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Showing papers on "Conformational change published in 1986"


Journal ArticleDOI
TL;DR: The structure of sugar-Induced Conformational Change in Proteins and its effects on Binding Sites and Bonds and van der Waals Contacts are illustrated.
Abstract: PERSPECTIVES AND SUMMARY . 287 TERTIARY STRUCTURES 288 PROTEIN-SUGAR INTERACTIONS 292 Locations of Sugar-Binding Sites in Proteins 294 Hydrogen Bonds and van der Waals Contacts , 300 Oligosaccharide Binding ... 310 Sugar-Induced Conformational Change 312 CONCLUDING REMARKS 313

466 citations


Journal ArticleDOI
TL;DR: It is shown that the human p21N-ras protein, isolated from an Escherichia coli expression system, can exist as a stable GDP complex which exchanges very slowly with exogenous GTP, but in low Mg2+ the exchange rate is dramatically increased, and the relative binding affinity of the protein for GTP as compared to GDP is increased 10-fold.

203 citations


Journal ArticleDOI
TL;DR: A tentative model of the change is proposed, with reference to the known X‐ray structure at neutral pH, in which major changes occur in the stem tertiary structure, while the top portion is only affected in its quaternary structure.
Abstract: Influenza virus haemagglutinin mediates infection of cells by fusion of viral and endosomal membranes, triggered by low pH which induces a conformational change in the protein. We report studies of this change by electron microscopy, neutron scattering, sedimentation and photon correlation on X-31 (H3N2) haemagglutinin, both intact and bromelain cleaved, in various assemblies. HAs in all preparations showed a thinning at low pH, and a marked elongation which was removed on tryptic digestion, revealing altered features in the remaining stem portion of the molecule. A tentative model of the change is proposed, with reference to the known X-ray structure at neutral pH, in which major changes occur in the stem tertiary structure, while the top portion is only affected in its quaternary structure.

139 citations


Journal ArticleDOI
TL;DR: In this paper, the authors studied the conformational behavior of α-lactalbumin (α-LACT) in hydrophobic interaction chromatography and derived the relationship of Z to other slopes, such as S (slope of the plot of 1n k'vs. o B f).

132 citations


Journal ArticleDOI
TL;DR: An ordered addition, essential activation model in which the enzyme undergoes two conformational transformations: one as a consequence of binding to tissue factor, resulting in a species which binds to and hydrolyzes its natural substrates, and the other which is induced by substrate.
Abstract: One way in which coagulation may be initiated is by the action of factor VIIa (a plasma serine protease) and tissue factor (a membrane-bound lipid-dependent glycoprotein). We show that in the absence of either factor VIIa or tissue factor, the activation of the natural coagulation substrates, factors IX and X, is not detectable; i.e., tissue factor is an essential activator. We propose that the reaction is fully ordered; that is, the enzyme-activator complex picks up substrate to form a ternary product forming species. Our model precludes the formation of enzyme-substrate and activator-substrate complexes. We have derived equations for the two possible variations of this model: one in which product formation is accompanied by the release of the enzyme-activator complex and the other in which product, free enzyme, and free activator are formed with each catalytic cycle. Our data support only the former which is consistent with both steady-state and rapid equilibrium assumptions. The model is supported by experiments using a monoclonal anti-tissue factor antibody, which affects only the Km app, and a modified form of factor VIIa, which, depending on the sequence in which reagents are added to the reaction, either decreases the Vmax or increases the Km app. We present equations describing the initial velocity of these reactions. Utilizing dilution-jump experiments, we show that the system is hysteretic and suggest that this phenomenon is due to a slow release of enzyme from activator. However, the kinetically determined dissociation constant of enzyme and activator, previously found to be 4.5 nM under equilibrium conditions, was estimated to be 0.04-0.09 nM. Accordingly, we examined other essential activation models in which the product-forming species consists of a complex of enzyme, activator, and substrate at a molar ratio of 1:1:1; none could account for the apparent tight binding of enzyme and activator. We therefore postulate an ordered addition, essential activation model in which the enzyme undergoes two conformational transformations: one as a consequence of binding to tissue factor, resulting in a species which binds to and hydrolyzes its natural substrates. The other conformational change in the enzyme is induced by substrate, resulting in a species which binds more tightly to its activator. Thus, we hypothesize a "conformational cage" which precludes the dissociation of enzyme from activator while significant concentrations of substrate are present.

