Influence of the g− conformation of Ser and Thr on the structure of transmembrane helices

In order to study the influence of Ser and Thr on the structure of transmembrane helices we have analyzed a database of helix stretches extracted from crystal structures of membrane proteins and an ensemble of model helices generated by molecular dynamics simulations. Both complementary analyses show that Ser and Thr in the g? conformation induce and/or stabilize a structural distortion in the helix backbone. Using quantum mechanical calculations, we have attributed this effect to the electrostatic repulsion between the side chain Oγ atom of Ser and Thr and the backbone carbonyl oxygen at position i ? 3. In order to minimize the repulsive force between these negatively charged oxygens, there is a modest increase of the helix bend angle as well as a local opening of the helix turn preceding Ser/Thr. This small distortion can be amplified through the helix, resulting in a significant displacement of the residues located at the other side of the helix. The crystal structures of aquaporin Z and the β₂-adrenergic receptor are used to illustrate these effects. Ser/Thr-induced structural distortions can be implicated in processes as diverse as ligand recognition, protein function and protein folding.     

Frequency of human Aγ75Thr globin chain in a population from Tunisia

Summary Cord blood samples, collected at Sousse and Monastir, from Tunisian newborns were focused on a thin layer of agarose in order to detect the carriers of the ^A75Thr chain (^A chain bearing a replacement IleThr at position 75). Nineteen individuals (10%) were positive for this variant. The frequency of the ^A75Thr gene in the Tunisian population (0.050) is compared with that of various ethnic populations.This work was supported in part by the Faculté de Pharmacie et de Médecine Dentaire of Monastir and by a grant from the Ambassade de France in Tunisia     

Lectinochemical studies on the glyco-recognition factors of a <Emphasis Type="Bold">Tn</Emphasis> (GalNAc<Emphasis Type="Bold">α</Emphasis>1→Ser/Thr) specific lectin isolated from the seeds of <Emphasis Type="Italic">Salvia sclarea</Emphasis>

The lectin extracted from the seeds of Salvia sclarea (SSL) recognizes the Tn antigen (GalNAc 1Ser/Thr) expressed in certain human carcinomas. In previous studies, knowledge of the binding properties of SSL was restricted to GalNAc1 related oligosaccharides and glycopeptides. Thus, the requirements of functional groups in monosaccharide and high-density polyvalent carbohydrate structural units for SSL binding and an updated affinity profile were further evaluated by enzyme-linked lectinosorbent (ELLSA) and inhibition assays. Among the glycoproteins (gps) tested for interaction, a high density of exposed Tn-containing glycoproteins such as in the armadillo salivary Tn glycoprotein and asialo ovine salivary glycoprotein reacted best with SSL. When the gps were tested for inhibition of SSL binding, which was expressed as 50% nanogram inhibition, the high density polyvalent Tn present in macromolecules was the most potent inhibitor. Among the monosaccharide and carbohydrate structural units studied, which were expressed as nanomole inhibition, GalNAc 13GalNAc 13Gal 14Gal 14Glc (Fp), GalNAc 13Gal 14Glc (A_L), GalNAc 13GalNAc 1Me (F), GalNAc 13GalNAc 1Me (F ) and GalNAc 1 Ser/Thr (Tn) were the most active ligands, being 2.5–5.0× 10³ and 1.25–2.5 times more active than Gal and GalNAc, respectively. From the results, it is suggested that the combining site of SSL is a shallow groove type, recognizing the monosaccharide of GalNAc as the major binding site or Tn up to the Forssman pentasaccharide (Fp). It can be concluded that the three critical factors for SSL binding are the –NH CH₃CO at carbon-2 in Gal, the configuration of carbon-3 in GalNAc, and the polyvalent Tn (GalNAc 1Ser/Thr) present in macromolecules. These results should assist in understanding the glyco-recognition factors involved in carbohydrate–lectin interactions in biological processes. The effect of the polyvalent F , F and GalNAc 13Gal 1 (P ) glycotopes on binding should be examined. However, this is hampered by the lack of availability of suitable reagents.     

