Adjacent Tetramers Research Articles

Unravelling the regulation pathway of photosynthetic AB-GAPDH.

Oxygenic phototrophs perform carbon fixation through the Calvin-Benson cycle. Different mechanisms adjust the cycle and the light-harvesting reactions to rapid environmental changes. Photosynthetic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key enzyme in the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant isoform is formed by A2B2 heterotetramers and the least abundant by A4 homotetramers. Regardless of thesubunit composition, GAPDH is the major consumer of photosynthetic NADPH and its activity is strictly regulated. While A4-GAPDH is regulated byCP12, AB-GAPDH is autonomously regulated through the C-terminal extension (CTE) of its B subunits. Reversible inhibition of AB-GAPDH occurs via the oxidation of a cysteine pair located in the CTE and the substitution of NADP(H) with NAD(H) in the cofactor-binding site. These combined conditions lead to a change in the oligomerization state and enzyme inhibition. SEC-SAXS and single-particle cryo-EM analysis were applied to reveal the structural basis of this regulatory mechanism. Both approaches revealed that spinach (A2B2)n-GAPDH oligomers with n = 1, 2, 4 and 5 co-exist in a dynamic system. B subunits mediate the contacts between adjacent tetramers in A4B4 and A8B8 oligomers. The CTE of each B subunit penetrates into the active site of a B subunit of the adjacent tetramer, which in turn moves its CTE in the opposite direction, effectively preventing the binding of the substrate 1,3-bisphosphoglycerate in the B subunits. The whole mechanism is made possible, and eventually controlled, by pyridine nucleotides. In fact, NAD(H), by removing NADP(H) from A subunits, allows the entrance of the CTE into the active site of the B subunit, hence stabilizing inhibited oligomers.

Synthesis and crystal structures of tetra-meric [2-(4,4-dimethyl-2-oxazolin-2-yl)anilido]sodium and tris-[2-(4,4-dimethyl-2-oxazolin-2-yl)anilido]ytterbium(III).

Reaction of 2-(4,4-dimethyl-2-oxazolin-2-yl)aniline (H2-L1) with one equivalent of Na[N(SiMe3)2] in toluene afforded pale-yellow crystals of tetra-meric poly[bis-[μ3-2-(4,4-dimethyl-2-oxazolin-2-yl)anilinido][μ2-2-(4,4-dimethyl-2-oxa-zolin-2-yl)aniline]tetra-sodium(I)], [Na4(C11H13N2O)4] n or [Na4(H-L1)4] n (2), in excellent yield. Subsequent reaction of [Na4(H-L1)4] n (2) with 1.33 equivalents of anhydrous YbCl3 in a 50:50 mixture of toluene-THF afforded yellow crystals of tris-[2-(4,4-dimethyl-2-oxazolin-2-yl)anilinido]ytterbium(III), [Yb(C11H13N2O)3] or Yb(H-L1)3 (3) in moderate yield. Direct reaction of three equivalents of 2-(4',4'-dimethyl-2'-oxazolin-yl)aniline (H2-L1) with Yb[N(SiMe3)2]3 in toluene resulted in elimination of hexa-methyl-disilazane, HN(SiMe3)2, and produced Yb(H-L1)3 (3) in excellent yield. The structure of 2 consists of tetra-meric Na4(H-L1)4 subunits in which each Na+ cation is bound to two H-L1 bridging bidentate ligands and these subunits are connected into a polymeric chain by two of the four oxazoline O atoms bridging to Na+ cations in the adjacent tetra-mer. This results in two 4-coordinate and two 5-coordinate Na+ cations within each tetra-meric unit. The structure of 3 consists of a distorted octa-hedron where the bite angle of ligand L1 ranges between 74.72 (11) and 77.79 (11) degrees. The oxazoline (and anilide) N atoms occupy meridional sites such that for one ligand an anilide nitro-gen is trans to an oxazoline nitro-gen while for the other two oxazoline N atoms are trans to each other. This results in a significantly longer Yb-N(oxazoline) distance [2.468 (3) Å] for the bond trans to the anilide compared to those for the oxazoline N atoms trans to one another [2.376 (3), 2.390 (3) Å].

The binding interactions that maintain excitation-contraction coupling junctions in skeletal muscle.

