Evolution of allosteric models for hemoglobin

We compare various allosteric models that have been proposed to explain cooperative oxygen binding to hemoglobin, including the two-state allosteric model of Monod, Wyman, and Changeux (MWC), the Cooperon model of Brunori, the model of Szabo and Karplus (SK) based on the stereochemical mechanism of Perutz, the generalization of the SK model by Lee and Karplus (SKL), and the Tertiary Two-State (TTS) model of Henry, Bettati, Hofrichter and Eaton. The preponderance of experimental evidence favors the TTS model which postulates an equilibrium between high (r)- and low (t)-affinity tertiary conformations that are present in both the T and R quaternary structures. Cooperative oxygenation in this model arises from the shift of T to R, as in MWC, but with a significant population of both r and t conformations in the liganded T and in the unliganded R quaternary structures. The TTS model may be considered a combination of the SK and SKL models, and these models provide a framework for a structural interpretation of the TTS parameters. The most compelling evidence in favor of the TTS model is the nanosecond - millisecond carbon monoxide (CO) rebinding kinetics in photodissociation experiments on hemoglobin encapsulated in silica gels. The polymeric network of the gel prevents any tertiary or quaternary conformational changes on the sub-second time scale, thereby permitting the subunit conformations prior to CO photodissociation to be determined from their ligand rebinding kinetics. These experiments show that a large fraction of liganded subunits in the T quaternary structure have the same functional conformation as liganded subunits in the R quaternary structure, an experimental finding inconsistent with the MWC, Cooperon, SK, and SKL models, but readily explained by the TTS model as rebinding to r subunits in T. We propose an additional experiment to test another key prediction of the TTS model, namely that a fraction of subunits in the unliganded R quaternary structure has the same functional conformation (t) as unliganded subunits in the T quaternary structure.     

Hemoglobin tertiary structural change on ligand binding its role in the co-operative mechanism

Analysis of the tertiary structural alterations in hemoglobin induced by ligand binding demonstrates that an allosteric core composed of the heme, histidine F8, the FG corner and part of the F-helix plays an essential role in co-operativity. This conclusion is based on structural and spectroscopic data and theoretical studies of hemoglobin chains. The methodology employed in the calculations is presented with details of the empirical energy function. Energy minimized structures of the unliganded hemoglobin chains, which serve as reference systems for the analysis, are described. To determine the structural changes induced by ligand binding, the effects of FeN bond shortening and of heme translation and tilting perturbations are examined. Energy minimization in the presence of the perturbations serves to provide information concerning the globin structural modifications produced by them. The validity of the results is supported by comparisons with the X-ray data of Anderson, Pulsinelli, Baldwin and Chothia on tertiary changes in the hemoglobin subunits.Internal to the allosteric core, there appear to be two stable positions for its elements: one of these corresponds to the liganded and the other to the unliganded species. The unliganded geometry fits without strain into the deoxy tetramer, while the liganded one fits without strain into the oxy tetramer. On ligation of a subunit in the deoxy tetramer, the structural changes within the allosteric core are in the direction of those found in going from the unliganded deoxy to the liganded oxy system, although they are reduced by the presence of constraints due to the other subunits in the deoxy tetramer. In addition, the quaternary constraints in the deoxy tetramer prevent the large overall displacement of the allosteric core that occurs in the transition to the liganded oxy tetramer. The coupling between the changes internal to the allosteric core, produced on ligation and the overall displacement of the core that accompanies the quaternary transition, is an essential element of the co-operative mechanism. As shown in previous work (Gelin & Karplus, 1977), the proximal histidine serves as the link between the position of the heme and the F-helix; the asymmetric orientation of the histidine in the deoxy structure, coupled with contributions from other heme-protein interactions, appears to initiate the tertiary structural changes induced by ligand binding. The reduced oxygen affinity of hemoglobin results not from tension on the heme in the unliganded structure (there is none) but instead from strain in the liganded subunit of the tetramer within the deoxy quaternary structure. Further, the changes in the allosteric core provide a relatively localized reaction path for transmitting information concerning ligand binding from the heme group to the surface of the subunit; particularly in the α-chain, the residue Val FG5 appears to play an important role in the reaction path.The present analysis has important implications for realistic statistical thermodynamic models of hemoglobin co-operativity. It suggests that the previously formulated model (Szabo & Karplus, 1972) should be generalized by the introduction of two different subunit tertiary structures in the deoxy and in the oxy tetramer; they would be associated with the unliganded and the liganded allosteric core, respectively, and would take account of steric constraints that reduce the ligand affinity of the deoxy tetramer.     

