Chain Helix Research Articles

Syndiotactic Poly(Substituted Methylene)s with Alkyloxycarbonyl Side Chains: Effects of Side Chain Chirality on the Main Chain Helix and Crystallinity

AbstractSyndiotactic poly(substituted methylene)s (st‐PMs) with alkyloxycarbonyl side chains form hexagonal columnar liquid crystals (Colh LCs), wherein the main chain adopting either right‐ or left‐handed helices is arranged in a 2D hexagonal lattice. This study investigates the main chain conformation for st‐PMs with chiral side chains, designated as st‐P8(3,7Me)‐fSS. st‐P8(3,7Me)‐fSS polymers possess 3,7‐dimethyloctyloxycarbonyl side chains at different S‐enantiomeric fractions, fS. In chiral st‐P8(3,7Me)‐fSS with an fS > 50%, the main chain adopts a one‐handed helical conformation along the longer contour with increasing fS. The fS influences the crystallinity of the polymers. Chiral P8(3,7Me)‐fSS polymers crystallize the Colh LCs upon cooling. The crystallite size, melting temperature, and fusion enthalpy increase with fS. Racemic st‐P8(3,7Me)‐50S does not crystallize.

Glycation restrains open-closed conformation of Insulin

In hyperglycemic conditions, the level of reactive dicarbonyl metabolites concentration is found to be high, which plays a significant role in protein glycation. Despite decades of research, the effect of methylglyoxal on the structure and function of insulin is still unknown. Through a shift in conformation at the B-chain C-terminal (BT-CT) hinge from an "open" to a "wide-open" conformation, insulin binds to the receptor and activates the signal cascade. Insulin resistance, which is the main sign of Type 2 Diabetes, can be caused by a lack of insulin signaling. Methylglyoxal site-specific glycation in residue R22 at B chain forms AGE product Methylglyoxal-hydroimidazolone (MGH1) in insulin. In this work, we present molecular dynamics study of this glycated insulin R22MGH1, which revealed new insights into the conformational and structural changes. We find the following key results: 1) B-chain in insulin undergoes a closed conformational change upon glycation. 2) Glycated insulin shows secondary structure alteration. 3) Glycated insulin retains its closed shape due to an unusually strong hydrophobic contact between B-chain residues. 4) Wide open native conformation of insulin allows the B chain helix to be surrounded by more water molecules compared to the closed conformation of glycated insulin. The closed conformation of glycated insulin impairs its binding to insulin receptor (IR).

The Structural Characteristics of Collagen in Swim Bladders with 25-Year Sequence Aging: The Impact of Age

Aged swim bladders from the yellow drum (Protonibea diacanthus) are considered collagen-based functional food with extremely high market value. The structural integrity of collagen may be crucial for its biological functions. In the current study, swim bladders with 25-year-old sequences were collected and found to be basically composed of collagen. Then, thermogravimetry (TG), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and attenuated total reflectance–Fourier transform infrared spectroscopy (ATR–FTIR) were conducted to evaluate the integrity of the peptide chain and triple helix in the collagen. The structures of microfibers and fiber bundles were revealed with atomic force microscopy (AFM), scanning electrical microscopy (SEM), and optical spectroscopy. The collagens in the aged swim bladders were found to have similar thermal properties to those of fresh ones, but the relative content of the triple helixes was found to be negatively correlated with aging. The secondary structure of the remaining triple helix showed highly retained characteristics as in fresh swim bladders, and the microfibrils also showed a similar D-period to that of the fresh one. However, the fiber bundles displayed more compact and thick characteristics after years of storage. These results indicate that despite 25 years of aging, the collagen in the swim bladders was still partially retained with structures.

Interactions Determining the Structural Integrity of the Trimer of Plant Light Harvesting Complex in Lipid Membranes.

