The interactions of horse heart apocytochromec with phospholipid vesicles and surfactant micelles: time-resolved fluorescence study of the single tryptophan residue (Trp-59)

The interactions of horse heart apocytochromec with membrane interfaces were studied on membrane models including micelles of the anionic surfactant sodium dodecyl sulfate (SDS), the micelle forming lipid analogs dodecylphosphoglycol (Cl2PG), tetradecylphosphoglycol (Cl4PG), and dodecylphosphocholine (Cl2PN), and the negatively charged phospholipid 1-palmitoyl-2-oleoylsn-glycero phosphocholine (POPS) forming small unilamellar vesicles (SUV). The time-resolved fluorescence of the single tryptophan residue (Trp-59) emission was monitored to characterize the modifications of the conformational equilibrium and of the internal dynamics of the protein, which can be brought about by its binding to these model membranes. In most of the cases, as for the protein in solution, the excited state lifetime distribution of the Trp emission was described by four discrete classes, whose relative proportions and barycenters vary significantly in the different complexes formed. In the complex with POPS, however, the decay analysis showed only 3 lifetime classes: the long lifetime class displayed a bary-center value smaller than that observed for the protein in aqueous solution but with a much higher proportion, indicating a stabilization of this conformer in the membrane-bound form of the protein. A similar sensitivity of theTrp-59 excited state to deactivation by thermal collisions in water and in the protein/POPS complex was observed, indicating a probable location ofTrp-59 at the membrane/water interface. The effects of protein binding to C12PN, C12PG and C14PG micelles on the long life-time class proportion were similar to that of POPS but, in addition, there was a large contribution of a short lifetime component which was absent in POPS vesicles. The bary-center values of the excited state lifetime classes were comparable in these membrane systems, suggesting thatTrp-59 is not transferred to a non-polar environment. Binding of apocytochrome c to SDS micelles strongly reduced the lifetime class barycenters and, in contrast to the other membrane systems, strongly favored the contribution of the shortest lifetime class at the expense of the c3 class. This suggests an interaction of theTrp-59 with the sulfur containing head-group of this surfactant. The indole ring mobility is reduced at the interface contacts. A fastTrp-59 mobility with a large amplitude is suggested in the complex with POPS by an initial anisotropy value lower than the expected one of 0.295 measured in vitrified medium. These observations can be correlated with the induction of -helical structure after interactions of apocytochromec with membrane model systems (de Jongh and de Kruijff 1990).Abbreviations AOT sodium bis-(2-ethylhexyl)sulfosuccinate - C12PG dodecylphosphoglycol - C14PG tetradecylphosphoglycol - C12PN dodecylphosphocholine - MEM Maximum Entropy Method - NMR nuclear magnetic resonance - POPS 1-palmitoyl, 2-oleoyl-sn-glycerophosphoserine - SUV small unilamellar vesicles - Trp tryptophan     

Influence of size and polarity of residue 31 in porcine pancreatic phospholipase A2 on catalytic properties

Residue 31 of porcine pancreatic phospholipase A2 (PLA2) is located at the entrance to the active site. To study the role of residue 31 in PLA2, six mutant enzymes were produced by site-directed mutagenesis, replacing Leu by either Trp, Arg, Ala, Thr, Ser or Gly. Direct binding studies indicated a three to six times greater affinity of the Trp31 PLA2 for both monomeric and micellar substrate analogs, relative to the wild-type enzyme. The other five mutants possess an unchanged affinity for monomers of the product analog n-decylphosphocholine and for micelles of the diacyl substrate analog rac-1,2-dioctanoylamino-dideoxy-glycero-3-phosphocholine. The affinities for micelles of the monoacyl product analog n-hexadecylphosphocholine were decreased 9-20 times for these five mutants. Kinetic studies with monomeric substrates showed that the mutants have Vmax values which range between 15 and 70% relative to the wild-type enzyme. The Vmax values for micelles of the zwitterionic substrate 1,2-dioctanoyl-sn-glycero-3-phosphocholine were lowered 3-50 times. The Km values for the monomeric substrate and the Km values for the micellar substrate were hardly affected in the case of five of the six mutants, but were considerably decreased when Trp was present at position 31. The results of these investigations point to a versatile role for the residue at position 31: involvement in the binding and orientating of monomeric substrate (analogs), involvement in the binding of the enzyme to micellar substrate analogs and possibly involvement in shielding the active site from excess water.     