Properties of human hemoglobin immobilized on Sepharose 4B.

This paper reports the properties of human hemoglobin covalently bound to Sepharose 4B both in 'high-affinity' and 'low-affinity' conformations. The results suggest that the coupling reaction is strongly affected by the conformational changes linked to oxygenation of the protein. The rate and the extent of the reaction are different for the oxy and deoxyderivatives, probably due to the change in reactivity of the amino groups in the liganded and unliganded tetramer. The data on the equilibrium which is established between matrix-bound and soluble subunits, measured by the 'subunit-exchange chromatography', indicate that the system displays a minimal heterogeneity when hemoglobin is coupled to the gel in the deoxy state at intermediate protein concentration and pH 8. Maxtrix-bound hemoglobin is characterized by a higher oxygen affinity and by decreased homotropic and heterotropic interactions with respect to hemoglobin in solution, but the changes depend strongly on the conditions used in the coupling procedure.     

Ligand-induced conformational changes in spin label-modified human hemoglobins and chains and their carboxypeptidase A-digested derivatives.

The reactive sulfhydryls of human adult and fetal hemoglobin and the single sulfhydryl of isolated gamma chains have been spin labeled with N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) iodoacetamide. Similar electron paramagnetic spectral differences between oxy- and deoxy-modified hemoglobins were observed for both these hemoglobins and for the isolated chains, indicating that ligand-induced conformational changes occur in isolated hemoglobin subunits as well as intact hemoglobin tetramers. Ligand induced changes in the reactivity of p-hydroxymercuribenzoate with the sulfhydryl groups of both intact hemoglobins and isolated subunits, observed by McDonald and Noble (1974) J. Biol. Chem. 249, 3161-3165), led them to draw a similar conclusion. Following carboxypeptidase A digestion of these modified hemoglobins and gamma chains, a procedure which specifically removes the two C-terminal residues of the beta or gamma chains, spectral differences between the liganded and unliganded spin-labeled derivatives still persisted. However, the magnitude of this difference was not only more reduced in the case of the hemoglobins than in that of the subunits but the spectra of both the oxy and deoxy derivatives of the hemoglobins were characteristic of the oxy derivative of a cooperative tetrameric hemoglobin. These findings support the premise that the COOH-terminal end of the beta or gamma chain contributes, although possibly to different extents, to the spectral differences exhibited by both the spin-labeled hemoglobins and chains.     

Functional and subunit assembly properties of hemoglobin Alberta (alpha 2 beta 2(101) Glu----Gly)

Hemoglobin Alberta has an amino acid substitution at position 101 (Glu----Gly), a residue involved in the alpha 1 beta 2 contact region of both the deoxy and oxy conformers of normal adult hemoglobin. Oxygen equilibrium measurements of stripped hemoglobin Alberta at 20 degrees C in the absence of phosphate revealed a high affinity (P50 = 0.75 mm Hg at pH 7), co-operative hemoglobin variant (n = 2.3 at pH 7) with a normal Bohr effect (- delta log P50/delta pH(7-8) = 0.65). The addition of inositol hexaphosphate resulted in a decrease in oxygen affinity (P50 = 8.2 mm Hg at pH 7), a slight increase in the value of n and an enhanced Bohr effect. Rapid mixing experiments reflected the equilibrium results. A rapid rate of carbon monoxide binding (l' = 7.0 X 10(5) M-1 S-1) and a slow rate of overall oxygen dissociation (k = 15 s-1) was seen at pH7 and 20 degrees C in the absence of phosphate. Under these experimental conditions the tetramer stability of liganded and unliganded hemoglobin Alberta was investigated by spectrophotometric kinetic techniques. The 4K4 value (the liganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta was found to be 0.83 X 10(-6) M compared to a 4K4 value for hemoglobin A of 2.3 X 10(-6) M, indicating that the Alberta tetramer was less dissociated into dimers than the tetramer of hemoglobin A. The values of 0K4 (the unliganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta and hemoglobin A were also measured and found to be 2.5 X 10(-8) M and 1.5 X 10(-10) M, respectively, demonstrating a greatly destabilized deoxyhemoglobin tetramer for hemoglobin Alberta compared to deoxyhemoglobin A. The functional and subunit dissociation properties of hemoglobin Alberta appear to be directly related to the dual role of the beta 101 residue in stabilizing the tetrameric form of the liganded structure, while concurrently destabilizing the unliganded tetramer molecule.     

