detection of tertiary structure modifications induced by an interaction between a protein and its specific ligand. Deuterium/hydrogen exchange kinetic measurements indicated that, in the absence of Ca2+, a large fraction of the protein (40%) is inaccessible to solvent. Addition of Ca2+ increased to 55% the inaccessibility to solvent, representing a major conformational change characterized by the shielding of at least 93 amino acids.
Animals↗
Membrane helix orientation from linear dichroism of infrared attenuated total reflection spectra.
Oriented multilamellar systems containing phospholipids and peptides have been formed on a germanium internal reflection element. Attenuated total reflection infrared spectra have been recorded and the linear dichroism of peptide amide I and amide II bands measured. Using peptides for which the orientation had been previously studied under similar experimental conditions by 15N solid-state nuclear magnetic resonance spectroscopy, important conclusions were drawn on the approach to be used to derive secondary structure orientation in a membrane from dichroic ratios. In particular, it is shown that the influence of the film thickness and refractive index on the orientation determination can be evaluated from the value of RATRiso, i.e., the dichroic ratio of a dipole oriented at the magic angle or with isotropic mobility. A series of peptides was used to test the validity of our suggestions on various helix orientations in the membrane. These include magainin 2 and hydrophobic (hPhi20) model peptides, the transmembrane segment of glycophorin (GLY), and LAH4, a designed peptide antibiotic that changes between a transmembrane and an in-plane orientation in a pH-dependent manner.
Amino Acid Sequence↗
Structural difference in the H+,K+-ATPase between the E1 and E2 conformations. An attenuated total reflection infrared spectroscopy, UV circular dichroism and raman spectroscopy study.
Conformational changes taking place in the gastric H+,K+-ATPase when shifting from the K+-induced E2 form to the E1 form upon replacing K+ ions by Na+ were investigated by different spectroscopic approaches. No significant secondary-structure change or secondary-structure reorientation with respect to the membrane plane could be measured by attenuated total reflection Fourier transform infrared spectroscopy of oriented films. Circular dichroism and Raman spectra obtained on tubulovesicle suspensions indicated no significant secondary structure or tyrosine and tryptophan side-chain environment changes in tubulovesicle suspensions. The smallest observable structural changes are discussed in term of the number of amino-acid residues involved for each technique.
Animals↗
Secondary structure of the membrane-bound domains of H+,K+-ATPase and Ca2+-ATPase, a comparison by FTIR after proteolysis treatment of the native membranes.
The sarcoplasmic reticulum Ca2+-ATPase and the gastric H+,K+-ATPase were cleaved under three different proteolysis conditions. After elimination of the protease and of the cleaved peptides, the vesicles containing the membrane-bound peptides of the ATPases were studied by Fourier transform attenuated total reflection infrared spectroscopy. In the harsher proteolysis conditions, the membrane-associated domain of the Ca2+-ATPase represented about 20% of the protein and was mainly constituted of alpha-helices. Polarized infrared spectroscopy showed that these alpha-helices were mainly oriented perpendicular to the membrane. However, only 10-20% of the H+,K+-ATPase was cleaved. The remaining, membrane-associated domain of the protein contained about 30% of alpha-helices and 30% of beta-sheet structures. The alpha-helices adopted a mainly transmembrane orientation. While the data on the Ca2+-ATPase are in general agreement with the current model of the protein, our results indicate that caution must be used in choosing this protein as a general structural model for all P-type ATPases. The protease-resistant, membrane-associated domain of the H+K+-ATPase is indeed much larger than predicted and also contained beta-sheet structures.
Adenosine Triphosphate↗
The low density lipoprotein receptor active conformation of apolipoprotein E. Helix organization in n-terminal domain-phospholipid disc particles.
