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PubMed1987

Properties of covalent benzo[a]pyrene diol epoxide-DNA adducts investigated by fluorescence techniques.

The spectroscopic absorption and fluorescence properties of adducts derived from the covalent binding of (+/-)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyre ne (BPDE) to DNA are re-examined in view of conflicting interpretations regarding the conformations of these adducts which currently exist in the literature. The fluorescence decay profiles were accurately determined utilizing synchrotron-pulsed light source excitation and the time-correlated single photon counting technique. The conformational properties of the adducts were probed by determining their accessibilities to acrylamide, a known fluorescence quencher, and by comparing the accessibilities of the BPDE-DNA adducts with those of known model systems with intercalative, partially intercalative and minor groove binding conformations. In contrast to any of these model systems, the fluorescence of the aromatic pyrenyl residues in the covalent BPDE-DNA adducts exhibit significant sensitivity to acrylamide, suggesting that these residues are located at binding sites with significant solvent exposure. A quantitative analysis of the acrylamide fluorescence quenching according to a dynamic Stern-Volmer quenching model suggests the following characteristics: the major (65%) component (1.4 ns lifetime) is characterized by significant exposure to the solvent environment; the second component (6-7 ns lifetime) can be subdivided into a solvent-accessible and a solvent-inaccessible component, the inaccessible fraction being attributed to minor adducts, possibly with a quasi-intercalative conformation. The amplitude of the third, long-lived (200-ns) component is variable; it arises from the photochemical decomposition of the adducts which gives rise to tetraols (7,8,9,10-tetrahydro-tetrahydroxybenzo[a]pyrene). The variable content of these degradation products accounts for most discrepancies in the fluorescence properties of the covalent BPDE-DNA adducts previously reported.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1994

Hydrophobic interactions of n-alkyl diamines with the N-methyl-D-aspartate receptor: voltage-dependent and -independent blocking sites.

We examined the block of N-methyl-D-aspartate (NMDA) receptors by n-alkyl (straight chain) diamines and related monoamines and triamines using whole-cell voltage clamp recording of NMDA receptor currents in cultured rat hippocampal neurons and [3H] dizocilpine binding to rat forebrain homogenates. At -60 mV, the diamines (carbon chain lengths 3-12) produced a concentration-dependent inhibition of NMDA receptor current (IC50 values, 6128-7.3 microM). For diamines of carbon chain lengths greater than 6, the inhibition was partially, but not completely, relieved by depolarization, indicating that the block occurs at distinct voltage-dependent and voltage-independent sites. The block produced by short-chain diamines (carbon chain lengths 3-6) was completely relieved by depolarization, indicating little or no interaction with the voltage-independent site. In comparison with the corresponding diamines, homologous monoamines exhibited very low potency, whereas homologous triamines were of equal or lower potency. For long-chain diamines, inhibitory potency at both the voltage-dependent and voltage-independent sites was correlated with carbon chain length (binding energy increasing 600-700 cal/mol-CH2), suggesting that binding to each of the sites is stabilized by a hydrophobic interaction. Affinities for the voltage-dependent blocking site (transformed to 0 mV) and for the voltage-independent blocking site were similar. These values were also similar to the inhibitory potencies of the diamines in the [3H]dizocilpine binding assay. Analysis of the voltage-dependence of block at the voltage-dependent site yielded z delta values for diamines of intermediate length (carbon chain lengths 7-9) that decreased with increasing length from 0.91 to 0.63 [approaching the z delta values of monovalent blockers (approximately 0.54) and one-half of the z delta values of shorter diamines (approximately 1.1)], suggesting that the intermediate length diamines block in a linear, extended chain conformation with one of the charges having incomplete access to a deep binding site. Longer chain diamines (carbon chain lengths 10 and 12) exhibited larger z delta values (0.78 and 0.98, respectively), presumably because enhanced conformational flexibility permitted a folded-over conformation. From the interchange distances of the intermediate length diamines in their lowest energy conformation, we estimated that the total voltage drop within the NMDA receptor channel occurs over a distance of approximately 20 A. The putative polyamine facilitatory site antagonist diethylenetriamine inhibited NMDA-induced currents at the voltage-dependent site (IC50, 654 microM; -60 mV).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2004

The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan-binding lectin.

Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures are present on both Igs. The relative proportion of the oligomannose glycans is consistent with the occupation of one N-linked site on each heavy chain. We evaluated the accessibility of the oligomannose glycans on serum IgD and IgE to mannan-binding lectin (MBL). MBL is a member of the collectin family of proteins, which binds to oligomannose sugars. It has already been established that MBL binds to other members of the Ig family, such as agalactosylated glycoforms of IgG and polymeric IgA. Despite the presence of potential ligands, MBL does not bind to immobilized IgD and IgE. Molecular modeling of glycosylated human IgD Fc suggests that the oligomannose glycans located at Asn(354) are inaccessible because the complex glycans at Asn(445) block access to the site. On IgE, the additional C(H)2 hinge domain blocks access to the oligomannose glycans at Asn(394) on one H chain by adopting an asymmetrically bent conformation. IgE contains 8.3% Man(5)GlcNAc(2) glycans, which are the trimmed products of the Glc(3)Man(9)GlcNAc(2) oligomannose precursor. The presence of these structures suggests that the C(H)2 domain flips between two bent quaternary conformations so that the oligomannose glycans on each chain become accessible for limited trimming to Man(5)GlcNAc(2) during glycan biosynthesis. This is the first study of the glycosylation of human serum IgD and IgE from nonmyeloma proteins.

Carbohydrate Conformation↗
PubMed1988

Physiology of plasminogen: with special reference to activation and degradation.

Plasminogen is said to be synthesized in many organs, but the major source of its production is considered to be the liver. The production of plasminogen is observed in fetal life, but its plasma level is low in new born infants, rising rapidly to attain adult levels in about 13 weeks. The plasma levels do not change much with age. Although the fibrinolytic enzyme system is influenced by many factors such as hormones, exercise, emotion, age, sex, nutritional states etc., the plasma levels of plasminogen are relatively stable. In this presentation we would like to discuss three important subjects related to the physiology of plasminogen. The first subject is the activation pathway of the native form of plasminogen (Glu-plg) by various activators in the plasma or clotted plasma. Glu-plg was not easily activated by activators such as urokinase (UK) or tissue plasminogen activator (t-PA), but activated very easily in the presence of fibrin clot. In the presence of purified clot, Glu-plg was partly activated by activators to Glu-plasmin, but also converted to Lys-plg by preformed plasmin, subsequently being activated to plasmin. Glu-plg I (containing two carbohydrate chains) changed conformation more easily upon interaction with fibrin than Glu-plg II (containing one carbohydrate chain) and Glu-plg I was also more easily activated by activators than Glu-plg II. Although Glu-plg was hardly activated by activators in the plasma, Glu-plg was activated easily in the presence of plasma clot. Results of immunoblotting experiments indicated that Glu-plg was mainly activated by activators directly to plasmin, not via Lys-plg in contrast to purified systems. The second subject is the degradation of plasminogen by elastase. Plasminogen may be degraded by proteolytic enzymes such as cathepsin or elastase. The degradation of plasminogen by elastase is shown to give rise to K1 to K3, K4, and mini-plg (containing K5). We have shown that the degradation rate to Glu-plg by elastase increased in the presence of tranexamic acid, indicating that the conformational change of Glu-plg in the presence of tranexamic acid resulted in the exposure of the hydrophobic regions connecting K3, K4 and K5, thus making them accessible to elastase. Consequently, Lys-plg or conformationally altered Glu-plg (possibly bound to fibrin or FDP) is more easily degraded in vivo. The last subject is relationship between plasma plasminogen levels and fibrinolytic activity in various ages. Although plasma plasminogen levels do not change with age, the fibrinolytic activity lowers.(ABSTRACT TRUNCATED AT 400 WORDS)

Enzyme Activation↗
PubMed1978

Influence of phosphate ligands in abolishing the conformational difference between ribonuclease A and its acid-denatured derivative.

The initial structural alteration of RNAase A due to acid denaturation (0.5 N HCl, 30 degrees C) that accompanies deamidation (without altering enzymic activity) has been dectected by spectrophotometric titration, fluorescence and ORD/CD measurements. It is shown that acid treated RNAase A has an altered conformation at neutral pH, 25 degrees C. This is characterized by the increased accessibility of buried tyrosine residue(s) towards the solvent. The most altered conformation of RNAase A is found in the 10 h acid-treated derivative. This has about 1.5 additional exposed tyrosine residues and a lesser amount of secondary structure than RNAase A. All three methods (titration, fluorescence and CD) established that the structural transition of RNAase A is biphasic. The first phase occurs within 1 h and the resulting subtle conformational change is constant up to 7 h. Following this, after the release of 0.55 mol of ammonia, the major conformational change begins. The altered conformation of the acid-denatured RNAase A could be reversed completely to the native state through a conformational change induced by substrate analogs like 2'- or 3'-CMP. Thus the monodeamidated derivative isolated from the acid-denatured RNAase A by phosphate is very similar to RNAase A in over-all conformation. The results suggest the possibility of flexibility in the RNAase A molecule that does not affect its catalytic activity, as probed through the tyrosine residues.

