Kinetics of the low pH-induced conformational changes and fusogenic activity of influenza hemagglutinin.

The decrease of the intrinsic tryptophan fluorescence intensity of purified influenza (X31 strain) hemagglutinin (HA) was used to monitor the low pH-induced conformational change of this protein. The kinetics of the fluorescence decrease depended strongly on the pH. At pH optimal for fusion, the change in tryptophan fluorescence was fast and could be fitted to a monoexponential function. We measured a rate constant of 5.78 s-1 (t1/2 = 120 ms) at pH 4.9 using rapid stopped-flow mixing. Under suboptimal conditions (higher pH), the rate constant was decreased by an order of magnitude. In addition, a slow component appeared and the fluorescence decrease followed a sum of two exponentials. The kinetics of conformational changes were compared with those of the fusion of influenza virus with red blood cell membranes as assessed by the R18-dequenching assay. At optimal pH the HA conformational change was not rate-limiting for the fusion process. However, at sub-optimal pH, the slow transition to the fusogenic conformational of HA resulted in slower kinetics and decreased extent of fusion.     

Conformational Stability and Hemolytic Activity of Actinoporin RTX-SII from the Sea Anemone <Emphasis Type="Italic">Radianthus macrodactylus</Emphasis>

The spatial organization of actinoporin RTX-SII from the sea anemone Radianthus macrodactylus on the level of tertiary and secondary structures was studied by UV and CD spectroscopy and intrinsic protein fluorescence. The specific and molar extinction coefficients of RTX-SII were determined. The percentages of canonical secondary structures of actinoporin were calculated. The tertiary structure of the polypeptide is well developed and its secondary structure is highly ordered and contains about 50% antiparallel folded beta-sheets. The irreversible thermal denaturation of RTX-SII was studied by CD spectroscopy; a conformational transition occurs at 53 degrees C. Above this temperature irreversible conformational changes are observed in the secondary and tertiary structures. This is accompanied by redistribution of the content of regular and distorted forms of beta-sheet and also by increase in the content of an unordered form. It is suggested that an intermediate is formed in the process of thermal denaturation. Acid-base titration of RTX-SII results in irreversible conformational changes at pH below 2.0 and above 12.0. As shown by intrinsic protein fluorescence, tyrosine residues of RTX-SII make a fundamental contribution to emission, and the total fluorescence depends more on temperature and ionic strength of the solution than tryptophan fluorescence. The data on conformational stability of actinoporin are correlated with data on its hemolytic activity. Activity of RTX-SII significantly decreases at increased temperature and slightly decreases at low pH. Hemolytic activity drastically increases at high pH. Increase in the actinoporin activity at pH above 10 seems to be caused by ionization of the molecule.     

Influenza hemagglutinin assumes a tilted conformation during membrane fusion as determined by attenuated total reflection FTIR spectroscopy.

Fusion of influenza virus with target membranes is mediated by an acid-induced conformational change of the viral fusion protein hemagglutinin (HA) involving an extensive reorganization of the alpha-helices. A 'spring-loaded' displacement over at least 100 A provides a mechanism for the insertion of the fusion peptide into the target membrane, but does not explain how the two membranes are brought into fusion contact. Here we examine, by attenuated total reflection Fourier transform infrared spectroscopy, the secondary structure and orientation of HA reconstituted in planar membranes. At neutral pH, the orientation of the HA trimers in planar membranes is approximately perpendicular to the membrane. However, at the pH of fusion, the HA trimers are tilted 55-70 degrees from the membrane normal in the presence or absence of bound target membranes. In the absence of target membranes, the overall secondary structure of HA at the fusion pH is similar to that at neutral pH, but approximately 50-60 additional residues become alpha-helical upon the conformational change in the presence of bound target membranes. These results are discussed in terms of a structural model for the fusion intermediate of influenza HA.     

pH-induced conformational changes of membrane-bound influenza hemagglutinin and its effect on target lipid bilayers.