Plasma membrane calcium pump (PMCA) differential exposure of hydrophobic domains after calmodulin and phosphatidic acid activation

The exposure of the plasma membrane calcium pump (PMCA) to the surrounding phospholipids was assessed by measuring the incorporation of the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16 to the protein. In the presence of Ca(2+) both calmodulin (CaM) and phosphatidic acid (PA) greatly decreased the incorporation of [(125)I]TID-PC/16 to PMCA. Proteolysis of PMCA with V8 protease results in three main fragments: N, which includes transmembrane segments M1 and M2; M, which includes M3 and M4; and C, which includes M5 to M10. CaM decreased the level of incorporation of [(125)I]TID-PC/16 to fragments M and C, whereas phosphatidic acid decreased the incorporation of [(125)I]TID-PC/16 to fragments N and M. This suggests that the conformational changes induced by binding of CaM or PA extend to the adjacent transmembrane domains. Interestingly, this result also denotes differences between the active conformations produced by CaM and PA. To verify this point, we measured resonance energy transfer between PMCA labeled with eosin isothiocyanate at the ATP-binding site and the phospholipid RhoPE included in PMCA micelles. CaM decreased the efficiency of the energy transfer between these two probes, whereas PA did not. This result indicates that activation by CaM increases the distance between the ATP-binding site and the membrane, but PA does not affect this distance. Our results disclose main differences between PMCA conformations induced by CaM or PA and show that those differences involve transmembrane regions.     

Interactions between mastoparan B and the membrane studied by 1H NMR spectroscopy

Mastoparan B (MP-B) is an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis. NMR spectroscopy was used to study the membrane associated structures of MP-B in various model membrane systems such as 120 mM DPC micelles, 200 mM SDS micelles, and 3%(w/v) DMPC/DHPC (1:2) bicelles. In all systems, MP-B has an amphiphilic alpha-helical structure from Lys2 to Leu14. NOESY experiments performed on MP-B in nondeuterated SDS micelles show that protons in the indole ring of Trp9 are in close contact with methylene protons of SDS micelles. T1 relaxation data and NOE data revealed that the bound form of MP-B may be dominant in SDS micelles. The interactions between MP-B and zwitterionic DPC micelles were much weaker than those between MP-B and anionic SDS micelles. By substitution of Trp9 with Ala9, the pore-forming activity of MP-B was decreased dramatically. All of these results imply that strong electrostatic interactions between the positively charged Lys residues in MP-B and the anionic phospholipid head groups must be the primary factor for MP-B binding to the cell membrane. Then, insertion of the indole ring of Trp9 into the membrane, as well as the amphiphilic alpha-helical structures of MP-B may allow MP-B to span the lipid bilayer through the C-terminal portion. These structural features are crucial for the potent antibiotic activities of MP-B.     

Heterogeneity in the binding of lipid molecules to the surface of a membrane protein: hot spots for anionic lipids on the mechanosensitive channel of large conductance MscL and effects on conformation

We have introduced single Trp residues into the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and used fluorescence quenching by brominated phospholipids to detect the presence of a binding site of high affinity for anionic phospholipids. A cluster of three positively charged residues, Arg-98, Lys-99, and Lys-100, is located on the cytoplasmic side of MscL, in a position where they could interact with the headgroup of an anionic phospholipid. Single mutations of these charged residues in the Trp-containing mutant F80W results in a decreased affinity for phosphatidic acid. Single mutations of the charged residues also result in a significant shift in the fluorescence emission spectrum in dioleoylphosphatidylcholine [di(C18:1)PC] but smaller shifts in dioleoylphosphatidic acid [di(C18:1)PA], suggesting that single mutations result in a conformational change for the protein that is reversed by interaction with anionic phospholipids. This is consistent with the observation that single mutations of the charged residues do not result in a gain of function phenotype. In contrast, simultaneous mutation of all three charged residues results in a gain of function phenotype, and a shift in fluorescence emission spectrum in di(C18:1)PC not reversed in di(C18:1)PA. The gain of function mutant F80W:V21K also shows a shifted fluorescence emission spectrum in both di(C18:1)PC and di(C18:1)PA and binds di(C18:1)PC and di(C18:1)PA with equal affinity, suggesting that the conformational change caused by the V21K mutation results in a breakup of the cluster of three positive charges. Experiments with the Trp mutants L69W and Y87W allow us to measure lipid binding constants on the periplasmic and cytoplasmic sides of the membrane, respectively. On both sides of the membrane the affinity for di(C18:1)PC is equal to that for dioleoylphosphatidylethanolamine. On the periplasmic side of the membrane, there is no selectivity for anionic phospholipids. In contrast, quenching data for Y87W provides evidence for the existence of two lipid binding sites on the cytoplasmic side of the membrane close to the Trp residue at position 87, with binding to one of these sites showing a marked preference for anionic lipid over zwitterionic lipid, presumably involving the charged cluster Arg-98, Lys-99, and Lys-100.     