123 citations


Journal ArticleDOI
TL;DR: The effects of the cation bound to the high-affinity binding site must, therefore, be considered in quantitatively analyzing the kinetics of polymerization of NBD-labeled actin and pyrenyl-labeling actin.

97 citations


Journal ArticleDOI
TL;DR: It is shown that the results obtained with different alcohols can be normalized by the use of a dielectric constant scale and this normalization corrects for the different molar volumes of differentcohols, allows comparison of results obtaining with differentalcohols, and should be useful in studying this phenomenon with different proteins.

97 citations


Journal ArticleDOI
TL;DR: Dynamic fluorescence measurements showed that incubating calcium at high S100a protein concentrations induces an apparent slow dimer-monomer equilibrium which might result in total subunit dissociation at lower protein concentrations, and confirmed that only the alpha-subunit exposes hydrophobic domains to solvent in the presence of calcium.

74 citations


Journal ArticleDOI
TL;DR: Hydrophobic labeling of Ca-ATPase in sarcoplasmic reticulum vesicles with the photoactivable reagent trifluoromethyl-[125I]iodophenyl-diazirine indicated that E2 and E2V states are more exposed to the membrane phase than E1 and E1P (Ca2+-occluded) states.
Abstract: Tryptic peptides of Ca-ATPase in Et and E2 conformational states (Andersen, J. P., Jorgensen, P. L.,J. Membrane Biol.88:187–198 (1985)) have been isolated by size exclusion high performance liquid chromatography in sodium dodecyl sulfate. This permitted unambiguous localization of a conformational sensitive tryptic split at Arg 198 by N-terminal amino acid sequence analysis. Other splits at Arg 505 and at Arg 819-Lys 825 were insensitive to E1–E2 transitions. Tryptic cleavage of Ca-ATPase after phosphorylation by inorganic phosphate showed that this enzyme form has a conformation similar to that of the vanadate-bound E2 state, both in membranous and in soluble monomeric Ca-ATPase.

58 citations


Journal ArticleDOI
17 Jul 1986-Nature
TL;DR: Electron transfer in cytochrome c is directional, with the protein envelope presumably involved in this directionality, and the question of whether electron transfer or protein conformational change is the rate limiting step in this process is raised.
Abstract: Cytochrome c can be modified by [(NH3)5RuII/III-] specifically at the imidazole moiety of histidine 33, and we have recently discussed the thermodynamics and kinetics of electron transfer within this modified protein1–5. X-ray crystal structures of the oxidized and reduced forms of tuna cytochrome c6 indicate that the separation between the haem group of cytochrome c and the ruthenium label is 12–16 A. Internal electron transfer from the [(NH3)5RuII-] centre to the Fe(III) haem centre occurs with a rate constant K ≃ 53s−1 (25 °C) (ΔH‡=3.5kcal mol−1, ΔS‡ = −39EU), as measured by pulse radiolysis. The measured unimolecular rate constant1, k ≃ 53 s−1, is on the same timescale as a number of conformational changes that occur within the cytochrome c molecule7–9. These results raise the question of whether electron transfer or protein conformational change is the rate limiting step in this process. We describe here an experiment that probes this intramolecular electron transfer step further. It involves reversing the direction of electron transfer by changing the redox potential of the ruthenium label. Electron transfer in the new ruthenium–cytochrome c derivative described here is from haem(II) to the RU(III) label, whereas in (NH3)5Ru–cytochrome c the electron transfer is from RU(II) to haem(III). Intramolecular electron transfer from haem(II) to RU(III) in the new ruthenium–cytochrome c described here proceeds much slower (> 105 times) than the electron transfer from Ru(II) to haem(III) in the (NH3)5Ru–cytochrome c. We therefore conclude that electron transfer in cytochrome c is directional, with the protein envelope presumably involved in this directionality.

56 citations


Journal ArticleDOI
TL;DR: There are a number of independent conformational changes that occur within the monomeric ER steroid-binding subunit upon ligand binding and exposure to elevated temperature in vitro, demonstrating that there are significant differences in the transformation process for receptors bound to 4-OH-tamoxifen versus estrogenic ligands.