Influenza virus hemagglutinin (HA) has served as a paradigm for both pH-dependent and -independent viral membrane fusion. Although large conformational changes were observed by X-ray crystallography when soluble fragments of HA were subjected to fusion-pH conditions, it is not clear whether the same changes occur in membrane-bound HA, what the spatial relationship is between the conformationally changed HA and the target and viral membranes, and in what way HA perturbs the target membrane at low pH. We have taken a spectroscopic approach using an array of recently developed FTIR techniques to address these questions. Difference attenuated total reflection FTIR spectroscopy was employed to reveal reversible and irreversible components of the pH-induced conformational change of the membrane-bound bromelain fragment of HA, BHA. Additional proteolytic fragments of BHA were produced which permitted a tentative assignment of the observed changes to the HA1 and HA2 subunits, respectively. The membrane-bound HA1 subunit undergoes a reversible conformational change, which most likely involves the loss of a small proportion of beta-sheet at low pH. BHA was found to undergo a partially reversible tilting motion relative to the target membrane upon exposure to pH 5, indicating a previously undescribed hinge near the anchoring point to the target membrane. Time-resolved amide H/D exchange experiments revealed a more dynamic (tertiary) structure of membrane-bound BHA and its HA2, but not its HA1, subunit. Finally BHA and, to a lesser degree, HA1 perturbed the lipid bilayer of the target membrane at the interface, as assessed by spectral changes of the lipid ester carbonyl groups. These results are discussed in the context of a complementary study of HA that was bound to viral membranes through its transmembrane peptide (Gray C, Tamm LK, 1997, Protein Sci 6:1993-2006). A distinctive role for the HA1 subunit in the conformational change of HA becomes apparent from these combined studies.     

Conformational stability of serine proteinase inhibitor from the sea anemone Heteractis crispa

The influence of different environmental values of the pH and temperature on the spatial organization of serine proteinase inhibitor from the sea anemone Heteractis crispa (=Radianthus macrodactylus) on the level of tertiary and secondary structure was studied by CD spectroscopy. The molecule InhVJ was shown to possess a high conformational thermo- and pH-stability. We determined the point of conformational thermotransition of polypeptide (70 degrees C) after which the molecule gets denaturational stable state with conservation of 80% proteinase inhibitory activity. The significant partial reversible changes of molecule spatial organization were established to occur at the level of tertiary structure in the process of acid-base titration in the range of pH 11.0-13.0. This can be explained by of ionization of tyrosine residues. The molecule InhVJ is conformationally stable at the low pH values (2.0). The quenching of tyrosine residues by acrylamide showed that two of these residues are accessible to the quencher in full, while the third part is available.     

Structural studies on membrane-embedded influenza hemagglutinin and its fragments.

The mechanism of influenza virus hemagglutinin (HA)-mediated membrane fusion has been inferred in part from studies examining pH-induced structural changes in soluble HA derivatives lacking the viral membrane anchor and, sometimes, the fusion peptide (the C- and N-terminal residues of the HA2 chain, respectively). To reconcile structure-based mechanisms of HA-mediated membrane fusion with structural implications of functional studies performed on membrane-embedded HA, we have undertaken attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic analyses of membrane-embedded HA (strain X:31) and its fragments reconstituted into supported lipid bilayers. The fragments correspond to proteolytic products with the majority of the HA1 chain and, in some cases, the fusion peptide removed (THA2 and THA2F-, respectively). In combination with R18 fluorescence dequenching to monitor the functional implications of HA1 subunit removal, we have assessed the influence of pH and target membrane presentation on the secondary structures, orientations relative to the membrane, and dynamics of these molecules. We find that X:31 HA is more tilted towards the plane of the membrane under fusion than under resting conditions, that the fitting of HA depends on the presence of the HA1 chain, that the residues connecting the membrane-inserted fusion peptide with the crystallographically determined coiled coil probably adopt an alpha-helical conformation, and that several changes in the secondary structure and the amide H/D exchange kinetics occur as a result of acidification and target membrane presentation, which can be interpreted as small changes and a release of strain in the static and dynamic structure of membrane-bound HA. THA2 mediatcs fusion, but less efficiently and with less pH-selectivity than HA.     