Calcium for contraction of skeletal muscles is released via tetrameric ryanodine receptor (RYR1) channels of the sarcoplasmic reticulum (SR), which are assembled in ordered arrays called couplons at junctions where the SR abuts T tubules or plasmalemma. Voltage-gated Ca2+ (CaV1.1) channels, found in tubules or plasmalemma, form symmetric complexes called CaV tetrads that associate with and activate underlying RYR tetramers during membrane depolarization by conveying a conformational change. Intriguingly, CaV tetrads regularly skip every other RYR tetramer within the array; therefore, the RYRs underlying tetrads (named V), but not the voltage sensor-lacking (C) RYRs, should be activated by depolarization. Here we hypothesize that the checkerboard association is maintained solely by reversible binary interactions between CaVs and RYRs and test this hypothesis using a quantitative model of the energies that govern CaV1.1-RYR1 binding, which are assumed to depend on number and location of bound CaVs. A Monte Carlo simulation generates large statistical samples and distributions of state variables that can be compared with quantitative features in freeze-fracture images of couplons from various sources. This analysis reveals two necessary model features: (1) the energy of a tetramer must have wells at low and high occupation by CaVs, so that CaVs positively cooperate in binding RYR (an allosteric effect), and (2) a large energy penalty results when two CaVs bind simultaneously to adjacent RYR protomers in adjacent tetramers (a steric clash). Under the hypothesis, V and C channels will eventually reverse roles. Role reversal justifies the presence of sensor-lacking C channels, as a structural and functional reserve for control of muscle contraction.

Protein-directed self-assembly of a fullerene crystal.

Learning to engineer self-assembly would enable the precise organization of molecules by design to create matter with tailored properties. Here we demonstrate that proteins can direct the self-assembly of buckminsterfullerene (C60) into ordered superstructures. A previously engineered tetrameric helical bundle binds C60 in solution, rendering it water soluble. Two tetramers associate with one C60, promoting further organization revealed in a 1.67-Å crystal structure. Fullerene groups occupy periodic lattice sites, sandwiched between two Tyr residues from adjacent tetramers. Strikingly, the assembly exhibits high charge conductance, whereas both the protein-alone crystal and amorphous C60 are electrically insulating. The affinity of C60 for its crystal-binding site is estimated to be in the nanomolar range, with lattices of known protein crystals geometrically compatible with incorporating the motif. Taken together, these findings suggest a new means of organizing fullerene molecules into a rich variety of lattices to generate new properties by design.

Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix.

MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNA molecules bound specifically to the tetramer and engaged with the enzyme's two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act.

A 1:1 co-crystal of the herbicide triflusulfuron-methyl and its degradation product triazine amine

The herbicide triflusulfuron-methyl (systematic name: methyl 2-{[4-dimethyl­amino-6-(2,2,2-trifluoro­eth­oxy)-1,3,5-triazin-2-yl]carbamoylsulfamo­yl}-3-methyl­benzoate) and its degradation product triazine amine [systematic name: 2-amino-4-dimethyl­amino-6-(2,2,2-trifluoro­eth­oxy)-1,3,5-triazine] form a triclinic 1:1 co-crystal of the title compound, C7H10F3N5O·C17H19F3N6O6S, in which its two components are connected via a pair of complementary N—H⋯N hydrogen bonds, similar to the monoclinic crystal structure of the parent compound triflusulfuron-methyl [Mereiter (2011 ▶). Acta Cryst. E67, o1778–o1779] in which a pair of mol­ecules related by a twofold axis are linked by two N—H⋯N bonds. The triflusulfuron-methyl mol­ecules of both crystal structures are similar in geometric parameters and conformation, which is due to stiffening by a short intra­molecular N—H⋯N bond [N⋯N = 2.620 (4) Å] and an intra­molecular dipole–dipole inter­action between the sulfamide and the carboxyl moieties, with Os⋯Cc = 2.802 (5) Å and Oc⋯Ns = 2.846 (4) Å. Inter­molecular N—H⋯O hydrogen bonds and slipped π–π stacking inter­actions between the diamino­triazine moieties [perpendicular distances of 3.25 Å within hydrogen-bonded tetra­mers and 3.27 Å between adjacent tetra­mers] link the two constituents of the co-crystal into columns parallel to the a axis. An intra­molecular C—H⋯O hydrogen bond occurs in the triflusulfuron-methyl mol­ecule and inter­molecular C—H⋯O inter­actions between triflusulfuron-methyl mol­ecules occur in the crystal structure. In the triflusulfuron-methyl molecule the dihedral angle between the least-squares planes of the two rings is 75.8 (1)°. In the triazine molecule, the CF3 group is partly orientationally disordered.

