Biomedical subjects

M A Olson

Publications and source records attributed to M A Olson.

At least 19 recordsLinked to original sources

PubMed2001

Electrostatic effects on the free-energy balance in folding a ribosome-inactivating protein.

Electrostatics of globular proteins provides structural integrity as well as specificity of biological function. This dual role is particularly striking for ricin A-chain (RTA), an N-glycosidase which hydrolyzes a single adenine base from a conserved region of rRNA. The reported X-ray crystallographic structure of the RTA mutant E177A demonstrated a remarkable rescue of charge balance in the active site, achieved by the rotation of a second glutamic acid (Glu-208) into the vacated space. To understand this conformational reorganization, molecular-dynamics simulations were applied to estimate relative free energies that govern the thermodynamic stability of E177A together with mutants E177Q and E177D. The simulations anticipate that while E177A is a non-conservative substitution, the protein is more stable than the other two mutants. However, the structural plasticity of the RTA active site is not obtained penalty-free, rather E177A among the mutants shows the largest unfavorable net change in the electrostatic contribution to folding. Of the E177A folded state, reorganization of Glu-208 lowers the electrostatic cost of the free-energy change, yet interestingly, protein interactions oppose the rotational shift, while solvent effects favor the transition.

Chemical Phenomena↗

PubMed2001

Calculations of free-energy contributions to protein-RNA complex stabilization.

The problem of calculating binding affinities of protein-RNA complexes is addressed by analyzing a computational strategy of modeling electrostatic free energies based on a nonlinear Poisson-Boltzmann (NLPB) model and linear response approximation (LRA). The underlying idea is to treat binding as a two-step process. Solutions to the NLPB equation calculate free energies arising from electronic polarizability and the LRA is constructed from molecular dynamics simulations to model reorganization free energies due to conformational transitions. By implementing a consistency condition of requiring the NLPB model to reproduce the solute-solvent free-energy transitions determined by the LRA, a "macromolecule dielectric constant" (epsilon(m)) for treating reorganization is obtained. The applicability of this hybrid approach was evaluated by calculating the absolute free energy of binding and free-energy changes for amino acid substitutions in the complex between the U1A spliceosomal protein and its cognate RNA hairpin. Depending on the residue substitution, epsilon(m) varied from 3 to 18, and reflected dipolar reorientation not included in the polarization modeled by epsilon(m) = 2. Although the changes in binding affinities from substitutions modeled strictly at the implicit level by the NLPB equation with epsilon(m) = 4 reproduced the experimental values with good overall agreement, substitutions problematic to this simple treatment showed significant improvement when solved by the NLPB-LRA approach.

Computational Biology↗

PubMed2001

Implicit attitude formation through classical conditioning.

We sought to demonstrate that attitudes can develop through implicit covariation detection in a new classical conditioning paradigm. In two experiments purportedly about surveillance and vigilance, participants viewed several hundred randomly presented words and images interspersed with critical pairings of valenced unconditioned stimuli (USs) with novel conditioned stimuli (CSs). Attitudes toward the novel objects were influenced by the paired USs: In a surprise evaluation task, the CS paired with positive items was evaluated more positively than the CS paired with negative items. This attitudinal conditioning effect was found using both an explicit measure (Experiments 1 and 2) and an implicit measure (Experiment 2). In a covariation estimation task involving the stimuli presented in the conditioning procedure, participants displayed no explicit memory for the pairings.

Adult↗

PubMed2000

Modeling implicit reorganization in continuum descriptions of protein-protein interactions.