Membrane binding of pH-sensitive influenza fusion peptides. positioning, configuration, and induced leakage in a lipid vesicle model

pH-sensitive HA2 fusion peptides from influenza virus hemagglutinin have potential as endosomal escape-inducing components in peptide-based drug delivery. Polarized light spectroscopy and tryptophan fluorescence were used to assess the conformation, orientation, effect on lipid order, and binding kinetics of wild-type peptide HA2(1-23) and a glutamic acid-enriched analogue (INF7) in large unilamellar POPC or POPC/POPG (4:1) lipid vesicles (LUVs). pH-sensitive membrane leakage was established for INF7 but not HA2(1-23) using an entrapped-dye assay. A correlation is indicated between leakage and a low degree of lipid chain order (assessed by linear dichroism, LD, of the membrane orientation probe retinoic acid). Both peptides display poor alignment in zwitterionic POPC LUVs compared to POPC/POPG (4:1) LUVs, and it was found that peptide-lipid interactions display slow kinetics (hours), resulting in reduced lipid order and increased tryptophan shielding. At pH 7.4, INF7 displays tryptophan emission and LD features indicative of a surface-orientated peptide, suggesting that its N-terminal glutamic acid residues prevent deep penetration into the hydrocarbon core. At pH 5.0, INF7 displays weaker LD signals, indicating poor orientation, possibly due to aggregation. By contrast, the orientation of the HA2(1-23) peptide backbone supports previously reported oblique insertion ( approximately 60-65 degrees relative to the membrane normal), and aromatic side-chain orientations are consistent with an interfacial (pH-independent) location of the C-terminus. We propose that a conformational change upon reduction of pH is limited to minor rearrangements of the peptide "hinge region" around Trp14 and repositioning of this residue.     

Anti-peptide antibodies detect steps in a protein conformational change: low-pH activation of the influenza virus hemagglutinin

At low pH, the hemagglutinin (HA) of influenza virus undergoes an irreversible conformational change that potentiates its essential membrane fusion function. We have probed the details of this conformational change using a panel of 14 anti-HA-peptide antibodies. Whereas some antibodies reacted equally well with both the neutral and low-pH HA conformations, others reacted to a significantly greater extent with the low-pH form. The locations of the peptides recognized by the latter antibodies in the three-dimensional HA structure indicated regions of the protein that change in response to low pH. Moreover, kinetic experiments suggested steps in the conformational change. In addition to their relevance to membrane fusion, our results show that anti-peptide antibodies can be used to study some types of biologically important protein conformational changes.     

Conformation of pepsin and pepsinogen: some aspects of the role of tyrosine residues and the 1-44 segment of pepsinogen on conformational stability

Conformational changes induced in pepsin and pepsinogen by iodination of tyrosine residues and the possible role of lysine residues on conformational stability of pepsinogen are investigated by circular dichroism (CD) studies in solution. At low degrees of iodination (6 I/molecule) the pepsin molecule denatured, with complete loss of β-structure at pH 5.5. Pepsinogen showed greater resistance to conformational change on iodination (10 I/molecule) and about 30% of its ordered structure is retained. In the aromatic region, the tyrosyl CD bands of iodinated pepsin decreased in intensity, indicating a change in the environment of tyrosine residues. A comparison with the CD spectra of expanded structures of pepsin in 6 m guanidine hydrochloride or alkaline solutions (pH 9.75) indicated retention of a significant amount of tertiary structure in iodinated pepsin. Changes in tertiary structures were marginal on iodination of pepsinogen. Less than 1% (residue moles) of poly-l-lysine, a known inhibitor, was found to destabilize the secondary and tertiary structure of pepsin at pH 6.75, although the lysine-rich 1–44 segment of pepsinogen tends to stabilize the conformation of the pepsin chain. This seems to suggest that the inhibitory effects of polylysine on pepsin occur by a mechanism different from that of the activity-limiting effect of the lysine-rich 1–44 segment of pepsinogen.     