Amides↗
PubMed1995

The inhibition mechanism of serpins. Evidence that the mobile reactive center loop is cleaved in the native protease-inhibitor complex.

Inhibitors that belong to the serine protease inhibitor or serpin family have reactive centers that constitute a mobile loop with P1-P1' residues acting as a bait for cognate protease. Current hypotheses are conflicting as to whether the native serpin-protease complex is a tetrahedral intermediate with an intact inhibitor or an acyl-enzyme complex with a cleaved inhibitor P1-P1' peptide bond. Here we show that the P1' residue of the plasminogen activator inhibitor type 1 mutant (P1' Cys) became more accessible to radiolabeling in complex with urokinase-type plasminogen activator (uPA) compared with its complex with catalytically inactive anhydro-uPA, indicating that complex formation with cognate protease leads to a conformational change whereby the P1' residue becomes more accessible. Analysis of chemically blocked NH2 termini of serpin-protease complexes revealed that the P1-P1' peptide bonds of three different serpins are cleaved in the native complex with their cognate protease. Complex formation and reactive center cleavage were found to be rapid and coordinated events suggesting that cleavage of the reactive center loop and the subsequent loop insertion induce the conformational changes required to lock the serpin-protease complex.

Autoradiography↗
PubMed1991

Molecular recognition. Conformational analysis of limited proteolytic sites and serine proteinase protein inhibitors.

The conformations of known tryptic limited proteolytic sites have been analysed and compared to the structures of the binding regions of serine proteinase inhibitors, as they are found when complexed to a serine proteinase. Conformational parameters studied include main-chain torsion angles, root-mean-square fits, accessibility, mobility and protrusion indices. As observed before, the inhibitors share a common main-chain conformation at the binding loop from P3-P'3 (Schechter & Berger notation), which is maintained throughout all the serine proteinase inhibitor families for which X-ray data is available, despite lack of similarity in the rest of the protein. This canonical structure is not found amongst the limited proteolytic sites (or nicksites), which differ markedly from the inhibitor binding loop conformation, and also amongst themselves. The experimentally determined nicksites are in general both accessible and protruding; as are the inhibitor binding loops, as well as being typically flexible regions of structure, as denoted by elevated temperature factors from crystallographic determinations. For cleavage by serine proteinases these loops must radically alter their local conformations and a large motion of the loop relative to the structure, in some cases, would be required to orientate these sites for cleavage.

Amino Acid Sequence↗
PubMed2000

Common themes and problems of bioenergetics and voltage-gated proton channels.

The existence of a proton-selective pathway through a protein is a common feature of voltage-gated proton channels and a number of molecules that play pivotal roles in bioenergetics. Although the functions and structures of these molecules are quite diverse, the proton conducting pathways share a number of fundamental properties. Conceptual parallels include the translocation by hydrogen-bonded chain mechanisms, problems of supply and demand, equivalence of chemical and electrical proton gradients, proton wells, alternating access sites, pK(a) changes induced by protein conformational change, and heavy metal participation in proton transfer processes. An archetypal mechanism involves input and output proton pathways (hydrogen-bonded chains) joined by a regulatory site that switches the accessibility of the bound proton from one 'channel' to the other, by means of a pK(a) change, molecular movement, or both. Although little is known about the structure of voltage-gated proton channels, they appear to share many of these features. Evidently, nature has devised a limited number of mechanisms to accomplish various design strategies, and these fundamental mechanisms are repeated with minor variation in many superficially disparate molecules.

Cell Membrane↗
PubMed2002

Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics.