Journal ArticleDOI
TL;DR: Results show that Nha 10, a short neurotoxin with weak neurotoxicity, causes no fluorescence change in the receptor but can still bind to the receptor with sufficiently high affinity, and the substitution of the conserved residue Asp-31 to Gly-31 in Nha is probably responsible for the reduced neurotoxicity.
Abstract: Acetylcholine receptor from Narke japonica electroplax exhibits a fluorescence change upon binding of snake neurotoxins. This fluorescence change primarily arises from the conformational change of the acetylcholine receptor and reflects the binding process of the toxin with the receptor. The time dependence of the fluorescence change has been monitored for 28 short neurotoxins and 8 long neurotoxins by using a stopped-flow technique. The steady-state fluorescence change is of the same order of magnitude for the short neurotoxins but varies among the long neurotoxins. Nha 10, a short neurotoxin with weak neurotoxicity, causes no fluorescence change in the receptor but can still bind to the receptor with sufficiently high affinity. The substitution of the conserved residue Asp-31 to Gly-31 in Nha is probably responsible for the reduced neurotoxicity. The rate constants for the binding of the neurotoxins to the receptor have been obtained by analyzing the transient fluorescence change. The rate constants show surprisingly a wide range of distribution: (1.0-20.5) X 10(6) M-1 s-1 for short neurotoxins and (0.26-1.9) X 10(6) M-1 s-1 for long neurotoxins. Examination of the relationship between the rate constants of fluorescence change of the short neurotoxins and their amino acid sequences, thermal stability, hydrogen-deuterium exchange behavior, overall net charge, etc. reveals the following. Positive charges on the side chains of residues 27 and 30 and overall net charge of the neurotoxin govern the magnitude of the binding rate of the neurotoxin with the receptor.

Journal ArticleDOI
TL;DR: The conformational change induced by Mg2+ and cytochalasin D precedes the formation of an actin dimer and direct binding studies show that actin which has not been labeled with the fluorophore binds cytochasin D more tightly.

Journal ArticleDOI
TL;DR: The slow increase of a cyanide‐induced optical change at 437 nm following rapid cyanide inhibition of cytochrome oxidase has been followed as a function of the number of electrons donated from ferrocytochrome c to cy tochrome a and Cu a .

Journal ArticleDOI
TL;DR: Five monoclonal antibodies recognizing five different epitopes of the native beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.2.1.20) were used to analyze the conformational changes occurring upon ligand binding or chemical modifications of the enzyme.
Abstract: Five monoclonal antibodies recognizing five different epitopes of the native beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.1.2.20) were used to analyze the conformational changes occurring upon ligand binding or chemical modifications of the enzyme. For this purpose, the affinities of each antibody for the different forms of the enzyme were determined by using an enzyme-linked immunosorbent assay which allows measurement of the dissociation constant of antigen-antibody equilibrium in solution. The fixation of the coenzyme pyridoxal 5'-phosphate and the substrate L-serine modifies the affinity constants of most of the antibodies for the enzyme, thus showing the existence of extended conformational rearrangements of the protein. The association of the alpha subunit with the beta 2 subunit, which brings about an increase of the tryptophan synthase activity and abolishes the serine deaminase activity of beta 2, is accompanied by an important conformational change of the N-terminal domain of beta 2 (F1) since none of the anti-F1 monoclonal antibodies can bind to alpha 2 beta 2. Similarly, chemical modifications of beta 2 which are known to produce significant effects on the enzymatic activities of beta 2 result in changes of the affinities of the monoclonal antibodies which can be interpreted as the acquisition of different conformational states of the enzyme.

Journal ArticleDOI
TL;DR: The conclusion was drawn that a polymer material that selectively adsorbs bovine serum albumin in a native state could be antithrombogenic.
Abstract: Polyetherurethaneureas carrying tertiary amino groups in the side chains were synthesized, quaternized, and heparinized. The adsorption of plasma proteins to the polyurethane derivatives was investigated using Fourier-transform infrared, surface fluorescence, ultraviolet, and circular dichroism spectroscopy. Bovine gamma-globulin and bovine plasma fibrinogen were easily adsorbed to hydrophobic polyurethanes and denatured. It was found that the beta structure was generated during the irreversible conformational change of the proteins on adsorption to hydrophobic polyurethane materials. On the other hand, bovine serum albumin was easily adsorbed to hydrophilic quaternized and heparinized polyurethanes without a serious conformational change. The conclusion was drawn that a polymer material that selectively adsorbs bovine serum albumin in a native state could be antithrombogenic.