Conformational stability of the serine protease inhibitor InhVJ from the sea anemone <Emphasis Type="Italic">Heteractis crispa</Emphasis>

The effect of temperature and pH of medium on the spatial organization of a molecule of the serine protease inhibitor InhVJ from the sea anemone Heteractis crispa (=Radianthus macrodactylus) at the level of the tertiary and secondary structures has been studied by CD spectroscopy. It has been shown that the conformation of an InhVJ molecule is highly stable to changes in temperature and pH. The point of the thermal conformational transition of the polypeptide (70°C) has been determined, after which the molecule turns into a denatured stable state with the retention of 80% of the inhibitory activity. It was found that significant, partially reversible changes in the spatial organization of the molecule occur on the level of the tertiary structure in the pH range 11.0–13.0, which may be explained by the ionization of tyrosine residues. At a low pH value (2.0), the InhVJ molecule is conformationally stable. The results of quenching of tyrosine residues by acrylamide showed that two residues are completely accessible for the quencher, whereas the third residue is partially accessible.     

Conformation ability test of human, rabbit and bovine plasminogens and their specific interaction with streptokinase.

Human, rabbit and bovine plasminogens, having different sensitivity to streptokinase-activating action, differ, according to spectrophotometric titration, tryptophan fluorescence and circular dichroism spectroscopy, in the state of tyrosine and tryptophan residues, and in secondary and tertiary structures. Human plasminogen-streptokinase equimolar complex formation (according to gel chromatography) is accompanied by a differential ultraviolet spectrum. Difference spectroscopy is a convenient and adequate means of studying the formation of the said complexes. Streptokinase-human plasminogen complex formation is not hindered by partial substitution of water (20%) with ethanol or dimethylsulphoxide or by addition of 0.001 M sodium dodecylsulphate. The complex is not formed in 6 M urea, in solution, at pH less than 2.0 or approximately 12.0-13.0, or with bovine plasminogen. Circular dichroism and tryptophan fluorescence spectral pattern changes during streptokinase-plasminogen complex formation enable us to conclude that streptokinase secondary and tertiary structures undergo certain rearrangements in the framework of the complex, while tryptophan-containing sites of the molecule are not drastically changed. The data obtained enable us to presuppose formation of streptokinase-rabbit plasminogen complexes which differ from human plasminogen complexes with streptokinase.     

The structure of human acidic fibroblast growth factor and its interaction with heparin.

The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo.     

Effect of an induced conformational change on the physical properties of two chemotactic receptor molecules.

The physical properties and conformational dynamics of the Salmonella typhimurium ribose and galactose receptors have been examined. Studies involving circular dichroism, fluorescence, absorption spectroscopy, and sedimentation analysis show that the two receptor proteins have different morphologies and exhibit diverse responses to sugar binding. The ribose receptor lacks both tryptophan and disulfide residues, and the galactose receptor lacks disulfides and has only a single tryptophan residue. By virtue of these fortuitous properties, the conformational changes induced in these proteins by sugar binding can be dissected by utilizing a variety of physical probes. A ligand-induced conformational change in the ribose receptor is shown by circular dichroism and fluorescence spectroscopy, which reveal spectral changes assignable to tyrosine, phenylalanine, and methionine residues. A conformational change in the galactose receptor has been demonstrated by fluorescence spectroscopy involving the distant reporter group method, which shows changes assignable to tryptophan and methionine sites and which is corroborated by sedimentation analysis. It is clear that there are extensive conformational changes in the two receptor proteins and that the different physical methods provide complementary information on the nature of these changes.     

Conformational change of influenza virus hemagglutinin is sensitive to ionic concentration

The homotrimeric spike glycoprotein hemagglutinin (HA) of influenza virus undergoes a low pH-mediated conformational change which mediates the fusion of the viral envelope with the target membrane. Previous approaches predict that the interplay of electrostatic interactions between and within HA subunits, HA 1 and HA2, are essential for the metastability of the HA ectodomain. Here, we show that suspension media of low ionic concentration promote fusion of fluorescent labelled influenza virus X31 with erythrocyte ghosts and with ganglioside containing liposomes. By measuring the low pH mediated inactivation of the fusion competence of HA and the Proteinase K sensitivity of low pH incubated HA we show that the conformational change is promoted by low ionic concentration. We surmise that electrostatic attraction within the HA ectodomain is weakened by lowering the ionic concentration facilitating the conformational change at low pH. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Membrane fusion activity of influenza virus.