Functional modulation of the Thr72→Ile mutant from Scapharca inaequivalvis homodimeric hemoglobin

 The pH and temperature dependence of both the kinetic and thermodynamic properties of the Thr72→Ile mutant of Scapharca inaequivalvis homodimeric hemoglobin were investigated between pH 2 and 10 and between 8  °C and 36  °C, in comparison with the wild-type recombinant protein. Results demonstrate pH-independent O₂-binding properties, at least between pH 5 and 10, with the higher affinity of the mutant being related to a less negative entropy change. This observation may relate to a variation in the number of water molecules involved in the intersubunit communication. Furthermore, the kinetic properties of ligand association and dissociation seem to be in keeping with possible structural alterations of water molecules at the subunit interface occurring in the Thr72→Ile mutant as well as with amino acid residues involved in the modulation of reactivity and cooperativity at the level of (1) the proximal side of the heme pocket and of (2) the heme propionates bridging the two subunits. Received: 25 February 1999 / Accepted: 9 August 1999     

Thr90 phosphorylation of Hsp90α by protein kinase A regulates its chaperone machinery

Hsp90 (heat-shock protein 90) is one of the most important molecular chaperones in eukaryotes. Hsp90 facilitates the maturation, activation or degradation of its client proteins. It is now well accepted that both ATP binding and co-chaperone association are involved in regulating the Hsp90 chaperone machinery. However, other factors such as post-translational modifications are becoming increasingly recognized as being involved in this process. Recent studies have reported that phosphorylation of Hsp90 plays an unanticipated role in this process. In the present study, we systematically investigated the impact of phosphorylation of a single residue (Thr90) of Hsp90α (pThr90-Hsp90α) on its chaperone machinery. We demonstrate that protein kinase A specifically phosphorylates Hsp90α at Thr90, and that the pThr9090-Hsp90α level is significantly elevated in proliferating cells. Thr90 phosphorylation affects the binding affinity of Hsp90α to ATP. Subsequent examination of the interactions of Hsp90α with co-chaperones reveals that Thr90 phosphorylation specifically regulates the association of a subset of co-chaperones with Hsp90α. The Hsp90α T90E phosphor-mimic mutant exhibits increased association with Aha1 (activator of Hsp90 ATPase homologue 1), p23, PP5 (protein phosphatase 5) and CHIP (C-terminus of Hsp70-interacting protein), and decreased binding affinity with Hsp70, Cdc37 (cell division cycle 37) and Hop [Hsc70 (heat-shock cognate protein 70)/Hsp90-organizing protein], whereas its interaction with FKBP52 (FK506-binding protein 4) is only moderately affected. Moreover, we find that the ability of the T90E mutant to form complexes with its clients, such as Src, Akt or PKCγ (protein kinase Cγ), is dramatically impaired, suggesting that phosphorylation affects its chaperoning activity. Taken together, the results of the present study demonstrate that Thr90 phosphorylation is actively engaged in the regulation of the Hsp90α chaperone machinery and should be a generic determinant for the cycling of Hsp90α chaperone function.     

Roles of β-Tubulin Residues Ala428 and Thr429 in Microtubule Formation in Vivo

The C termini of β-tubulin isotypes are regions of high sequence variability that bind to microtubule-associated proteins and motors and undergo various post-translational modifications such as polyglutamylation and polyglycylation. Crystallographic analyses have been unsuccessful in resolving tubulin C termini. Here, we used a stepwise approach to study the role of this region in microtubule assembly. We generated a series of truncation mutants of human βI and βIII tubulin. Transient transfection of HeLa cells with the mutants shows that mutants with deletions of up to 22 residues from βIII and 16 from βI can assemble normally. Interestingly, removal of the next residue (Ala⁴²⁸) results in a complete loss of microtubule formation without affecting dimer formation. C-terminal tail switching of human βI and βIII tubulin suggests that C-terminal tails are functionally equivalent. In short, residues outside of 1–429 of human β-tubulins make no contribution to microtubule assembly. Ala⁴²⁸, in the C-terminal sequence motif N-QQYQDA⁴²⁸, lies at the end of helix H12 of β-tubulin. We hypothesize that this residue is important for maintaining helix H12 structure. Deletion of Ala⁴²⁸ may lead to unwinding of helix H12, resulting in tubulin dimers incapable of assembly. Thr⁴²⁹ plays a more complex role. In the βI isotype of tubulin, Thr⁴²⁹ is not at all necessary for assembly; however, in the βIII isotype, its presence strongly favors assembly. This result is consistent with a likely more complex function of βIII as well as with the observation that evolutionary conservation is total for Ala⁴²⁸ and frequent for Thr⁴²⁹.Microtubules are involved in a great variety of cellular functions. Their constituent protein tubulin is an αβ heterodimer, both α- and β-tubulin existing as multiple isotypes, encoded by different genes and differing in amino acid sequence (1). The differences among the isotypes are highly conserved in evolution. In mammals, the β isotypes are βIa, βIb, βII, βIII, βIVa, βIVb, βV, and βVI. There is evidence that the isotype differences have functional significance. For instance, the βIV isotype is found in all axonemes (2).Structurally, both α- and β-tubulin consist of a globular region of 427 amino acids followed by a C-terminal region of 17–24 amino acids (3–5). The C-terminal region is highly negatively charged, being especially rich in glutamate residues and lacking in basic residues, and is likely to project outward from the rest of the molecule, because of its high negative charge and the electrostatic repulsion among the glutamate residues (3). The three-dimensional structure of the globular domain has been determined by electron and x-ray crystallography (4, 5). However, the C-terminal region has never been localized in the three-dimensional reconstructions except by computer modeling. The probable reasons for this are 1) that, if the C-terminal region projects out from the rest of molecule, it is likely to be very flexible with respect to the rest of the molecule and 2) the C-terminal region undergoes post-translational modification. Both of these can lead to structural heterogeneity and cause the C terminus to be invisible to crystallographic techniques.In this work, we examine the role of the C termini of human β-tubulins to determine the minimal sequence requirement for microtubule incorporation through structure/function analyses. The human βI and βIII tubulin isotypes were utilized based on their high degree of sequence variability clustered at the C terminus (Fig. 1) and the fact that βI is broadly distributed among normal tissues, whereas βIII has a very narrow tissue distribution. These two isotypes share 92% sequence identity, with differences among these isotypes occurring in both the globular domain and the C-terminal region (1).Open in a separate windowFIGURE 1.Sequence alignment of human βIa and βIII tubulin isotypes. Human βIa and βIII tubulin isotypes were aligned with ClustalX 1.83 and processed with BioEdit. Hyphens denote identical residues between sequences.