Influence of the Cytoplasmic Domains of Aquaporin-4 on Water Conduction and Array Formation

Phosphorylation of Ser180 in cytoplasmic loop D has been shown to reduce the water permeability of aquaporin (AQP) 4, the predominant water channel in the brain. However, when the structure of the S180D mutant (AQP4M23S180D), which was generated to mimic phosphorylated Ser180, was determined to 2.8 Å resolution using electron diffraction patterns, it showed no significant differences from the structure of the wild-type channel. High-resolution density maps usually do not resolve protein regions that are only partially ordered, but these can sometimes be seen in lower-resolution density maps calculated from electron micrographs. We therefore used images of two-dimensional crystals and determined the structure of AQP4M23S180D at 10 Å resolution. The features of the 10-Å density map are consistent with those of the previously determined atomic model; in particular, there were no indications of any obstruction near the cytoplasmic pore entrance. In addition, water conductance measurements, both in vitro and in vivo, show the same water permeability for wild-type and mutant AQP4M23, suggesting that the S180D mutation neither reduces water conduction through a conformational change nor reduces water conduction by interacting with a protein that would obstruct the cytoplasmic channel entrance. Finally, the 10-Å map shows a cytoplasmic density in between four adjacent tetramers that most likely represents the association of four N termini. This finding supports the critical role of the N terminus of AQP4 in the stabilization of orthogonal arrays, as well as their interference through lipid modification of cysteine residues in the longer N-terminal isoform.

Effects of cross-linking and zero-link polymerization on oxygen transport and redox chemistry of bovine hemoglobin

Cross-linked hemoglobins (Hbs) were found to have enhanced oxidative reactions which compromise the ability of cell-free Hb to carry oxygen. Zero-link bovine Hb (ZL-HbBv), also known as OxyVita™, a large polymer held together by pseudopeptide bonds on the surface of adjacent tetramers, provides a model in which these reactions can be evaluated. The oxygen affinity of ZL-HbBv was greatly increased, whereas the oxygen binding cooperativity ( n 50) as well as the regulatory responses to pH and chloride ions was diminished. Rapid mixing kinetic studies revealed faster carbon monoxide (CO) and nitric oxide (NO) binding to ZL-HbBv, consistent with a more accessible heme pocket conformation. The rate of autoxidation of ferrous ZL-HbBv was 3 folds faster than the unmodified HbBv (control) but only slightly suppressed by the presence of superoxide dismutase and catalase enzymes. The peroxide (H 2O 2) reaction rates of ferric ZL-HbBv and its degradation were comparable to that of the control. The rate of heme loss from ZL-HbBv to a mutant apomyoglobin (H64Y/V68F) was also very close to that of the control. Taken together, allosteric and redox reactions of this protein are altered due to heme accessibility to solvent, however, the compact tetramer to tetramer interaction of the ZL-HbBv polymer appears to restrict heme loss even in the presence of excess H 2O 2.

Structure and function of the visual arrestin oligomer

A distinguishing feature of rod arrestin is its ability to form oligomers at physiological concentrations. Using visible light scattering, we show that rod arrestin forms tetramers in a cooperative manner in solution. To investigate the structure of the tetramer, a nitroxide side chain (R1) was introduced at 18 different positions. The effects of R1 on oligomer formation, EPR spectra, and inter-spin distance measurements all show that the structures of the solution and crystal tetramers are different. Inter-subunit distance measurements revealed that only arrestin monomer binds to light-activated phosphorhodopsin, whereas both monomer and tetramer bind microtubules, which may serve as a default arrestin partner in dark-adapted photoreceptors. Thus, the tetramer likely serves as a 'storage' form of arrestin, increasing the arrestin-binding capacity of microtubules while readily dissociating to supply active monomer when it is needed to quench rhodopsin signaling.