The structural basis for the stability of the trimeric form of the light harvesting complex (LHCII), a pigmented protein from green plants pivotal for photosynthesis, remains elusive till date. The protein embedded in a dipalmitoylphosphatidylcholine (DPPC) lipid membrane is investigated using all-atom molecular dynamics simulations to find out the interactions responsible for the structural integrity of the trimer and its relation to antenna function. Central association of chlorophyll a (CLA) molecules near the LHCII chains is attributed to a conserved coordination between the Mg of CLA and the oxygen of a specific residue of the first helix of a chain. The residue forms a salt-bridge with the fourth helix of the same chain of the trimer, not of the monomer. In an earlier experiment, three residues (WYR) at each chain of the trimer have been found indispensable for the trimerization and referred to as trimerization motif. We find that the residues of the trimerization motif are connected to the lipids or pigments by a chain of interactions rather than a direct contact. Synergistic effects of sequentially located hydrogen bonds and salt-bridges within monomers of the trimer keep the trimer conformation stable in association with the pigments or the lipids. These interactions are exclusively present in the pigmented trimer and not present in the monomer or in the unpigmented trimer. Thus, our results provide a molecular basis for the inherent stability of the LHCII trimer in a lipid membrane and explain many pre-existing experimental data.

Recyclable Helical Poly(phenyl isocyanide)-Supported l-Proline Catalyst for Direct Asymmetric Aldol Reaction in Brine

A novel helical poly(phenyl isocyanide) bearing Boc protected l-proline pendants (poly-1m) was designed and synthesized. Removed the protecting Boc groups on the l-proline pendants led to the formation of helical polymer poly-2m, which showed high optical activity owing to the preferred right-handed helix of polyisocyanide main chain. Optically active helical poly-2m showed excellent catalytic ability on asymmetric aldol reaction. Helical polymer catalysts exhibited enhanced stereoselectivity in aldol reaction compared to small molecule l-proline. Under the optimized aldol reaction condition, the enantiomeric excess (ee) and diastereomeric ratio (dr) values of the aldol reaction product were respectively up to 90% and > 20/1. Moreover, the helical polyisocyanide catalyst Poly-2m can be easily recovered and reused in the aldol reaction for at least five cycles with maintained its activity and stereoselectivity. Enantioselective aldol reaction catalyzed by poly-2m.

High-resolution structure of a partially folded insulin aggregation intermediate.

Insulin has long been served as a model for protein aggregation, both due to the importance of aggregation in the manufacture of insulin and because the structural biology of insulin has been extensively characterized. Despite intensive study, details about the initial triggers for aggregation have remained elusive at the molecular level. We show here that at acidic pH, the aggregation of insulin is likely initiated by a partially folded monomeric intermediate. High-resolution structures of the partially folded intermediate show that it is coarsely similar to the initial monomeric structure but differs in subtle details-the A chain helices on the receptor interface are more disordered and the B chain helix is displaced from the C-terminal A chain helix when compared to the stable monomer. The result of these movements is the creation of a hydrophobic cavity in the center of the protein that may serve as nucleation site for oligomer formation. Knowledge of this transition may aid in the engineering of insulin variants that retain the favorable pharamacokinetic properties of monomeric insulin but are more resistant to aggregation.

Fabrication of surface charge pH-sensitive multi-bumpy small magnetic bead with ultrahigh magnetic content and its ultrahigh loading capacity and salt-free rapid isolation for DNA