Lipid association is a prerequisite for receptor interactions of apolipoprotein E (apoE). Disc complexes of the N-terminal 22-kDa apoE3 receptor binding domain and dimyristoylphosphatidylcholine display full receptor binding activity. Studies have been performed to characterize conformational adaptations of the globular, lipid-free four-helix bundle structure that culminate in stable association of its amphipathic alpha-helices with a lipid surface. Helix-lipid interactions in bilayer disc complexes can conceivably adopt two orientations: parallel or perpendicular to the phospholipid acyl chains. Evidence based on infrared dichroism, geometrical arguments, and x-ray crystallography support the view that defined helical segments in the four-helix bundle realign upon lipid association, orienting perpendicular to the phospholipid fatty acyl chains, circumscribing the bilayer disc. Thus, it is likely that paired helical segments align in tandem, presenting a convex receptor-active surface.
Apolipoprotein E3↗
Lipid membrane binding of NK-lysin.
The membrane-binding properties and pore-forming potential of the tumor-lysing and antibacterial polypeptide NK-lysin were investigated. Fluorescence quenching experiments show a drastic change of accessibility to Trp58 in solution and in association with a lipid membrane. Calcein release from large unilamellar vesicles and fluctuating conductivity observed across a planar lipid bilayer of asolectin show that NK-lysin renders lipid bilayers permeable in a transient fashion, indicating a nonspecific lipid interaction as the mechanism underlying the biological activity. FTIR experiments show the same amount and type of regular secondary structure of NK-lysin in the membrane as in aqueous solution and exclude a structural rearrangement into a set of parallel or antiparallel alpha-helices as the predominant conformation. The molecular mechanism of the membrane-destabilizing effect of NK-lysin is discussed.
Anti-Infective Agents↗
Secondary structure of the intact H+,K+-ATPase and of its membrane-embedded region. An attenuated total reflection infrared spectroscopy, circular dichroism and Raman spectroscopy study.
Models of P-type ATPase predict that membrane-embedded fragments represent about 20% of the protein and adopt an all-alpha-helical structure. While this prediction was confirmed for the Ca2-ATPase [Corbalan-Garcia, S., Teruel, J., Villalain, J. & Gomez-Fernandez, J. (1994) Biochemistry 33, 8247-8254], it is at odds with recent experimental evidence gathered on the Neurospora crassa plasma membrane H+-ATPase [Vigneron, L., Ruysschaert, J.-M. & Goormaghtigh, E. (1995) J. Biol. Chem. 270, 17685-17696] and on the gastric H+,K+-ATPase [Raussens, V., Ruysschaert, J.-M. & Goormaghtigh, E. (1997) J. Biol. Chem. 276, 262-270]. Extensive proteinase K proteolysis of open gastric tubulovesicles was performed here to generate the membrane-protected fragments of the H+,K+-ATPase. Secondary structure of the intact and of the membrane-protected segments was compared for oriented membrane films by attenuated total-reflection Fourier-transform infrared spectroscopy and by circular dichroism and for vesicles suspension by circular dichroism and Raman spectroscopy. All the spectroscopic data indicate that the protease-resistant membrane-bound residue of the H+,K+-ATPase contains significant amount of beta-sheet structure, both on films and in membrane suspensions. Polarized attenuated total-reflection infrared spectroscopy indicates that only the alpha-helical content of protease-resistant membrane-bound residue of the H+,K+-ATPase is oriented (parallel) with respect to the membrane normal. Raman spectroscopy reveals that Phe residues are preferentially removed by protease activity. Evaluation of the amount of removed Phe and Tyr residues places constraints on the model of membrane insertion of the H+,K+-ATPase.
Animals↗
Transmembrane helix stability: the effect of helix-helix interactions studied by Fourier transform infrared spectroscopy.
