Negatively-charged Residues Research Articles

Layer-by-layer assembled thin films composed of carboxyl-terminated poly(amidoamine) dendrimer as a pH-sensitive nano-device

Dendrimer-containing multilayer thin films have successfully been prepared by a layer-by-layer deposition of carboxyl-terminated poly(amidoamine) dendrimer (PAMAM–COOH) and poly(methacrylic acid) (PMA) on a solid surface at pH 4.0, while the multilayer film did not form at pH 7.0. The PMA/PAMAM–COOH multilayer films prepared at pH 4.0 are decomposed at neutral pH due to electrostatic repulsion between negatively-charged carboxylate residues. The results suggest that the primary force for the successful deposition of PAMAM–COOH and PMA at pH 4.0 is hydrogen bonding between COOH residues on the surface of the dendrimer and PMA. The multilayer films are decomposed also at strongly acidic pH, suggesting an electrostatic force of attraction between the protonated tertiary amino groups in PAMAM–COOH and a small fraction of COO − residues in PMA contributes in part to the multilayer formation at pH 4.0. The PMA/PAMAM–COOH thin films can accommodate model dyes, Rose Bengal and 5,10,15,20-tetraphenyl-21H,23H-porphinetetrasulfonate, and the release can be controlled by changing pH.

Identification and Structural Characterization of a CBP/p300-Binding Domain from the ETS Family Transcription Factor GABPα

Using NMR spectroscopy, we identified and characterized a previously unrecognized structured domain near the N-terminus (residues 35–121) of the ETS family transcription factor GABPα. The monomeric domain folds as a five-stranded β-sheet crossed by a distorted helix. Although globally resembling ubiquitin, the GABPα fragment differs in its secondary structure topology and thus appears to represent a new protein fold that we term the OST (On-SighT) domain. The surface of the GABPα OST domain contains two predominant clusters of negatively-charged residues suggestive of electrostatically driven interactions with positively-charged partner proteins. Following a best-candidate approach to identify such a partner, we demonstrated through NMR-monitored titrations and glutathione S-transferase pulldown assays that the OST domain binds to the CH1 and CH3 domains of the co-activator histone acetyltransferase CBP/p300. This provides a direct structural link between GABP and a central component of the transcriptional machinery.

Modulation of receptor phosphorylation contributes to activation of peroxisome proliferator activated receptor alpha by dehydroepiandrosterone and other peroxisome proliferators.

Dehydroepiandrosterone (DHEA), a C19 human adrenal steroid, activates peroxisome proliferator-activated receptor alpha (PPARalpha) in vivo but does not ligand-activate PPARalpha in transient transfection experiments. We demonstrate that DHEA regulates PPARalpha action by altering both the levels and phosphorylation status of the receptor. Human hepatoma cells (HepG2) were transiently transfected with the expression plasmid encoding PPARalpha and a plasmid containing two copies of fatty acyl coenzyme oxidase (FACO) peroxisome-proliferator activated receptor responsive element consensus oligonucleotide in a luciferase reporter gene. Nafenopin treatment increased reporter gene activity in this system, whereas DHEA treatment did not. Okadaic acid significantly decreased nafenopin-induced reporter activity in a concentration-dependent manner. Okadaic acid treatment of primary rat hepatocytes decreased both DHEA- and nafenopin-induced FACO activity in primary rat hepatocytes. DHEA induced both PPARalpha mRNA and protein levels, as well as PP2A message in primary rat hepatocytes. Western blot analysis showed that the serines at positions 12 and 21 were rapidly dephosphorylated upon treatment with DHEA and nafenopin. Results using specific protein phosphatase inhibitors suggested that protein phosphatase 2A (PP2A) is responsible for DHEA action, and protein phosphatase 1 might be involved in nafenopin induction. Mutation of serines at position 6, 12, and 21 to an uncharged alanine residue significantly increased transcriptional activity, whereas mutation to negative charged aspartate residues (mimicking receptor phosphorylation) decreased transcriptional activity. DHEA action involves induction of PPARalpha mRNA and protein levels as well as increased PPARalpha transcriptional activity through decreasing receptor phosphorylation at serines in the AF1 region.

