Chimeric beta-EF3-alpha hemoglobin (Psi): energetics of subunit interaction and ligand binding

Among the numerous strategies to design an oxygen carrier, we outline in this work the engineering of a stable homotetrameric hemoglobin, expressed in Escherichia coli. The chimeric globin (Psi) consists of the first 79 residues of human beta globin (corresponding to positions NA1 --> EF3) followed by the final 67 residues of human alpha globin (corresponding to positions EF3 --> HC3). The molecular mass for beta-EF3-alpha (Psi) globin was measured using mass spectrometry to be equal to its theoretical value: 15782 Da. Correct protein folding was assessed by UV/visible and fluorescence spectra. The subunit interaction free energies were estimated by HPLC gel filtration. In the cyanometHb species, the formation of the dimer-tetramer interface is 2 kcal/mol less favorable (Delta G = -7 kcal/mol) than that of Hb A (Delta G = -9 kcal/mol), whereas the dimer-monomer interface is tightly assembled (< -10 kcal/mol) as for the Hb A alpha 1 beta 1 interface. In contrast to Hb A, oxygen binding to Psi Hb is not cooperative. The free energy for binding four oxygen molecules to a Psi homotetramer is slightly increased compared to a Hb A heterotetramer (-28 and -27.5 kcal/4 mol of O2, respectively). The intrinsic O2 affinity of a Psi homodimer is 6-fold higher than that of a homotetramer. The linkage scheme between dimer-tetramer subunit assembly and the noncooperative oxygenation of Psi Hb predicts a stabilization of the tetramer after ligand release. This protein mechanism resembles that of Hb A for which the dimers exhibit a 100-fold higher O2 affinity relative to deoxy tetramers (which are 10(5) times more stable than oxy tetramers). A potent allosteric effector of Hb A, RSR4, binds to Psi Hb tetramers, inducing a decrease of the overall O2 affinity. Since RSR4 interacts specifically with two binding sites of deoxy Hb A, we propose that the chimeric tetramer folding is close to this native structure.     

The TATA-binding protein from Saccharomyces cerevisiae oligomerizes in solution at micromolar concentrations to form tetramers and octamers

Equilibrium analytical ultracentrifugation has been used to determine the stoichiometry and energetics of the self-assembly of the TATA-binding protein of Saccharomyces cerevisiae at 30 degreesC, in buffers ranging in salt concentration from 60 mM KCl to 1 M KCl. The data are consistent with a sequential association model in which monomers are in equilibrium with tetramers and octamers at protein concentrations above 2.6 microM. Association is highly cooperative, with octamer formation favored by approximately 7 kcal/mol over tetramers. At high [KCl], the concentration of tetramers becomes negligible and the data are best described by a monomer-octamer reaction mechanism. The equilibrium association constants for both monomer <--> tetramer and tetramer <--> octamer reactions change with [KCl] in a biphasic manner, decreasing with increasing [KCl] from 60 mM to 300 mM, and increasing with increasing [KCl] from 300 mM to 1 M. At low [KCl], approximately 3 mole equivalents of ions are released at each association step, while at high [KCl], approximately 3 mole equivalents of ions are taken up at each association step. These results suggest that there is a salt concentration-dependent change in the assembly mechanism, and that the mechanistic switch takes place near 300 mM KCl. The possibility that this self-association reaction may play a role in the activity of the TATA-binding protein in vivo is discussed.     

Hb Sinai-Baltimore or alpha 2 beta (2)18(A15)Val->Gly, a silent, mildly unstable beta chain variant detected by isoelectrofocusing and high performance liquid chromatography

Isoelectrofocusing and high performance liquid chromatographic methods were used to study an abnormal hemoglobin present in a Black male infant and his mother. The variant, named Hb Sinai-Baltimore, focused slightly behind Hb A and separated incompletely from Hb A by cation exchange high performance liquid chromatography, while the separation of the beta A and beta X chains by reversed phase high performance liquid chromatography was complete. The variant was identified through an analysis of peptides in a tryptic digest of the isolated beta X chain and by sequencing of amplified DNA which included the beta-globin gene. The Val->Gly replacement at position beta 18 (codon 18; GTG->GGG) or at the last position of the A helix decreases the stability of the variant without affecting the hematological parameters of its carrier. The propositus was a compound heterozygote for Hb Sinai-Baltimore and Hb S; the relative quantities of the two variant chains were somewhat different from those of the beta X and beta A chains in the mother with the simple Hb Sinai-Baltimore heterozygosity. An uncertainty about the alpha-globin gene status in the child prevented a further evaluation of these differences.     