TABLE 1

The C-terminal amino acid sequences of the human β-tubulin isotypes

PPARGC1A基因Thr394Thr/Gly482Ser多态性与2型糖尿病的关联研究

对344例2型糖尿病患者和307名正常人的PPARGC1A基因单核苷酸多态性rs2970847(Thr394Thr)和rs8192678(Gly482Ser)与2型糖尿病的关系进行了单标记和单体型关联分析以及Logistic回归分析。在单标记分析中,对照组与病例组Thr394Thr的基因型和等位基因频率有显著差异(基因型, P =0.006; 等位基因, P < 0.001); Logistic回归和单体型分析表明, Thr394Thr的AA基因型及Thr394(ACA)-Ser482单体型增加患2型糖尿病的风险。Gly482Ser的基因型和等位基因频率在对照组与病例组间无显著差异。PPARGC1A基因是湖北汉人的一个2型糖尿病易感基因。     

Structure of the δ-Chain of Chimpanzee Haemoglobin A<Subscript>2</Subscript>

STUDIES of adult¹ and foetal² haemoglobin from the chimpanzee (Pan troglodytes) have shown that the amino-acid compositions of tryptic and chymotryptic peptides of the α, β and γ-chains are indistinguishable from those of man. The primary structures of chimpanzee α, β and γ-chains are therefore almost certainly identical to the homologous human chains. The two types of γ-chains found in man³, ^Gγ and ^Aγ, with glycine and alanine in position γ136, respectively, are likewise present in the chimpanzee².     

Effects of antimicrobial activity of silver nanoparticles on in vitro establishment of G × N15 (hybrid of almond × peach) rootstock

In the present investigation were evaluated the antifungal and antibacterial activities of Nano-silver (NS). Two separate experiments were done to evaluate the potential of silver nanoparticles in controlling the contamination of G × N15 micro-propagation. In the first experiment, a factorial experiment based on a completely randomized design with 15 treatments including five different NS concentrations (0, 50, 100, 150 and 200 ppm) and three soaking time of explants (3, 5 and 7 min) with five replications was conducted. In the other experiment, NS was added to Murashige and Skoog (MS) medium with concentrations of 0, 50, 100, 150 and 200 ppm in a completely randomized design. Results showed that the application of 100 and 150 ppm NS both as an immersion and as added directly to the culture medium significantly reduces internal and external contaminations compared with the control group. Using NS in culture medium was more efficient to reduce fungal and bacterial contamination than immersion. High concentrations of NS had an adverse effect on the viability of G × N15 single nodes and this effect was more serious in immersed explants in NS than directly added NS ones regarding the viability of buds and plantlet regeneration. In conclusion, due to high contamination of woody plants especially fruit trees and also adverse environmental effects of mercury chloride, the NS solution can be used as a low risk bactericide in micro-propagation of G × N15 and can be an appropriate alternative to mercury chloride in the future.     