Insight into the conformational dynamics of specific regions of porcine pancreatic phospholipase A2 from a time-resolved fluorescence study of a genetically inserted single tryptophan residue

The effects of Ca2+ and substrate analogue binding on the conformational dynamics of porcine pancreas phospholipase A2 (PLA2) in different regions was explored by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) of the wild-type protein (W3), in the alpha-helix A, was replaced by a phenylalanine residue (W3F), whereafter Trp was substituted either for leucine-31 (W31), located in the calcium binding loop, or for phenylalanine-94 (W94), located at the "back side" of the enzyme. Furthermore, mutants lacking the 62-66 sequence were constructed with the Trp at position 3 (delta W3) or 31 (delta W31). The total fluorescence intensity decays of Trp in each protein, in the protein-calcium and the protein-calcium-substrate analogue complexes, analyzed by the maximum entropy method (MEM) can be interpreted as distributions of separated lifetime classes. In the case of the W94 mutant, a major short-lived excited-state population (tau approximately 50 ps) is observed, probably deactivated by the interaction with two proximate disulfide bridges via a radiationless process. For the four other mutants, the respective barycenters of the four lifetime classes display comparable values, but the amplitude distributions are different for Trp-3 and Trp-31. The rotational mobility of the Trp residue varies along the peptide chain. Trp-3 experiences only a fast hindered motion. Trp-31 is sensitive to an additional local flexibility that is absent in the N-terminal part of the protein. The largest wobbling angle is observed at position 94. No effect of calcium binding occurs on the lifetime distribution of the Trp-3 and Trp-94 residues. Their mobilities are not affected. In contrast, calcium binding displays a strong influence on the excited-state population distribution of Trp-31. A major population decaying with the longest lifetime is selected in the W31 protein and contributes to approximately 50% of the decay. The local flexibility and the amplitude of motion of Trp-31 is wider in the protein-calcium complex than in the unliganded protein. Binding of the monomeric substrate analogue n-dodecylphosphocholine (C12PN) in the presence of calcium slightly affects the Trp-3 excited-state population distribution and its mobility. Trp-31 is more sensitive to this binding. In particular, a more restricted rotation of the Trp-31 residue and a decrease of the peptide local flexibility as protein-calcium complexes are observed in both the W31 and delta W31 mutants.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sensitivity of tryptophan and related compounds to oxidation induced by lipid autoperoxidation. Application to human serum low- and high-density lipoproteins

Tryptamine, serotonin and tryptophan are readily oxidized during the Cu2+-catalyzed peroxidation of arachidonic acid (AA) at neutral pH and under certain experimental conditions which determine their relative susceptibility to oxidation. Thus, in AA micelles, fluorescence spectroscopy demonstrates that positively-charged indoles interact with negatively-charged micelles while Trp remains in the aqueous phase. As a result, serotonin and tryptamine are preferentially oxidized. In egg phosphatidylcholine liposomes loaded with AA, the three substrates interact with vesicles and undergo lipid-induced oxidation. EDTA inhibits the formation of thiobarbituric-reactive substances (TBARS) and prevents the indoles from oxidation. Owing to the intricate contact between the lipidic core and the apolipoproteins, the Trp residues of human serum LDL and HDL3 are very rapidly oxidized, i.e., at least one order of magnitude faster than Tyr HDL and Lys LDL, which are believed to be involved in the binding of these lipoproteins to their cell receptors. Cupric ions are rather specific for the lipid-induced autoxidation of Trp residues of lipoproteins whereas in micelles and liposomes, Mn2+ and Fe2+ can lead to TBARS production and to oxidation of indoles. This specificity is surprising considering the known ability of Fe2+ to catalyze LDL modification (measured by TBARS production) during their incubation with various cells. Biological consequences of the easy lipid-induced oxidation of biologically important indoles are discussed.     