Solution Structures and Backbone Dynamics of a Flavodoxin MioC from Escherichia coli in both Apo- and Holo-forms

Flavodoxins play central roles in the electron transfer involving various biological processes in microorganisms. The mioC gene of Escherichia coli encodes a 16-kDa flavodoxin and locates next to the chromosomal replication initiation origin (oriC). Extensive researches have been carried out to investigate the relationship between mioC transcription and replication initiation. Recently, the MioC protein was proposed to be essential for the biotin synthase activity in vitro. Nevertheless, the exact role of MioC in biotin synthesis and its physiological function in vivo remain elusive. In order to understand the molecular basis of the biological functions of MioC and the cofactor-binding mechanisms of flavodoxins, we have determined the solution structures of both the apo- and holo-forms of E. coli MioC protein at high resolution by nuclear magnetic resonance spectroscopy. The overall structures of both forms consist of an alpha/beta sandwich, which highly resembles the classical flavodoxin fold. However, significant diversities are observed between the two forms, especially the stabilization of the FMN-binding loops and the notable extension of secondary structures upon FMN binding. Structural comparison reveals fewer negative charged and aromatic residues near the FMN-binding site of MioC, as compared with that of flavodoxin 1 from E. coli, which may affect both the redox potentials and the redox partner interactions. Furthermore, the backbone dynamics studies reveal the conformational flexibility at different time scales for both apo- and holo-forms of MioC, which may play important roles for cofactor binding and electron transfer.

C-terminal Domain of Kv4.2 and Associated KChIP2 Interactions Regulate Functional Expression and Gating of Kv4.2

The Kv4.2 transient voltage-dependent potassium current contributes to the morphology of the cardiac action potential as well as to neuronal excitability and firing frequency. Here we report profound effects of the Kv4.2 C terminus on the surface expression and activation gating properties of Kv4.2 that are modulated by the direct interaction between KChIP2, an auxiliary regulatory subunit, and the C terminus of Kv4.2. We show that increasingly large truncations of the C terminus of rat Kv4.2 (wild type) cause a progressive decrease of Kv4.2 current along with a shift in voltage-dependent activation that is closely correlated with negative charge deletion. Co-expression of more limited Kv4.2 C-terminal truncation mutants (T588 and T528) with KChIP2 results in a doubling of Kv4.2 protein expression and up to an 8-fold increase in Kv4.2 current amplitude. Pulsechase experiments show that co-expression with KChIP2 slows Kv4.2 wild type degradation 8-fold. Co-expression of KChIP2 with an intermediate-length C-terminal truncation mutant (T474) shifts Kv4.2 activation voltage dependence and enhances expression of Kv4.2 current. The largest truncation mutants (T417 and DeltaC) show an intracellular localization with no measurable currents and no response to KChIP2 co-expression. Co-immunoprecipitation and competitive glutathione S-transferase-binding assays indicate a direct interaction between KChIP2 and the Kv4.2 C terminus with a relative binding affinity comparable with that of the N terminus. Overall, these results suggest that the C-terminal domain of Kv4.2 plays a critical role in voltage-dependent activation and functional expression that is mediated by direct interaction between the Kv4.2 C terminus and KChIP2.

N-terminal positively charged amino acids, but not their exact position, are important for apicoplast transit peptide fidelity in Toxoplasma gondii

The non-photosynthetic plastid – or apicoplast – of Toxoplasma gondii and other apicomplexan parasites is an essential organelle and promising drug target. Most apicoplast proteins are encoded in the nucleus and targeted into the organelle through the apicoplast's four membranes courtesy of a bipartite N-terminal leader sequence comprising of an endomembrane signal peptide followed by a plastid transit peptide. Apicoplast transit peptides, like plant plastid transit peptides, have no primary consensus, are variable in length and may be distinguishable only by a relative depletion of negative charged residues and consequent enrichment in basic residues. In this study we examine the role of charged residues within an apicoplast transit peptide in T. gondii by point mutagenesis. We demonstrate that positive charged residues, combined with the absence of negatively charged amino acids, are essential for apicoplast transit peptide fidelity, as also observed in P. falciparum. Furthermore, we show that positive charge is more important at the transit peptide's N-terminus than its C-terminus, and that the nature of the positive residue and the exact position of the N-terminal positive charge are not important. These results suggest that a simple, rule-based prediction for T. gondii transit peptides, similar to that successfully implemented for P. falciparum should help to identify apicoplast proteins and facilitate the identification of drug targets in this important human pathogen.