We have measured, using infrared spectroscopy, the hydrogen/deuterium exchange rates of the amide protons in the photosynthetic antenna of Rhodospirillum rubrum. These measurements were made not only on the intact protein in detergent solution but also on two dissociated forms (B820 and B777). We have, on the basis of our knowledge of the structure of this protein, been able to assign the various groups of amide protons that exchange with different time constants to distinct regions of the protein. The most protected group of protons that we observe exchanging with time constants near 6000 min we assign to the transmembrane helices. The slow exchange rates measured for the amide protons of the transmembrane helices of this protein in detergent solution may indicate a destabilization of the helices in detergent solution compared with the membrane. This group of protons is progressively destabilized by stepwise dissociation of the antenna protein, and this destabilization is greater than we can account for by increases in solvent accessibility. We suggest that the observed loss of amide proton protection in the transmembrane helices as they are dissociated might be due to an increase in the helix flexibility and breathing motions as interactions between helices are reduced.
Bacterial Chromatophores↗
Monitoring structural stability of trypsin inhibitor at the submolecular level by amide-proton exchange using Fourier transform infrared spectroscopy: a test case for more general application.
Combining the information on the secondary structure content as present in the shape of a protein amide I infrared band with the approach of monitoring amide-proton exchange using infrared spectroscopy, we have been able to investigate the structural stability of different components present in a protein, which are shown to be correlated to the different classes of secondary structures. For this purpose, the changes in intensity in different regions of the amide I have been detected upon exposure of the protein to a 2H2O environment, revealing four separate classes of exchanging components. As a test case for the approach described in this work, the amide-proton exchange of hydrated protein films of bovine pancreatic trypsin inhibitor has been studied using infrared spectroscopy, and is compared to literature data obtained by other techniques. A slow amide-proton exchange is observed for a class correlated to the beta-strands present in the protein, with protection of amide-protons for more than 19 h. Another class, which has been assigned to mainly helical residues, shows much less protection from exchange. The distribution function of the exchange rates of a class linked to the beta-turns displays five times faster exchange rates compared to those found for the majority of the helical residues, but they are still ten times slower compared to a class which we defined to represent the nonstructured parts of the protein.
Absorption↗
Amide-proton exchange of water-soluble proteins of different structural classes studied at the submolecular level by infrared spectroscopy.
For eleven films of various water-soluble alpha-, beta-, alpha-/beta-, and alpha-+beta-proteins, the amide-proton exchange, initiated by exposure of the protein film to 2H2O, has been monitored using infrared spectroscopy. The approach to obtain the kinetics of exchange for four different classes of amide protons, correlating to the different secondary structure types, has been described in detail in the preceding paper. In this work the more general applicability of the approach is illustrated by testing it for different types of proteins. The results obtained are shown not only to be comparable to reported time-resolved nuclear magnetic resonance data (as in the case of myoglobin, phospholipase A2, lysozyme, and cytochrome c), or to the more qualitative data obtained by neutron diffraction (trypsin, ribonuclease S, papain, and subtilisin BPN'), but the infrared approach us also provides with quantitative detailed insight on the distribution of exchange rate constants at the submolecular level of proteins, too complex to be studied by other techniques, as for tetrameric hemoglobin, and of proteins in which exchange is too fast to be detected by these other techniques, as is shown in this work for alpha-casein and apocytochrome c.
Amides↗
Structural domain organization of gastric H+,K+-ATPase and its rearrangement during the catalytic cycle.
Differential scanning calorimetry has been used to characterize the thermal denaturation of gastric (H+,K+)-ATPase. The excess heat capacity function of (H+,K+)-ATPase in highly oriented gastric vesicles displays two peaks at 53.9 degrees C (Tm1) and 61.8 degrees C (Tm2). Its thermal denaturation is an irreversible process that does not exhibit kinetic control and can be resolved in two independent two-state processes. They can be assigned to two cooperative domains located in the cytoplasmic loops of the alpha-subunit, according to the disappearance of the endothermic signal upon removal of these regions by proteinase K digestion. Analysis of the thermal-induced unfolding of the enzyme trapped in different catalytic cycle intermediates has allowed us to get insight into the E1-E2 conformational change. In the E1 forms both transitions are always observed. As Tm1 is shifted to Tm2 by vanadate and ATP interaction, the unfolding mechanism changes from two independent to two sequential two-state transitions, revealing interdomain interactions. Stabilization of the E2 forms results in the disappearance of the second transition at saturation by K+, Mg2+-ATP, and Mg2+-vanadate as well as in significant changes in Tm2 and DeltaH1. The catalytic domain melts following a process in which intermolecular interactions either in the native or in the unfolded state might be involved. Interestingly, the E2-vanadate-K+ form displays intermediate properties between the E1 and E2 conformational families.