A simple assay is described to monitor fusion between fowl plague virus (FPV, an avian influenza A virus) and liposomes which allows the simultaneous quantitation of both lytic and non-lytic fusion events. As in fusion between viruses and the plasma membrane and in FPV-induced cell-cell fusion, the reaction only occurs at pH 5.5 or below, and it is fast, highly efficient, and essentially non-lytic when fresh virus and liposomes are used. The fusion occurs over a broad temperature range, and has no requirement for divalent cations. The fusion factor of influenza virus is a hemagglutinin (HA) spike which protrudes from the virus membrane and which is also responsible for virus binding to the host cell. The finding that fusion occurs as efficiently with liposomes containing or lacking virus receptor structures, further emphasizes the remarkable division of labor in the HA molecule: the receptor-binding sites are located in the globular HA1 domains and the fusion activation peptide is found at the N-terminal of HA2 in the stem region of the protein. The mechanism of fusion is discussed in terms of the three-dimensional structure of the HA and the conformational change which the protein undergoes at the fusion pH optimum.     

Structural characterization of an early fusion intermediate of influenza virus hemagglutinin

The hemagglutinin (HA) envelope protein of influenza virus mediates viral entry through membrane fusion in the acidic environment of the endosome. Crystal structures of HA in pre- and postfusion states have laid the foundation for proposals for a general fusion mechanism for viral envelope proteins. The large-scale conformational rearrangement of HA at low pH is triggered by a loop-to-helix transition of an interhelical loop (B loop) within the fusion domain and is often referred to as the "spring-loaded" mechanism. Although the receptor-binding HA1 subunit is believed to act as a "clamp" to keep the B loop in its metastable prefusion state at neutral pH, the "pH sensors" that are responsible for the clamp release and the ensuing structural transitions have remained elusive. Here we identify a mutation in the HA2 fusion domain from the influenza virus H2 subtype that stabilizes the HA trimer in a prefusion-like state at and below fusogenic pH. Crystal structures of this putative early intermediate state reveal reorganization of ionic interactions at the HA1-HA2 interface at acidic pH and deformation of the HA1 membrane-distal domain. Along with neutralization of glutamate residues on the B loop, these changes cause a rotation of the B loop and solvent exposure of conserved phenylalanines, which are key residues at the trimer interface of the postfusion structure. Thus, our study reveals the possible initial structural event that leads to release of the B loop from its prefusion conformation, which is aided by unexpected structural changes within the membrane-distal HA1 domain at low pH.     

Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers.

The amino-terminal segment of the membrane-anchored subunit of influenza hemagglutinin (HA) plays a crucial role in membrane fusion and, hence, has been termed the fusion peptide. We have studied the secondary structure, orientation, and effects on the bilayer structure of synthetic peptides corresponding to the wild-type and several fusogenic and nonfusogenic mutants with altered N-termini of the influenza HA fusion peptide by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. All peptides contained segments of alpha-helical and beta-strand conformation. In the wild-type fusion peptide, 40% of all residues were in alpha-secondary and 30% in beta-secondary structures. By comparison, the nonfusogenic peptides exhibited larger beta/alpha secondary structure ratios. The order parameters of the helices and the amide carbonyl groups of the beta-strands of the wild-type fusion peptide were measured separately, based on the infrared dichroism of the respective absorption bands. Order parameters in the range 0.1-0.7 were found for both segments of the wild-type peptide, which indicates that they are most likely aligned at oblique angles to the membrane normal. The nonfusogenic but not the fusogenic peptides induced splitting of the infrared absorption band at 1735 cm(-1), which is assigned to stretching vibrations of the lipid ester carbonyl bond. This splitting, which reports on an alteration of the hydrogen bonds formed between the lipid ester carbonyls and water and/or hydrogen-donating groups of the fusion peptides, correlated with the beta/alpha ratio of the peptides, suggesting that unpaired beta-strands may replace water molecules and hydrogen-bond to the lipid ester carbonyl groups. The profound structural changes induced by single amino acid replacements at the extreme N-terminus of the fusion peptide further suggest that tertiary or quaternary structural interactions may be important when fusion peptides bind to lipid bilayers.     

