PubMed Health⌕ Search

Biomedical subjects

C E Dempsey

Publications and source records attributed to C E Dempsey.

At least 19 recordsLinked to original sources
PubMed2001

Structure, location, and lipid perturbations of melittin at the membrane interface.

Melittin is arguably the most widely studied amphipathic, membrane-lytic alpha-helical peptide. Although several lines of evidence suggest an interfacial membrane location at low concentrations, melittin's exact position and depth of penetration into the hydrocarbon core are unknown. Furthermore, the structural basis for its lytic action remains largely a matter of conjecture. Using a novel x-ray absolute-scale refinement method, we have now determined the location, orientation, and likely conformation of monomeric melittin in oriented phosphocholine lipid multilayers. Its helical axis is aligned parallel to the bilayer plane at the depth of the glycerol groups, but its average conformation differs from the crystallographic structure. As observed earlier for another amphipathic alpha-helical peptide, the lipid perturbations induced by melittin are remarkably modest. Small bilayer perturbations thus appear to be a general feature of amphipathic helices at low concentrations. In contrast, a dimeric form of melittin causes larger structural perturbations under otherwise identical conditions. These results provide direct structural evidence that self-association of amphipathic helices may be the crucial initial step toward membrane lysis.

Animals↗
PubMed2000

The asparagine-stabilized beta-turn of apamin: contribution to structural stability from dynamics simulation and amide hydrogen exchange analysis.

Molecular dynamics simulations of bee venom apamin, and an analogue having an Asn to Ala substitution at residue 2 (apamin-N2A), were analyzed to explore the contribution of hydrogen bonds involving Asn2 to local (beta-turn residues N2, C3, K4, A5) and global stability. The wild-type peptide retained a stable conformation during 2.4 ns of simulation at 67 degrees C, with high beta-turn stability characterized by backbone-side chain hydrogen bonds involving beta-turn residues K4 and A5, with the N2 side chain amide carbonyl. The loss of stabilizing interactions involving the N2 side chain resulted in the loss of the beta-turn conformation in the apamin N2A simulations (27 or 67 degrees C). This loss of beta-turn stability propagates throughout the peptide structure, with destabilization of the C-terminal helix connected to the N-terminal region by two disulfide bonds. Backbone stability in a synthetic peptide analogue (apamin-N2A) was characterized by NMR and amide hydrogen exchange measurements. Consistent with the simulations, loss of hydrogen bonds involving the N2 side chain resulted in destabilization of both the N-terminal beta-turn and the C-terminal helix. Amide exchange protection factors in the C-terminal helix were reduced by 9-11-fold in apamin N2A as compared with apamin, corresponding to free energy (deltaDeltaG(uf)) of around 1.5 kcal M(-1) at 20 degrees C. This is equivalent to the contribution of hydrogen bond interactions involving the N2 side chain to the stability of the beta-turn. Together with additional measures of exchange protection factors, the three main contributions to backbone stability in apamin that account for virtually the full thermodynamic stability of the peptide have been quantitated.

Amides↗
PubMed2000

Alternative splicing and gene structure of the transforming growth factor beta-activated kinase 1.

We have identified a fourth splice variant of the TGF beta-activated kinase (TAK1), called TAK1-d, and identified an error in the previously published TAK1-c sequence. Our data shows that the c and d variants encode proteins whose carboxyl ends differ markedly from those of variants a and b. Analysis of the human TAK1 gene sequence, located at 6q16.1-q16.3, shows that the coding sequence is organised in 17 exons. The four splice variants result from alternative splicing of exons 12 and 16, the reading frame of exon 17 being determined by the presence or absence of exon 16. Study of the relative levels of expression of the four splice variants showed significant variations between tissues. Our evidence suggests that the alternative splicing of the TAK1 mRNA may have important functional implications.

Alternative Splicing↗
PubMed2000

Structural characterization of a methionine-rich, emulsifying protein from sunflower seed.

The 2 S seed storage protein, sunflower albumin 8, contains an unusually high proportion of hydrophobic residues including 16 methionines in a mature protein of 103 amino acids. A structural model, based on the known structure of a related protein, has been constructed as a four-helix bundle cross-linked by four disulphide bonds. This model structure is consistent with data from circular dichroism and nuclear magnetic resonance experiments. Analysis of the model's surface shows the presence of a large hydrophobic face that may be responsible for the highly stable emulsions this protein is known to form with oil/water mixtures.

2S Albumins, Plant↗
PubMed1999

Intrinsic helical propensities and stable secondary structure in a membrane-bound fragment (S4) of the shaker potassium channel.

