RESUMEN
The intracellular polymerization and the concomitant sickling processes, central to the pathology of sickle cell disease, can be mitigated by increasing the oxygen affinity of sickle hemoglobin (HbS). Attempts to develop azolylacryloyl derivatives to covalently interact with ßCys93 and destabilize the low-O2-affinity T-state (deoxygenated) HbS to the polymer resistant high-O2-affinity R-state (liganded) HbS were only partially successful. This was likely due to the azolylacryloyls carboxylate moiety directing the compounds to also bind in the central water cavity of deoxygenated Hb and stabilizing the T-state. We now report a second generation of KAUS compounds (KAUS-28, KAUS-33, KAUS-38, and KAUS-39) without the carboxylate moiety designed to bind exclusively to ßCys93. As expected, the compounds showed reactivity with both free amino acid l-Cys and the Hb ßCys93. At 2 mM concentrations, the compounds demonstrated increased Hb affinity for oxygen (6% to 15%) in vitro, while the previously reported imidazolylacryloyl carboxylate derivative, KAUS-15 only showed 4.5% increase. The increased O2 affinity effects were sustained through the experimental period of 12 h for KAUS-28, KAUS-33, and KAUS-38, suggesting conserved pharmacokinetic profiles. When incubated at 2 mM with red blood cells from patients with homozygous SS, the compounds inhibited erythrocyte sickling by 5% to 9%, respectively in correlation with the increase Hb-O2 affinity. These values compare to 2% for KAUS-15. When tested with healthy mice, KAUS-38 showed very low toxicity.
RESUMEN
Aromatic aldehydes and ethacrynic acid (ECA) exhibit antipolymerization properties that are beneficial for sickle cell disease therapy. Based on the ECA pharmacophore and its atomic interaction with hemoglobin, we designed and synthesized several compounds - designated as KAUS (imidazolylacryloyl derivatives) - that we hypothesized would bind covalently to ßCys93 of hemoglobin and inhibit sickling. The compounds surprisingly showed weak allosteric and antisickling properties. X-ray studies of hemoglobin in complex with representative KAUS compounds revealed an unanticipated mode of Michael addition between the ß-unsaturated carbon and the N-terminal αVal1 nitrogen at the α-cleft of hemoglobin, with no observable interaction with ßCys93. Interestingly, the compounds exhibited almost no reactivity with the free amino acids, L-Val, L-His and L-Lys, but showed some reactivity with both glutathione and L-Cys. Our findings provide a molecular level explanation for the compounds biological activities and an important framework for targeted modifications that would yield novel potent antisickling agents.
Asunto(s)
Acrilatos/farmacología , Anemia de Células Falciformes/tratamiento farmacológico , Antidrepanocíticos/farmacología , Hemoglobina Falciforme/antagonistas & inhibidores , Imidazoles/farmacología , Acrilatos/síntesis química , Acrilatos/química , Anemia de Células Falciformes/patología , Animales , Antidrepanocíticos/síntesis química , Antidrepanocíticos/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hemoglobina Falciforme/química , Humanos , Imidazoles/síntesis química , Imidazoles/química , Ratones , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The liganded hemoglobin (Hb) high-salt crystallization condition described by Max Perutz has generated three different crystals of human adult carbonmonoxy hemoglobin (COHbA). The first crystal is isomorphous with the "classical" liganded or R Hb structure. The second crystal reveals a new liganded Hb quaternary structure, RR2, that assumes an intermediate conformation between the R form and another liganded Hb quaternary structure, R2, which was discovered more than a decade ago. Like the R2 structure, the diagnostic R state hydrogen bond between beta2His97 and alpha1Thr38 is missing in the RR2 structure. The third crystal adopts a novel liganded Hb conformation, which we have termed R3, and it shows substantial quaternary structural differences from the R, RR2, and R2 structures. The quaternary structure differences between T and R3 are as large as those between T and R2; however, the T --> R3 and T --> R2 transitions are in different directions as defined by rigid-body screw rotation. Moreover, R3 represents an end state. Compared to all known liganded Hb structures, R3 shows remarkably reduced strain at the alpha-heme, reduced steric contact between the beta-heme ligand and the distal residues, smaller alpha- and beta-clefts, and reduced alpha1-alpha2 and beta1-beta2 iron-iron distances. Together, these unique structural features in R3 should make it the most relaxed and/or greatly enhance its affinity for oxygen compared to the other liganded Hbs. The current Hb structure-function relationships that are now based on T --> R, T -->R --> R2, or T --> R2 --> R transitions may have to be reexamined to take into account the RR2 and R3 liganded structures.