Charged residues are involved in membrane fusion mediated by a hydrophilic peptide located in vesicular stomatitis virus G protein

Membrane fusion is an essential step of the internalization process of the enveloped animal viruses. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in VSV G protein, comprising the residues 145 to 164, directly involved in membrane interaction and fusion. Unlike fusion peptides from other viruses, this sequence is very hydrophilic, containing six charged residues, but it was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Using a carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), and several synthetic mutant peptides, we demonstrated that the negative charges of peptide acidic residues, especially Asp153 and Glu158, participate in the formation of a hydrophobic domain at pH 6.0, which is necessary to the peptide-induced membrane fusion. The formation of the hydrophobic region and the membrane fusion itself were dependent on peptide concentration in a higher than linear fashion, suggesting the involvement of peptide oligomerization. His148 was also necessary to hydrophobicity and fusion, suggesting that peptide oligomerization occurs through intermolecular electrostatic interactions between the positively-charged His and a negatively-charged acidic residue of two peptide molecules. Oligomerization of hydrophilic peptides creates a hydrophobic region that is essential for the interaction with the membrane that results in fusion.

Identification of a Functional Phosphatidylinositol 3,4,5-Trisphosphate Binding Site in the Epithelial Na+ Channel

Membrane phospholipids, such as phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)), are signaling molecules that can directly modulate the activity of ion channels, including the epithelial Na(+) channel (ENaC). Whereas PI(3,4,5)P(3) directly activates ENaC, its binding site within the channel has not been identified. We identify here a region of gamma-mENaC just following the second trans-membrane domain (residues 569-583) important to PI(3,4,5)P(3) binding and regulation. Deletion of this track decreases activity of ENaC heterologously expressed in Chinese hamster ovary cells. K-Ras and its first effector phosphoinositide 3-OH kinase (PI3-K), as well as RhoA and its effector phosphatidylinositol 4-phosphate 5-kinase increase ENaC activity. Whereas the former, via generation of PI(3,4,5)P(3), increases ENaC open probability, the latter increases activity by increasing membrane levels of the channel. Deletion of the region just distal to the second trans-membrane domain disrupted regulation by K-Ras and PI3-K but not RhoA and phosphatidylinositol 4-phosphate 5-kinase. Moreover, PI(3,4,5)P(3) binds ENaC with deletion of the region following the second transmembrane domain disrupting this interaction and disrupting direct activation of the channel by PI(3,4,5)P(3). Mutation analysis revealed the importance of conserved positive and negative charged residues as well as bulky amino acids within this region to modulation of ENaC by PI3-K. The current results identify the region just distal to the second trans-membrane domain within gamma-mENaC as being part of a functional PI(3,4,5)P(3) binding site that directly impacts ENaC activity. Phospholipid binding to this site is probably mediated by the positively charged amino acids within this track, with negatively charged and bulky residues also influencing specificity of interactions.

3D Solution Structure of [Tyr3]Octreotate Derivatives in DMSO: Structure Differentiation of Peptide Core Due to Chelate Group Attachment and Biologically Active Conformation