Journal ArticleDOI
TL;DR: The assumption that monoclonal antibodies, which failed to inhibit hemagglutination of the virus yet neutralized viral infectivity, inhibited the fusion step in the viral replication process by interfering with a low pH-induced conformational change in the HA molecule is supported.

Journal ArticleDOI
TL;DR: The nonlinear increase of the elongation rate of actin filaments above the critical monomer concentration was investigated by nucleated polymerization of actIn, and the conformational change model turned out to be the simplest assembly mechanism by which all available experimental data could be explained.
Abstract: The nonlinear increase of the elongation rate of actin filaments above the critical monomer concentration was investigated by nucleated polymerization of actin. Significant deviations from linearity were observed when actin was polymerized in the presence of magnesium ions. When magnesium ions were replaced by potassium or calcium ions, no deviations from linearity could be detected. The nonlinearity was analyzed by two simple assembly mechanisms. In the first model, if the ATP hydrolysis by polymeric actin is approximately as fast as the incorporation of monomers into filaments, terminal subunits of lengthening filaments are expected to carry to some extent ADP. As ADP-containing subunits dissociate from the ends of actin filaments faster than ATP-containing subunits, the rate of elongation of actin filaments would be nonlinearly correlated with the monomer concentration. In the second model (conformational change model), actin monomers and filament subunits were assumed to occur in two conformations. The association and dissociation rates of actin molecules in the two conformations were thought to be different. The equilibrium distribution between the two conformations was assumed to be different for monomers and filament subunits. The ATP hydrolysis was thought to lag behind polymerization and conformational change. As under the experimental conditions the rate of ATP hydrolysis by polymeric actin was independent of the concentration of filament ends, the observed nonlinear increase of the rate of elongation with the monomer concentration above the critical monomer concentration was unlikely to be caused by ATP hydrolysis at the terminal subunits. The conformational change model turned out to be the simplest assembly mechanism by which all available experimental data could be explained.

Journal ArticleDOI
TL;DR: Spectral correlations are given which strongly support the postulation of a metal-bound alkoxide in the closed structure of the enzyme as an essential feature of the catalytic mechanism of horse liver alcohol dehydrogenase.
Abstract: The conformational change of horse liver alcohol dehydrogenase induced by binding of NAD+ was studied by electronic absorption spectroscopy using cobalt as a spectroscopic probe in the active site. The complex of the enzyme with NAD+ exists in an acidic and an alkaline form. The transition between the two forms proceeds through several intermediates and is controlled by an apparent pKa of 6.9. Only at pH values below this pKa can a complex between enzyme, NAD+, and Cl- be formed. The spectral changes indicate that chloride displaces the cobalt-bound water molecule in a tetracoordinate structure. We conclude that a negative charge at the active site is necessary to stabilize the closed conformation of the enzyme in the presence of NAD+. Spectral correlations are given which strongly support the postulation of a metal-bound alkoxide in the closed structure of the enzyme as an essential feature of the catalytic mechanism of horse liver alcohol dehydrogenase.

Journal ArticleDOI
TL;DR: Deuterated phosphatidylcholine used here was proved to be more suitable for the direct detection of the amount of the trans conformation by Raman spectroscopy than the nondeuterated one, and spectra of the deuterated compound in the gel and liquid-crystalline states confirmed that the trans Conformation of the choline group does not appear at all in both states.

Journal ArticleDOI
TL;DR: Thermodynamic studies indicated that the aflatoxin-albumin interaction was exothermic (delta H = -29.3 kJ X mol-1), with a delta S value of -13.8 J Xmol-1 X K-1.

Journal ArticleDOI
TL;DR: After purification from extracts of whole cells, M1 RNA, the catalytic subunit of ribonuclease P from Escherichia coli, apparently must undergo a change in conformation before it can function catalytically.
Abstract: After purification from extracts of whole cells, M1 RNA, the catalytic subunit of ribonuclease P from Escherichia coli, apparently must undergo a change in conformation before it can function catalytically. The rate of this conformational change is dependent upon the duration of incubation at various temperatures and pH. delta E of the transition at pH 7.5 is approximately 36 kcal/mol. The change in conformation is not sensitive to Mg2+ concentration between 10 and 100 mM. A decrease in A260 of M1 RNA in solution has been observed during the incubation period that potentiates the conformational change at 30 degrees C, but no direct correlation can yet be made to specific structural rearrangements.