Poly[di-μ2-methanolato-di-μ2-tetramethoxyborato-disodium(I)

In the polymeric title compound, [Na2(C4H12BO4)2(CH4O)2]n, the two independent sodium cations are bound by five O atoms. All the O atoms of one tetramethoxyborate anion bind cations, forming a tetrameric cluster around a tetragonal inversion centre [Na-O = 2.2777 (18)-2.3907 (16) A]. Two methanol O atoms bridge the two Na atoms [Na-O = 2.3590 (15)-2.4088 (18) A] and provide the hydrogen-bonding H atoms. The second tetramethoxyborate anion provides two O atoms to one Na atom [mean Na-O = 2.31 (2) A] and two O atoms as donors for crosslinking hydrogen bonds to adjacent tetramers, which complete the three-dimensional packing. The crystal was a treated as a racemic twin.

Structure of the Complex of Maclura pomiferaAgglutinin and the T-antigen Disaccharide, Galβ1,3GalNAc

Maclura pomifera agglutinin is a tetrameric plant seed lectin with high affinity for the tumor-associated T-antigen disaccharide, Galbeta1,3GalNAcalpha, and hence for many O-linked glycopeptide structures. Unlike members of most lectin families, it lacks both metal ions and Cys residues. The structure of its complex with Galbeta1,3GalNAc was determined to 2.2 by first using multiwavelength anomalous diffraction with a lead derivative of the native protein, and then using molecular replacement with the unrefined structure as a model to solve the structure of the complex. The subunits share the beta-prism architecture and three-fold pseudo-symmetry of the related lectin jacalin, with the 21-residue beta-chains in the center of the tetramer. Interactions with the GalNAc predominate in the binding of the disaccharide. It forms a network of H-bonds with only one side chain, from an Asp residue, the amino group of the N-terminal Gly of the alpha-chain, and peptide backbone atoms of two aromatic residues. The Gal moiety does not H-bond directly with residues in the same monomer, i.e. there is no true subsite for it, but there are interactions through two water molecules. In the crystal, it interacts with residues in the binding site of an adjacent tetramer. The minimum energy conformation expected for the disaccharide is retained, despite its mediating the tetramer-tetramer interactions in the crystal packing. The resulting lattice is comparable to those seen for complexes of other lectins with branched glycopeptides.

Polymerization of Recombinant Hemoglobin F γE6V and Hemoglobin F γE6V, γQ87T Alone, and in Mixtures With Hemoglobin S

To further understand determinants for Hemoglobin (Hb) S polymerization, as well as the inhibitory mechanism of Hb F on Hb S polymerization, Hb F variants containing Val-gamma 6 (Hb F gamma E6V) or Val-gamma 6, Thr-gamma 87 (Hb F gamma E6V, gamma Q87T) were expressed in yeast. The oxy form of Hb F gamma E6V was about 10-fold less stable to mechanical agitation than native oxy Hb F, which is similar to stability differences comparing oxy Hb S and oxy Hb A. Deoxy Hb F gamma E6V showed approximately 20-fold decreased solubility compared with native deoxy Hb F in high phosphate buffer and formed gels like deoxy Hb S in low phosphate buffer, indicating that the Val-gamma 6 substitution decreases solubility of Hb F like Val-beta 6 in deoxy Hb S. Oversaturated deoxy Hb F gamma E6V polymerized without a delay time in low and high phosphate buffers, in contrast to deoxy Hb S, which is accompanied by a distinct delay time before polymerization. Deoxy Hb F gamma E6V, gamma Q87T also polymerized without a delay time like deoxy Hb F gamma E6V. These results suggest that deoxy Hb F gamma E6V gamma Q87T polymers are different from those of deoxy Hb S, and that contact sites differ from those of deoxy Hb S, even though both have the same primary donor (A3) and acceptor sites in the EF helix. These results also suggest that other amino acids in addition to beta 6 Val and amino acids in the F helix are critical for nucleation-controlled polymerization of deoxy Hb S. 1:1 mixtures of deoxy Hb S and either Hb F variant polymerized with a delay time when the concentrations for the Hb S/Hb F gamma E6V and Hb S/Hb F gamma E6V, gamma Q87T mixtures were about 2- and 1.5-fold, respectively, higher than that for Hb S. Logarithmic plots of delay time versus concentration for Hb S/Hb F gamma E6V mixtures showed the same straight line as the line for Hb S/Hb S beta T87Q mixtures, but values for Hb S/Hb F gamma E6V, gamma Q87T mixtures were intermediate between those for Hb S and Hb S/Hb F gamma E6V mixtures. A 1:1 mixture of deoxy Hb A and Hb F gamma E6V, gamma Q87T also polymerized, but exhibited biphasic kinetics, when the concentration was increased to more than 3.5-fold higher than that required for Hb S polymer formation. These results suggest that Gin-gamma 87 is a critical amino acid for exclusion of FS hybrids (alpha 2 beta S gamma) from nuclei formation with Hb S. Our findings also show that Val-gamma 6 in hybrids that form in mixtures of the Hb F variants with either Hb S or Hb A interacts with the hydrophobic acceptor pocket on the EF helix of an adjacent tetramer containing Thr-beta 87.