The solution models of [Tyr3]octreotate (DPhe1-Cys2-Tyr3-DTrp4-Lys5-Thr6-Cys7-Thr8-COOH, disulfide bridged) (I), its analogs functionalized with an open chain tetraamine chelator, N4-[Tyr3]octreotate (II), and the N4-(Asp)2-[Tyr3]octreotate (III) peptide have been determined through 2D 1H NMR spectroscopy in DMSO. Chemical shift analysis has been performed in an attempt to elucidate structural changes occurring during attachment of the tetraamine to the peptide backbone. NMR-derived geometrical constraints have been used in order to calculate high resolution conformers of the above peptides. Conformational analysis of the three synthetic analogues, have shown that these somatostatin analoges adopt a predominant antiparallel beta-sheet conformation characterized by a beta-like turn spanning residues DTrp4 and Lys5 which is supported in the case of N4-(Asp)2-[Tyr3]octreotate and N4-[Tyr3]octreotate by medium range NOEs. These data indicate that the above-mentioned molecules adopt a rather constrained structure in the 4-residue loop Tyr3-Thr6. Additionally, the C-terminal of [Tyr3]octreotate, comprising Cys7 and Thr8, appears to form a turn-like structure manifested by characteristic side-chain NOEs between Lys5 and Thr8, which have not been detected for the other two compounds. These data are discussed in the light of previous structural data of Sandostatin (octreotide) and suggest that attachment of the N4-chelator and two Asp residues at the N-end of [Tyr3]octreotate impose considerable structural changes and affect the binding properties of these peptides. Indeed, the IC50 values determined during competition binding assays against the sst2 (somatostatin subtype 2 receptor) suggest that the presence of the N4 group enhances receptor affinity, while extension of peptide chain by two negatively-charged Asp residues impairs receptor affinity at approximately one order of magnitude.

Role of Thrombin Exosites in Protection from Inhibition by Antithrombin in the Presence of Heparin and Fibrin.

The γ-chain of fibrinogen exists in two forms, γA and γ′, such that circulating fibrinogen consists of two populations, γA/γA (90%) and γA/γ′ (10%). The 16 amino acid extension at the COOH-terminus of the γ′ chain contains numerous negatively-charged residues. This alteration endows γA/γ′-fibrin (Fn) with a greater capacity to bind thrombin (IIa), a feature that may render thrombi prothrombotic. The purpose of this study was to explore how the various mechanisms by which IIa binds to Fn impact on IIa protection from inactivation by antithrombin/heparin. IIa binds weakly to γA/γA-Fn utilizing exosite 1. IIa binds with higher affinity to γA/γ′-Fn due to the additional exosite 2/γ′ interaction. In the presence of heparin, IIa can bind to γA/γA-Fn with high affinity by forming a ternary complex wherein heparin bridges IIa to Fn via exosite 2, thereby heightening the interaction of IIa with Fn via exosite 1. Consequently, the amount of IIa bound to γA/γA-Fn clots increases in the presence of heparin. Formation of the γA/γA-Fn/heparin/IIa ternary complex reduces the second order rate constant of IIa inhibition by antithrombin (AT) 11-fold compared with the heparin-catalyzed rate of inhibition of free IIa (1.1x108 M−1 min−1). This reduction reflects the inability of AT-bound heparin to access exosite 2. When γA/γ′-Fn is used in place of γA/γA-Fn, the heparin-catalyzed rate is reduced 55-fold. The enhanced protection with γA/γ′-Fn is due to the exosite 2-mediated interaction of IIa with the γ′-chain because addition of an antibody against the γ′ sequence that blocks this interaction reduces the protection from AT to the level seen with γA/γA-Fn. Thus, both γA/γ′-Fn/IIa and γA/γA-Fn/heparin/IIa complexes restrict the access of heparin to exosite 2, thereby impairing inhibition by heparin/AT. Heparin cofactor II (HCII) also utilizes heparin to bridge to IIa, but, unlike AT, HCII must also directly engage exosite 1 to effect IIa inhibition. Consequently, the heparin-catalyzed rate of IIa inhibition by HCII is reduced 27-fold in the presence of γA/γA-Fn compared with the 11-fold reduction with AT. To examine the contribution of the heparin-Fn interaction to this phenomenon, dermatan sulfate (DS) was used to catalyze HCII because, unlike heparin, DS does not bind Fn. γA/γA-Fn produced only a 5-fold reduction in the DS-catalyzed rate of IIa inhibition by HCII. This suggests that in the absence of heparin, occupation of exosite 1 by γA/γA-Fn only modestly impairs inhibition by HCII. In contrast, γA/γ′-Fn produced a 28-fold reduction in the DS-catalyzed rate of IIa inhibition by HCII. This protection is abolished by addition of the antibody that blocks binding of IIa to the γ′ chain. Therefore, although exosite 1 mediates IIa binding to both γA/γA- and γA/γ′-Fn, interaction of exosite 2 with the γ′-chain in γA/γ′-Fn heightens exosite 1-mediated binding. These findings provide independent confirmation that ligation of both exosites on IIa accentuates the affinity of the individual exosite 1 and 2 interactions. Thus, reactants that require access to either exosite will be restricted when IIa binding is mediated by both exosites as occurs with γA/γA-Fn/heparin or with γA/γ′-Fn. These results confirm that Fn can serve as a reservoir of active IIa and that bound IIa is protected from inhibition by circulating inhibitors. Our data also highlight the limitations of physiological inhibitors of IIa and validate the need for development of direct thrombin inhibitors.