The determination of free energies that govern protein-protein recognition is essential for a detailed molecular understanding of biological specificity. Continuum models of macromolecular interactions, in which the solvent is treated by an implicit representation and the proteins are treated semi-microscopically, are computationally tractable for estimating free energies, yet many questions remain concerning their accuracy. This article reports a continuum analysis of the free-energy changes underlying the binding of 31 interfacial alanine substitutions of two complexes of the antihen egg white lysozyme (HEL) antibody D1.3 bound with HEL or the antibody E5.2. Two implicit schemes for modeling the effects of protein and solvent relaxation were examined, in which the protein environment was treated as either homogeneous with a "protein dielectric constant" of epsilon(p) = 4 or inhomogeneous, with epsilon(p) = 4 for neutral residues and epsilon(p) = 25 for ionized residues. The results showed that the nonuniform dielectric model reproduced the experimental differences better, with an average absolute error of +/-1.1 kcal/mol, compared with +/-1.4 kcal/mol for the uniform model. More importantly, the error for charged residues in the nonuniform model is +/-0.8 kcal/mol and is nearly half of that corresponding to the uniform model. Several substitutions were clearly problematic in determining qualitative trends and probably required explicit structural reorganization at the protein-protein interface.

Amino Acid Substitution↗

PubMed2000

In vivo 4-androstene-3,17-dione and 4-androstene-3 beta,17 beta-diol supplementation in young men.

To determine if known androgenic hormone precursors for testosterone in the androgen pathway would be readily transformed to testosterone, eight male subjects [mean age 23.8 (SEM 3) years, bodymass 83.1 (SEM 8.7) kg, height 175.6 (SEM 8.5) cm] underwent a randomized, double-blind, cross-over, placebo-controlled oral treatment with 200 mg of 4-androstene-3,17-dione (delta 4), 4-androstene-3 beta,17 beta-diol (delta 4 Diol), and placebo (PL). The periods of study were separated by 7 days of washout. Blood was drawn at baseline and subsequently every 30 min for 90 min after treatment. Analysis revealed mean area-under-the-curve (AUC) serum delta 4 concentrations to be higher during delta 4 treatment [2177 (SEM 100) nmol.l-1] than delta 4Diol [900 (SEM 96) nmol.l-1] or PL [484 (SEM 82) nmol.l-1; P < 0.0001]. The delta 4 treatment also revealed a significant effect on total testosterone with a mean AUC [1632.5 (SEM 121) nmol.l-1] that was greater than PL [1418.5 (SEM 131) nmol.l-1; P < 0.05] but not significantly different from those observed after delta 4Diol treatment [1602.9 (SEM 119) nmol.l-1; P = 0.77]. Free testosterone concentrations followed a similar pattern where mean AUC for the delta 4 treatment [6114.0 (SEM 600) pmol.l-1] was greater than after PL [4974.6 (SEM 565) pmol.l-1; P < 0.06] but not significantly different from those observed after delta 4Diol [5632.0 (SEM 389) pmol.l-1; P = 0.48]. The appearance and apparent conversion to total and free testosterone over 90 min was stronger for the delta 4 treatment (r = 0.91, P < 0.045) than for delta 4Diol treatment (r = 0.69, NS) and negatively correlated for PL (r = -0.90, P < 0.02). These results would suggest that delta 4, and perhaps delta 4Diol, taken by month are capable of producing in vivo increases in testosterone concentrations in apparently healthy young men as has already been observed in women after treatment with delta 4.

Adult↗

PubMed1999

Free energy determinants of binding the rRNA substrate and small ligands to ricin A-chain.

A continuum model is provided of the free energy terms that contribute to the molecular association of ricin A-chain (RTA) with the rRNA substrate and several small ligands. The model for RTA interactions with the RNA was taken from a previously proposed complex containing a 29-mer oligonucleotide hairpin (. Proteins 27:80-95), and models for the ligands were constructed from x-ray crystallographic structures. The calculated absolute free energies of complex formation for the RTA-RNA assembly and several single-residue substitutions are in good agreement with experimental data, given the approximations of evaluating the strain energy and conformational entropy. The free energy terms were found to resemble those of protein-protein complexes, with the net unfavorable electrostatic contribution offset by the favorable nonspecific hydrophobic effect. Decomposition of the RTA-RNA binding free energy into individual contributions revealed the electrostatic "hot" spots arising from charge-charge complementarity of the interfacial arginines with the RNA phosphate backbone. Base interactions of the GAGA loop structure dominate the hydrophobic complementarity. A linear-scaling model was parametrized for evaluating the binding of small ligands against the rRNA substrate and illustrates the free energy determinant required for designing specific RTA inhibitors.