Dimer-dimer interfaces of the lambda-repressor are different in liganded and free states.

Lambda-repressor dimers associate in solution to form tetramers and higher order structures. Dimer-dimer contact is also crucial in cooperative binding to adjacent operators. Fluorescence quenching studies indicate that the tryptophan 230 environment is significantly different in unliganded and adjacent operator-bound tetramers. Acrylodan attached to Cys 235, in a mutant F235C repressor, is also in different environments in the unliganded and adjacent operator bound tetramers. Thermodynamics of protein association, measured by fluorescence anisotropy, indicate that, whereas free repressor dimer association is strongly enthalpy driven, the single-operator (OR1)-bound repressor dimer association is largely entrophy driven with little change in enthalpy. Single-operator-bound dimer association to the corresponding tetramer does not lead to any significant change in tryptophan 230 environment, as was seen in the case of the free repressor. Data are also presented to support the contention that, under the conditions of this study, the free repressor association is predominantly from dimer to tetramer and then to octamer, unlike the dimer to octamer transition observed under a different condition. The results presented here point toward the conclusion that the lambda-repressor dimer-dimer interface is significantly different in the free and operator-bound states and that operator binding plays a crucial role in changing the nature of the dimer-dimer interface.

The Macromolecular Structure of Type-VI Collagen. Formation and Stability of Filaments

Type-VI collagen microfilaments were directly isolated from human amnion without using strong denaturing reagents. The microfilaments were characterized by electron microscopy, SDS/PAGE and immunoprecipitation. There was no evidence of other components bound to the isolated filaments and no covalent bonds between adjacent tetramers. The association between tetramers was further analyzed by studying the affinity between globular domains and the helix of type-VI collagen. Solid-phase-binding assays and conventional column chromatography showed that the globular domains have a high affinity for each other and for the helices of type-VI collagen, indicating that filaments may be assembled and stabilized in the absence of additional components. Hyaluronan did not stabilize the filaments nor facilitate the assembly of tetramers into filaments. The interaction between domains was also studied after modifying the sugar moieties of type-VI collagen globular domains and monomeric triple-helical domains. The oligosaccharides are involved in helix-helix interactions but not in interactions of the globular domains with each other or with the triple helix.

The Macromolecular Structure of Type-VI Collagen. Formation and Stability of Filaments

Type-VI collagen microfilaments were directly isolated from human amnion without using strong denaturing reagents. The microfilaments were characterized by electron microscopy, SDS/PAGE and immunoprecipitation. There was no evidence of other components bound to the isolated filaments and no covalent bonds between adjacent tetramers. The association between tetramers was further analyzed by studying the affinity between globular domains and the helix of type-VI collagen. Solid-phase-binding assays and conventional column chromatography showed that the globular domains have a high affinity for each other and for the helices of type-VI collagen, indicating that filaments may be assembled and stabilized in the absence of additional components. Hyaluronan did not stabilize the filaments nor facilitate the assembly of tetramers into filaments. The interaction between domains was also studied after modifying the sugar moieties of type-VI collagen globular domains and monomeric triple-helical domains. The oligosaccharides are involved in helix-helix interactions but not in interactions of the globular domains with each other or with the triple helix.