REM-sleep motor disorder in children

Beta-thalassemia (thal) is a common single-gene disease worldwide. However, the prevalence of beta-thal and the spectrum of beta-globin gene mutations in Filipinos remain unclear. This study sought to answer these two questions. A total of 2954 apparently healthy Filipinos in Taiwan were recruited for a prevalence study. A complete blood count was done in every subject. Those with microcytosis were studied with hemoglobin (Hb) high-performance liquid chromatography to determine the levels of Hb A2 and Hb F. Twenty-seven subjects had elevated levels of Hb A2 (>4.0%). These 27 suspected beta-thal carriers and another 16 beta-thal major patients who were being treated in the Philippines were studied to determine the spectrum of beta-globin gene mutations. Gap-PCR was used to detect the Filipino deletion of beta-thal, and direct sequencing was used to detect point or small mutations in the beta-globin gene. All of the 27 suspected beta-thal carriers had one mutation in the beta-globin gene, resulting in an overall prevalence of 0.9%. The spectrum of beta-thal mutations was similar in the carrier and patient groups. Analysis of the pooled identified seven different mutations in the study population. The Filipino deletion was the most common mutation, accounting for 45.8% (27/59) of the alleles, followed by codon 67 (-TG) (16 alleles), and Hb E (11 alleles). These three mutations accounted for 92% of the Filipino beta-thal alleles. Elucidation of the beta-thal mutations in Filipinos is useful for the genetic counseling and prenatal diagnosis of this disease.     

A new hemoglobin variant found during Hb A1c measurement: Hb Hokusetsu [beta52(D3)Asp-->Gly]

A new beta chain variant was accidentally found through the assay of Hb A1c in a diabetic patient. The variant was detected by polyacrylamide gel isoelectrofocusing and electrospray ionization mass spectrometry. For sequence determination, globin was cleaved with combination of trypsin and lysyl endopeptidase and analyzed by high performance liquid chromatography connected to electrospray ionization mass spectrometry. An abnormal betaT-5 peptide was found by reconstructed selected ion monitoring. The collision-induced dissociation spectrum of an ion derived from the abnormal betaT-5 peptide revealed a new substitution, [beta52(D3)Asp-->Gly], named Hb Hokusetsu. The sequence was confirmed with an automatic sequencer using peptides isolated by reversed phase high performance liquid chromatography. Amplification of the beta-globin exon 2 and nucleotide sequencing revealed a GAT-->GGT mutation in codon 52 corresponding to an Asp-->Gly replacement. Electrospray ionization mass spectrometry analysis of the hemolysate showed a reasonable value of 10.4% for glycated globin. The variant migrated as Hb S on isoelectrofocusing. Hematological analysis revealed normal parameters. The patient's hemolysate showed normal stability in the isopropanol test. Oxygen equilibrium studies on the patient's red blood cells and hemolysate showed no significant change in oxygen affinity or cooperativity.     