HIV entry inhibitors include coreceptor antagonists and the fusion inhibitor T-20. T-20 binds the first helical region (HR1) in the gp41 subunit of the viral envelope (Env) protein and prevents conformational changes required for membrane fusion. HR1 appears to become accessible to T-20 after Env binds CD4, whereas coreceptor binding is thought to induce the final conformational changes that lead to membrane fusion. Thus, T-20 binds to a structural intermediate of the fusion process. Primary viruses exhibit considerable variability in T-20 sensitivity, and determinants outside of HR1 can affect sensitivity by unknown mechanisms. We studied chimeric Env proteins containing different V3 loop sequences and found that gp120coreceptor affinity correlated with T-20 and coreceptor antagonist sensitivity, with greater affinity resulting in increased resistance to both classes of entry inhibitors. Enhanced affinity resulted in more rapid fusion kinetics, reducing the time during which Env is sensitive to T-20. Reduced coreceptor expression levels also delayed fusion kinetics and enhanced virus sensitivity to T-20, whereas increased coreceptor levels had the opposite effect. A single amino acid change (K421D) in the bridging sheet region of the primary virus strain YU2 reduced affinity for CCR5 and increased T-20 sensitivity by about 30-fold. Thus, mutations in Env that affect receptor engagement and membrane fusion rates can alter entry inhibitor sensitivity. Because coreceptor expression levels are typically limiting in vivo, individuals who express lower coreceptor levels may respond more favorably to entry inhibitors such as T-20, whose effectiveness we show depends in part on fusion kinetics.

3T3 Cells↗
PubMed2006

Probing the architecture of an L-type calcium channel with a charged phenylalkylamine: evidence for a widely open pore and drug trapping.

Voltage-gated calcium channels are in a closed conformation at rest and open temporarily when the membrane is depolarized. To gain insight into the molecular architecture of Ca(v)1.2, we probed the closed and open conformations with the charged phenylalkylamine (-)devapamil ((-)qD888). To elucidate the access pathway of (-)D888 to its binding pocket from the intracellular side, we used mutations replacing a highly conserved Ile-781 by threonine/proline in the pore-lining segment IIS6 of Ca(v)1.2 (1). The shifted channel gating of these mutants (by 30-40 mV in the hyperpolarizing direction) enabled us to evoke currents with identical kinetics at different potentials and thus investigate the effect of the membrane potentials on the drug access per se. We show here that under these conditions the development of channel block by (-)qD888 is not affected by the transmembrane voltage. Recovery from block at rest was, however, accelerated at more hyperpolarized voltages. These findings support the conclusion that Ca(v)1.2 must be opening widely to enable free access of the charged (-)D888 molecule to its binding site, whereas drug dissociation from the closed channel conformation is restricted by bulky channel gates. The functional data indicating a location of a trapped (-)D888 molecule close to the central pore region are supported by a homology model illustrating that the closed Ca(v)1.2 is able to accommodate a large cation such as (-)D888.

Binding Sites↗
PubMed1993

Structural and functional studies on the interaction of sodium dodecyl sulfate with beta-galactosidase.

The effect of sodium dodecyl sulfate (SDS) on enzyme activity, electrophoretic behavior, and conformation of Escherichia coli beta-galactosidase is presented. Fourier-transform infrared spectroscopy (FT-IR), previously used to study the structure of native beta-galactosidase has been applied to examine the detergent effects on the enzyme. At 20 degrees C, the presence of 1% SDS does not cause appreciable changes in the secondary structure, and enzyme activity is preserved; however, 10% SDS produces complete enzyme inactivation and FT-IR spectroscopy indicates a concomitant change in conformation. Thermal denaturation of beta-galactosidase starts at approximately 53 degrees C in the absence and at approximately 46 degrees C in the presence of 1% SDS, indicating tertiary structure changes; also, a good correlation between structural (FT-IR) and functional (Arrhenius plots) data is observed. The secondary structure of thermally denatured beta-galactosidase contains mainly extended structures, and intermolecular interactions produce protein aggregation. In the presence of 10% SDS, however, the hydrophobic segments of the protein are stabilized by SDS into helical structures without protein aggregation. At 30 degrees C, in the presence of 1% SDS, two protein bands are resolved by gel electrophoresis, only one of them being active. A model for SDS-galactosidase interaction is proposed, according to which, at low surfactant concentrations, SDS molecules bind the outer surface of the protein, without affecting the protein core. Higher detergent concentrations produce a larger conformational change involving enzyme inactivation and increased accessibility of the solvent to the protein core. Increasing temperature in the presence of 10% SDS leads to a facilitated access of surfactant molecules to the inner protein regions and to an increase of the beta-galactosidase alpha-helical content.