Polymeric [{ cyclo{bis- μ3( η2-S, η1-N)-(1,3-thiazolidine-2-thionato)-bis- μ4 ( η3-S, η1-N)-(1,3-thiazolidine-2-thionato) tetracopper (I) } monotoluene solvate]: electrochemical synthesis, characterisation and crystal structure at 160 K

The preparation of the title compound, [{Cu(tzdt)} 4], as the monotoluene solvate, has been achieved electrochemically, in toluene, with a copper anode, a platinum cathode and the parent ligand, 1,3-thiazolidine-2-thione, (tzdtH), with tetrabutylammonium tetrafluoroborate as supporting electrolyte. The colourless, diamagnetic complex crystallises in a triclinic cell with a = 11.51 (2), b = 11.80(2), c = 12.59(2) Å, α = 116.03(6), β = 100.42, γ = 107.67(6)°, Z = 2, space group P 1 , final R = 0.0331 from 3805 observed reflections with F 0 2 > 2 σ( F 0 2). The structure consists of the self-assembled [{Cu(tzdt)} 4] tetramers which are sulfur bridged through the ‘wing tip’ copper(I) atoms into polymeric chains; the toluene molecules occupy space between the polymeric chains. The central cyclic Cu 4 cluster is butterfly shaped with CuCu distances in the range 2.692(4)–2.882(5) Å. The ‘spinal’ copper atoms have distorted trigonal S 2N coordination from three different ligands and the ‘wing tip’ atoms have distorted tetrahedral S 3N coordination from three different ligands within an individual tetramer plus an additional contact from a thionato sulfur atom in an adjacent tetramer. The latter contacts generate asymetric, centrosymmetrically related Cu 2S 2 cores (CuS = 2.539(3) and 2.790(3) Å), which link the ‘wing tip’ atoms of the tetrameric units into a polymeric chain. The remaining CuS distances are in the range 2.222(3)–2.461(4) Å with the Cu distances in the range 1.993(4)–2.023(4) Å. The four ligands in each tetramer consist of two μ 3-S,N( η 2-S, η 1-N) and two μ 4-S,N( η 3-S, η 1-N) bridging species. The complex exhibits ligand centred irreversible redox behaviour, in acetonitrile, and is stable to atmospheric oxidation at room temperature. Thermal decomposition of the complex, to the metal, occurs in flowing air (60–1000 °C), with endothermic solvent desorption, intermediate phase change and melting processes as well as exothermic decomposition in static air (60–850 °C).

Properties of a recombinant human hemoglobin double mutant: sickle hemoglobin with Leu-88(beta) at the primary aggregation site substituted by Ala.

A recombinant double mutant of hemoglobin (Hb), E6V/L88A(beta), was constructed to study the strength of the primary hydrophobic interaction in the gelation of sickle Hb, i.e., that between the mutant Val-6(beta) of one tetramer and the hydrophobic region between Phe-85(beta) and Leu-88(beta) on an adjacent tetramer. Thus, a construct encoding the donor Val-6(beta) of the expressed recombinant HbS and a second mutation encoding an Ala in place of Leu-88(beta) was assembled. The doubly mutated beta-globin gene was expressed in yeast together with the normal human alpha-chain, which is on the same plasmid, to produce a soluble Hb tetramer. Characterizations of the Hb double mutant by mass spectrometry, by HPLC, and by peptide mapping of tryptic digests of the mutant beta-chain were consistent with the desired mutations. The absorption spectra in the visible and the ultraviolet regions were practically superimposable for the recombinant Hb and the natural Hb purified from human red cells. Circular dichroism studies on the overall structure of the recombinant Hb double mutant and the recombinant single mutant, HbS, showed that both were correctly folded. Functional studies on the recombinant double mutant indicated that it was fully cooperative. However, its gelation concentration was significantly higher than that of either recombinant or natural sickle Hb, indicating that the strength of the interaction in this important donor-acceptor region in sickle Hb was considerably reduced even with such a conservative hydrophobic mutation.