Identifying the conformational state of bi-liganded haemoglobin

While most researchers agree on the global features of cooperative ligand binding to haemoglobin (Hb), the internal mechanisms remain open to debate. This is not due to inaccurate measurements, but is rather a consequence of the cooperative ligand binding that decreases the equilibrium populations of the partially liganded states and makes observation of the transitions between these substates more difficult. For example, the equilibrium population of the doubly liganded tetramers is typically less than 5% of the total Hb. As a result many models with widely varying mechanisms may fit the oxygen equilibrium curve, but may not be consistent with observations of other parameters, such as ligand-binding kinetics or subunit association equilibria. The wide range of methods and models has led to divergent conclusions about the properties of specific substates. One notable debate concerns the properties of the doubly liganded forms. The simple two-state model predicts a shift in the allosteric equilibrium based on the number of ligands bound, but not on their distribution within the tetramer. From studies of dimer-tetramer equilibria of various pure and hybrid forms, it was concluded that a tetramer with two ligands bound on the same alpha beta dimer (species 21, an asymmetric hybrid) shows an enhanced tetramer stability, similar to singly liganded Hb, relative to the other three types of doubly liganded tetramers which resemble the triply liganded forms [Ackers et al. (1992). Science 255: 54-63]. The implications of this model and the relevant experiments will be reviewed here.     

Heterometallic hybrids of homometallic human hemoglobins

Hybridization experiments between normal Hb tetramers (Fe2+ Hb) and those with four metal-substituted hemes (i.e., replacement of Fe2+ by Co2+, Mg2+, Mn2+, Mn3+, Ni2+, or Zn2+) have revealed unexpected behavior. These homometallic Hbs have previously served as models that mimic the deoxy or oxy properties of normal Fe2+ Hb. In this study, hybrids were composed of one alpha 1 beta 1 dimer that is metal-substituted at both hemes, in association with a second dimer alpha 2 beta 2 that has normal Fe2+ hemes. Both metal-substituted subunits are unligated, whereas the two Fe2+ subunits either are both unligated or both ligated with O2, CO, or CN. It was found that four of the metal-substituted Hbs (Mg2+ Hb, Mn2+ Hb, Ni2+ Hb, and Zn2+ Hb) did not form detectable amounts of heterometallic hybrids with normal Fe2+ Hb even though (i) their homometallic parents formed tight tetrameric complexes with stabilities similar to that of Fe2+ Hb and (ii) hybrids with metal substitution at both alpha sites or both beta sites are known to form readily. This striking positional effect was independent of whether the normal Fe2+ hemes were ligated and of which ligand was used. These findings indicate that surprisingly large changes in tetramer behavior can arise from small and subtle perturbations at the heme sites. Possible origins of these effects are considered.     

Role of beta87 Thr in the beta6 Val acceptor site during deoxy Hb S polymerization

Three new Hb S variants containing beta87 Leu, Trp, or Asp instead of Thr were expressed in yeast in order to further define the role of the beta87 position in stability and polymerization of deoxy Hb S. Previous studies showed that hydrophobicity at beta85 Phe and beta88 Leu is critical for stabilization of hemoglobin. Results with the three Hb S beta87 variants, however, showed minimal differences in stability, suggesting that beta87 amino acid hydrophobicity is not critical for stabilization of hemoglobin. Polymerization properties of the variants in the deoxy form, however, were affected by the beta87 amino acid. Polymerization of Hb S beta87 Thr --> Leu and Hb S beta87 Thr --> Trp was preceded by a delay time like Hb S, while Hb S beta87 Thr --> Asp did not show a delay time. In addition, changes in time required for half polymer formation (T1/2) as a function of hemoglobin concentration for Hb S beta87 Thr --> Asp were similar to that for beta87 Thr --> Gln. Hb S beta87 Thr --> Leu polymerized at a lower hemoglobin concentration than Hb S while beta87 Thr --> Trp and Hb S beta87 Thr --> Asp required much higher hemoglobin concentrations for polymer formation. Critical concentration required for deoxy Hb S beta87 Thr --> Asp polymerization was 6- and 2.3-fold greater than that for Hb S beta85 Phe --> Glu and Hb S beta88 Leu --> Glu, respectively. These results suggest that even though beta87 Thr is not a direct interaction site for beta6 Val in deoxy Hb S polymers, it does play a critical role in formation of the hydrophobic acceptor pocket which then promotes protein-protein interactions facilitating formation of stable nuclei and polymers of deoxy Hb S.     