Gene transfection vector polyethyleneimine (PEI) was used as a cross-linking agent to crosslink the surface epoxidized magnetic nanoparticles and aggregate them to form a small magnetic bead (MB) with multiple nanoscale bumps on its surface (i.e. the multi-bumpy small magnetic bead, mbsMB). As there is a very low content of non-magnetic components (the cross-linking agent) in the magnetic bead, the mbsMB has an ultrahigh magnetic content of 81.95 % and a smaller particle size of 1.4 μm when compared with the usual medical MB. Such a small MB also has a strong magnetic force allowing it to reach the rapid separating ability of the commonly used larger medical MB which has 8 times its volume. The mbsMB has an obvious pH sensitivity of positive and negative surface charges and the salt-free isolation of DNA has been achieved based on the electrostatic interactions between mbsMB and DNA. This avoids the desalting of the isolated DNA as well as the effects of high salt concentration on its long chain helix structure. Whether in an acidic absorbing medium, an alkalinous desorbing one or a near neutral particle-storing one, the mbsMB will have obvious surface electrostatic charges. There is also its good suspension stability in an aqueous medium which provides a good condition for isolating of DNA suitable for efficiently adsorbing and desorbing. The as-prepared MB has a unique surface structure and some excellent properties, all suitable for adsorbing DNA. In addition, a large amount of commonly used gene transfection vector PEI can be cross-linked and bonded on the surface of mbsMB, whilst still having an excellent DNA-loading ability. In summary, the mbsMB has an ultrahigh capacity of 629.49 mg/g for DNA load.

A variant of human growth differentiation factor-9 that improves oocyte developmental competence

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are co-expressed exclusively in oocytes throughout most of folliculogenesis and play central roles in controlling ovarian physiology. Although both growth factors exist as homodimers, recent evidence indicates that GDF9 and BMP15 can also heterodimerize to form the potent growth factor cumulin. Within the cumulin complex, BMP15 "activates" latent GDF9, enabling potent signaling in granulosa cells via type I receptors (i.e. activin receptor-like kinase-4/5 (ALK4/5)) and SMAD2/3 transcription factors. In the cumulin heterodimer, two distinct type I receptor interfaces are formed compared with homodimeric GDF9 and BMP15. Previous studies have highlighted the potential of cumulin to improve treatment of female infertility, but, as a noncovalent heterodimer, cumulin is difficult to produce and purify without contaminating GDF9 and BMP15 homodimers. In this study we addressed this challenge by focusing on the cumulin interface formed by the helix of the GDF9 chain and the fingers of the BMP15 chain. We demonstrate that unique BMP15 finger residues at this site (Arg301, Gly304, His307, and Met369) enable potent activation of the SMAD2/3 pathway. Incorporating these BMP15 residues into latent GDF9 generated a highly potent growth factor, called hereafter Super-GDF9. Super-GDF9 was >1000-fold more potent than WT human GDF9 and 4-fold more potent than cumulin in SMAD2/3-responsive transcriptional assays in granulosa cells. Our demonstration that Super-GDF9 can effectively promote mouse cumulus cell expansion and improve oocyte quality in vitro represents a potential solution to the current challenges of producing and purifying intact cumulin.

Fast Living Polymerization and Helix-Sense-Selective Polymerization of Diazoacetates Using Air-Stable Palladium(II) Catalysts.

In this work, air-stable palladium(II) catalysts bearing bidentate phosphine ligands were designed and prepared, which could initiate fast and living polymerizations of various diazoacetate monomers under mild conditions. The polymerization afforded the desired polymers in high yields with controlled molecular weights ( Mns) and narrow molecular weight distributions ( Mw/ Mns). The Mns of the isolated polymers were linearly correlated to the initial feed ratios of monomer to catalyst, confirming the living/controlled manner of the polymerizations. The Mn also increased linearly with the monomer conversion, and all of the isolated polymers showed narrow Mw/ Mns. The polymerization was relatively fast and could be accomplished within several minutes. Such fast living polymerization method can be applied to a wide range of diazoacetate monomers in various organic solvents at room temperature in air. Taking advantage of the living nature, we facilely prepared a series of block copolymers through chain extension reactions. The amphiphilic block copolymers synthesized by this method exhibited interesting self-assembly properties. Moreover, polymerization of achiral bulky diazoacetate by Pd(II) catalysts bearing a chiral bidentate phosphine ligand leads to the formation of polymers with high optical activity due to the formation of the predominantly one-handed helix of the main chain. The helix sense of the polymers was determined by the chirality of the Pd(II) catalysts.

