Structural characterization of two papaya chitinases, a family GH19 of glycosyl hydrolases.

Two chitinases, able to use tetra-N-acetylglucosamine, chitin and chitosan as substrates, were characterized after purification from Carica papaya latex. The complete amino acid sequence of the major form and about 40% of the minor one were determined through proteolytic digestions and mass spectroscopy analysis. Sequencing demonstrated that both papaya chitinases are members of the family 19 of glycosyl hydrolases (GH19). Based on the known 3-D structures of other members of family GH19, it was expected that papaya chitinases would adopt all-alpha structures. However, circular dichroism and infrared spectroscopy indicated, for the papaya chitinases, a content of 15-20% of extended structures besides the expected 40% of alpha helices. Since the fully sequenced papaya chitinase contains a large number of proline residues the possibility that papaya chitinase contains polyproline II stretches was examined in the context of their resistance against proteolytic degradation.

Hydrogen-deuterium exchange in membrane proteins monitored by IR spectroscopy: a new tool to resolve protein structure and dynamics.

As more and more high-resolution structures of proteins become available, the new challenge is the understanding of these small conformational changes that are responsible for protein activity. Specialized difference Fourier transform infrared (FTIR) techniques allow the recording of side-chain modifications or minute secondary structure changes. Yet, large domain movements remain usually unnoticed. FTIR spectroscopy provides a unique opportunity to record (1)H/(2)H exchange kinetics at the level of the amide proton. This approach is extremely sensitive to tertiary structure changes and yields quantitative data on domain/domain interactions. An experimental setup designed for attenuated total reflection and a specific approach for the analysis of the results is described. The study of one membrane protein, the gastric H(+),K(+)-ATPase, demonstrates the usefulness of (1)H/(2)H exchange kinetics for the understanding of the molecular movement related to the catalytic activity.

Structural modifications in the membrane-bound regions of the gastric H+/K+-ATPase upon ligand binding.

Extensive trypsin proteolysis was used to examine the accessibility of membrane bound segments of the gastric H+/K+-ATPase under different experimental conditions known to induce either the E1 or the E2 conformation. Membrane-anchored peptides were isolated after trypsinolysis and identified by sequencing. We show that several membrane bound segments are involved in the conformational change. In the N-terminal region, a M1-M2 peptide (12 kDa) was found to be associated with the membrane fraction after digestion in the presence of K+ or in the presence of vanadate (12 kDa and 15 kDa). In the M3 and M4 region, no difference was observed for the peptide obtained in E1 or E2-K conformations, but the peptide generated in the presence of vanadate begins 12 amino-acid residues earlier in the sequence. Cytoplasmic loop region: we show here that a peptide beginning at Asp574 and predicted to end at Arg693 is associated with the membrane for a vanadate-induced conformation. In the M5-M6 region, the membrane-anchored peptide obtained on E1 is 39 amino acids shorter than the E2 peptide. In the M7-M8 region, the same peptide encompassing the M7 and M8 transmembrane segments was produced for E1 and E2 conformations.

Difference between the E1 and E2 conformations of gastric H+/K+-ATPase in a multilamellar lipid film system. Characterization by fluorescence and ATR-FTIR spectroscopy under a continuous buffer flow.

A liquid flow cell was used for an attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) study of conformational changes taking place in the gastric H+/K+-ATPase. Shifting from E1 to E2 form is induced by replacing Na+ by K+ ions. Introducing ions through a flow passing over a protein multilayer film induced the conformational change without cell manipulations. Measurement sensitivity was thereby improved by about one order of magnitude. The detection threshold allowed the possibility to detect a change affecting five amino acids out of the 1324 that compose the H+/K+-ATPase molecule. It appeared that fewer than five amino-acid residues undergo a conformational change upon replacing Na+ by K+ ions in the medium. Evidence that conformational changes occur in an identical system was brought by monitoring the fluorescence of fluorescein isothiocyanate-labeled H+/K+-ATPase in similar conditions. Our data suggest that essentially the tertiary structure of the protein is modified.

Structural characterization of a low density lipoprotein receptor-active apolipoprotein E peptide, ApoE3-(126-183).

Apolipoprotein E (apoE) plays a critical role in lipoprotein particle clearance from blood plasma through its interaction with the low density lipoprotein (LDL) receptor and other related receptors. Here, we studied a 58-residue peptide encompassing the receptor binding region of apoE. ApoE3-(126-183) was generated by cyanogen bromide cleavage of recombinant apoE3-(1-183), purified by reversed-phase high pressure liquid chromatography, and characterized by mass spectrometry. Far UV CD spectroscopy of the peptide showed that it is unstructured in aqueous solution. The addition of trifluoroethanol or dodecylphosphocholine induces the peptide to adopt an alpha-helical conformation. ApoE3-(126-183) efficiently transforms dimyristoylphosphatidylglycerol (DMPG) vesicles into peptide-lipid complexes. Analysis of apoE3-(126-183). DMPG complexes by electron microscopy revealed disc-shaped particles with an average diameter of 13 +/- 3 nm. Flotation equilibrium analysis yielded a particle molecular mass of 252 kDa. Far UV CD analysis of apoE3-(126-183).DMPG discs provided evidence that the peptide adopts a helical conformation. Competition binding experiments with (125)I-labeled low density lipoprotein (LDL) were conducted to assess the ability of apoE3-(126-183).DMPG complexes to bind to the LDL receptor. Both N-terminal apoE and the peptide, when complexed with DMPG, competed with (125)I-LDL for binding sites on the surface of cultured human skin fibroblasts. Under the conditions employed, apoE3-(126-183).DMPG complexes were similar to apoE3-(1-183).DMPG discs in their ability to bind to the receptor, demonstrating that the peptide represents a good model to study the interaction between apoE and the LDL receptor. Preliminary NMR results indicated that a high resolution structure of the apoE3-(126-183) peptide is obtainable.

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.

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.

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.

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.

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.

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.

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.

Alignment of the apolipophorin-III alpha-helices in complex with dimyristoylphosphatidylcholine. A unique spatial orientation.

Apolipophorin-III (apoLp-III) from Manduca sexta can exist in two alternate states: as a globular, lipid-free helix bundle or a lipid surface-associated apolipoprotein. Previous papers (Ryan R.O., Oikawa K., and Kay C. M. (1993) J. Biol. Chem. 268, 1525-1530; Wientzek M., Kay C.M., Oikawa K., and Ryan R.O. (1994) J. Biol. Chem. 269, 4605-4612) have investigated the structures and properties of apolipophorin-III from M. sexta in the lipid-free state and associated to lipids. Association of apoLp-III with dimyristoylphosphatidylcholine vesicles leads to the formation of uniform lipid discs with an average diameter and thickness of 18.5 +/- 2.0 and 4.8 +/- 0.8 nm, respectively. These discs contain six molecules of apoLp-III. Geometrical calculations based on these data, together with x-ray crystallographic data from the homologous L. migratoria apoLp-III (Breiter D. R., Kanost M.R., Benning M.M., Wesenberg G., Law J.H., Wells M.A., Rayment I., and Holden H.M. (1991) Biochemistry 30, 603-608), have allowed the presentation of a model of lipid-protein interaction, in which the alpha-helices of the apoLp-III orient perpendicular to the phospholipid chains and surround the lipid disc. Here, using polarized Fourier transform-attenuated total reflection infrared spectroscopy, we provide the first experimental evidence of a unique perpendicular orientation of the alpha-helices with respect to the fatty acyl chains of the phospholipids in the disc.