Base Sequence↗

PubMed1999

Prediction of a conserved, neutralizing epitope in ribosome-inactivating proteins.

The secondary structures, side-chain solvent accessibilities, and superpositioned crystal structures of the A-chain of ricin and four other plant rRNA N-glycosidases (ribosome-inactivating proteins, RIPs) were examined. Previously, a 26-residue fragment from the A-chain of ricin was determined to bind to a neutralizing monoclonal antibody. The region in the native ricin A-chain, to which this peptide corresponds, is solvent-exposed and contains a negatively charged residue that has been hypothesized to participate in the toxin's function, namely, rRNA binding and/or enzymatic activity. This region appears to be conserved in all of the structurally defined plant RIPs examined. Moreover, other plant RIPs, whose tertiary structures are, as yet, unknown, were predicted to have an analogous, solvent-exposed region containing a conserved, negatively charged residue. By analogy, these conserved structural and functional features lead to the suggestion that this exposed region represents a logical starting point for experiments designed to locate neutralizing epitopes in these RIPs. In contrast, the tertiary structure of the analogous region in a bacteria-derived RIP (Shiga toxin) is a less solvent-exposed, truncated loop and is a structure that is not as likely to be a neutralizing epitope. Because most of the amino acid residues are not conserved within this exposed region, these RIPs are predicted to be antigenically distinct.

Abrin↗

PubMed1998

Identifying the principal protective antigenic determinants of type A botulinum neurotoxin.

The neurotoxins from Clostridium botulinum (BoNT serotypes A-G) exert their lethal effect by preventing the release of acetylcholine at the neuromuscular junction. As with tetanus toxin, immunization with a non-toxic fragment, the 50 kDa C-terminal portion of BoNT/A (Hc; residues 861-1296), protects mice against lethal challenges with the intact toxin. To locate the neutralizing epitopes, several protective monoclonal antibodies (mAbs) against BoNT/A-Hc were isolated and cloned. Specific binding of the mAbs to BoNT/A-Hc was demonstrated by surface plasmon resonance, with Kas in the range of 10(-10) to 10(-11) M. These antibodies recognized a genetically engineered polypeptide (1150-1289) that was previously shown to induce protective immunity. Prior to the determination of the X-ray crystal structure of the tetanus neurotoxin Hc fragment, molecular modelling studies indicated that it contained two highly solvent-exposed loops. Based on these predictions, two 25-mer Hc-peptides corresponding to these two regions were synthesized and were demonstrated to bind the neutralizing mAbs. Mice immunized with the Hc-peptides had high levels of antibodies that recognized BoNT/A-Hc. However, immunizations with only one of the Hc peptides protected when mice were challenged with BoNT/A. On the basis of these analyses, it should be possible to develop small peptides that could be useful in the design of future vaccines against these neurotoxins.

Amino Acid Sequence↗

PubMed1998

Development of engineered vaccines effective against structurally related bacterial superantigens.

Staphylococcal and streptococcal superantigens are exotoxins that may be linked to many human pathologies involving impaired immune functions. Despite considerable sequence divergence, bacterial superantigens share extensive secondary and tertiary structure and use similar structural strategies to bind major histocompatibility complex class II receptors. We produced by site-directed mutagenesis of the conserved receptor-binding surfaces of the superantigens staphylococcal enterotoxins A and B. These vaccines protected immunized mice and rhesus monkeys from lethal toxic shock. In addition, antibodies produced against each superantigen recognized and neutralized distantly related superantigens. This antibody cross-reactivity was additive in that mixtures of superantigens used in immunization were more effective than single-component vaccines in protecting mice from challenges with individual or mixed superantigens. We conclude that an optimal combination of these genetically attenuated superantigen vaccines may protect against all structurally related superantigens.

Animals↗

PubMed1998

Accuracy of secondary structure and solvent accessibility predictions for a clostridial neurotoxin C-fragment.