Adenosine Triphosphate↗
Fourier transform infrared spectroscopy study of the secondary structure of the gastric H+,K+-ATPase and of its membrane-associated proteolytic peptides.
Membrane topology of the H+,K+-ATPase has been studied after proteolytic degradation of the protein by proteinase K. Proteinase K had access to either the cytoplasmic part of the protein or to both sides of the membrane. Fourier transform infrared attenuated total reflection spectroscopy indicated that membrane-associated domain of the protein represented about 55% of the native protein, meanwhile the cytoplasmic part represented only 27% of the protein. The secondary structure of the ATPase and of its membrane-associated domains was investigated by infrared spectroscopy. The secondary structure of the membrane-associated structures and of the entire protein was quite similar (alpha-helices, 35%; beta-sheets, 35%; turns, 20%; random, 15%). These data were in agreement with 10 alpha-helical transmembrane segments but suggested a participation of beta-sheet structures in the membrane-associated part of the protein. Polarized infrared spectroscopy indicated that the alpha-helices were oriented nearly perpendicular to the membrane plane. No preferential orientation could be attributed to the beta-sheets. Monitoring the amide hydrogen/deuterium exchange kinetics demonstrated that the membrane associated part of the ATPase molecule is characterized by a relatively high accessibility to the solvent, quite different from that observed for bacteriorhodopsin membrane segments.
Animals↗
The different molar absorptivities of the secondary structure types in the amide I region: an attenuated total reflection infrared study on globular proteins.
Differences in molar absorptivity of the various secondary structures in the amide I region of infrared protein spectra would have a great impact on the interpretation of the data published thus far on protein films studied by attenuated total reflection infrared spectroscopy. In this work, representative values for amide I absorptivities are obtained for 15 different films of globular proteins spread from H2O solutions. The observed intensities are corrected for variations in film thickness and for contributions of hydration water, atmospheric water, and side chains. These absorptivities, together with the reported secondary structure of the proteins investigated, are used to deduce the molar absorptivities of the individual secondary structure types. It is found that the molar absorptivity of beta-strands is 1.4-1.6 times larger than that of alpha-helices, which in turn is 1.3-2.1 times larger than those found for beta-turns or random coiled structures. The implications of our findings for spectral analysis currently used in literature are discussed.
Deuterium↗
Secondary and tertiary structure changes of reconstituted P-glycoprotein. A Fourier transform attenuated total reflection infrared spectroscopy analysis.
The structure of purified P-glycoprotein functionally reconstituted into liposomes was investigated by attenuated total reflection Fourier transform infrared spectroscopy. A quantitative evaluation of the secondary structure and a kinetic of 2H/H exchange of the P-glycoprotein were performed both in the presence and in the absence of MgATP, MgATP-verapamil, and MgADP. This approach was previously shown to be a useful tool to detect tertiary structure changes resulting from the interaction between a protein and its specific ligands, as established for the Neurospora crassa H+-ATPase. 2H/H exchange measurements provided evidence that a large fraction of the P-glycoprotein is poorly accessible to the aqueous medium. Addition of MgATP induced an increased accessibility to the solvent of a population of amino acids, while addition of MgATP-verapamil resulted in a subtraction of a part of the protein from access to the aqueous solvent. No significant changes were observed upon addition of MgADP or verapamil alone. The secondary structure of P-glycoprotein was not affected by addition of ligands. The variations observed in the 2H/H exchange rate when P-glycoprotein interacted with the above ligands therefore represented tertiary structure changes. Fluorescence quenching experiments confirmed that MgATP-induced changes are to be found in the tertiary structure of the enzyme.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Hydrogen/deuterium exchange kinetics of apolipophorin-III in lipid-free and phospholipid-bound states. An analysis by Fourier transform infrared spectroscopy.