Respiratory Properties of the Neonatal Blood of the Common Brushtail Possum (Trichosurus vulpecula)

The respiratory properties of the blood of a newborn marsupial, the common brushtail possum (Trichosurus vulpecula of the family Phalangeridae), have been examined. The O₂ equilibrium curves (OECs) under standard conditions (36° C and partial pressure of CO₂ [Pco₂] = 42 mmHg) were right shifted, being to the right of the maternal curves. The partial pressure of O₂ (Po₂) at which blood is 50% saturated with oxygen (P50) under these conditions was 42 mmHg in the neonatal blood. All the neonatal OECs had a Hill plot bent at about 50% saturation, and the mean value of the Hill coefficient (nH) was 5.96 for the upper part of the curve, indicating aggregation of Hb tetramers or interaction between adjacent tetramers. Apart from previous findings in another marsupial (Macropus eugenii), the first of these properties had not been shown before in any species, the second had not been shown before in any normal mammalian blood. At higher and lower Pco₂ (72 and 21 mmHg) the neonatal OECs were of the same shape and were also to the right of the maternal OECs under equivalent conditions. The Bohr effect of the neonatal blood was lower than that of adult blood; the neonatal red blood cells were nearly all nucleated and contained four Hb types, each different from adult Hbs. In these respects it was characteristic of embryonic blood and similar to the blood of the prenatal and neonatal tammar wallaby of the family Macropodidae

Effects of beta 6 aromatic amino acids on polymerization and solubility of recombinant hemoglobins made in yeast.

Valine, leucine, tryptophan, and phenylalanine substitutions at the beta 6 position of hemoglobin (Hb) were made using a yeast expression system coupled with a polymerase chain reaction-based mutagenesis strategy. The oxygen affinity and absorption spectra of these mutants were similar to recombinant Hb A except for Hb beta E6W which had a higher absorbance at approximately 280 nm. The deoxy forms of Hb beta E6L and Hb S showed characteristic delay times prior to polymerization. Tetrameric deoxy-Hbs containing tryptophan or phenylalanine at the beta 6 position had higher solubilities and polymerized less readily compared with deoxy-Hb S. However, when oversaturated, these Hbs polymerized without a delay time. These results suggest that Hb beta E6W and Hb beta E6F form polymers upon deoxygenation by a linear polymerization mechanism without nuclei formation. During polymerization, bulky hydrophobic amino acids, like phenylalanine and tryptophan at the beta 6 position, might interact with the acceptor pocket on the surface of an adjacent Hb molecule but may not be able to form stable hydrophobic interactions like beta 6 valine and leucine. Difficulty in insertion of the bulky side chains of these aromatic amino acids into the hydrophobic acceptor pocket on an adjacent tetramer may inhibit nuclei formation prior to polymerization.

Polylysine increases the number of insulin binding sites in soluble insulin receptor preparations.

The effects of cationic polyamino acids on insulin binding to soluble insulin receptor preparations were studied. Incubation of partially or fully purified receptor preparations with polylysine (pLys) increased by several-fold the amount of [125I]insulin that remained associated with the receptor, as determined both by precipitation of receptor-insulin complexes by polyethylene glycol or by separation of the complexes from the free hormone by gel filtration. This elevation in the amount of bound insulin resulted from increased number of insulin binding sites, and could not be attributed to an increased affinity of the receptors to insulin. In fact, pLys reduced 2-3-fold the affinity of insulin binding to its receptor as determined by equilibrium binding studies, and by monitoring the rate of exchange of bound [125I]insulin with unlabeled hormone. pLys induced specific interactions between insulin and its native receptor since other basic compounds such as histone, spermidine, polymixin B, compound 48/80, lysine, and arginine failed to reproduce its effects. pLys did not interact with the free ligand, nor did it promote interactions between insulin and denatured receptor forms. Furthermore, pLys did not induce binding of insulin to other proteins present in the partially purified receptor preparations. The effects of pLys were time and dose-dependent and were proportional to the pLys chain length. The longer the chain, the greater was the effect. Enhanced insulin binding and receptor beta-subunit autophosphorylation (in the presence of insulin) exhibited a similar dependency on the chain length of pLys. pLys effects on insulin binding were associated with formation of large protein aggregates that remained trapped at the top of Sephacryl S-300 columns. These aggregates contained substantial amounts of receptor-insulin complexes. Our results suggest that pLys induces formation of receptor clusters that create de novo insulin binding sites among adjacent receptor tetramers. Alternatively, formation of receptor aggregates might facilitate insulin binding to a soluble receptor subfraction that otherwise fails to bind the hormone.