Polymerization Amplified Stereoselectivity (PASS) of Asymmetric Michael Addition Reaction and Aldol Reaction Catalyzed by Helical Poly(phenyl isocyanide) Bearing Secondary Amine Pendants

A novel enantiopure phenyl isocyanide (1) carrying tert-butyloxycarboryl (Boc) protected l-prolinol ester was designed and synthesized. Living polymerization of 1 using a alkyne–Pd(II) catalyst afforded helical poly-1ms in high yields with controlled molecular weights (Mns) and narrow molecular weight distributions (Mw/Mns). Removing the protecting Boc groups on the l-prolinol ester pendants lead to the formation of helical poly-2m, which showed high optical activity owing to the preferred left-handed helix of polyisocyanide main chain. The poly-2ms showed excellent catalytic ability on asymmetric Michael addition reaction. Both the enantiomeric excess (ee) and diastereomeric ratio (dr) values of the product were linearly correlated to the Mn and optical activity of poly-2m. Increasing the Mn of poly-2ms will amplify the stereoselectivity of the asymmetric Michael addition reaction until the Mn reached to 44.9 kDa, revealing the polymerization amplified stereoselectivity (PASS) behavior of the asymmetric ...

The Possible Mechanism of Idiosyncratic Lapatinib-Induced Liver Injury in Patients Carrying Human Leukocyte Antigen-DRB1*07:01.

Idiosyncratic lapatinib-induced liver injury has been reported to be associated with human leukocyte antigen (HLA)-DRB1*07:01. In order to investigate its mechanism, interaction of lapatinib with HLA-DRB1*07:01 and its ligand peptide derived from tetanus toxoid, has been evaluated in vitro. Here we show that lapatinib enhances binding of the ligand peptide to HLA-DRB1*07:01. Furthermore in silico molecular dynamics analysis revealed that lapatinib could change the β chain helix in the HLA-DRB1*07:01 specifically to form a tightly closed binding groove structure and modify a large part of the binding groove. These results indicate that lapatinib affects the ligand binding to HLA-DRB1*07:01 and idiosyncratic lapatinib-induced liver injury might be triggered by this mechanism. This is the first report showing that the clinically available drug can enhance the binding of ligand peptide to HLA class II molecules in vitro and in silico.

Modeling of starch retrogradation onset in its aqueous solution using thermoreversible gelation concept

A model was developed to predict starch retrogradation onset in its aqueous solution and verified with the selected literature data. The most probable chain helix distribution was first estimated via minimizing the system free energy with respect to chain helix length. Later, the calculation was extended to double helix formation among single polymer chains with ζ¯2=10 as the retrogradation criterion. ζ¯2 is the average double helix length usually equals 10 implying the number of participating residues in the thickness of the thinnest reported starch lamella in the literature. The model prediction namely the retrogradation onset temperature, showed quite reasonable agreement with the selected literature data. Equal chain conformational entropy loss, Δs/kB≈−0.7, was inferred for various starch aqueous solutions due to the double helix kind of associations. Nonetheless, the studied systems showed distinct restrictions, σ=9×10−4–4.7×10−3, against association.

Helix-sense-selective Polymerization of Substituted Acetylenes by Using an Isolated Rh Chiral Initiator with an Amino Acid Ligand

Abstract Helix-sense-selective polymerization (HSSP) of substituted acetylenes by using an isolated chiral Rh initiator with an amino acid ligand has been achieved. The sense of the helix of the main chain of the resulting polymer was controlled by the sign of the chirality of the ligand. The isolated initiator needed less of the chiral ligand and gave polymers having a higher [θ] value than the two component chiral initiator systems we reported previously. The resulting polymers had a higher Mw.