The mutation beta 99 Asp-Tyr stabilizes Y--a new, composite quaternary state of human hemoglobin

Carbonmonoxy hemoglobin Ypsilanti (beta 99 Asp-Tyr) exhibits a quaternary form distinctly different from any structures previously observed for human hemoglobins. The relative orientation of alpha beta dimers in the new quaternary form lies well outside the range of values observed for normal unliganded and liganded tetramers (Baldwin, J., Chothia, C., J. Mol. Biol. 129:175-220, 1979). Despite this large quaternary structural difference between carbonmonoxy hemoglobin Ypsilanti and the two canonical structures, the new quaternary structure's hydrogen bonding interactions in the "switch" region, and packing interactions in the "flexible joint" region, show noncovalent interactions characteristic of the alpha 1 beta 2 contacts of both unliganded and liganded normal hemoglobins. In contrast to both canonical structures, the beta 97 histidine residue in carbonmonoxy hemoglobin Ypsilanti is disengaged from quaternary packing interactions that are generally believed to enforce two-state behavior in ligand binding. These features of the new quaternary structure, denoted Y, may therefore be representative of quaternary states that occur transiently along pathways between the normal unliganded, T, and liganded, R, hemoglobin structures.     

Solution structures of chicken parvalbumin 3 in the Ca(2+)-free and Ca(2+)-bound states

Birds express two β-parvalbumin isoforms, parvalbumin 3 and avian thymic hormone (ATH). Parvalbumin 3 from chicken (CPV3) is identical to rat β-parvalbumin (β-PV) at 75 of 108 residues. CPV3 displays intermediate Ca(2+) affinity--higher than that of rat β-parvalbumin, but lower than that of ATH. As in rat β-PV, the attenuation of affinity is associated primarily with the CD site (residues 41-70), rather than the EF site (residues 80-108). Structural data for rat α- and β-parvalbumins suggest that divalent ion affinity is correlated with the similarity of the unliganded and Ca(2+)-bound conformations. We herein present a comparison of the solution structures of Ca(2+)-free and Ca(2+)-bound CPV3. Although the structures are generally similar, the conformations of residues 47 to 50 differ markedly in the two protein forms. These residues are located in the C helix, proximal to the CD binding loop. In response to Ca(2+) removal, F47 experiences much greater solvent accessibility. The side-chain of R48 assumes a position between the C and D helices, adjacent to R69. Significantly, I49 adopts an interior position in the unliganded protein that allows association with the side-chain of L50. Concomitantly, the realignment of F66 and F70 facilitates their interaction with I49 and reduces their contact with residues in the N-terminal AB domain. This reorganization of the hydrophobic core, although less profound, is nevertheless reminiscent of that observed in rat β-PV. The results lend further support to the idea that Ca(2+) affinity correlates with the structural similarity of the apo- and bound parvalbumin conformations.     