Journal ArticleDOI
TL;DR: Steady-state fluorescence anisotropy measurements indicate that the loss of Ca2+ leads to a significant increase in internal mobility of previously buried tryptophan residues.

Journal ArticleDOI
TL;DR: Both alpha and beta subunits of the soluble 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole-modified enzyme were labeled by the fluorescent thiol reagents, suggesting that the soluble enzyme undergoes a conformational change on binding to the membrane.
Abstract: The sulfhydryl groups of soluble and membrane-bound F1 adenosine triphosphatase of Escherichia coli were modified by reaction with the fluorescent thiol reagents 5-iodoacetamidofluorescein, 2-[(4′-iodoacetamido)anilino]naphthalene-6-sulfonic acid, 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-diazole and 2-[(4′-maleimidyl)anilino]naphthalene-6-sulfonic acid. Whereas γ and δ subunits were always labeled by these reagents, the β subunit reacted preferentially in the soluble enzmye, and the α subunit in the membrane-bound enzyme. This suggests that the soluble enzyme undergoes a conformational change on binding to the membrane. The three β subunits of the soluble ATPase did not react with chemical reagents in a similar manner. One β subunit was cross-linked to the ɛ subunit on treatment of the ATPase with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide, as observed previously by Lotscher et al. [Biochemistry (1984) 23, 4134–4140]. A second β subunit, which did not cross-link to the ɛ subunit, was modified preferentially by the fluorescent thiol reagents and by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The third β subunit was less chemically reactive than the others. Both α and β subunits of the soluble 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole-modified enzyme were labeled by the fluorescent thiol reagents. Thus, the modified enzyme, which is inactive, probably has a different conformation from the native soluble ATPase.

Journal ArticleDOI
TL;DR: It was concluded that the enzyme assumes a highly anionic form in the presence of a low GSH concentration, whereas it is converted into relatively weaker anionic shape when its immediate environment contains a high GSH concentrations.
Abstract: Glutathione transferase (GST) from human placenta was purified by affinity chromatography and anion-exchange h.p.l.c. The enzyme exhibited different chromatographic and electrophoretic behaviours according to the concentration of GSH, suggesting a possible change in the net charge of the molecule and a concomitant conformational change due to ligand binding. Two interconvertible forms were quantitatively separated into distinct catalytically active states by h.p.l.c. Depending upon the GSH concentration, polyacrylamide-gel electrophoresis revealed the presence of one or two bands. A Kd of 0.42 mM for GSH was determined fluorimetrically. The loss in intrinsic fluorescence also suggested a conformational change in the enzyme. Kinetic studies using ethacrynic acid were conducted to determine whether the presumed conformational change could effect the catalytic capability of placental GST. A biphasic response in initial velocities was observed with increasing concentrations of GSH. Two apparent Km values of 0.38 and 50.27 mM were obtained for GSH, whereas Vmax. values showed a 46-fold difference. It was concluded that the enzyme assumes a highly anionic form in the presence of a low GSH concentration, whereas it is converted into relatively weaker anionic form when its immediate environment contains a high GSH concentration. Since the average tissue concentration of total GSH was estimated at 0.11 mM for term placenta, the results suggest that the high-affinity-low-activity conformer would predominate in vivo.