Electrophoresis, Polyacrylamide Gel↗
PubMed1999

Ligand-induced conformational changes in the crystal structures of Pneumocystis carinii dihydrofolate reductase complexes with folate and NADP+.

Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary complex and from the ternary complex with the oxidized coenzyme, NADP+, and recombinant Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to an average of 2.15 A resolution, show the first evidence of ligand-induced conformational changes in the structure of pcDHFR. These data are also compared with the crystal structure of the ternary complex of methotrexate (MTX) with NADPH and pcDHFR in the monoclinic lattice with data to 2.5 A resolution. Comparison of the data for the FA binary complex of pcDHFR with those for the ternary structures reveals significant differences, with a >7 A movement of the loop region near residue 23 that results in a new "flap-open" position for the binary complex, and a "closed" position in the ternary complexes, similar to that reported for Escherichia coli (ec) DHFR complexes. In the orthorhombic lattice for the binary FA pcDHFR complex, there is also an unwinding of a short helical region near residue 47 that places hydrophobic residues Phe-46 and Phe-49 toward the outer surface, a conformation that is stabilized by intermolecular packing contacts. The pyrophosphate moiety of NADP+ in the ternary folate pcDHFR complexes shows significant differences in conformation compared with that observed in the MTX-NADPH-pcDHFR ternary complex. Additionally, comparison of the conformations among these four pcDHFR structures reveals evidence for subdomain movement that correlates with cofactor binding states. The larger binding site access in the new "flap-open" loop 23 conformation of the binary FA complex is consistent with the rapid release of cofactor from the product complex during catalysis as well as the more rapid release of substrate product from the binary complex as a result of the weaker contacts of the closed loop 23 conformation, compared to ecDHFR.

Binding Sites↗
PubMed1999

Action of phosphatidylinositol-specific phospholipase Cgamma1 on soluble and micellar substrates. Separating effects on catalysis from modulation of the surface.

The kinetics of PI-PLCgamma1 toward a water-soluble substrate (inositol 1,2-cyclic phosphate, cIP) and phosphatidylinositol (PI) in detergent mixed micelles were monitored by 31P NMR spectroscopy. That cIP is also a substrate (Km = approximately 15 mM) implies a two-step mechanism (intramolecular phosphotransferase reaction to form cIP followed by cyclic phosphodiesterase activity to form inositol-1-phosphate (I-1-P)). PI is cleaved by PI-PLCgamma1 to form cIP and I-1-P with the enzyme specific activity and ratio of products (cIP/I-1-P) regulated by assay temperature, pH, Ca2+, and other amphiphilic additives. Cleavage of both cIP and PI by the enzyme is optimal at pH 5. The effect of Ca2+ on PI-PLCgamma1 activity is unique compared with other isozymes enzymes: Ca2+ is necessary for the activity and low Ca2+ activates the enzyme; however, high Ca2+ inhibits PI-PLCgamma1 hydrolysis of phosphoinositides (but not cIP) with the extent of inhibition dependent on pH, substrate identity (cIP or PI), substrate presentation (e.g. detergent matrix), and substrate surface concentration. This inhibition of PI-PLCgamma1 by high Ca2+ is proposed to derive from the divalent metal ion-inducing clustering of the PI and reducing its accessibility to the enzyme. Amphiphilic additives such as phosphatidic acid, fatty acid, and sodium dodecylsulfate enhance PI cleavage in micelles at pH 7.5 but not at pH 5.0; they have no effect on cIP hydrolysis at either pH value. These different kinetic patterns are used to propose a model for regulation of the enzyme. A key hypothesis is that there is a pH-dependent conformational change in the enzyme that controls accessibility of the active site to both water-soluble cIP and interfacially organized PI. The low activity enzyme at pH 7.5 can be activated by PA (or phosphorylation by tyrosine kinase). However, this activation requires lipophilic substrate (PI) present because cIP hydrolysis is not enhanced in the presence of PA.

Animals↗
PubMed1995

Susceptibility of beta-lactoglobulin and sodium caseinate to proteolysis by pepsin and trypsin.