Asunto(s)
Carboxihemoglobina/química , Hemoglobinas/química , Modelos Moleculares , Estructura Cuaternaria de Proteína , Adulto , Regulación Alostérica/fisiología , Carboxihemoglobina/fisiología , Cristalización , Cristalografía por Rayos X , Hemo/química , Hemoglobinas/fisiología , Humanos , Hierro/química , Ligandos , Modelos QuímicosRESUMEN
Escherichia coli pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate by the FMN oxidation of pyridoxine 5'-phosphate forming FMNH(2) and H(2)O(2). Recent studies have shown that in addition to the active site, pyridoxine 5'-phosphate oxidase contains a non-catalytic site that binds pyridoxal 5'-phosphate tightly. The crystal structure of pyridoxine 5'-phosphate oxidase from E. coli with one or two molecules of pyridoxal 5'-phosphate bound to each monomer has been determined to 2.0 A resolution. One of the pyridoxal 5'-phosphate molecules is clearly bound at the active site with the aldehyde at C4' of pyridoxal 5'-phosphate near N5 of the bound FMN. A protein conformational change has occurred that partially closes the active site. The orientation of the bound pyridoxal 5'-phosphate suggests that the enzyme catalyzes a hydride ion transfer between C4' of pyridoxal 5'-phosphate and N5 of FMN. When the crystals are soaked with excess pyridoxal 5'-phosphate an additional molecule of this cofactor is also bound about 11 A from the active site. A possible tunnel exists between the two sites so that pyridoxal 5'-phosphate formed at the active site may transfer to the non-catalytic site without passing though the solvent.
Asunto(s)
Escherichia coli/enzimología , Fosfato de Piridoxal/metabolismo , Piridoxaminafosfato Oxidasa/química , Piridoxaminafosfato Oxidasa/metabolismo , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Dimerización , Mononucleótido de Flavina/metabolismo , Enlace de Hidrógeno , Ligandos , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/química , Agua/metabolismoRESUMEN
Crystallographic studies of the intermediate states between unliganded and fully liganded hemoglobin (Hb) have revealed a large range of subtle but functionally important structural differences. Only one T state has been reported, whereas three other quaternary states (the R state, B state, and R2 or Y state) for liganded Hb have been characterized; other studies have defined liganded Hbs that are intermediate between the T and R states. The high-salt crystal structure of bovine carbonmonoxy (CO bovine) Hb has been determined at a resolution of 2.1 A and is described here. A detailed comparison with other crystallographically solved Hb forms (T, R, R2 or Y) shows that the quaternary structure of CO bovine Hb closely resembles R state Hb. However, our analysis of these structures has identified several important differences between CO bovine Hb and R state Hb. Compared with the R state structures, the beta-subunit N-terminal region has shifted closer to the central water cavity in CO bovine Hb. In addition, both the alpha- and beta-subunits in CO bovine Hb have more constrained heme environments that appear to be intermediate between the T and R states. Moreover, the distal pocket of the beta-subunit heme in CO bovine Hb shows significantly closer interaction between the bound CO ligand and the Hb distal residues Val 63(E11) and His 63(E7). The constrained heme groups and the increased steric contact involving the CO ligand and the distal heme residues relative to human Hb may explain in part the low intrinsic oxygen affinity of bovine Hb.