Journal ArticleDOI
TL;DR: A photochemically induced dynamic nuclear polarization study of yeast and horse muscle phosphoglycerate kinase with flavin dyes was undertaken to identify the histidine, tryptophan, and tyrosine resonances in the aromatic region of the simplified 1H NMR spectra of these enzymes and to investigate the effect of substrates on the resonances observable by CIDNP.
Abstract: A photochemically induced dynamic nuclear polarization (photo-CIDNP) study of yeast and horse muscle phosphoglycerate kinase with flavin dyes was undertaken to identify the histidine, tryptophan, and tyrosine resonances in the aromatic region of the simplified /sup 1/H NMR spectra of these enzymes and to investigate the effect of substrates on the resonances observable by CIDNP. Identification of the CIDNP-enhanced resonances with respect to the type of amino acid residue has been achieved since only tyrosine yields emission peaks and the dye 8-aminoriboflavin enhances tryptophan but not histidine. By use of the known amino acid sequences and structures derived from X-ray crystallographic studies of the enzymes from the two species, assignment of the specific residues in the protein sequences giving rise to the CIDNP spectra was partially achieved. In addition, flavin dye accessibility was used to probe any changes in enzyme structure induced by substrate binding. The accessibility of a tyrosine to photoexcited flavin is reduced in the presence of MgATP. Since the tyrosine residues are located some distance from the MgATP binding site of the catalytic center, it is proposed either that this change is due to a distant conformational change or that a second metal-ATP site inferred from othermore » studies lies close to one of the tyrosines. Horse muscle phosphoglycerate kinase exhibits seven resonances by CIDNP NMR. The addition of 3-phosphoglycerate and MgATP results in the appearance of two additional resonances in the CIDNP spectrum due to a histidine residue that is inaccessible to flavin in both the enzyme alone and its binary complex with 3-phosphoglycerate. The CIDNP spectra are consistent with the suggestions that binding of 3-phosphoglycerate alone is insufficient to effect domain movement and that binding of both substrates are required for conversion of the horse muscle enzyme to its catalytically active form.« less

Journal ArticleDOI
TL;DR: Adding Zn(II) to the copper enzyme reveals a slow exchange process that correlates with an increase in peptidase activity and with changes in the Cu(II)'s electron paramagnetic resonance spectra.
Abstract: Cu(II)-substituted carboxypeptidase A catalyzes the hydrolysis of oligopeptides and their depsipeptide (ester) analogues. Stopped-flow fluorescence assays demonstrate that relative to the zinc enzyme the Cu enzyme can have kcat/Km values up to 24% toward esters but only up to 2.5% toward the corresponding peptides. Adding Zn(II) to the copper enzyme reveals a slow exchange process that correlates with an increase in peptidase activity and with changes in the Cu(II) electron paramagnetic resonance spectra. Low concentrations of 1,10-phenanthroline (OP) (0.1-2.5 microM) markedly increase activity toward furanacryloyl-Phe-Phe (up to 8% of the zinc enzyme), but higher concentrations inhibit, resulting in complete inhibition at 0.8 mM OP. The non-metal-binding, hydrophobic analogues m- and p-phenanthroline are only activators of peptide hydrolysis, even at 1 mM. Activation is likely due to a modifier binding to a hydrophobic locus and either displacing an inhibitory peptide binding mode or inducing a conformational change in the active site.

Journal ArticleDOI
TL;DR: It is decided that binding of colchicine to tubulin induces a conformational change, which affects the accessibility of the most reactive SH-groups, and sterically isolated from the binding sites for GTP, Mg(II), Zn(II) and the two essential SH- groups.

Journal ArticleDOI
TL;DR: It is suggested that F1-ATPase exists in two temperature dependent conformational states to which different catalytic properties may be assigned.

Journal ArticleDOI
TL;DR: The heme peripheral modification resulted in the preferential downfield shift of the proximal histidine N1H signal for the beta subunit, indicating nonequivalence of the structural change induced by the heme modification in the alpha and beta subunits of Hb.
Abstract: The effect of heme modification on the tertiary and quaternary structures of hemoglobins was examined by utilizing the NMR spectra of the reconstituted [mesohemoglobin (mesoHb), deuterohemoglobin (deuteroHb)] and hybrid heme (meso-proto, deutero-proto) hemoglobins (Hbs). The heme peripheral modification resulted in the preferential downfield shift of the proximal histidine N1H signal for the beta subunit, indicating nonequivalence of the structural change induced by the heme modification in the alpha and beta subunits of Hb. In the reconstituted and hybrid heme Hbs, the exchangeable proton resonances due to the intra- and intersubunit hydrogen bonds, which have been used as the oxy and deoxy quaternary structural probes, were shifted by 0.2-0.3 ppm from that of native Hb upon the beta-heme substitution. This suggests that, in the fully deoxygenated form, the quaternary structure of the reconstituted Hbs is in an "imperfect" T state in which the hydrogen bonds located at the subunit interface are slightly distorted by the conformational change of the beta subunit. Moreover, the two heme orientations are found in the alpha subunit of deuteroHb, but not in the beta subunit of deuteroHb, and in both the alpha and beta subunits of mesoHb. The tertiary and quaternary structural changes in the Hb molecule induced by the heme peripheral modification were also discussed in relation to their functional properties.