The location and stability of helical secondary structure in a fragment comprising an extended sequence of the S4 transmembrane segment of the Shaker potassium channel was determined in methanol, and when bound to vesicles composed of egg phosphatidylcholine: egg phosphatidylglycerol (4:1; mol:mol) in water. The N-acetylated, C-amidated peptide corresponds to the sequence comprising residues A355-I384 in the Shaker potassium channel. Although NOEs characteristic of helical structure encompass essentially the full peptide sequence in methanol, analysis of amide and CH(alpha) chemical shifts, and amide exchange protection factors establish that stable helical structure comprises only around the first 22 amino acids of the 30 residue peptide. This sequence corresponds to that predicted to have the highest helical stability in water, indicating that while helical structure is considerably stabilised in methanol, the relative helical propensities of amino acids in methanol may be similar to those in water. In the presence of vesicles containing negatively charged lipids, helical structure corresponding to a maximum of around 40 % of the extended S4 peptide is induced; no helical structure is induced in the presence of vesicles composed only of neutral lipids. The location of stable helical structure in the membrane-bound peptide was determined by amide hydrogen-deuterium exchange trapping, and was shown to encompass the sequence between residues near M2 and I18. This sequence is similar to that having high helix propensity in water and methanol, supporting the idea that intrinsic helical propensities are important in defining the location of stable helical structure in polypeptides bound in the interfacial region of lipid bilayers. The study defines an approach to determining the location of, and contributions to, the stability of helical secondary structure in membrane-reconstituted polypeptides.

Amino Acid Sequence↗
PubMed1999

Direct kinetic evidence for folding via a highly compact, misfolded state.

The 2 S seed storage protein, sunflower albumin 8 (SFA-8), contains an unusually high proportion of hydrophobic residues including 16 methionines (some of which may form a surface hydrophobic patch) in a disulfide cross-linked, alpha-helical structure. Circular dichroism and fluorescence spectroscopy show that SFA-8 is highly stable to denaturation by heating or chaotropic agents, the latter resulting in a reversible two-state unfolding transition. The small m(U) (-4.7 M(-1) at 10 degrees C) and DeltaC(p) (-0.95 kcal mol(-1) K(-1)) values indicate that relatively little nonpolar surface of the protein is exposed during unfolding. Commensurate with the unusual distribution of hydrophobic residues, stopped-flow fluorescence data show that the folding pathway of SFA-8 is highly atypical, in that the initial product of the rapid collapse phase of folding is a compact nonnative state (or collection of nonnative states) that must unfold before acquiring the native conformation. The inhibited folding reaction of SFA-8, in which the misfolded state (m(M) = -0.95 M(-1) at 10 degrees C) is more compact than the transition state for folding (m(T) = -2.5 M(-1) at 10 degrees C), provides direct kinetic evidence for the transient misfolding of a protein.

2S Albumins, Plant↗
PubMed1999

Generalised bilayer perturbation from peptide helix dimerisation at membrane surfaces: vesicle lysis induced by disulphide-dimerised melittin analogues.

The effects of covalent dimerisation of melittin by disulphide formation in cysteine-substitution analogues, (melittin K23C)2 and (melittin K23Q,Q25C)2, on the kinetics of pore formation in phosphatidylcholine small unilamellar vesicles was measured under low ionic strength conditions. The initial rate of melittin-induced pore formation increased with the square of the peptide concentration, whereas both disulphide-dimerised melittin analogues showed a first-order dependence of pore formation rates on peptide concentration. These results indicate that peptide dimerisation is rate-limiting for pore formation under these conditions. A model for a generalised bilayer perturbation resulting from the self-association of a pair of peptide helices at the membrane surface is proposed which may have implications for a number of biological processes that involve the interaction of helical polypeptides with membranes.

Animals↗
PubMed1999

A general method for relieving substrate inhibition in lactate dehydrogenases.

The mutation S163L in human heart lactate dehydrogenase removes substrate inhibition while only modestly reducing the turnover rate for pyruvate. Since this is the third enzyme to show this behaviour, we suggest that the S163L mutation is a general method for the removal of substrate inhibition in L-LDH enzymes. Engineering such enzymatic properties has clear industrial applications in the use of these enzymes to produce enantiomerically pure alpha-hydroxy acids. The mutation leads to two principal effects. (1) Substrate inhibition is caused by the formation of a covalent adduct between pyruvate and the oxidized form of the cofactor. The inability of S163L mutants to catalyse the formation of this inhibitory adduct is demonstrated. However, NMR experiments show that the orientation of the nicotinamide ring in the mutant NAD+ binary complex is not perturbed. (2) The mutation also leads to a large increase in the KM for pyruvate. The kinetic and binding properties of S163L LDH mutants are accounted for by a mechanism which invokes a non-productive, bound form of the cofactor. Molecular modelling suggests a structure for this non-productive enzyme-NADH complex.