Asunto(s)
Carboxihemoglobina/química , Hemoglobinas/química , Animales , Bovinos , Cristalografía por Rayos X/métodos , Hemo/química , Humanos , Ligandos , Modelos Moleculares , Oxígeno/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The crystal structure of human deoxy hemoglobin (Hb) complexed with a potent allosteric effector (2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-2-methylpropionic acid) = RSR-13) is reported at 1.85 A resolution. Analysis of the hemoglobin:effector complex indicates that two of these molecules bind to the central water cavity of deoxy Hb in a symmetrical fashion, and that each constrains the protein by engaging in hydrogen bonding and hydrophobic interactions with three of its four subunits. Interestingly, we also find that water-mediated interactions between the bound effectors and the protein make significant contributions to the overall binding. Physiologically, the interaction of RSR-13 with Hb results in increased oxygen delivery to peripheral tissues. Thus, this compound has potential therapeutic application in the treatment of hypoxia, ischemia, and trauma-related blood loss. Currently, RSR-13 is in phase III clinical trials as a radiosensitizing agent in the treatment of brain tumors. A detailed structural analysis of this compound complexed with deoxy Hb has important implications for the rational design of future analogs.
Asunto(s)
Compuestos de Anilina/química , Compuestos de Anilina/farmacología , Hemoglobinas/antagonistas & inhibidores , Hemoglobinas/química , Propionatos/química , Propionatos/farmacología , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Compuestos de Anilina/metabolismo , Compuestos de Anilina/uso terapéutico , Antidrepanocíticos/química , Antidrepanocíticos/metabolismo , Antidrepanocíticos/farmacología , Antidrepanocíticos/uso terapéutico , Ensayos Clínicos Fase III como Asunto , Cristalografía por Rayos X , Diseño de Fármacos , Hemoglobinas/aislamiento & purificación , Hemoglobinas/metabolismo , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Propionatos/metabolismo , Propionatos/uso terapéutico , Conformación Proteica/efectos de los fármacos , Relación Estructura-Actividad , Agua/química , Agua/metabolismoRESUMEN
The structure of catalase from human erythrocytes (HEC) was determined in tetragonal crystals of space group I4(1) by molecular-replacement methods, using the orthorhombic crystal structure as a search model. It was then refined in a unit cell of dimensions a = b = 203.6 and c = 144.6 A, yielding R and R(free) of 0.196 and 0.244, respectively, for all data at 2.4 A resolution. A major difference of the HEC structure in the tetragonal crystal compared with the orthorhombic structure was the omission of a 20-residue N-terminal segment corresponding to the first exon of the human catalase gene. The overall structures were otherwise identical in both crystal forms. The NADPH-binding sites were empty in all four subunits and bound water molecules were observed at the active sites. The structure of the C-terminal segment, which corresponds to the last exon, remained undetermined. The tetragonal crystals showed a pseudo-4(1)22 symmetry in molecular packing. Two similar types of lattice contact interfaces between the HEC tetramers were observed; they were related by the pseudo-dyad axes.
Asunto(s)
Catalasa/química , Eritrocitos/enzimología , Cristalografía por Rayos X , Análisis de Fourier , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
A series of allosteric effectors of hemoglobin, 2-(aryloxy)-2-alkanoic acids, was prepared to investigate the effect of the stereocenter on allosteric activity. The chiral analogues were based on the lead compound, RSR13 (3b), with different alkyl/alkanoic and cycloalkyl/cycloalkanoic groups positioned at the acidic chiral center. Of the 23 racemic molecules synthesized, 5 were selected for resolution based on structure-activity relationships. One chiral analogue, (-)-(1R,2R)-1-[4-[[(3, 5-dimethylanilino)carbonyl]methyl]phenoxy]-2-methylcyclopentane carbox ylic acid (11), exhibited greater in vitro activity in hemoglobin solutions than its antipode, racemate, and RSR13. Compound (-)-(1R, 2R)-11 was equipotent with RSR13 in whole blood, is a candidate for in vivo animal studies, and if efficacious and safe has a potential for use in humans. In general, it was found that chirality affects allosteric effector activity with measurable differences observed between enantiomers and the racemates.