Crystal structure of the TM1442 protein from Thermotoga maritima, a homolog of the Bacillus subtilis general stress response anti-anti-sigma factor RsbV.

Crystal structure of the TM1442 protein from Thermotoga maritima, a homolog of the Bacillus subtilis general stress response anti-anti-sigma factor RsbV.

A brownian dynamics study of evolotionary cahnges in electrostatic interactions between plastocyanin and cytochrome f in cyanobacteria and plants.

Brownian Dynamics (BD) were used to model the electrostatic interactions of Phormidium plastocyanin cytochrome f (cyt f) with Phormidium and Prochlorothrix plastocyanin (PC) in order to explore evolutionary relationships with proteins from higher plants such as poplar (Pearson and Gross, Biophys. J. 75: 2698-2711 (1998) and spinach. Cyt f?s from higher plants such as turnip typically contain five conserved positively charged residues (#58, #65, #66, #189 and #209) that produces a positively-charged electrostatic field which attracts negatively-charged residues on PC (positions #42-45 and #59-61). In Phormidium cyt f, all but residues #66 is replaced by a negatively-charged residue For Phormidium PC, #44 and #45. are the only negative charges that remain and a Zn2+ ion lies adjacent to these residues in the crystal structure. All cyanobacterial PC contain an arginine at position #86 replacing a glutamine in other PC?s. BD simulations of Phormdiium PC with Phormidium cyt f show a significant number of formed in the presence of the Zn2+ ion compared to almost none in its absence suggesting that a divalent cation may occupy that position in vivo.. A greater number of close complexes was observed using Prochlorothrix PC. Replacing Tyr 12 with a glycine (found in all other PC?s) decreased the Cu-Fe distance for maximal complex formation due to a decrease in steric hindrance. In conclusion, electrostatic interactions occur between PC and cyt f in all species but the signs of the electrostatic fields are reversed in cyanobacteria.

In Vivo Mitochondrial Import: A COMPARISON OF LEADER SEQUENCE CHARGE AND STRUCTURAL RELATIONSHIPS WITH THE IN VITRO MODEL RESULTING IN EVIDENCE FOR CO-TRANSLATIONAL IMPORT

The positive charges and structural properties of the mitochondrial leader sequence of aldehyde dehydrogenase have been extensively studied in vitro. The results of these studies showed that increasing the helicity of this leader would compensate for reduced import from positive charge substitutions of arginine with glutamine or the insertion of negative charged residues made in the native leader. In this in vivo study, utilizing the green fluorescent protein (GFP) as a passenger protein, import results showed the opposite effect with respect to helicity, but the results from mutations made within the native leader sequence were consistent between the in vitro and in vivo experiments. Leader mutations that reduced the efficiency of import resulted in a cytosolic accumulation of a truncated GFP chimera that was fluorescent but devoid of a mitochondrial leader. The native leader efficiently imported before GFP could achieve a stable, import-incompetent structure, suggesting that import was coupled with translation. As a test for a co-translational mechanism, a chimera of GFP that contained the native leader of aldehyde dehydrogenase attached at the N terminus and a C-terminal endoplasmic reticulum targeting signal attached to the C terminus of GFP was constructed. This chimera was localized exclusively to mitochondria. The import result with the dual signal chimera provides support for a co-translational mitochondrial import pathway.