A single H:CDR3 residue in the anti-digoxin antibody 26-10 modulates specificity for C16-substituted digoxin analogs

We constructed Fab libraries of bacteriophage-displayed H:CDR3 mutants in the high-affinity anti-digoxin antibody 26-10 to determine structural constraints on affinity and specificity for digoxin. Libraries of mutant Fabs randomized at five or 10 contiguous positions were panned against digoxin and three C16-substituted analogs, gitoxin (16-OH), 16-formylgitoxin and 16-acetylgitoxin. The sequence data from 83 different mutant Fabs showed highly restricted consensus patterns at positions H:100, 100a and 100b for binding to digoxin; these residues contact digoxin in the 26-10:digoxin co-crystal structure. Several mutant Fabs obtained following panning on digoxin-BSA showed increased affinity for digoxin compared with 26-10 and retained the wild-type (wt) Trp at position 100. Those Fabs selected following panning on C16-substituted analogs showed enhanced binding to the analogs. Replacement of H:Trp100 by Arg resulted in mutants that bound better to the analogs than to digoxin. This specificity change was unexpected, as C16 lies on the opposite side of digoxin from H:CDR3. Substitution of wt Trp by Arg appears to alter specificity by allowing the hapten to shift toward H:CDR3, thereby providing room for C16 substituents in the region of H:CDR1.     

Lipid-protein interactions studied by introduction of a tryptophan residue: the mechanosensitive channel MscL

Trp fluorescence spectroscopy is a powerful tool to study the structures of membrane proteins and their interactions with the surrounding lipid bilayer. Many membrane proteins contain more than one Trp residue, making analysis of the fluorescence data more complex. The mechanosensitive channels MscL's of Mycobacterium tuberculosis (TbMscL) and Escherichia coli (EcMscL) contain no Trp residues. We have therefore introduced single Trp residues into the transmembrane regions of TbMscL and EcMscL to give the Trp-containing mutants F80W-TbMscL and F93W-EcMscL, respectively, which we show are highly suitable for measurements of lipid binding constants. In vivo cell viability assays in E. coli show that introduction of the Trp residues does not block function of the channels. The Trp-containing mutants have been reconstituted into lipid bilayers by mixing in cholate followed by dilution to re-form membranes. Cross-linking experiments suggest that the proteins retain their pentameric structures in phosphatidylcholines with chain lengths between C14 and C24, phosphatidylserines, and phosphatidic acid. Quenching of Trp fluorescence by brominated phospholipids suggests that the Trp residue in F80W-TbMscL is more exposed to the lipid bilayer than the Trp residue in F93W-EcMscL. Binding constants for phosphatidylcholines change with changing fatty acyl chain length, the strongest interaction for both TbMscL and EcMscL being observed with a chain of length C16, corresponding to a bilayer of hydrophobic thickness ca. 24 A, compared to a hydrophobic thickness for TbMscL of about 26 A estimated from the crystal structure. Lipid binding constants change by only a factor of 1.5 in the chain length range from C12 to C24, much less than expected from theories of hydrophobic mismatch in which the protein is treated as a rigid body. It is concluded that MscL distorts to match changes in bilayer thickness. The binding constants for dioleoylphosphatidylethanolamine for both TbMscL and EcMscL relative to those for dioleoylphosphatidylcholine are close to 1. Quenching experiments suggest a single class of binding sites for phosphatidylserine, phosphatidylglycerol, and cardiolipin on TbMscL; binding constants are greater than those for phosphatidylcholine and decrease with increasing ionic strength, suggesting that charge interactions are important in binding these anionic phospholipids. Quenching experiments suggest two classes of lipid binding sites on TbMscL for phosphatidic acid, binding of phosphatidic acid being much less dependent on ionic strength than binding of phosphatidylserine.     