Asunto(s)
Acetatos/síntesis química , Compuestos de Anilina/química , Hemoglobinas/química , Propionatos/química , Acetatos/sangre , Acetatos/química , Regulación Alostérica , Compuestos de Anilina/sangre , Derivados del Benceno , Cristalografía por Rayos X , Hemoglobinas/metabolismo , Humanos , Modelos Moleculares , Oxígeno/metabolismo , Propionatos/sangre , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate (PLP), a cofactor used by many enzymes involved in amino acid metabolism. The enzyme oxidizes either the 4'-hydroxyl group of pyridoxine 5'-phosphate (PNP) or the 4'-primary amine of pyridoxamine 5'-phosphate (PMP) to an aldehyde. PNPOx is a homodimeric enzyme with one flavin mononucleotide (FMN) molecule non-covalently bound to each subunit. A high degree of sequence homology among the 15 known members of the PNPOx family suggests that all members of this group have similar three-dimensional folds. RESULTS: The crystal structure of PNPOx from E. coli has been determined to 1.8 A resolution. The monomeric subunit folds into an eight-stranded beta sheet surrounded by five alpha-helical structures. Two monomers related by a twofold axis interact extensively along one-half of each monomer to form the dimer. There are two clefts at the dimer interface that are symmetry-related and extend from the top to the bottom of the dimer. An FMN cofactor that makes interactions with both subunits is located in each of these two clefts. CONCLUSIONS: The structure is quite similar to the recently deposited 2.7 A structure of Saccharomyces cerevisiae PNPOx and also, remarkably, shares a common structural fold with the FMN-binding protein from Desulfovibrio vulgaris and a domain of chymotrypsin. This high-resolution E. coli PNPOx structure permits predictions to be made about residues involved in substrate binding and catalysis. These predictions provide testable hypotheses, which can be answered by making site-directed mutants.
Asunto(s)
Proteínas Bacterianas/química , Escherichia coli/enzimología , Mononucleótido de Flavina/química , Piridoxaminafosfato Oxidasa/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Sitios de Unión , Quimotripsina/química , Cristalografía por Rayos X , Desulfovibrio vulgaris/enzimología , Dimerización , Transporte de Electrón , Mononucleótido de Flavina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fosfatos/metabolismo , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Piridoxaminafosfato Oxidasa/metabolismo , Proteínas Recombinantes de Fusión/química , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Catalase (E.C. 1.11.1.6) was purified from human erythrocytes and crystallized in three different forms: orthorhombic, hexagonal and tetragonal. The structure of the orthorhombic crystal form of human erythrocyte catalase (HEC), with space group P2(1)2(1)2(1) and unit-cell parameters a = 84.9, b = 141.7, c = 232.5 A, was determined and refined with 2.75 A resolution data. Non-crystallographic symmetry restraints were employed and the resulting R value and R(free) were 0.206 and 0.272, respectively. The overall structure and arrangement of HEC molecules in the orthorhombic unit cell were very similar to those of bovine liver catalase (BLC). However, no NADPH was observed in the HEC crystal and a water was bound to the active-site residue His75. Conserved lattice interactions suggested a common growth mechanism for the orthorhombic crystals of HEC and BLC.
Asunto(s)
Catalasa/sangre , Catalasa/química , Eritrocitos/enzimología , Animales , Catalasa/aislamiento & purificación , Bovinos , Cristalización , Cristalografía por Rayos X , Humanos , Estructura Secundaria de Proteína , SolventesRESUMEN
Pyridoxine 5'-phosphate oxidase (PNP Ox) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate. The 53-kDa homodimeric enzyme contains a noncovalently bound flavin mononucleotide (FMN) on each monomer. Three crystal forms of Escherichia coli PNP Ox complexed with FMN have been obtained at room temperature. The first crystal form belongs to trigonal space group P3(1)21 or P3(2)21 with unit cell dimensions a = b = 64.67A, c = 125.64A, and has one molecule of the complex (PNP Ox-FMN) per asymmetric unit. These crystals grow very slowly to their maximum size in about 2 to 4 months and diffract to about 2.3 A. The second crystal form belongs to tetragonal space group P4(1) or P4(3) with unit cell dimensions a = b = 54.92A, c = 167.65A, and has two molecules of the complex per asymmetric unit. The crystals reach their maximum size in about 5 weeks and diffract to 2.8 A. A third crystal form with a rod-like morphology grows faster and slightly larger than the other two forms, but diffracts poorly and could not be characterized by X-ray analysis. The search for heavy-atom derivatives for the first two crystal forms to solve the structure is in progress.