Attenuated total reflection Fourier transform infrared spectroscopy was used to probe the kinetics of hydrogen/deuterium exchange in Manduca sexta apolipophorin-III (apoLp-III). ApoLp-III is an exchangeable apolipoprotein that is made up of five elongated amphipathic alpha-helices in a helical bundle conformation in the monomeric lipid-free form. Upon interaction with phospholipids, it is postulated to undergo a large conformational change whereby the hydrophobic interior is exposed, facilitating binding to the lipid surfaces. We have used the lipid-free and dimyristoylphosphatidylcholine-bound apoLp-III to study the dynamically variable domains in the two forms. Three populations of amide protons varying in their hydrogen/deuterium exchange rates were found to exist: slow, intermediate, and fast exchanging, which could correspond to completely buried, partially buried, and solvent-exposed domains on the protein in both the states. In lipid-free apoLp-III, 36, 12, and 52% of the total residues contributed to the slow, intermediate, and fast exchanging populations, respectively. In the dimyristoylphosphatidylcholine-bound form, the corresponding distribution was 20, 16, and 64%, representing a 12% increase in the number of exposed residues. The results are discussed in terms of increased solvent accessibility due to gross tertiary structural reorganization.
Amides↗
The N-terminal half of a mitochondrial presequence peptide inserts into cardiolipin-containing membranes. Consequences for the action of a transmembrane potential.
The orientation of a mitochondrial-presequence peptide, associated with anionic lipid-containing model membranes, was investigated. The peptide inserts with its N-terminal alpha-helical part into cardiolipin (CL) monolayers so that the N-terminal 14 residues are protected from proteinase K. In phosphatidylglycerol (PG) monolayers the inserted peptide was fully accessible to the protease. A consequence of the different orientations of the peptide was that membrane potential-dependent protection from trypsin was much faster for the peptide bound to PG-containing vesicles compared to CL-containing membranes, suggesting that in the mitochondrial protein import process other components of the import apparatus are involved in the efficient potential-driven translocation of presequences across the inner mitochondrial membranes.
Amino Acid Sequence↗
Fourier transform infrared spectroscopy study of the secondary structure of the reconstituted Neurospora crassa plasma membrane H(+)-ATPase and of its membrane-associated proteolytic peptides.
We reconstituted purified plasma membrane H(+)-ATPase from Neurospora crassa into soybean phospholipid vesicles (lipid/ATPase ratio of 5:1 w/w). The proteoliposomes contained an active ATPase, oriented inside-out. They were subjected to proteolysis by using Pronase, proteinase K, trypsin, and carboxypeptidase Y. Fourier transform infrared attenuated total reflection spectroscopy indicates that the amount of protein remaining after hydrolysis and elimination of the extramembrane domain of ATPase represents about 43% of the intact protein. The secondary structure of intact ATPase and of the membrane-associated domain of ATPase was determined by infrared spectroscopy. The membrane domain shows a typical alpha-helix and beta-sheet absorption. Polarized infrared spectroscopy reveals that the orientation of the helices is about perpendicular to the membrane. Amide hydrogen/deuterium exchange kinetics performed for the intact H(+)-ATPase and for the membrane-associated domain demonstrate that this part of ATPase shows less accessibility to the solvent than the entire protein but remains much more accessible to the solvent than bacteriorhodopsin membrane segments.
Bacteriorhodopsins↗