Identification of common binding sites for calmodulin and inositol 1,4,5-trisphosphate receptors on the carboxyl termini of trp channels

Homologues of Drosophila Trp (transient receptor potential) form plasma membrane channels that mediate Ca(2+) entry following the activation of phospholipase C by cell surface receptors. Among the seven Trp homologous found in mammals, Trp3 has been shown to interact with and respond to IP(3) receptors (IP(3)Rs) for activation. Here we show that Trp4 and other Trp proteins also interact with IP(3)Rs. The IP(3)R-binding domain also interacts with calmodulin (CaM) in a Ca(2+)-dependent manner with affinities ranging from 10 nm for Trp2 to 290 nm for Trp6. In addition, other binding sites for CaM and IP(3)Rs are present in the alpha but not the beta isoform of Trp4. In the presence of Ca(2+), the Trp-IP(3)R interaction is inhibited by CaM. However, a synthetic peptide representing a Trp-binding domain of IP(3)Rs inhibited the binding of CaM to Trp3, -6, and -7 more effectively than that to Trp1, -2, -4, and -5. In inside-out membrane patches, Trp4 is activated strongly by calmidazolium, an antagonist of CaM, and a high (50 microm) but not a low (5 microm) concentration of the Trp-binding peptide of the IP(3)R. Our data support the view that both CaM and IP(3)Rs play important roles in controlling the gating of Trp-based channels. However, the sensitivity and responses to CaM and IP(3)Rs differ for each Trp.     

pH effects on binding between the anthrax protective antigen and the host cellular receptor CMG2

The anthrax protective antigen (PA) binds to the host cellular receptor capillary morphogenesis protein 2 (CMG2) with high affinity. To gain a better understanding of how pH may affect binding to the receptor, we have investigated the kinetics of binding as a function of pH to the full-length monomeric PA and to two variants: a 2-fluorohistidine-labeled PA (2-FHisPA), which is ～1 pH unit more stable to variations in pH than WT, and an ～1 pH unit less stable variant in which Trp346 in the domain 2β(3) -2β(4) loop is substituted with a Phe (W346F). We show using stopped-flow fluorescence that the binding rate increases as the pH is lowered for all proteins, with little influence on the rate of dissociation. In addition, we have crystallized PA and the two variants and examine the influence of pH on structure. In contrast to previous X-ray studies, the domain 2β(3) -2β(4) loop undergoes little change in structure from pH ～8 to 5.5 for the WT protein, but for the 2-FHis labeled and W346F mutant there are changes in structure consistent with previous X-ray studies. In accord with pH stability studies, we find that the average B-factor values increase by ～20-30% for all three proteins at low pH. Our results suggest that for the full-length PA, low pH increases the binding affinity, likely through a change in structure that favors a more "bound-like" conformation.     

Estimation of helix-helix association free energy from partial unfolding of bacterioopsin

To obtain thermodynamic information about interactions between transmembrane helices in integral membrane proteins, partial unfolding of bacterioopsin in ethanol/water mixtures was studied by F?rster-type resonance energy transfer (FRET) from tryptophan to a dansyl group on Lys 41. Tryptophan to dansyl FRET was detected by measuring sensitized emission at 490-500 nm from 285 nm excitation. FRET was observed in dansylbacterioopsin in apomembranes and in detergent micelles but not in 90% ethanol/water or in the chymotrypsin fragment C2 (residues 1-71). The main fluorescence donors are Trp 86 and Trp 182. Increase of FRET from C2 with added chymotrypsin fragment C1 (residues 72-248) provides an estimate of the C1-C2 association constant as 7.7 x 10(6) M(-1). With increasing ethanol concentration, the FRET signal from dansylbacterioopsin in detergent micelles disappeared with a sharp transition above 60% ethanol. No transition occurred in Trp fluorescence from bacterioopsin lacking the dansyl acceptor, nor did dansyl model compounds undergo a similar transition. Light scattering measurements show that the detergent micelles dissipate below 50% ethanol. Thus the observed transition is likely to be a partial unfolding of bacterioopsin. Assuming a two-state unfolding model, the free energy of unfolding was obtained by extrapolation as 9.0 kcal/mol. The slope of the transition (m-value) was -0.8 kcal mol(-1) M(-1). The unfolding process probably involves dissociation of several helices. The rate of association was measured by stopped-flow fluorometry. Two first-order kinetic processes were observed, having approximately equal weights, with rate constants of 2.32 s (-1) and 0.185 s(-1).     