Photoinduced Electron Transfer between CytochromecPeroxidase (D37K) and Zn-Substituted Cytochromec: Probing the Two-Domain Binding and Reactivity of the Peroxidase

Cytochrome c peroxidase (CcP) binds cytochrome c (Cc) at two distinct surface binding domains, one having high affinity for Cc and the other having low affinity. The identity of the surface binding domains on CcP has been probed by studying photoinduced interprotein heme−heme electron transfer (ET) between Zn-substituted horse Cc, ZnCc, and a cloned cytochrome c peroxidase, CcP(D37K). Charge-reversal substitution of the negatively-charged residue Asp 37 of CcP by a positively-charged residue lysine greatly decreases the stoichiometric constant for 1:1 binding of Cc, from 8.5 × 105 M-1 for the wild-type CcP to 1.2 × 104 M-1 for CcP(D37K) (μ = 18 mM), thereby identifying residue 37 as part of the high-affinity binding domain of CcP(WT) (domain 1). This assignment is consistent with domain 1 as being that observed in a crystal of the Cc−CcP complex. The diminished ability of CcP(D37K) to bind Cc at domain 1 also suppresses binding of a second Cc molecule to form a ternary complex. However, the mutation sharp...

Genetic Diagnosis of Dysplasminogenemia: Detection of an Ala601 -Thr Mutation in 118 out of 125 Families and Identification of a New Asp676-Asn Mutation

Dysplasminogenemia (plasminogen abnormality) is frequently found in association with thrombosis. Two types of mutation, Ala601-Thr and Val355-Phe, have already been identified; the precise genetic defects of most of these patients, however, remain unknown. In this study, we examined the genetic DNAs of two unrelated cases by single-strand conformational polymorphism and nucleotide sequencing analysis. A new mutation, designated as Asp676-Asn, has been identified in these cases. This mutation leads to the loss of a negatively-charged residue and the creation of a potential carbohydrate attachment site, which may impair the enzymatic properties of plasminogen. As many as 158 individuals with dysplasminogenemia were analyzed by a combination of in vitro amplification and restriction digestion. Among 125 unrelated families, the Ala601-Thr mutation accounted for about 94% of cases. The Val355-Phe mutation was found in four unrelated families, indicating that it is a recurring mutation and is not very rare.

A structure-based multiple sequence alignment of all class I aminoacyl-tRNA synthetases

The superimposable dinucleotide fold domains of MetRS, GlnRS and TyrRS define structurally equivalent amino acids which have been used to constrain the sequence alignments of the 10 class I aminoacyl-tRNA synthetases (aaRS). The conservation of those residues which have been shown to be critical in some aaRS enables to predict their location and function in the other synthetases, particularly: i) a conserved negatively-charged residue which binds the α-amino group of the amino acid substrate; ii) conserved residues within the inserted domain bridging the two halves of the dinucleotide-binding fold; and iii) conserved residues in the second half of the fold which bind the amino acid and ATP substrate. The alignments also indicate that the class I synthetases may be partitioned into two subgroups: a) MetRS, IleRS, LeuRS, ValRS, CysRS and ArgRS; b) GlnRS, GluRS, TyrRS and TrpRS.

Exploring the interface between the N- and C-terminal helices of cytochrome c by random mutagenesis within the C-terminal helix.

Buried within cytochrome c lies a highly-conserved helix-helix interface formed by the perpendicular packing of the C-terminal helix against the N-terminal helix. This interface involves a peg-in-hole interaction between Gly-6 and Leu-94 and an aromatic-aromatic interaction between Phe-10 and Tyr-97. To gain insight into protein design, we investigated the relationship between the sequence of the interface and the physiological function of yeast iso-1-cytochrome c. A library of mutants at positions 94 and 97 of the C-terminal helix was created to examine the effect of novel amino acid combinations. We isolated 45 of the 400 possible amino acid combinations, 32 of which result in a functional cytochrome c. Contrary to evolutionary conservation of the peg-in-hole and aromatic-aromatic interactions, we find that side-chain volume and conservation of aromatic residues do not play an essential role in determining function. Additionally, we find negatively-charged residues within the interface that result in a functional cytochrome c. Examination of the 45 missense mutants indicates that approximately 120 unique combinations are compatible with function. These results show that the interface is flexible. However, truncation of the C-terminal helix at position 94 abolishes function, suggesting that the interface is essential. The correlation observed between our library of mutants and the mutation matrix compiled by Gonnet et al. [Gonnet, G. H., Cohen, M. A., & Benner, S. A. (1992) Science 256, 1443-1445] demonstrates the potential use of the matrix to predict the effect of sequence changes on natural proteins and to optimize the design of novel proteins.