Coupling of tertiary and quaternary changes in human hemoglobin: a 1D and 2D NMR study of hemoglobin Saint Mandé (beta N102Y)

Hemoglobin Saint Mandé (beta N102Y) is a low-affinity mutant with the substitution site situated in the quaternary-sensitive alpha 1 beta 2 interface. In adult hemoglobin the Asn102 beta contributes to the stability of the liganded (R) state, forming a hydrogen bond with Asp94 alpha. The quaternary and tertiary perturbations subsequent to the Tyr for Asn substitution in monocarboxylated hemoglobin Saint Mandé have been investigated by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis of the one-dimensional NMR spectra of the liganded and unliganded samples in 1H2O provides evidence that both R and T quaternary structures of Hb Saint Mandé are different from the corresponding ones in HbA. In the monocarboxylated form of the mutant hemoglobin, at acid pH, we have observed the disappearance of an R-type hydrogen bond and the appearance of a new one whose proton resonates like a deoxy T marker. Using two-dimensional NMR methods and on the basis of previous results on the monocarboxylated HbA, we have obtained a significant number of resonance assignments in the spectra of monocarboxylated Hb Saint Mandé at pH 5.6 in the presence or absence of a strong allosteric effector, inositol hexaphosphate. This enabled us to characterize the tertiary conformational changes (relative to the liganded normal hemoglobin) triggered by the quaternary-state modification. The observed structural variations are confined within the heme pocket regions but concern both the alpha and beta subunits. Most of them, localized in the C, F, G, and FG segments, could result directly from the side-chain substitution, while others, such as Leu141 beta, could be explained only by long-range interactions.     

Proton NMR studies of human hemoglobin variants modified in the proximal side of beta heme pocket. Implications for the affinity control and cooperative mechanism

Using high resolution proton NMR spectroscopy, we have investigated 10 human hemoglobin variants modified in the proximal side of the heme pocket in beta subunits. Comparative observation of several resonances in the spectra of liganded and unliganded hemoglobins allowed us to characterize the localization and nature of the structural perturbations induced by amino acid substitutions or chemical modification. The present data indicate that the structural perturbations are localized in the beta subunits, mainly in the tertiary domain surrounding the modification site. Analysis of the aromatic region of the liganded hemoglobin spectra gives substantial information for the assignment of the His-beta 97 C-2H resonance. Correlation of the spectroscopic observations with the functional characteristics of the studied hemoglobins demonstrates that structural factors localized in the proximal side of the heme pocket can control the ligand-iron interaction taking place on the other heme side. The structural perturbations induced by the modifications in the F or FG segments of the beta subunits do not extend to the distal side but rather to the alpha 1 beta 2 interface. This argues the existence of a gradient of tertiary structural stability, indicating a possible structural pattern of heme-heme interaction in the cooperativity control.     

Slowly interchanging conformers of bovine neurophysin-I in the unliganded dimeric state.

The effect of neurophysin dimerization on Tyr-49, a residue adjacent to the hormone-binding site, was investigated by proton NMR in order to analyze the basis of the dimerization-induced increase in neurophysin hormone affinity. Dimerization-induced changes in Tyr-49 resonances, in two unliganded bovine neurophysins, suggested that Tyr-49 perturbation is an intrinsic consequence of dimerization, although Tyr-49 is distant from the monomer-monomer interface in the crystalline liganded state. To determine whether this perturbation reflects a conformational difference between liganded and unliganded states that places Tyr-49 at the interface in the unliganded state, or a dimerization-induced change in secondary (2 degrees) or tertiary (3 degrees) structure, the more general structural consequences of dimerization were further analyzed. No change in 2 degrees structure upon dimerization was demonstrable by CD. On the other hand, a general similarity of regions involved in dimerization in unliganded and liganded states was indicated by NMR evidence of participation of His-80 and Phe-35 in dimerization in the unliganded state; both residues are at the interface in the crystal structure and distant from Tyr-49. Consistent with a lack of direct participation of Tyr-49 at the monomer-monomer interface, dimerization induced at least two distinct slowly exchanging environmental states for the 3.5 ring protons of Tyr-49 without significantly increased dipolar broadening relative to the monomer. Two environments were also found in the dimer of des-1-8 neurophysin-I for the methyl protons of Thr-9, another residue distant from the monomer-monomer interface and close to the binding site in the liganded state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cooperative fluctuations of unliganded and substrate-bound HIV-1 protease: a structure-based analysis on a variety of conformations from crystallography and molecular dynamics simulations

