Familial secondary erythrocytosis due to increased oxygen affinity is caused by destabilization of the T state of hemoglobin Brigham (α(2) β(2) (Pro100Leu) )

Hemoglobin Brigham (β Pro100 to Leu) was first reported in a patient with familial erythrocytosis. Erythrocytes of an affected individual from the same family contain both HbA and Hb Brigham and exhibit elevated O(2) affinity compared with normal cells (P(50) = 23 mm Hg vs. 31 mmHg at pH 7.4 at 37°C). O(2) affinities measured for hemolysates were sensitive to changes in pH or chloride concentrations, indicating little change in the Bohr and Chloride effects. Hb Brigham was separated from normal HbA by nondenaturing cation exchange liquid chromatography, and the amino acid substitution was verified by mass spectrometry. The properties of Hb Brigham isolated from the patient's blood were then compared with those of recombinant Hb Brigham expressed in Escherichia coli. Kinetic experiments suggest that the rate constants for ligand binding and release in the high (R) and low (T) affinity quaternary states of Hb Brigham are similar to those of native hemoglobin. However, the Brigham mutation decreases the T to R equilibrium constant (L) which accelerates the switch to the R state during ligand binding to deoxy-Hb, increasing the rate of association by approximately twofold, and decelerates the switch during ligand dissociation from HbO(2) , decreasing the rate approximately twofold. These kinetic data help explain the high O(2) affinity characteristics of Hb Brigham and provide further evidence for the importance of the contribution of Pro100 to intersubunit contacts and stabilization of the T quaternary structure.     

Sequence Effects On The Propeller Twist Of Base Pairs In DNA Helices

Abstract

Molecular mechanics calculations are performed on all the ten base pair steps (duplex dimers) and also a number of trimer and tetramer duplexes comprising them, in an attempt to systematically examine the possible base sequence effects on the magnitudes of propeller twists of base pairs at a given step. The analysis reveals that though propeller twist is a base pair property, it behaves very much like other base step parameters such as slide, roll, twist etc., Hence, it may be necessary to monitor the nature and variation of magnitudes of pt at a step. Calculations performed on 45 out of the 136 unique tetramer combinations involving all the ten unique base steps show that the difference in magnitudes of propeller twists of the base pairs of a given step has been found to be either steep or moderate depending on base pairs that flank the base step. These observations compare very well with the available experimental data. Tetramer sequences, wherein abase pair of a base step repeats in the same direction, exhibit a relatively steep difference in propeller twist at the step. Tetramers other than these exhibit moderate difference in propeller twist Such sequences are broadly classified as type-I and type-II respectively. Practically all the tetrads considered in the study, excepting those with GT step and a few involving CG and GC steps, conform to the above classification.     

Current issues in the chemistry of cytochromec oxidase

Some contemporary issues relevant to the chemistry of mammalian cytochromec oxidase are discussed. These include the optical properties of heme A and the spectroscopic consequences of the differences in side-chain substitution compared to heme B; a common fallacy concerning the electrostatic exchange interaction between cytochromea ₃ and Cu_B; the question of the number and location of the copper components of the enzyme; and the mode of binding of ligands such as cyanide and azide.     

Zinc–Ligand Interactions Modulate Assembly and Stability of the Insulin Hexamer – A Review

Zinc and calcium ions play important roles in the biosynthesis and storage of insulin. Insulin biosynthesis occurs within the β-cells of the pancreas via preproinsulin and proinsulin precursors. In the golgi apparatus, proinsulin is sequestered within Zn²⁺- and Ca²⁺-rich storage/secretory vesicles and assembled into a Zn²⁺ and Ca²⁺ containing hexameric species, (Zn²⁺)₂(Ca²⁺)(Proin)₆. In the vesicle, (Zn²⁺)₂(Ca²⁺)(Proin)₆ is converted to the insulin hexamer, (Zn²⁺)₂(Ca²⁺)(In)₆, by excision of the C-peptide through the action of proteolytic enzymes. The conversion of (Zn²⁺)₂(Ca²⁺)(Proin)₆to (Zn²⁺)₂(Ca²⁺)(In)₆ significantly lowers the solubility of the hexamer, causing crystallization within the vesicle. The (Zn²⁺)₂(Ca²⁺)(In)₆ hexamer is an allosteric protein that undergoes ligand-mediated interconversion among three global conformation states designated T₆, T₃R₃ and R₆. Two classes of allosteric sites have been identified; hydrophobic pockets (3 in T₃R₃ and 6 in R₆) that bind phenolic ligands, and anion sites (1 in T₃R₃ and 2 in R₆) that bind monovalent anions. The allosteric states differ widely with respect to the physical and chemical stability of the insulin subunits. Fusion of the vesicle with the plasma membrane results in the expulsion of the insulin crystals into the intercellular fluid. Dissolution of the crystals, dissociation of the hexamers to monomer and transport of monomers to the liver and other tissues then occurs via the blood stream. Insulin action then follows binding to the insulin receptors. The role of Zn²⁺ in the assembly, structure, allosteric properties, and dynamic behavior of the insulin hexamer will be discussed in relation to biological function.     