A Role for von Willebrand Factor Proline Residues 702-704 in Ristocetin-Mediated Binding to Platelet Glycoprotein Ib

Mutant domains of von Willebrand factor (vWF) were constructed to determine the effects of altering net charge, and presumably conformation, within a peptide sequence (residues 694-708) previously shown to be involved in the platelet receptor glycoprotein (GP) Ib binding function of vWF. Non-conservative substitutions replaced a triplet of proline residues (proline702-704) with either a triplet of arginine (positively-charged) or aspartic acid (negatively-charged) residues. After establishing stable CHO cell transformants, we observed the secretion of covalently-linked dimeric molecules analogous to a domain with native sequence. Functional assays using immunopurified molecules revealed that the ristocetin-dependent binding to GP Ib was abolished with both charge mutants. However, in the absence of disulfide-bond dependent conformation both mutant molecules and the molecule with native sequence interacted with GP Ib. The results demonstrate that vWF proline702-704 are important for the ristocetin-mediated interaction between vWF and GP Ib, but are not essential residues of the GP Ib binding site within vWF.

Hemoglobin variants and activity of the (K+Cl-) cotransport system in human erythrocytes

To determine if the activation of the (K+Cl-) cotransport system observed in hemoglobin (Hb) S- or C-containing erythrocytes is related either to a global change of isoelectric point of the Hb molecule or to the specific location of these mutations on the position 6 of the beta chain of Hb, we studied the (K+Cl-) cotransport system in erythrocytes containing beta chain variants exhibiting either the Glu----Lys substitution observed in position beta 6 in Hb C (Hb E: beta 26 Glu---- Lys; Hb O-Arab: beta 121 Glu----Lys; Hb Siriraj:beta 7 Glu----Lys) or the Glu----neutral residue substitution observed in position beta 6 in Hb S (Hb G-San Jose: beta 7 Glu----Gly; Hb D Punjab or D-Los Angeles: beta 121 Glu----Gln). The K transport mediated by the (K+Cl-) cotransport was increased in AC, AS and A-Siriraj and A-San Jose red blood cells and was similar to AA control in the other variants. These results indicate that an enhanced (K+Cl-) cotransport is not a property of all positively charged Hb variants, but it is mainly associated with mutations occurring at the beta 6 or beta 7 residues. An interaction of Hb with the cell membrane mediated by the disappearance of one of the negative charged residues (Glu) at this site of the A helix of the beta chain is the most likely candidate for the persistent activation of the (K+Cl-) cotransport system in these Hb variants.

Hemoglobin Variants and Activity of the (K+C1-) Cotransport System in Human Erythrocytes

To determine if the activation of the (K+Cl-) cotransport system observed in hemoglobin (Hb) S- or C-containing erythrocytes is related either to a global change of isoelectric point of the Hb molecule or to the specific location of these mutations on the position 6 of the beta chain of Hb, we studied the (K+Cl-) cotransport system in erythrocytes containing beta chain variants exhibiting either the Glu----Lys substitution observed in position beta 6 in Hb C (Hb E: beta 26 Glu----Lys; Hb O-Arab: beta 121 Glu----Lys; Hb Siriraj:beta 7 Glu----Lys) or the Glu----neutral residue substitution observed in position beta 6 in Hb S (Hb G-San Jose: beta 7 Glu----Gly; Hb D Punjab or D-Los Angeles: beta 121 Glu----Gln). The K transport mediated by the (K+Cl-) cotransport was increased in AC, AS and A-Siriraj and A-San Jose red blood cells and was similar to AA control in the other variants. These results indicate that an enhanced (K+Cl-) cotransport is not a property of all positively charged Hb variants, but it is mainly associated with mutations occurring at the beta 6 or beta 7 residues. An interaction of Hb with the cell membrane mediated by the disappearance of one of the negative charged residues (Glu) at this site of the A helix of the beta chain is the most likely candidate for the persistent activation of the (K+Cl-) cotransport system in these Hb variants.