The susceptibility of beta-LG and sodium caseinate to proteolysis by pepsin and trypsin was investigated using SDS or urea-PAGE. The effects were studied of heat, urea, and 2-mercaptoethanol on proteolysis. Native beta-LG was resistant to hydrolysis by pepsin or trypsin because of its compact globular structure. Heat treatment of beta-LG solutions at 90 to 100 degrees C for 5 or 10 min caused changes in the structure or conformation of the protein that rendered it accessible to pepsin and enhanced the extent of proteolysis by trypsin. The susceptibility of beta-LG to proteolysis by pepsin was markedly increased in the presence of urea (3 to 6 M), and the effect was reversible after removal of urea by dialysis. Proteolysis by trypsin was also increased by the presence of 2% 2-mercaptoethanol. Sodium caseinate was very accessible to pepsin without pretreatment and was extensively hydrolyzed at pH 1 to 5 in the presence of 5 M urea (which prevented the protein from precipitation in the isoelectric region); optimal pH was about 2. The activity of pepsin on sodium caseinate at pH 2 was not significantly affected by urea concentration up to about 8 M. The results indicated that the changes in conformation and structure of beta-LG that were induced by heating, reduction, or urea rendered the protein susceptible to peptic hydrolysis.

Animals↗
PubMed1986

ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins.

The MgATP binding site of adenylate kinase, located by a combination of NMR and x-ray diffraction, is near three protein segments, five to seven amino acids in length, that are homologous in sequence to segments found in other nucleotide-binding phosphotransferases, such as myosin and F1-ATPase, ras p21 and transducin GTPases, and cAMP-dependent and src protein kinases, suggesting equivalent mechanistic roles of these segments in all of these proteins. Segment 1 is a glycine-rich flexible loop that, on adenylate kinase, may control access to the ATP-binding site by changing its conformation. Segment 2 is an alpha-helix containing two hydrophobic residues that interact with the adenine-ribose moiety of ATP, and a lysine that may bind to the beta- and gamma-phosphates of ATP. Segment 3 is a hydrophobic strand of parallel beta-pleated sheet, terminated by a carboxylate, that flanks the triphosphate binding site. The various reported mutations of ras p21 that convert it to a transforming agent all appear to involve segment 1, and such substitutions may alter the properties of p21 by hindering a conformational change at this segment. In F1-ATPase, the flexible loop may, by its position, control both the accessibility and the ATP/ADP equilibrium constant on the enzyme.

Adenosine Triphosphate↗
PubMed2002

The somatostatin-28(1-12)-NPAMAP sequence: an essential helical-promoting motif governing prosomatostatin processing at mono- and dibasic sites.

Proline residues, known to have special structural properties, induce particular conformations which participate in some biological functions. Two prolines (Pro(-9), Pro(-5)) located near the processing sites (Arg(-15) and Arg(-2)Lys(-)(1)) of human prosomatostatin were previously shown to be important for cleavage of the precursor into somatostatin-28 (S-28) and somatostatin-14 (S-14) [Gomez et al. (1989) EMBO J. 8, 2911-2916]. In this study, the importance of the pentapeptide P-A-M-A-P sequence (P-(X)(3)-P pattern), located in the S-28(1-12) segment connecting the mono- and dibasic cleavage sites, was investigated by using site-directed mutagenesis. Analysis of prosomatostatin-derived peptides produced by expression of mutated cDNA species in Neuro2A cells indicated that (i) deletion of PAMAP decreased S-14 production, (ii) deletion of the two Pro residues almost abolished the cleavage at the dibasic site, and (iii) Pro displacement generating the AMAPP motif resulted in a decrease of S-28 production. Moreover, both theoretical and spectroscopic studies of synthetic peptides reproducing the S-28(1-12) sequence bearing critical mutations showed that this sequence can organize as an alpha helical structure. These observations demonstrate that NPAMAP constitutes an accurate alpha-helix nucleation motif allowing for the generation of equal amounts of S-28 and S-14 from their common precursor in Neuro2A cells. Moreover, they emphasize the importance of the S-28(1-12) segment joining Arg(-15) and Arg(-2)Lys(-1) cleavage sites whose conformational organization is essential for controlling their accessibility to the appropriate processing proteases.

Amino Acid Motifs↗
PubMed1984

Conformational changes in bacterial polysomes induced by amino acid starvation.

The effects of amino acid starvation on polysome conformation were analyzed comparatively in stringent (relA+) and relaxed (relA) bacteria by measuring the accessibility in vitro of ribosomal proteins to reductive methylation. In polysomes of stringent cells, the conformational state of two proteins (L13 and L29) appeared significantly changed by starvation. In polysomes isolated from relaxed mutants, the accessibility of five proteins (L5, L13, L29, L31 and L32) was found modified.

Amino Acids↗