Oligomeric states of bacteriophage T7 gene 4 primase/helicase

Electron microscopic and crystallographic data have shown that the gene 4 primase/helicase encoded by bacteriophage T7 can form both hexamers and heptamers. After cross-linking with glutaraldehyde to stabilize the oligomeric protein, hexamers and heptamers can be distinguished either by negative stain electron microscopy or electrophoretic analysis using polyacrylamide gels. We find that hexamers predominate in the presence of either dTTP or beta,gamma-methylene dTTP whereas the ratio between hexamers and heptamers is nearly the converse in the presence of dTDP. When formed, heptamers are unable to efficiently bind either single-stranded DNA or double-stranded DNA. We postulate that a switch between heptamer to hexamer may provide a ring-opening mechanism for the single-stranded DNA binding pathway. Accordingly, we observe that in the presence of both nucleoside di- and triphosphates the gene 4 protein exists as a hexamer when bound to single-stranded DNA and as a mixture of heptamer and hexamer when not bound to single-stranded DNA. Furthermore, altering regions of the gene 4 protein postulated to be conformational switches for dTTP-dependent helicase activity leads to modulation of the heptamer to hexamer ratio.     

CHAPS对M_(412)的动力学和共振拉曼光谱的影响

研究了表面活性剂CHAPS对紫膜中菌紫质在光循环过程中M_(412)的衰减速率及质子泵功能的影响.光循环中间体M_(412)快、慢衰减组分的衰减速率及质子衰减速率均受到CHAPS的影响,综合激光拉曼光谱对M_(412)和其它光循环中间体相对含量的测定,表明CHAPS对紫膜的影响是通过影响其膜脂完整的液晶结构,而使紫膜光循环动力学过程及质子泵功能发生变化的.     

Hemoglobin site-mutants reveal dynamical role of interhelical H-bonds in the allosteric pathway: time-resolved UV resonance Raman evidence for intra-dimer coupling

The dynamical effect of eliminating specific tertiary H-bonds in the hemoglobin (Hb) tetramer has been investigated by site-directed mutagenesis and time-resolved absorption and ultraviolet resonance Raman (UVRR) spectroscopy. The Trp alpha 14...Thr alpha 67 and Trp beta 15...Ser beta 72 H-bonds connect the A and E helices in the alpha and beta chains, and are proposed to break in the earliest protein intermediate (Rdeoxy) following photo-deligation of HbCO, along with a second pair of H-bonds involving tyrosine residues. Mutation of the acceptor residues Thr alpha 67 and Ser beta 72 to Val and Ala eliminates the A-E H-bonds, but has been shown to have no significant effect on ligand-binding affinity or cooperativity, or on spectroscopic markers of the T-state quaternary interactions. However, the mutations have profound and unexpected effects on the character of the Rdeoxy intermediate, and on the dynamics of the subsequent steps leading to the T state. Formation of the initial quaternary contact (RT intermediate) is accelerated, by an order of magnitude, but the locking-in of the T state is delayed by a factor of 2. These rate effects are essentially the same for either mutation, or for the double mutation, suggesting that the alpha beta dimer behaves as a mechanically coupled dynamical unit. Further evidence for intra-dimer coupling is provided by the Rdeoxy UVRR spectrum, in which either or both mutations eliminate the tyrosine difference intensity, although only tryptophan H-bonds are directly affected. A possible mechanism for mechanical coupling is outlined, involving transmission of forces through the alpha(1)beta(1) (and alpha(2)beta(2)) interface. The present observations establish that quaternary motions can occur on the approximately 100 ns time-scale. They show also that a full complement of interhelical H-bonds actually slows the initial quaternary motion in Hb, but accelerates the locking in of the T-contacts.     

A highly sensitive photometric method for proton release or uptake: Difference protometry

A highly sensitive quantitative method was developed to detect protons released or taken up upon ligand binding. A small change in pH due to proton release or uptake was detected by measuring the difference in the absorbance of a pH indicator upon ligand addition. Owing to the difference detection of protons, the uncertainty of pH due to CO₂ dissolution and unknown buffering capacities of sample solutes could be compensated with easy manipulations. Precise calibration of the absolute amount of protons could also be made very easily. The amount of protons measurable by the method is as small as 0.5 nmol that is 10 to 30 times more sensitive than the pH-stat method. We measured the Mg²⁺ ion-induced proton releases of ADP to confirm the accuracy and reliability of the method and of Escherichia coli ribosomes to show the improvement in sensitivity. The method is useful for protometric studies of biomolecules that are difficult to obtain in large amount.     

Binding site amino acid residues of jack fruit (artocarpus integrifolia) seed lectin: chemical modification and protein difference spectral studies

The effect of chemical modification of amino acid residues essential for sugar binding in the α-D-galactoside specific jack fruit (Artocarpus integrifolia) seed lectin and the protection of the residues by specific sugar from modification were studied. Citraconylation or maleylation of 75 % of its lysyl residues or acetylation of 70 % of the tyrosyl residues completely abolished sugar binding and agglutination without dissociation of subunits. 1-O-methyl α-D-galactoside could protect its essential lysyl and tyrosyl groups from modification. Tryptophan could not be detected in the protein. Difference absorption spectra on binding of the above sugar confirmed the role of tyrosine residues and showed an association constantK = 0.4 × 10³ M⁻¹. Data suggests that the lectin could be immobilized without any loss of sugar binding activity     

Antigen-Induced Allosteric Changes in a Human IgG1 Fc Increase Low-Affinity Fcγ Receptor Binding

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