Asunto(s)
Cristalización , Mononucleótido de Flavina/química , Piridoxaminafosfato Oxidasa/química , Cristalografía por Rayos X , Escherichia coli/química , Escherichia coli/enzimología , Mononucleótido de Flavina/metabolismo , Unión Proteica , Conformación Proteica , Piridoxaminafosfato Oxidasa/metabolismo , Proteínas RecombinantesRESUMEN
The Fis protein regulates site-specific DNA inversion catalyzed by a family of DNA invertases when bound to a cis-acting recombinational enhancer. As is often found for transactivation domains, previous crystal structures have failed to resolve the conformation of the N-terminal inversion activation region within the Fis dimer. A new crystal form of a mutant Fis protein now reveals that the activation region contains two beta-hairpin arms that protrude over 20 A from the protein core. Saturation mutagenesis identified the regulatory and structurally important amino acids. The most critical activating residues are located near the tips of the beta-arms. Disulfide cross-linking between the beta-arms demonstrated that they are highly flexible in solution and that efficient inversion activation can occur when the beta-arms are covalently linked together. The emerging picture for this regulatory motif is that contacts with the recombinase at the tip of the mobile beta-arms activate the DNA invertase in the context of an invertasome complex.
Asunto(s)
Proteínas Portadoras/química , Estructura Secundaria de Proteína , Recombinación Genética , Transactivadores/química , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Simulación por Computador , Cristalografía por Rayos X , Cisteína/química , Análisis Mutacional de ADN , ADN Nucleotidiltransferasas/metabolismo , Factor Proteico para Inverción de Estimulación , Factores de Integración del Huésped , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
The immunity protein of colicin E7 (ImmE7) can bind specifically to the DNase-type colicin E7 and inhibit its bactericidal activity. Here we report the 1.8-angstrom crystal structure of the ImmE7 protein. This is the first x-ray structure determined in the superfamily of colicin immunity proteins. The ImmE7 protein consists of four antiparallel alpha-helices, folded in a topology similar to the architecture of a four-helix bundle structure. A region rich in acidic residues is identified. This negatively charged area has the greatest variability within the family of DNase-type immunity proteins; thus, it seems likely that this area is involved in specific binding to colicin. Based on structural, genetic, and kinetic data, we suggest that all the DNase-type immunity proteins, as well as colicins, share a "homologous-structural framework" and that specific interaction between a colicin and its cognate immunity protein relies upon how well these two proteins' charged residues match on the interaction surface, thus leading to specific immunity of the colicin.
Asunto(s)
Proteínas Bacterianas/química , Colicinas/química , Proteínas de Escherichia coli/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Enlace de Hidrógeno , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
The ImmE7 protein, which can bind specifically to the DNase colicin E7 and neutralize its bactericidal activity, has been purified and crystallized in two different crystal forms by vapor diffusion method. The orthorhombic crystals belong to space group I222 or I2(1)2(1)2(1) and have unit cell dimensions a = 75.1 A, b = 50.5 A, and c = 45.4 A. The second form is monoclinic space group P2(1) with cell dimensions a = 29.3 A, b = 102.7 A, c = 53.0 A, and beta = 91.5 degrees. The orthorhombic crystals diffract to 1.8 A resolution, and are suitable for high-resolution X-ray analysis.