Highly efficient technetium-99m labeling procedure based on the conjugation of N-[N-(3-diphenylphosphinopropionyl)glycyl]cysteine ligand with poly(ethylene glycol)

The PN(2)S N-(N-(3-diphenylphosphinopropionyl)glycyl)cysteine ligand was conjugated to methoxy-poly(ethylene glycol)-amino (mPEG-NH(2)) 5 and 20 kDa to yield PN(2)S(Trt)-PEG(5000) 1 and PN(2)S(Trt)-PEG(20000) 2, and then detritylated to PN(2)S-PEG(5000) 4 and PN(2)S-PEG(20000) 5. When an acidic solution of (99m)TcO(4)(-) is added to 4 or 5 in solid form, a quantitative yield in a single labeled species, (99m)Tc-labeled PN(2)S-PEG(5000) 9 and (99m)Tc-labeled PN(2)S-PEG(20000) 10, respectively, is obtained. The reaction occurs in less than 15 min at room temperature for 4 and 35 degrees C for 5. This labeling procedure avoids the use of an external reducing agent, and it is based on the amphiphilic properties of PN(2)S-PEGs. Once in water, 4 and 5 self-assemble in micelles, which catalyze the metal reduction by means of an electron pair transfer from the phosphorus to technetium. The [(99m)TcO](3+) species is then coordinated, and at micelle level, both the (P)ON(2)S and the PN(2)S coordinations are possible, as demonstrated by reacting (99m)Tc-gluconate and ReOCl(3)(PPh(3))(2) with 4 and 5 and with the oxidized analogous (P)ON(2)S-PEG(5000) 6. Compounds 9 and 10 exhibited a high stability both in vitro and in vivo. Biodistribution studies in mice also indicated that PN(2)S linking and (99m)Tc labeling do not modify PEG behavior in water and in vivo since the polymer dictates the fate of the conjugate.     

NMR analysis of bovine tRNATrp: conformation dependence of Mg2+ binding

NMR was used to study the solution structure of bovine tRNA(Trp) hyperexpressed in Escherichia coli. With the use of (15)N labeling and site-directed mutagenesis to assign overlapping resonances through the base pair replacement of U(71)A(2) by G(2)C(71), U(27)A(43) by G(27)C(43), and G(12)C(23) by U(12)A(23), the resonances of all 26 observable imino protons in the helical regions and in the tertiary interactions were assigned unambiguously by means of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear single quantum coherence methods. When the discriminator base A(73) and the G(12)C(23) base pair on the D stem, two identity elements on bovine tRNA(Trp) that are important for effective recognition by tryptophanyl-tRNA synthetase, were mutated to the ineffective forms of G(73) and U(12)A(23), respectively, NMR analysis revealed an important conformational change in the U(12)A(23) mutant but not in the G(73) mutant molecule. Thus A(73) appears to be directly recognized by tryptophanyl-tRNA synthetase, and G(12)C(23) represents an important structural determinant. Mg(2+) effects on the assigned resonances of imino protons allowed the identification of strong, medium, and weak Mg(2+) binding sites in tRNA(Trp). Strong Mg(2+) binding modes were associated with the residues G(7), s(4)U(8) (where s(4)U is 4-thiouridine), G(12), and U(52). The observations that G(42) was associated with strong Mg(2+) binding in only the U(12)A(23) mutant tRNA(Trp) but not the wild type or G(73) mutant tRNA(Trp) and that the G(7), s(4)U(8), G(24), and G(22) imino protons are associated with a two-site Mg(2+) binding mode in wild type and G(73) mutant but only a one-site mode in the U(12)A(23) mutant established the occurrence of conformational change in the U(12)A(23) mutant tRNA(Trp). These observations also established the dependence of Mg(2+) binding on tRNA conformation and the usefulness of Mg(2+) binding sites as conformational probes. The thermal titration of tRNA(Trp) in the presence and absence of 10 mm Mg(2+) indicated that overall tRNA(Trp) structure stability was increased by more than 15 degrees C by the presence of Mg(2+).     