Tetramer-dimer equilibrium of oxyhemoglobin mutants determined from auto-oxidation rates

One of the main difficulties with blood substitutes based on hemoglobin (Hb) solutions is the auto-oxidation of the hemes, a problem aggravated by the dimerization of Hb tetramers. We have employed a method to study the oxyHb tetramer-dimer equilibrium based on the rate of auto-oxidation as a function of protein concentration. The 16-fold difference in dimer and tetramer auto-oxidation rates (in 20 mM phosphate buffer at pH 7.0, 37 degrees C) was exploited to determine the fraction dimer. The results show a transition of the auto-oxidation rate from low to high protein concentrations, allowing the determination of the tetramer-dimer dissociation coefficient K4,2 = [Dimer] 2/[Tetramer]. A 14-fold increase in K4,2 was observed for addition of 10 mM of the allosteric effector inositol hexaphosphate (IHP). Recombinant hemoglobins (rHb) were genetically engineered to obtain Hb with a lower oxygen affinity than native Hb (Hb A). The rHb alpha2beta2 [(C7) F41Y/(G4) N102Y] shows a fivefold increase in K4,2 at pH 7.0, 37 degrees C. An atmosphere of pure oxygen is necessary in this case to insure fully oxygenated Hb. When this condition is satisfied, this method provides an efficient technique to characterize both the tetramer-dimer equilibrium and the auto-oxidation rates of various oxyHb. For low oxygen affinity Hb equilibrated under air, the presence of deoxy subunits accelerates the auto-oxidation. Although a full analysis is complicated, the auto-oxidation studies for air equilibrated samples are more relevant to the development of a blood substitute based on Hb solutions. The double mutants, rHb alpha2beta2 [(C7) F41Y/(G4) N102A] and rHb alpha2beta2 [(C7) F41Y/(E10) K66T], show a lower oxygen affinity and a higher rate of oxidation than Hb A. Simulations of the auto-oxidation rate versus Hb concentration indicate that very high protein concentrations are required to observe the tetramer auto-oxidation rate. Because the dimers oxidize much more rapidly, even a small fraction dimer will influence the observed oxidation rate.     

ATP-induced tetramerization and cooperativity in hemoglobin of lower vertebrates

The importance of intraerythrocytic organic phosphates in the allosteric control of oxygen binding to vertebrate hemoglobin (Hb) is well recognized and is correlated with conformational changes of the tetramer. ATP is a major allosteric effector of snake Hb, since the absence of this nucleotide abolishes the Hb cooperativity. This effect may be related to the molecular weight of about 32,000 for this Hb, which is compatible with the dimeric form. ATP induces a pH-dependent tetramerization of deoxyHb that leads to the recovery of cooperativity. This phenomenon may be partially explained by two amino acid replacements in the beta chains (CD2 Glu-43 --> Thr and G3 Glu-101 --> Val), which result in the loss of two negative charges at the alpha1beta2 interface and favors the dissociation into dimers. The ATP-dependent dimer left arrow over right arrow tetramer may be physiologically important among ancient animal groups that have similar mutations and display variations in blood pH that are governed by these animals' metabolic state. The enormous loss of free energy of association that accompanies Hb oxygenation, and which is also observed at a much lower intensity in higher vertebrate Hbs, must be taken into consideration in allosteric models. We propose that the transition from a myoglobin-like protein to an allosteric one may be of evolutionary significance.     

Identification of Hb C [beta 6(A3)Glu-->Lys] in a Thai male

The propositus was a 29-year-old Thai male, whose electrophoretic pattern showed Hb A (58%) plus an abnormal hemoglobin (42%) with mobility identical to Hb A2 and Hb E. Protein sequencer analysis and tryptic peptide mapping of the beta chain indicated that the abnormal hemoglobin was Hb C [beta 6(A3)Glu-->Lys], rather than Hb E which is more commonly found in South East Asia. This conclusion was confirmed by direct sequence analysis of the propositus' DNA, which showed AAG as well as GAG at codon 6 of the beta gene, in agreement with heterozygosity for Hb C and Hb A. Furthermore, the beta gene framework (Ava II-, Bam HI+) of the propositus suggested that the beta C gene may have arisen from an independent mutation. Since Hb C and Hb E have the same mutation (Glu-->Lys) in the beta chain, although at different positions, and behave similarly in electrophoresis, cases of Hb C and Hb E may sometimes have been mistakenly identified for each other, based on whichever variant is most prevalent in the particular population.     