Early steps of the conformational change of influenza virus hemagglutinin to a fusion active state: stability and energetics of the hemagglutinin

A conformational change of the homotrimeric glycoprotein hemagglutinin (HA) of influenza virus mediates fusion between the viral envelope and the endosome membrane. The conformational change of the HA ectodomain is triggered by the acidic pH of the endosome lumen. An essential step of the conformational change is the formation of an extended coiled-coil motif exposing the hydrophobic fusion peptide toward the target membrane. The structures of the neutral-pH, non-fusion active conformation of the HA ectodomain and of a fragment of the ectodomain containing the coiled-coil motif are known. However, it is not known by which mechanism protonation triggers the conformational change of the stable neutral-pH conformation of the ectodomain. Here, recent studies on the stability of the HA ectodomain at neutral pH, the energetics of the conformational change toward the fusion-active state and of the unfolding of the HA ectodomain are summarised. A model for the early steps of the conformational change of the HA ectodomain is presented. The model implicates that protonation leads to a partial dissociation of the distal domains of the HA monomers that is driven by electrostatic repulsion. The opening of the ectodomain enables water to enter the ectodomain. The interaction of water with respective sequences originally shielded from contact with water drives the formation of the coiled-coil structure.     

Characterization of a partially folded intermediate of stem bromelain at low pH.

Equilibrium studies on the acid included denaturation of stem bromelain (EC 3.4.22.32) were performed by CD spectroscopy, fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino 8-naphthalene sulfonic acid (ANS). At pH 2.0, stem bromelain lacks a well defined tertiary structure as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of some native like secondary structure at pH 2.0. The mean residue ellipticities at 208 nm plotted against pH showed a transition around pH 4.5 with loss of secondary structure leading to the formation of an acid-unfolded state. With further decrease in pH, this unfolded state regains most of its secondary structure. At pH 2.0, stem bromelain exists as a partially folded intermediate containing about 42.2% of the native state secondary structure Enhanced binding of ANS was observed in this state compared to the native folded state at neutral pH or completely unfolded state in the presence of 6 m GdnHCl indicating the exposure of hydrophobic regions on the protein molecule. Acrylamide quenching of the intrinsic tryptophan residues in the protein molecule showed that at pH 2.0 the protein is in an unfolded conformation with more tryptophan residues exposed to the solvent as compared to the native conformation at neutral pH. Interestingly, stem bromelain at pH 0.8 exhibits some characteristics of a molten globule, such as an enhanced ability to bind the fluorescent probe as well as considerable retention of secondary structure. All the above data taken together suggest the existence of a partially folded intermediate state under low pH conditions.     

Tryptophan residues at subunit interfaces used as fluorescence probes to investigate homotropic and heterotropic regulation of aspartate transcarbamylase

The homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) are accompanied by various structure modifications. The large quaternary structure change associated with the T to R transition, promoted by substrate binding, is accompanied by different local conformational changes. These tertiary structure modifications can be monitored by fluorescence spectroscopy, after introduction of a tryptophan fluorescence probe at the site of investigation. To relate unambiguously the fluorescence signals to structure changes in a particular region, both naturally occurring Trp residues in positions 209c and 284c of the catalytic chains were previously substituted with Phe residues. The regions of interest were the so-called 240's loop at position Tyr240c, which undergoes a large conformational change upon substrate binding, and the interface between the catalytic and regulatory chains in positions Asn153r and Phe145r supposed to play a role in the different regulatory processes. Each of these tryptophan residues presents a complex fluorescence decay with three to four independent lifetimes, suggesting that the holoenzyme exists in slightly different conformational states. The bisubstrate analogue N-phosphonacetyl-L-aspartate affects mostly the environment of tryptophans at position 240c and 145r, and the fluorescence signals were related to ligand binding and the quaternary structure transition, respectively. The binding of the nucleotide activator ATP slightly affects the distribution of the conformational substates as probed by tryptophan residues at position 240c and 145r, whereas the inhibitor CTP modifies the position of the C-terminal residues as reflected by the fluorescence properties of Trp153r. These results are discussed in correlation with earlier mutagenesis studies and mechanisms of the enzyme allosteric regulation.