The dynamics of HIV-1 protease, both in unliganded and substrate-bound forms have been analyzed by using an analytical method, Gaussian network model (GNM). The method is applied to different conformations accessible to the protein backbone in the native state, observed in crystal structures and snapshots from fully atomistic molecular dynamics (MD) simulation trajectories. The modes of motion obtained from GNM on different conformations of HIV-1 protease are conserved throughout the MD simulations. The flaps and 40's loop of the unliganded HIV-1 protease structure are identified as the most mobile regions. However, in the liganded structure these flaps lose mobility, and terminal regions of the monomers become more flexible. Analysis of the fast modes shows that residues important for stability are in the same regions of all the structures examined. Among these, Gly86 appears to be a key residue for stability. The contribution of residues in the active site region and flaps to the stability is more pronounced in the substrate-bound form than in the unliganded form. The convergence of modes in GNM to similar regions of HIV-1 protease, regardless of the conformation of the protein, supports the robustness of GNM as a potentially useful and predictive tool.     

Ligand-linked changes at the subunit interfaces in Scapharca hemoglobins probed through the sulfhydryl infrared absorption.

FTIR spectra of native Scapharca homodimeric hemoglobin (HbI) and of the Phe97-->Ile mutant have been measured in the region 2400-2700 cm(-1) where the absorption of the sulfhydryl groups can be observed. In native HbI, the two Cys92 residues give rise to a relatively intense band centered at 2559 cm(-1) that is shifted to 2568 cm(-1) and strongly quenched upon ligand binding. In the Phe97-->Leu mutant, such ligand-linked changes are not observed and the strong peak at around 2560 cm(-1) persists in the liganded derivatives. In native HbI, the observed changes have been attributed to the decrease in polarity of the interface due to the ligand-induced extrusion of the Phe97 phenyl ring from the heme pocket to the interface and the subsequent release of several water molecules that are clustered in the vicinity of Cys92. In contrast, in the Phe97-->Leu mutant, the Leu residue does not leave the heme pocket upon ligand binding and the interface is unaltered. The Cys92/S-H infrared band, therefore, represents a sensitive probe of the structural rearrangements that take place in the intersubunit interface upon ligand binding to HbI. The heterotetrameric Scapharca hemoglobin HbII contains, in addition to the Cys92 residues in the interfaces, two extra sulfhydryl groups per tetramer (Cys9 in the B chain) that are exposed to solvent in the A helix. The frequency of the Cys9/S-H stretching vibration occurs at 2582 cm(-1) in the unliganded and at 2586 cm(-1) in the liganded derivative, pointing to the involvement of the A helix in the ligand-linked polymerization characteristic of HbII.     

Carbon dioxide binding to human hemoglobin cross-linked between the alpha chains

The binding of carbon dioxide to human hemoglobin cross-linked between Lys alpha 99 residues with bis(3,5-di-bromosalicyl) fumarate was measured using manometric techniques. The binding of CO2 to unmodified hemoglobin can be described by two classes of sites with high and low affinities corresponding to the amino-terminal valines of the beta and alpha chains, respectively (Perrella, M., Kilmartin, J. V., Fogg, J., and Rossi-Bernardi, L. (1975b) Nature 256, 759-761. The cross-linked hemoglobin bound less CO2 than native hemoglobin at all CO2 concentrations in deoxygenated and liganded conformations, and the ligand-linked effect was reduced. Fitting the data to models of CO2 binding suggests that only half of the expected saturation with CO2 is possible. The remaining binding is described by a single affinity constant that for cross-linked deoxyhemoglobin is about two-thirds of the high affinity constant for deoxyhemoglobin A and that for cross-linked cyanomethemoglobin is equal to the high affinity constant for unmodified cyanomethemoglobin A or carbonmonoxyhemoglobin A. The low affinity binding constant for cross-linked hemoglobin in both the deoxygenated and liganded conformations is close to zero, which is significantly less than the affinity constants for either subunit binding site in unmodified hemoglobin. Comparing the low affinity sites in this modified hemoglobin to native hemoglobin suggests that cross-linking hemoglobin between Lys alpha 99 residues prevents CO2 binding at the alpha-subunit NH2 termini.     