Glycerol kinase from Escherichia coli and an Ala65-->Thr mutant: the crystal structures reveal conformational changes with implications for allosteric regulation

BACKGROUND: Glycerol kinase (GK) from Escherichia coli is a velocity-modulated (V system) enzyme that has three allosteric effectors with independent mechanisms: fructose-1,6-bisphosphate (FBP); the phosphocarrier protein IIAGlc; and adenosine nucleotides. The enzyme exists in solution as functional dimers that associate reversibly to form tetramers. GK is a member of a superfamily of ATPases that share a common ATPase domain and are thought to undergo a large conformational change as an intrinsic step in their catalytic cycle. Members of this family include actin, hexokinase and the heat shock protein hsc70. RESULTS: We report here the crystal structures of GK and a mutant of GK (Ala65-->Thr) in complex with glycerol and ADP. Crystals of both enzymes contain the same 222 symmetric tetramer. The functional dimer is identical to that described previously for the IIAGlc-GK complex structure. The tetramer interface is significantly different, however, with a relative 22.3 degrees rotation and 6.34 A translation of one functional dimer. The overall monomer structure is unchanged except for two regions: the IIAGlc-binding site undergoes a structural rearrangement and residues 230-236 become ordered and bind orthophosphate at the tetramer interface. We also report the structure of a second mutant of GK (IIe474-->Asp) in complex with IIAGlc; this complex crystallized isomorphously to the wild type IIAGlc-GK complex. Site-directed mutants of GK with substitutions at the IIAGlc-binding site show significantly altered kinetic and regulatory properties, suggesting that the conformation of the binding site is linked to the regulation of activity. CONCLUSIONS: We conclude that the new tetramer structure presented here is an inactive form of the physiologically relevant tetramer. The structure and location of the orthophosphate-binding site is consistent with it being part of the FBP-binding site. Mutational analysis and the structure of the IIAGlc-GK(IIe474-->Asp) complex suggest the conformational transition of the IIAGlc-binding site to be an essential aspect of IIAGlc regulation.     

Crystal structure of a complex of Escherichia coli glycerol kinase and an allosteric effector fructose 1,6-bisphosphate

The three-dimensional structures of Escherichia coli glycerol kinase (GK) with bound glycerol in the presence and absence of one of the allosteric regulators of its activity, fructose 1,6-bisphosphate (FBP), at 3.2 and 3.0 A, are presented. The molecule crystallized in space group P41212, and the structure was solved by molecular replacement. The models were refined with good stereochemistry to final R-factors of 21.1 and 21.9%, respectively. A tetrameric arrangement of monomers was observed which was essentially identical to the proposed inactive tetramer II previously described [Feese, M. D., Faber, H. R., Bystrom, C. E., Pettigrew, D. W., and Remington, S. J. (1998) Structure (in press)]. However, the crystal packing in this form was especially open, permitting the FBP binding site to be occupied and identified. The crystallographic data revealed a most unusual type of FBP binding site formed between two glycine-arginine loops (residues 234-236) where one-half of the binding site is donated by each monomer at the regulatory interface. The molecule of FBP binds in two mutually exclusive modes on a noncrystallographic 2-fold axis at 50% occupancy each; thus, a tetramer of GK binds two molecules of FBP. Ionic interactions between the 1- and 6-phosphates of FBP and Arg 236 were observed in addition to hydrogen bonding interactions between the backbone amide of Gly 234 and the 6-phosphate. No contacts between the protein and the furanose ring were observed. Mutagenesis of Arg 236 to alanine drastically reduced the extent of inhibition of GK by FBP and lowered, but did not eliminate, the ability of FBP to promote tetramer association. These observations are consistent with the previously characterized mechanism of FBP inhibition of GK, in which FBP acts both to promote dimer-tetramer assembly and to inactivate the tetramers.     