A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC

Self-reactive T cells that escape elimination in the thymus can cause autoimmune pathology, and it is therefore important to understand the structural mechanisms of self-antigen recognition. We report the crystal structure of a T cell receptor (TCR) from a patient with relapsing-remitting multiple sclerosis that engages its self-peptide-major histocompatibility complex (pMHC) ligand in an unusual manner. The TCR is bound in a highly tilted orientation that prevents interaction of the TCR-α chain with the MHC class II β chain helix. In this structure, only a single germline-encoded TCR loop engages the MHC protein, whereas in most other TCR-pMHC structures all four germline-encoded TCR loops bind to the MHC helices. The tilted binding mode also prevents peptide contacts by the short complementarity-determining region (CDR) 3β loop, and interactions that contribute to peptide side chain specificity are focused on the CDR3α loop. This structure is the first example in which only a single germline-encoded TCR loop contacts the MHC helices. Furthermore, the reduced interaction surface with the peptide may facilitate TCR cross-reactivity. The structural alterations in the trimolecular complex are distinct from previously characterized self-reactive TCRs, indicating that there are multiple unusual ways for self-reactive TCRs to bind their pMHC ligand.

Visualizing key hinges and a potential major source of compliance in the lever arm of myosin

We have determined the 2.3-Å-resolution crystal structure of a myosin light chain domain, corresponding to one type found in sea scallop catch ("smooth") muscle. This structure reveals hinges that may function in the "on" and "off" states of myosin. The molecule adopts two different conformations about the heavy chain "hook" and regulatory light chain (RLC) helix D. This conformational change results in extended and compressed forms of the lever arm whose lengths differ by 10 Å. The heavy chain hook and RLC helix D hinges could thus serve as a potential major and localized source of cross-bridge compliance during the contractile cycle. In addition, in one of the molecules of the crystal, part of the RLC N-terminal extension is seen in atomic detail and forms a one-turn alpha-helix that interacts with RLC helix D. This extension, whose sequence is highly variable in different myosins, may thus modulate the flexibility of the lever arm. Moreover, the relative proximity of the phosphorylation site to the helix D hinge suggests a potential role for conformational changes about this hinge in the transition between the on and off states of regulated myosins.

Solid-State NMR Investigation of Membrane-Associated Peptides and Proteins

Solid-state NMR spectroscopy has a proven record during the investigation of the structure and dynamics of membrane-associated polypeptides. In particular from oriented samples considerable details on the structure and topology of the protein is obtained. A solid-state NMR approach which allows for the accurate determination of the tilt and rotational pitch angles of peptides reconstituted into uniaxially oriented membranes will be presented. Proton-decoupled 15N and 2H solid-state NMR spectroscopy have been used to characterize the tilt and rotational pitch angle of several peptides in considerable detail. Furthermore, valuable information on the rotational diffusion constants in membranes and thereby the size of peptide complexes is obtained.Here we present solid-state NMR results that have been obtained from the heterodimeric antimicrobial peptide distinctin as well as from the two chains independently. The data indicate that upon membrane insertion the protein unfolds, that the amphipathic helix of chain 2 orients parallel to the membrane surface and stably anchors the polypeptide into the membrane. In contrast chain 1 remains more loosely associated and changes its alignment by about 5 degrees when in contact with chain 2. Figure from PNAS, 106, 16639 (2009)View Large Image | View Hi-Res Image | Download PowerPoint Slide

Solution Structure of a Conformationally Restricted Fully Active Derivative of the Human Relaxin-like Factor

Analogous to insulin, the relaxin-like factor (RLF) must undergo a structural transition to the active form prior to receptor binding. Thus, the C-terminus of the B chain of RLF folds toward the surface of the central B chain helix, causing partial obliteration of the two essential RLF receptor-binding site residues, valine B19 and tryptophan B27. Via comparison of the solution structure of a fully active C-terminally cross-linked RLF analogue with the native synthetic human RLF (hRLF), it became clear that the cross-linked analogue largely retains the essential folding of the native protein. Both proteins exist in a major and minor conformation, as revealed by multiple resonances from tryptophan B27 and adjacent residues on the B chain helix. Notably, the minor conformation is significantly more highly populated in the chemically cross-linked RLF than it is in the hRLF. In addition, compared to the unmodified molecule, subtle differences are observed within the B chain helix whereby the cross-linked derivative shows a reduced level of hydrogen bonding and significant peak broadening at the binding site residue ValB19. On the basis of these observations, we suggest that the solution structure of the native hormone represents an inactive conformer and that a dynamic equilibrium exists between the C-terminally unfolded binding conformation and the inactive conformation of the RLF.