The effect of a Pro²⁸Thr point mutation on the local structure and stability of human galactokinase enzyme-a theoretical study

Galactokinase is responsible for the phosphorylation of α-d-galactose, which is an important step in the metabolism of the latter. Malfunctioning of galactokinase due to a single point mutation causes cataracts and, in serious cases, blindness. This paper reports a study of the Pro²⁸Thr point mutation using a variety of theories including molecular dynamics (MD), MM-PBSA/GBSA calculations and AIM analysis. Altered H-bonding networks were detected based on geometric and electron density criteria that resulted in local unfolding of the β-sheet secondary structure. Another consequence was the decrease in stability (5–7 kcal mol⁻¹) around this region, as confirmed by ΔG_bind calculations for the extracted part of the whole system. Local unfolding was verified by several other MD simulations performed with different duration, initial velocities and force field. Based on the results, we propose a possible mechanism for the unfolding caused by the Pro²⁸Thr point mutation.     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

Can the combination of electrochemical regeneration of NAD+, selectivity of L‐α‐amino‐acid dehydrogenase,and reductive amination of α‐keto‐acid be applied to the inversion of configuration of a L‐α‐amino‐acid?

The inversion of configuration of L‐alanine can be carried out by combining its selective oxidation in the presence of NAD⁺ and L‐alanine dehydrogenase, electrochemical regeneration of the NAD⁺ at a carbon felt anode, and reductive amination of pyruvate, i.e., reduction of its imino derivative at a mercury cathode, the reaction mixture being buffered with concentrated ammonium/ammonia (1.28M / 1.28M). The dehydrogenase exhibits astonishing activity and stability under such extreme conditions of pH and ionic strength. The main drawback of the process is its slowness. At best, the complete inversion of a 10 mM solution of L‐alanine requires 140 h. A careful and detailed quantitative analysis of each of the key steps involved shows that the enzyme catalyzed oxidation is so thermodynamically uphill that it can be driven efficiently to completion only when both the coenzyme regeneration and the pyruvate reduction are very effective. The first condition is easily fulfilled. Under the best conditions, it is the rate of the chemical reaction producing the imine which controls the whole process kinetically. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 101–107, 1999.     

Study of the Mechanism of Interaction of Oligonucleotides with the 3′-Terminal Region of tRNA<Superscript>Phe</Superscript> by Computer Modeling

Three-dimensional atomic models of complexes between yeast tRNA^Phe and 10- or 15-mer oligonucleotides complementary to the 3′-terminal tRNA sequence have been constructed using computer modeling. It has been found that rapidly formed primary complexes appear when an oligonucleotide binds to the coaxial acceptor and T stems of the tRNA^Phe along the major groove, which results in the formation of a triplex. Long stems allow the formation of a sufficiently strong complex with the oligonucleotide, which delivers its 3′-terminal nucleotides to the vicinity of the T loop adjoining the stem. These nucleotides destabilize the loop structure and initiate conformational rearrangements involving local tRNA^Phe destruction and formation of the final tRNA^Phe-oligonucleotide complementary complex. The primary complex formation and the following tRNA^Phe destruction constitute the “molecular wedge” mechanism. An effective antisence oligonucleotide should consist of three segments—(1) complex initiator, (2) primary complex stabilizer, and (3) loop destructor—and be complementary to the (free end)/loop-stem-loop tRNA structural element.     

Interaction between the <Emphasis Type="Italic">UCP2</Emphasis>–866G/A,mtDNA 10398G/A and <Emphasis Type="Italic">PGC1α</Emphasis> p.Thr394Thr and p.Gly482Ser polymorphisms in type 2 diabetes susceptibility in North Indian population

In the recent past, we have observed a possible role of 10398A and 16189C mtDNA and PGC1α p.Thr394Thr (rs2970847) and p.Gly482Ser (rs8192673) variant genotypes providing susceptibility/protection against type 2 diabetes mellitus (T2DM) in two North Indian population groups. These initial observations encouraged us to look at the candidate genes in combination with –866G/A (rs659366) polymorphism in uncoupling protein 2 (UCP2) in a single study of a relatively large sample size, constituted of both the cohorts, to unravel an interesting outcome of an additive interaction in-between the studied genes. In a total of 1,686 individuals (762 cases and 924 controls) belonging to Indo-European linguistic group from North India, a comparison of risk genotype combinations of: UCP2–866GG, mtDNA 10398A and PGC1α p.Thr394Thr or p.Gly482Ser against the protective genotypes: UCP2–866XA, mtDNA 10398G and PGC1α p.Thr394Thr (nominal P value = 1.75 × 10⁻¹⁴, Odds ratio, OR = 5.29, 3.40–8.22 at 95% CI) or PGC1α p.Gly482Ser (nominal p value = 4.42 × 10⁻²⁴, OR = 8.59, 5.53–13.35 at 95% CI), showed a highly significant difference and increased ORs. In a complex disease, it is always encouraging to find an additive interaction of multiple small effects of the studied candidate gene variations. An erratum to this article is available at .