Hemodynamic and inotropic effects of milrinone after heart transplantation in the setting of recipient pulmonary hypertension

The complementary fragments of human Hb alpha, alpha1-30, and alpha31-141 are spliced together by V8 protease in the presence of 30% n-propanol to generate the full-length molecule (Hb alpha-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb alpha is facilitated by the organic cosolvent induced alpha-helical conformation of product acting as the "molecular trap" of the splicing reaction. The segments alpha24-30 and alpha31-40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) alpha24-40 has been manipulated by engineering the amino acid replacements to the positions alpha27 and alpha31 to delineate the structural basis of the molecular trap. The location of Glu27 and Arg31 residues in the contiguous segment alpha24-40 (as well as in other larger segments) is ideal to generate (i, i + 4) side-chain carboxylate-guanidino interaction in its alpha-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at alpha27 and alpha31 facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The gamma-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an alpha-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu30-Arg31 peptide bond. The second structural aspect is a site-specific event, an i, i + 4 side-chain interaction in the alpha-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven "molecular traps" of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

Role of Leu-beta 88 in the hydrophobic acceptor pocket for Val-beta 6 during hemoglobin S polymerization

X-ray crystallographic studies indicate that the hydrophobic acceptor pocket made by E and F helices involving Leu-beta 88 and Phe-beta 85 is critical for the formation of stable hydrophobic interactions with Val-beta 6 on an adjacent deoxy-hemoglobin (Hb) S tetramer. Ala and Phe substitutions at the beta 88 position in Hb S were made using a yeast expression system in an effort to clarify the role of Leu-beta 88 in creating a suitable acceptor site for Val-beta 6 during polymerization of Hb S. Both Ala- and Phe-beta 88 substitutions in Hb S inhibited polymerization compared with Hb S. Critical concentrations for polymerization of alpha 2 beta 2 Val-6,Ala-88 and alpha 2 beta 2Val-6,Phe-88 were 6- and 10-fold higher, respectively, than that of Hb S (alpha 2 beta 2Val-6,Leu-88). Deoxy-Hb S containing Phe-beta 88 polymerized without a delay time like Trp-beta 6- and Phe-beta 6-substituted hemoglobins (Adachi, K., Konitzer, P., Kim, J., Welch, N., and Surrey, S. (1993) J. Biol. Chem. 268, 21650-21656). In contrast, oversaturated deoxy-Hb S containing Ala-beta 88 also polymerized without a delay time; however, with decreasing hemoglobin concentrations, the kinetics of polymerization were biphasic. At lower hemoglobin concentrations, closer to the critical concentration for polymerization, deoxy-Hb S containing Ala-beta 88 polymerized after a distinct delay time. These results suggest that bulky beta 88 hydrophobic replacements like Phe may sterically inhibit insertion of Val-beta 6 into the acceptor pocket. In contrast, smaller sized, less hydrophobic amino acids like Ala compared with Leu-beta 88 may allow insertion of Val-beta 6 into the acceptor pocket but may not promote stable protein-protein interactions with an adjacent Hb molecule. Stereospecificity and hydrophobicity of the Val-beta 6 hydrophobic acceptor pocket as well as the beta 6 amino acid are, therefore, critical for polymerization of deoxy-Hb S.     

Alteration of an intersubunit contact in hemoglobin variants: comparative study of modifications at position alpha 126 Asp (H9)