Enzyme Inhibitors↗
PubMed1998

Self-association of disulfide-dimerized melittin analogues.

Two cysteine substitutions of bee venom melittin have been synthesized to investigate the effects of disulfide cross-linking on the self-association properties of the peptide in solution. K23C melittin (mltK23C) was designed to link nonpolar surfaces of the amphipathic melittin helix on the basis of the close juxtaposition of pairs of K23 side chains in the crystal of the native melittin tetramer. K23Q/Q25C melittin (mltQ25C) was designed to link the polar surfaces of the peptide such that self-association in membrane bound states might be stabilized. The mltK23C disulfide dimer, (mltK23C)2, is highly structured at low pH under conditions where native melittin, and the mltK23C monomer, are unstructured. High-resolution NMR, circular dichroism, and fluorescence spectroscopy established that (mltK23C)2 is a helical monomer (pseudodimer) with stable helical segments between residues 2-13 and 15-25. Although the symmetrical nature of the pseudodimer prevented high-resolution structure determination, analysis of calculated hydrogen bond lengths, chemical shifts, near-UV circular dichroism, and urea denaturation demonstrated similarities with alpha-helical coiled coils and with the structure of native melittin in methanol. Stopped flow fluorescence showed that (mltK23C)2 underwent pH- and divalent anion-linked dimerization to a melittin-like pseudotetramer, indicating that a pair of disulfide bonds could be accommodated in a structure similar to the native melittin crystal structure. Despite incorporation of two disulfide bonds into the melittin tetramer, the folding free energy (DeltaGw) of [(mltK23C)2]2 was similar to that for the native melittin tetramer under the condition used. Incorporation of a disulfide bond on the polar helix face in melittin did not stabilize helical structure in the absence of self-association. Instead, this molecule underwent pH- and divalent anion-linked self-association to an ill-defined aggregate which precipitated.

Amino Acid Sequence↗
PubMed1998

Hydrogen bonding in helical polypeptides from molecular dynamics simulations and amide hydrogen exchange analysis: alamethicin and melittin in methanol.

Molecular dynamics simulations of ion channel peptides alamethicin and melittin, solvated in methanol at 27 degrees C, were run with either regular alpha-helical starting structures (alamethicin, 1 ns; melittin 500 ps either with or without chloride counterions), or with the x-ray crystal coordinates of alamethicin as a starting structure (1 ns). The hydrogen bond patterns and stabilities were characterized by analysis of the dynamics trajectories with specified hydrogen bond angle and distance criteria, and were compared with hydrogen bond patterns and stabilities previously determined from high-resolution NMR structural analysis and amide hydrogen exchange measurements in methanol. The two alamethicin simulations rapidly converged to a persistent hydrogen bond pattern with a high level of 3(10) hydrogen bonding involving the amide NH's of residues 3, 4, 9, 15, and 18. The 3(10) hydrogen bonds stabilizing amide NH's of residues C-terminal to P2 and P14 were previously proposed to explain their high amide exchange stabilities. The absence, or low levels of 3(10) hydrogen bonds at the N-terminus or for A15 NH, respectively, in the melittin simulations, is also consistent with interpretations from amide exchange analysis. Perturbation of helical hydrogen bonding in the residues before P14 (Aib10-P14, alamethicin; T11-P14, melittin) was characterized in both peptides by variable hydrogen bond patterns that included pi and gamma hydrogen bonds. The general agreement in hydrogen bond patterns determined in the simulations and from spectroscopic analysis indicates that with suitable conditions (including solvent composition and counterions where required), local hydrogen-bonded secondary structure in helical peptides may be predicted from dynamics simulations from alpha-helical starting structures. Each peptide, particularly alamethicin, underwent some large amplitude structural fluctuations in which several hydrogen bonds were cooperatively broken. The recovery of the persistent hydrogen bonding patterns after these fluctuations demonstrates the stability of intramolecular hydrogen-bonded secondary structure in methanol (consistent with spectroscopic observations), and is promising for simulations on extended timescales to characterize the nature of the backbone fluctuations that underlie amide exchange from isolated helical polypeptides.

Alamethicin↗
PubMed1997

Distinct subcellular localisations of the putative inositol 1,3,4,5-tetrakisphosphate receptors GAP1IP4BP and GAP1m result from the GAP1IP4BP PH domain directing plasma membrane targeting.