Intra and intermolecular charge effects on the reaction of the superoxide radical anion with semi-oxidized tryptophan in peptides and N-acetyl tryptophan

The reaction of the superoxide radical anion (O^-·₂), with the semi-oxidized tryptophan neutral radical (Trp^·) generated from tryptophan (Trp) by pulse radiolysis has been observed in a variety of functionalized Trp derivatives including peptides. It is found that the reaction proceeds 4-5 times faster in positively charged peptides, such as Lys-Trp-Lys, Lys-Gly-Trp-Lys and Lys-Gly-Trp-Lys-O-tert-butyl, than in solutions of the negatively charged N-acetyl tryptophan (NAT). However, the reactivity of O^-·₂ with the Trp^· radical is totally inhibited upon binding of these peptides to micelles of negatively charged SDS and is reduced upon binding to native DNA. By contrast, no change in reactivity is observed in a medium containing CTAB, where the peptides cannot bind to the positively charged micelles. On the other hand, the reactivity of the Trp^· radical formed from NAT with O^-·₂ is reduced to half that of the free Trp^· in buffer but is markedly increased in CTAB micelles. The models studied here incorporate elements of the complex environment in which Trp^· and O^-·₂ may be concomitantly formed in biological system and demonstrate the magnitude of the influence such elements may have on the kinetics of reactions involving these two species.     

Identification of the binding sites and selectivity of sarpogrelate,a novel 5-HT2 antagonist,to human 5-HT2A, 5-HT2B and 5-HT2C receptor subtypes by molecular modeling

The aim of the present study was to investigate the binding sites interactions and the selectivity of sarpogrelate to human 5-HT(2) receptor family (5-HT(2A), 5-HT(2B) and 5-HT(2C) receptor subtypes) using molecular modeling. Rhodopsin (RH) crystal structures were used as template to build structural models of the human serotonin-2A and -2C receptors (5-HT(2A)R, 5-HT(2C)R), whereas for 5-HT(2B)R, we used our previously published three-dimensional (3D) models based on bacteriorhodopsin (BR). Sarpogrelate, a novel 5-HT(2)R antagonist, was docked to the receptors. Molecular dynamics (MD) simulations produced the strongest interaction for 5-HT(2A)R/sarpogrelate complex. Upon binding, sarpogrelate constraints aromatic residues network (Trp(3.28), Phe(5.47), Trp(6.48), Phe(6.51), Phe(6.52) in 5-HT(2A)R; Phe(3.35), Phe(6.51), Trp(7.40) in 5-HT(2B)R; Trp(3.28), Phe(3.35), Phe(5.47), Trp(6.48), Phe(6.51), Phe(6.52) in 5-HT(2C)R) in a stacked configuration, preventing activation of the receptor. The models suggest that the structural origin of the selectivity of sarpogrelate to 5-HT(2A)R vs both 5-HT(2B)R and 5-HT(2C)R comes from the following results: (1) The tight interaction between the antagonist and the transmembrane domain (TMD) 3. Asp(3.32) neutralizes the cationic head and interacts simultaneously with carboxylic group hydrogen of the antagonist molecule. (2) Due to steric hindrance, Ser(5.46) (vs Ala(5.46) in 5HT(2B) and 5HT(2C)) prevents sarpogrelate to enter deeply inside the hydrophobic core of the helix bundle and to interact with Pro(5.50). (3) The side chain of Ile(4.56) (vs Ile(4.56) in 5HT(2B)R and Val(4.56) in 5HT(2C)R) constraints sarpogrelate to adjust its position by translating toward the strongly attractive Asp(3.32). These results are in good agreement with binding affinities (pKi) of sarpogrelate for 5-HT(2) receptor family expressed in transfected cell.     

Orientations of the tryptophan 9 and 11 side chains of the gramicidin channel based on deuterium nuclear magnetic resonance spectroscopy.