Contributions of the interdomain loop, amino terminus, and subunit interface to the ligand-facilitated dimerization of neurophysin: crystal structures and mutation studies of bovine neurophysin-I

Current evidence indicates that the ligand-facilitated dimerization of neurophysin is mediated in part by dimerization-induced changes at the hormone binding site of the unliganded state that increase ligand affinity. To elucidate other contributory factors, we investigated the potential role of neurophysin's short interdomain loop (residues 55-59), particularly the effects of loop residue mutation and of deleting amino-terminal residues 1-6, which interact with the loop and adjacent residues 53-54. The neurophysin studied was bovine neurophysin-I, necessitating determination of the crystal structures of des 1-6 bovine neurophysin-I in unliganded and liganded dimeric states, as well as the structure of its liganded Q58V mutant, in which peptide was bound with unexpectedly increased affinity. Increases in dimerization constant associated with selected loop residue mutations and with deletion of residues 1-6, together with structural data, provided evidence that dimerization of unliganded neurophysin-I is constrained by hydrogen bonding of the side chains of Gln58, Ser56, and Gln55 and by amino terminus interactions, loss or alteration of these hydrogen bonds, and probable loss of amino terminus interactions, contributing to the increased dimerization of the liganded state. An additional intersubunit hydrogen bond from residue 81, present only in the liganded state, was demonstrated as the largest single effect of ligand binding directly on the subunit interface. Comparison of bovine neurophysins I and II indicates broadly similar mechanisms for both, with the exception in neurophysin II of the absence of Gln55 side chain hydrogen bonds in the unliganded state and a more firmly established loss of amino terminus interactions in the liganded state. Evidence is presented that loop status modulates dimerization via long-range effects on neurophysin conformation involving neighboring Phe22 as a key intermediary.     

Electrostatic effects in hemoglobin: electrostatic energy associated with allosteric transition and effector binding

The pH dependence of the summed electrostatic stabilization for deoxy- and liganded hemoglobin was computed for several ionic strength values. The computed contribution to the stabilization of deoxyhemoglobin by binding of 2,3-diphosphoglycerate in the beta cleft compared well with experimental binding behavior for human hemoglobin A0 and hemoglobin F. The contribution of diphosphoglycerate binding to the alkaline Bohr effect was computed correctly for both hemoglobins A0 and F. The computed effects of simultaneous binding of diphosphoglycerate and formation of Val-1 beta carbamino adducts suggested a competition between these effectors. A direct competition was formulated between these two effectors, with extension to include a simple anion such as chloride or bicarbonate binding in competition with diphosphoglycerate but not with Val-1 beta carbamino formation. This model was found to hold at pH 7.3-7.4 over a range of concentrations of the effectors involved and to predict the pH dependence of Val-1 beta carbamino formation over the pH range 7.0-8.0. The pH dependence of the computed differential stability of liganded vs. unliganded hemoglobin A compared well with observation.     

Cooperativity in Scapharca dimeric hemoglobin: simulation of binding intermediates and elucidation of the role of interfacial water

Cooperative binding of ligands to proteins can serve to increase their efficiency and to regulate their activity. Thus, understanding of the mechanism of cooperativity is one of the central concerns of molecular biology. For the tetrameric human hemoglobin (HbA), the cooperative mechanism involves a reasonably well understood combination of tertiary and quaternary changes that occur during the binding process. The dimeric hemoglobin of Scapharca (HbI), which is composed of subunits with the same fold as in HbA, is also highly cooperative but the structural changes on ligand binding are small. A re-orientation of Phe97 in the binding pocket and changes in the number of interfacial water molecules have been implicated in the cooperative mechanism. To explore the role of these factors, we have investigated models of partially liganded intermediate states of HbI with molecular dynamics simulation methods. Since, unlike HbA, no structures for intermediates are available, they were constructed by combining subunits from the unliganded and liganded dimers. Two structurally distinct intermediates were examined, and it was shown that the transition between the two intermediates is directly coupled to the number of interfacial water molecules. Further, it was found that there is a well-defined water channel that connects the interface between the subunits to bulk water. The bottleneck (gate) of the channel, which can be open or closed, is made of hydrophilic residues. The implication of the present results for the cooperative mechanism of HbI is discussed.     