Inositol 1,3,4,5-tetrakisphosphate (IP4), is a ubiquitous inositol phosphate that has been suggested to function as a second messenger. Recently, we purified and cloned a putative IP4 receptor, termed GAP1(IP4BP)[1], which is also a member of the GAP1 family of GTPase-activating proteins for the Ras family of GTPases. A homologue of GAP1(IP4BP), called GAP1(m), has been identified [2] and here we describe the cloning of a GAP1(m) cDNA from a human circulating-blood cDNA library. We found that a deletion mutant of GAP1(m), in which the putative phospholipid-binding domains (C2A and C2B) have been removed, binds to IP4 with a similar affinity and specificity to that of the corresponding GAP1(IP4BP) mutant. Expression studies of the proteins in either COS-7 or HeLa cells showed that, whereas GAP1(IP4BP) is located solely at the plasma membrane, GAP1(m) seems to have a distinct perinuclear localisation. By mutational analysis, we have shown that the contrast in subcellular distribution of these two closely related proteins may be a function of their respective pleckstrin homology (PH) domains. This difference in localisation has fundamental significance for our understanding of the second messenger functions of IP4.

Animals↗
PubMed1997

Acquisition of native beta-strand topology during the rapid collapse phase of protein folding.

The 98 residue C-terminal domain of the cell-surface receptor protein CD2 (CD2.D1) has a beta-sandwich fold belonging to the immunoglobulin superfamily but lacking the usual disulfide bridges. Kinetic studies on the folding/unfolding of CD2.D1 reveal that folding proceeds through a rapidly formed intermediate state [Parker, M. J., & Clarke, A. R. (1997) Biochemistry 36, 5786-5794]. To characterize the structural properties of this intermediate we have performed a series of amide hydrogen exchange studies using the pH competition method, in which folding and exchange are initiated simultaneously. The complex beta-sheet topology of this molecule makes it an ideal object for examining the acquisition of backbone hydrogen bonds made between sequence-local and sequence-distant segments of the chain during folding. The pattern of protected amides in the intermediate reveal that the essential features of the beta-sheet topology of CD2.D1 are defined early in the folding pathway, before the development of intimate side chain interactions characteristic of the native state. The results are discussed in light of current issues concerning the mechanistic relevance of kinetic protein folding intermediates.

Amides↗
PubMed1997

Solution structure of the actinorhodin polyketide synthase acyl carrier protein from Streptomyces coelicolor A3(2).

The solution structure of the actinorhodin acyl carrier protein (act apo-ACP) from the polyketide synthase (PKS) of Streptomyces coelicolor A3(2) has been determined using 1H NMR spectroscopy, representing the first polyketide synthase component for which detailed structural information has been obtained. Twenty-four structures were generated by simulated annealing, employing 699 distance restraints and 94 dihedral angle restraints. The structure is composed, principally, of three major helices (1, 2, and 4), a shorter helix (3) and a large loop region separating helices 1 and 2. The structure is well-defined, except for a portion of the loop region (residues 18-29), the N-terminus (1-4), and a short stretch (57-61) in the loop connecting helices 2 and 3. The RMS distribution of the 24 structures about the average structure is 1.47 A for backbone atoms, 1.84 A for all heavy atoms (residues 5-86), and 1.01 A for backbone atoms over the helical regions (5-18, 41-86). The tertiary fold of act apo-ACP shows a strong structural homology with Escherichia coli fatty acid synthase (FAS) ACP, though some structural differences exist. First, there is no evidence that act apo-ACP is conformationally averaged between two or more states as observed in E. coli FAS ACP. Second, act apo-ACP shows a disordered N-terminus (residues 1-4) and a longer flexible loop (19-41 with 19-29 disordered) as opposed to E. coli FAS ACP where the N-terminal helix starts at residue 3 and the loop region is three amino acids shorter (16-35). Most importantly, however, although the act apo-ACP structure contains a hydrophobic core, there are in addition a number of buried hydrophilic groups, principally Arg72 and Asn79, both of which are 100% conserved in the PKS ACPs and not the FAS ACPs and may therefore play a role in stabilizing the growing polyketide chain. The structure-function relationship of act ACP is discussed in the light of these structural data and recent genetic advances in the field.

Acyl Carrier Protein↗
PubMed1997

Helix bending in alamethicin: molecular dynamics simulations and amide hydrogen exchange in methanol.