Four human hemoglobin variants have already been described at position alpha 126 (H9), which is normally occupied by an aspartate: Hb Montefiore (-->Tyr), Hb Tarrant (-->Asn), Hb Fukutomi (-->Val), Hb Sassari (-->His). An additional variant, Hb West One (alpha 126 (H9) Asp-->Gly) is herein described. Aspartate alpha 126 (H9) is involved in a set of hydrogen bonds and salt bridges located at the C-terminal portion of the alpha-chains and of the C-helix of the beta-chains, which are broken in the oxy conformer, providing one of the most important sources of the difference in free energy between the T- and R-state in hemoglobin. A comparative study of four of these alpha 126 Hb variants is presented. An identical degree of alteration of the oxygen binding properties (increased oxygen affinity and decreased cooperativity) was found in all cases, when measured under standard experimental conditions (pH 7.2, 0.1 M NaCl). In contrast, the effect of L345 (a derivative of bezafibrate, which is a specific alpha-chain binding effector) on oxygen binding to Hb differed from one variant to another. When a bulky Tyr or His residue occupied the alpha 126 (H9) position, little effect of L345 was observed. Conversely, when this position was occupied by a residue of smaller size (Gly or Asn), normal heterotropic effects were observed. Molecular graphic modelling indicates that two classes of three-dimensional structure modifications may occur.     

Chromatin assembly in a yeast whole-cell extract

A simple in vitro system that supports chromatin assembly was developed for Saccharomyces cerevisiae. The assembly reaction is ATP-dependent, uses soluble histones and assembly factors, and generates physiologically spaced nucleosomes. We analyze the pathway of histone recruitment into nucleosomes, using this system in combination with genetic methods for the manipulation of yeast. This analysis supports the model of sequential recruitment of H3/H4 tetramers and H2A/H2B dimers into nucleosomes. Using a similar approach, we show that DNA ligase I can play an important role in template repair during assembly. These studies demonstrate the utility of this system for the combined biochemical and genetic analysis of chromatin assembly in yeast.     

Thermodynamic studies on the equilibrium properties of a series of recombinant betaW37 hemoglobin mutants

In human hemoglobin (Hb) the beta37 tryptophan residue (betaW37), located at the hinge region of the alpha1beta2 interface, forms many contacts with alpha subunit residues of the opposite dimer, in both the T and R quaternary structures. We have carried out equilibrium O2 binding studies on a series of recombinant Hbs that have mutations at this residue site: betaW37Y, betaW37A, betaW37G, and betaW37E. Binding isotherms measured at high concentrations of these mutants were found to be shifted toward increased affinity and decreased cooperativity from that of the normal HbA0 tetramer. Analysis of these binding isotherms indicated that amino acid substitutions at the beta37 position could both destabilize the tetrameric form of the mutants relative to their constituent dimers and also alter cooperativity of the intact tetrameric species. These alterations from wild-type function are dependent on the particular side chain substituted, with the magnitude of change increasing as Trp is substituted by Tyr, Ala, Gly, and Glu. The dimer to tetramer assembly free energy of deoxy-betaW37E, the most perturbed mutant in the series, was measured using analytical gel chromatography to be 9 kcal/tetramer less favorable than that of deoxy HbA0. Stabilizing the betaW37E tetramer by addition of IHP, or by cross-linking at the alphaK99 positions, does not restore normal O2 binding behavior. Thermodynamic parameters of all the mutants were found to correlate with their CO binding rates and with their high-resolution X-ray crystal structures (see accompanying papers: Kwiatkowski et al. (1998) Biochemistry 37, 4325-4335; Peterson & Friedman (1998) Biochemistry 37, 4346-4357; Kavanaugh et al. (1998) Biochemistry 37, 4358-4373].     

Cloning of a new allelic variant of a Saccharomyces diastaticus glucoamylase gene and its introduction into industrial yeasts

A new allelic variant of the STA2 gene, designated as STA2K, coding for a secreted glucoamylase, was cloned. Differences were revealed both in the structural gene and in the promoter region, as compared to other STA genes. The most peculiar structural features of STA2K are 1. a 1.1-kb natural deletion in its promoter located 189 nucleotides upstream of the translation start codon; and 2. an Asn-->Asp single amino acid change within the putative active site of the encoded glucoamylase. Neither the presence of glucose in the medium nor the host cell's mating type constellation affected the expression level of STA2K in S. cerevisiae. Self-replicating yeast plasmids containing STA2K were constructed and used to transform a laboratory yeast strain and various brewing strains. Pilot brewing tests with glucoamylase-secreting transformants of a brewing strain produced superattenuated beers at accelerated fermentation rates.