Deuterium nuclear magnetic resonance spectroscopy was used to investigate the orientations of the indole rings of Trp9 and Trp11 in specific indole-d5-labeled samples of gramicidin A incorporated into dimyristoyl phosphatidylcholine bilayers in the beta 6.3 channel conformation. The magnitudes and signs of the deuterium quadrupolar splittings were fit to the rings and assigned to specific ring bonds, using a full rotation search of the chi 1 and chi 2 angles of each Trp and a least-squares method. Unique assignments were obtained. The data and assignments are in close agreement with four sets of (chi 1, chi 2) angles for each Trp in which the indole N-H is oriented toward the membrane's exterior surface. (Four additional sets of (chi 1, chi 2) angles with the N-H's pointing toward the membrane interior are inconsistent with previous observations.) One of the sets of (chi 1, chi 2) angles for each Trp is consistent with the corresponding Trp orientation found by Arsen'ev et al. (1986. Biol. Membr. 3:1077-1104) for gramicidin in sodium dodecyl sulfate micelles. Together, the 1H and 2H nuclear magnetic resonance methods suggest that the Trp9 and Trp11 side chain orientations could be very similar in dimyristoyl phosphatidylcholine membranes and in sodium dodecyl sulfate micelles. The data for Trp11 could be fit using a static quadrupolar coupling constant of 180 kHz under the assumption that the ring is essentially immobile. By contrast, Trp9 could be fit only under the assumption that the quadrupolar splittings for ring 9 are reduced by approximately 14% due to motional averaging. Such a difference in motional averaging between rings 11 and 9 is also consistent with the 15N data of Hu et al. (1993. Biochemistry. 32:7035-7047).     

Binding of phosphatidic acid to the protein-tyrosine phosphatase SHP-1 as a basis for activity modulation.

Activation of the SH2 domain-possessing protein-tyrosine phosphatase SHP-1 by acidic phospholipids as phosphatidic acid (PA) has been described earlier and suggested to participate in regulation of SHP-1 activity toward cellular substrates. The mechanism of this activation is poorly understood. Direct binding of phosphatidic acid to recombinant SHP-1 could be demonstrated by measuring the extent of [(14)C]PA binding in a chromatographic assay, by measuring the extent of binding of SHP-1 to PA-coated ELISA plates or silica beads (TRANSIL), and by spectroscopic assays employing fluorescently labeled PA liposomes. In addition to PA, phosphatidylinositol 3,4, 5-trisphosphate (PIP3), dipalmitoylphosphatidylglycerol, phosphatidylinositol 4,5-bisphosphate, and phosphatidylserine (PS) were found to bind to SHP-1, albeit to a lesser extent. A high-affinity binding site for PA and PIP3 was mapped to the 41 C-terminal amino acids of SHP-1. This site was absent from the related protein-tyrosine phosphatase SHP-2 and conferred activation of SHP-1 by PA toward two different substrates at low lipid concentrations. A SHP-1 mutant missing this binding site could, however, still be activated toward phosphorylated myelin basic protein as a substrate at high PA concentrations. This activation is likely to be mediated by a second, low-affinity binding site for PA in the N-terminal part of SHP-1 within the SH2 domains. High-affinity phospholipid binding to the C-terminus of SHP-1 may present a specific mechanism of regulating activity and/or cellular localization.     

TAPAS-1, a novel microdomain within the unique N-terminal region of the PDE4A1 cAMP-specific phosphodiesterase that allows rapid, Ca2+-triggered membrane association with selectivity for interaction with phosphatidic acid

Here we identify an 11-residue helical module in the unique N-terminal region of the cyclic AMP-specific phosphodiesterase PDE4A1 that determines association with phospholipid bilayers and shows a profound selectivity for interaction with phosphatidic acid (PA). This module contains a core bilayer insertion unit that is formed by two tryptophan residues, Trp(19) and Trp(20), whose orientation is optimized for bilayer insertion by the Leu(16):Val(17) pairing. Ca(2+), at submicromolar levels, interacts with Asp(21) in this module and serves to gate bilayer insertion, which is completed within 10 ms. Selectivity for interaction with PA is suggested to be achieved primarily through the formation of a charge network of the form (Asp(21-):Ca(2+):PA(2-):Lys(24+)) with overall neutrality at the bilayer surface. This novel phospholipid-binding domain, which we call TAPAS-1 (tryptophan anchoring phosphatidic acid selective-binding domain 1), is here identified as being responsible for membrane association of the PDE4A1 cAMP-specific phosphodiesterase. TAPAS-1 may not only serve as a paradigm for other PA-binding domains but also aid in detecting related phospholipid-binding domains and in generating simple chimeras for conferring membrane association and intracellular targeting on defined proteins.