Molecular dynamics simulations of alamethicin in methanol were carried out with either a regular alpha-helical conformation or the x-ray crystal structure as starting structures. The structures rapidly converged to a well-defined hydrogen-bonding pattern with mixed alpha-helical and 3(10)-helical hydrogen bonds, consistent with NMR structural characterization, and did not unfold throughout the 1-ns simulation, despite some sizable backbone fluctuations involving reversible breaking of helical hydrogen bonds. Bending of the helical structure around residues Aib10-Aib13 was associated with reversible flips of the peptide bonds involving G11 (Aib10-G11 or G11-L12 peptide bonds), yielding discrete structural states in which the Aib10 carbonyl or (rarely) the G11 carbonyl was oriented away from the peptide helix. These peptide bond reversals could be accommodated without greatly perturbing the adjacent helical structure, and intramolecular hydrogen bonding was generally maintained in bent states through the formation of new (non-alpha or 3[10]) hydrogen bonds with good geometries: G11 NH-V9 CO (inverse gamma turn), Aib13 NH-Aib8 CO (pi-helix) and, rarely, L12 NH- Q7 NH (pi-helix). These observations may reconcile potentially conflicting NMR structural information for alamethicin in methanol, in which evidence for conformational flexibility in the peptide sequence before P14 (G11-Aib13) contrasts with the stability of backbone amide NH groups to exchange with solvent. Similar reversible reorientation of the Thr11-Gly12 peptide bond of melittin is also observed in dynamics simulations in methanol (R. B. Sessions, N. Gibbs, and C. E. Dempsey, submitted). This phenomenon may have some role in the orientation of the peptide carbonyl in solvating the channel lumen in membrane ion channel states of these peptides.

Alamethicin↗
PubMed1996

Conserved secondary structure in the actinorhodin polyketide synthase acyl carrier protein from Streptomyces coelicolor A3(2) and the fatty acid synthase acyl carrier protein from Escherichia coli.

The acyl carrier protein (ACP) of Streptomyces coelicolor A3(2) functions as a molecular chaperone during the biosynthesis of the polyketide actinorhodin (act). Here we compare structural features of the polyketide synthase (PKS) ACP, determined by two-dimensional 1H-NMR, with the Escherichia coli fatty acid synthase (FAS) ACP. The PKS ACP contains four helices (residues 7-16 [A], 42-53 [B], 62-67 [C], 72-86 [D]), and a large loop (residues 17-41) having no defined secondary structure with the exception of a turn between residues 21 and 24. The act ACP shows 47% sequence similarity with the E. coli FAS ACP and the results demonstrate that the sequence homology is extended to the secondary structure of the proteins.

Acyl Carrier Protein↗
PubMed1996

Hydrogen bond stabilities in membrane-reconstituted alamethicin from amide-resolved hydrogen-exchange measurements.

Amide-resolved hydrogen-deuterium exchange-rate constants were measured for backbone amides of alamethicin reconstituted in dioleoylphosphatidylcholine vesicles by an exchange-trapping method combined with high-resolution nuclear magnetic resonance spectroscopy. In vesicles containing alamethicin at molar ratios between 1:20 and 1:100 relative to lipid, the exchange-rate constants increased with increasing volume of the D20 buffer in which the vesicles were suspended, indicating that exchange under these conditions is dominated by partitioning of the peptide into the aqueous phase. This was supported by observation of a linear relationship between the exchange-rate constants for amides in membrane-reconstituted alamethicin and those for amides in alamethicin dissolved directly into D2O buffer. Significant protection of amides from exchange with D2O buffer in membrane-reconstituted alamethicin is interpreted in terms of stabilization by helical hydrogen bonding. Under conditions in which amide exchange occurred by partitioning of the peptide into solution, only lower limits for hydrogen-bond stabilities in the membrane were determined; all the potentially hydrogen-bonded amides of alamethicin are at least 1000-fold exchange protected in the membrane-bound state. When partitioning of alamethicin into the aqueous phase was suppressed by hydration of reconstituted vesicles in a limiting volume of water [D2O:dioleoylphosphatidylcholine:alamethicin; 220:1:0.05; (M:M:M)], the exchange-protection factors exhibited helical periodicity with highly exchange-protected, and less well-protected, amides on the nonpolar and polar helix faces, respectively. The exchange data indicate that, under the conditions studied, alamethicin adopts a stable helical structure in DOPC bilayers in which all the potentially hydrogen-bonded amides are stabilized by helical hydrogen bonds. The protection factors define the orientation of the peptide helix with respect to an aqueous phase, which is either the bulk solution or water within parallel or antiparallel transmembrane arrays of reconstituted alamethicin.

Alamethicin↗