DNA-induced conformational changes in type II restriction endonucleases: the structure of unliganded HincII

The 2.1A crystal structure of the unliganded type II restriction endonuclease, HincII, is described. Although the asymmetric unit contains only a single monomer, crystal lattice contacts bring two monomers together to form a dimer very similar to that found in the DNA bound form. Comparison with the published DNA bound structure reveals that the DNA binding pocket is expanded in the unliganded structure. Comparison of the unliganded and DNA liganded structures reveals a simple rotation of subunits by 11 degrees each, or 22 degrees total, to a more closed structure around the bound DNA. Comparison of this conformational change to that observed in the other type II restriction endonucleases where DNA bound and unliganded forms have both been characterized, shows considerable variation in the types of conformational changes that can occur. The conformational changes in three can be described by a simple rotation of subunits, while in two others both rotation and translation of subunits relative to one another occurs. In addition, the endonucleases having subunits that undergo the greatest amount of rotation upon DNA binding are found to be those that distort the bound DNA the least, suggesting that DNA bending may be less facile in dimers possessing greater flexibility.     

X-ray diffraction studies of a partially liganded hemoglobin, [alpha(FeII-CO)beta(MnII)]2

We have applied single-crystal X-ray diffraction methods to analyze the structure of [alpha(FeII-CO)beta(MnII)]2, a mixed-metal hybrid hemoglobin that crystallizes in the deoxyhemoglobin quaternary structure (the T-state) even though it is half liganded. This study, carried out at a resolution of 3.0 A, shows that (1) the Mn(II)-substituted beta subunits are structurally isomorphous with normal deoxy beta subunits, and (2) CO binding to the alpha subunits induces small, localized changes in the T-state that lack the main directional component of the corresponding larger structural changes in subunit tertiary structure that accompany complete ligand binding to all four subunits and the deoxy to oxy quaternary structure change. Specifically, in the T-state, CO binding to the alpha heme group draws the iron atom toward the heme plane, and this in turn pulls the last turn of the F helix (residues 85 through 89) closer to the heme group. The direction of these small movements is almost perpendicular to the axis of the F helix. In contrast, when the structures of fully liganded and deoxyhemoglobin are compared, extensive structural changes occur throughout the F helix and FG corner, and the main component of the atomic movements in the F helix (in addition to the smaller component toward the heme) is in a direction parallel to the heme plane and toward the alpha 1 beta 2 interface. These findings are discussed in terms of the current stereochemical theories of co-operative ligand binding and the Bohr effect.     

Conformational changes in the human estrogen receptor observed by (19)F NMR

The (19)F NMR spectra of the 5F-Trp labeled glutathione-S-transferase fusion protein with residues 282-595 of the human estrogen receptor show that there is a distinct conformational change in the protein when estradiol is added to the unliganded protein. Our studies show the empty receptor to have more conformational flexibility than the liganded form. This study shows the applicability of (19)F NMR to study conformational change in large protein systems.     

The interaction of organic phosphates with human and chicken hemoglobin.

In this study of the binding properties of inositol hexaphosphate and 2,3-bisphosphoglycerate to chicken and human deoxyhemoglobin and carboxyhemoglobin were compared. It appeared that in all cases the binding to chicken hemoglobin is much stronger than to human hemoglobin. This is very probably due to the fact that 4 out of the 12 residues, responsible for the binding of phosphates in chicken hemoglobin, are arginines. These are absent in human hemoglobin, where the binding site is made up to only 8 residues. For chicken hemoglobin one strong binding site could be observed in both unliganded and liganded hemoglobin. From these observations we conclude that the same binding site is involved in both the oxy- and deoxy structure showing different affinity to phosphates in the two conformational states. For human hemoglobin we reached the same conclusion.