Hb Dothan [β25/26 (B7/B8)/(-GTG/-GLY)/Gly+Glu→Glu]; A Novel Mechanism Leading to a M-Hemoglobin.

Hereditary methemoglobinemia is a relatively rare disorder usually manifesting with cyanosis at birth. The more common form results from the deficiency of the enzyme, NADH-Cytochrome b5 reductase (methemoglobin reductase, diaphorase) and displays an autosomal recessive inheritance pattern. Less common are the so-called M-hemoglobins with an autosomal dominant pattern, which result from amino acid substitutions in the heme binding pocket of α, β, or less commonly γ-globin chains. The majority of the M-hemoglobin (Hb) variants occur from substitutions in the E or F-helices, which constitute the heme binding pocket, most commonly from amino acid substitutions involving the conserved proximal (F8) or distal (E11) histidine residues. Here we report a new Hb variant due to a three nucleotide deletion (-GTG between codons 25 and 26 of the β globin gene causing a single amino acid (-Gly) deletion in the B helix (B7/B8) of the β-globin chain that leads to methemoglobinemia with a novel mechanism. The propositus is a 9 month old Caucasian boy from Dothan AL who was found to have a low O2 saturation prior to an ENT procedure. He was referred to cardiology at Children's Health System, Birmingham, AL to rule out a congenital heart disease. A low O2 saturation (85–86%) was confirmed. Cardiac catheterization excluded the structural abnormality of the heart. Cooximetry showed a normal PaO2 but confirmed a low O2 saturation. Methemoglobin level was 20%, while methemoglobin reductase activity was in the low–normal range but when repeated was found to be normal. His growth and development have been normal. On alkaline electrophoresis an abnormal hemoglobin band was observed. The patient's blood was sent to the Hemoglobinopathy Laboratory of the Sickle Cell Center at MCG, Augusta, GA for definite identification of the variant. CBC revealed a RBC of 4.3 M/mm3, HGB 13.6 g/dL, HCT 40.8 %, MCV 95.2 fl, MCH 31.8 pg MCHC 33.4 g/dL, Retics 4.4 %. Isoelectrofocusing (IEF) on agarose showed the presence of an abnormal Hb with approximately the same isoelectric point (pI) as Hb F. Quantitation of Hb components by Cation Exchange HPLC revealed 62.7% Hb A, 27.9% Hb X, 3.0% Hb A2, and 6.4% Hb F. By globin chain analyses with reversed phase HPLC, βχ was detected as 37.6% of the total beta chains. Isopropanol stability test gave strongly positive results. P50 was found to be 24.8 mm Hg in the patient and 26.4 in the control (slightly increased oxygen affinity). Peptide analysis was done using mass spectrometry (Alphalyse, Palo Alto, CA) where tryptic digests of purified Hb X (95.0% enriched) and normal control (97.0% Hb A) were analyzed and compared. Peptide 19–30 of helix-B fragment revealed 1314 Da mass in control, whereas peptide 19–29 (with –Gly) of helix-B fragment of Hb X gave 1257 Da mass, confirming the deletion of a Gly residue. The corresponding deletion of three nucleotides (-GTG) in the genomic DNA (codons 25–26: GTGGAG→GAG ) was demonstrated by polymerase chain reaction (PCR) amplification and direct sequencing of β-globin gene and confirmed by cDNA sequencing of β-globin mRNA. No abnormality was detected in the sequences of δ, Gγ, Aγ, α1 and α2 globin genes. The three nucleotide deletion between codons 25 and 26 (-GTG) of the β-globin gene causes a one amino acid (-Gly) deletion in the B helix (B7/B8) of the β-globin chain, however does not alter the amino acid composition of β-globin chain after the deletion point but results in a shorter (145 AA, instead of the normal 146) mutant β-globin chain. As a result close spatial contact of amino acids in tertiary structure of hemoglobin is altered completely. Most importantly, distal histidine at residue 63 of E7 helix now becomes Gly leading to methemoglobin formation. A similar variant was previously reported in a Japanese baby, Hb Higashitochigi (Fujisawa et al, Hemoglobin , 17:467, 1993) where a three nucleotide deletion in codons 24/25 also resulted in the loss of a single Gly residue with a similar outcome. These two cases differ from the known M-hemoglobins all of which result from single amino acid substitution in the E or F-helices thus altering the heme pocket. Hb Dothan and Hb Higashitochigi represent a novel mechanism for M-hemoglobin generation where an in frame deletion alters the tertiary structure of the globin chain with alterations in the structure of E-helix and loss of the distal histidine residue.