Asunto(s)
Proteínas Bacterianas/química , Colicinas/química , Conformación Proteica , Proteínas Bacterianas/aislamiento & purificación , Colicinas/antagonistas & inhibidores , Colicinas/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , Escherichia coli , Sustancias MacromolecularesRESUMEN
Four new series of monoaldehyde bisacids and bisaldehyde bisacids with varying chain lengths have been synthesized and evaluated as allosteric effectors of hemoglobin. Molecular modeling, oxygen equilibrium, and crystallographic studies were combined for structure/function studies. Crystallographic analyses of the bisaldehydes reveal that Schiff base interaction occurred exclusively between Val 1 alpha and Lys 99 alpha of the opposite alpha chain even though the two terminal Val 1 alpha nitrogens are ideally spaced to also form cross-links. The reason for the observed mode of binding appears to be the influence of chain direction set by key substitutions on the bisaldehyde molecule. Even longer chain derivatives that could overcome the direction set by the key functional groups bind in the same manner. These studies support the general conclusion that long flexible molecules prefer to bind along cavity walls, like double-sided molecular sticky tape, rather than span large open spaces with few chances for interaction. The cross-linked bisaldehydes bind at the same site when incubated under both allosteric states and exhibit reduced cooperativity with a significant decrease in oxygen affinity. The chain length acts as a molecular ratchet and dictates the degree of allosteric effect observed. The tighter the cross-link, the greater the constraint on the tense- (T-) state and the stronger the allosteric effect that is produced. The monoaldehyde bisacids bind in the same fashion with Schiff base formation at Val 1 alpha while the acid that replaces the second aldehyde moiety forms a salt bridge with Lys 99 alpha of the opposite subunit. This class of molecules has weaker allosteric effector activity as would be expected with replacement of one covalent bond by a salt bridge. The importance of Lys 99 alpha on the allosteric equilibrium is confirmed.
Asunto(s)
Aldehídos/síntesis química , Aldehídos/metabolismo , Hemoglobina A/metabolismo , Regulación Alostérica , Sitio Alostérico , Fenómenos Químicos , Química Física , Reactivos de Enlaces Cruzados , Cristalización , Cristalografía por Rayos X , Humanos , Lisina/química , Modelos Moleculares , Estructura Molecular , Oxígeno/metabolismo , Salicilatos/química , Salicilatos/metabolismo , Bases de Schiff , Relación Estructura-Actividad , Valina/químicaRESUMEN
Monoaldehyde allosteric effectors of hemoglobin were designed, using molecular modeling software (GRID), to form a Schiff base adduct with the Val 1 alpha N-terminal nitrogens and interact via a salt bridge with Arg 141 alpha of the opposite subunit. The designed molecules were synthesized if not available. It was envisioned that the molecules, which are aldehyde acids, would produce a high-affinity hemoglobin with potential interest as antisickling agents similar to other aldehyde acids reported earlier. X-ray crystallographic analysis indicated that the aldehyde acids did bind as modeled de novo in symmetry-related pairs to the alpha subunit N-terminal nitrogens. However, oxygen equilibrium curves run on solutions obtained from T- (tense) state hemoglobin crystals of reacted effector molecules produced low-affinity hemoglobins. The shift in the allosteric equilibrium was opposite to that expected. We conclude that the observed shift in allosteric equilibrium was due to the acid group on the monoaldehyde aromatic ring that forms a salt bridge with the guanidinium ion of Arg 141 alpha on the opposite subunit. This added constraint to the T-state structure that ties two subunits across the molecular symmetry axis shifts the equilibrium further toward the T-state. We tested this idea by comparing aldehydes that form Schiff base interactions with the same Val 1 alpha residues but do not interact across the dimer subunit symmetry axis (a new one in this study with no acid group and others that have had determined crystal structures). The latter aldehydes shift the allosteric equilibrium toward the R-state. A hypothesis to predict the direction in shift of the allosteric equilibrium is made and indicates that it is not exclusively where the molecule binds but how it interacts with the protein to stabilize or destabilize the T- (tense) allosteric state.