The Mode of Interaction of the Relaxin-like Factor (RLF) with the Leucine-rich Repeat G Protein-activated Receptor 8

The relaxin-like factor (RLF, also named INSL3) is a critical component in the chain of events that lead to the normal positioning of the gonads in the male fetus. RLF and relaxin share features of the secondary structure to the extent that relaxin cross-reacts with the LGR8, the RLF receptor. Although both hormones interact with their receptors essentially via the B chain, the sharply defined binding cassette of relaxin is not present in RLF. Structure and function analysis of RLF derivatives with single amino acid replacements revealed that the most important binding residues are tryptophan B27, followed by arginine B16 and valine B19. Single alanine replacements for each individual position resulted in a relative receptor affinity of 4.0% (B16), 6.1% (B19), and 0.5% (B27). Tryptophan B27 is located on an extended structure, and arginine B16 and valine B19 are positioned on the exposed surface of the B chain helix. The 3 residues could be brought together to form a contiguous binding area if the C-terminal end of the B chain were free to fold back against the central portion of the B chain helix. Such a movement depends critically on the flexibility of the C-terminal end, which is controlled by positions B23-25. In as much as these major binding residues seem hardly sufficient to explain the strong binding of RLF to LGR8 we searched for and found an extended region where little contributions by individual residues added up to a strong receptor affinity. This mode of interaction could drive the binding energy sufficiently high to account for the picomolar binding constant of RLF and its receptor.

Hb Bleuland [α108(G15)Thr→Asn, ACC→AAC (α2)]: A New Abnormal Hemoglobin Associated with a Mild α-Thalassemia Phenotype

We report a new structural defect of the α2-globin chain, not detectable on high performance liquid chromatography (HPLC) or electrophoresis, characterized in a 12-year-old boy of Surinamese-Hindustani origin. The child was suspected to be a carrier of α-thalassemia (thal) because of microcytic hypochromic parameters in the absence of iron depletion. Gap-polymerase chain reaction (gap-PCR) revealed only normal fragments in the proband, and the pattern of a −α4.2 (leftward) deletion in his father and sister. Direct sequencing of the α-globin genes revealed an ACC→AAC transversion at codon 108 of the α2-globin gene in the proband, in his mother and in a younger sister. The new mutation predicts a Thr →Asn amino acid substitution at the corresponding residue. Threonine, a covalent binder with an R-active OH group, situated in the G helix of the α-globin chain, is involved in α1β1 contacts. Asparagine, being an equally covalent binder but with a different R-active H2N-C=O group, could make the mutated chain less suitable for tetramer cooperation. Alternatively, an absent or reduced interaction with the α hemoglobin (Hb) stabilizing protein (AHSP) could lead to loss of α chains. Hb Bleuland is the first mutation described at codon 108 and is therefore interesting in regard to the possible effects and genetic risk. The nearest variant, Hb Suan-Dok [α109(G16)Leu →Arg, CTG→CGG (α2)] was originally observed in a Thai patient affected with Hb H, in combination with an α0-thal allele. The same Hb Suan-Dok mutation, recently described in our laboratory in a carrier of African ancestry, was also not detectable as a protein and presented with an α-thal phenotype similar to Hb Bleuland.