Earlier studies used Rost and Sander's artificial neural network [(1993a), J. Mol. Biol. 232, 584-599] to predict the secondary structures [Lebeda and Olson (1994), Proteins 20, 293-300] and residue solvent accessibilities [Lebeda and Olson (1997), J. Protein Chem. 16, 607-618] of the clostridial neurotoxins. Because the X-ray crystal structure of the 50-kDa C-terminal half of the heavy chain of tetanus toxin was recently determined, this report evaluates the accuracy of these network-derived predictions. For this predominantly beta-strand-containing fragment, predictions, on a per-residue basis, for both secondary structure and solvent accessibility were about 70% accurate. A more flexible and realistic analysis based on overlapping segments yielded accuracies of over 80% for the three-state secondary structure and for the two-state accessibility predictions. Because the accuracies of these predictions are comparable to those made by Rost and Sander using a dataset of 126 nonhomologous globular proteins, our predictions provide a quantitative foundation for gauging the results when building by homology the structures of related proteins.

Botulinum Toxins↗

PubMed1998

Predictions of secondary structure and solvent accessibility of the light chain of the clostridial neurotoxins.

Predictions were made of the secondary, two-dimensional (2-D) structures and side-chain solvent accessibilities of the light (L) chains of the clostridial neurotoxins (botulinum neurotoxin serotypes A-G and tetanus neurotoxin). An artificial neural network was used to make these predictions from a multiple alignment of their primary structures and was the approach used in making successful predictions for the C-fragments of these neurotoxins (Lebeda et al., J. Prot. Chem., 17:311, 1998). We also exploited the fact that the L-chains are Zn-dependent proteases. Although no other metalloproteases were found to be sequentially homologous to these neurotoxin L-chains, a sequence clustering algorithm showed that several bacterially derived Zn-dependent proteases, including thermolysin, were the most similar. A 2-D structure topology map for the type A L-chain was constructed by using thermolysin as a design template. As in thermolysin, the region containing the Zn-binding sequence motif, which is part of the active site in these neurotoxins, was predicted to be minimally solvent accessible. On the other hand, the locations of residues with highly exposed side chains were predicted to occur in non-periodic structure elements. Together, these 2-D structure and solvent accessibility predictions can be used to identify important solvent-exposed regions of the L-chain. These regions may include sites that interact with residues of the neurotoxin heavy chain, sites that bind to vesicle-docking substrates or sites that form antibody epitopes.

Algorithms↗

PubMed1997

Site-directed mutagenesis of dendrotoxin K reveals amino acids critical for its interaction with neuronal K+ channels.

Dendrotoxin K (DTXK) is a 57-residue protein from mamba venom that blocks certain non-inactivating, voltage-activated K+ currents in neurones. In order to pinpoint the residues responsible for its specificity, structure-activity relations of DTX(K) were investigated by mutagenesis. A previously cloned gene encoding this toxin [Smith et al. (1993) Biochemistry 32, 5692-5697] was used to make single mutations; after expression in Escherichia coli as fusion proteins and enzymatic cleavage of the conjugates isolated from the periplasmic space, nine toxins were purified. Structural analysis of the native DTXK and representative mutants by circular dichroism showed that no significant differences were detectable in their folded structures. The biological activity of the mutants, which contained alterations of positively charged and other amino acids, was determined from their abilities to compete for the binding of 125I-labeled DTX(K) to K+ channels in synaptic plasma membranes from rat cerebral cortex. Mutants with residues substituted in the alpha-helix near the C-terminus (R52A or R53A) yielded binding parameters similar to those of wild-type and native DTX(K). In the case of the beta-turn (residues 24-28), however, altering single amino acids reduced binding to the high-affinity site of K+ channels, with the rank order of decreases being K26A >> W25A > K24A = K28A. Also, substitutions made in the 3(10)-helix (residues 3-7), a region located close to the beta-turn, produced equivalent effects (K3A > K6A). Thus, it is deduced that residues in the distorted beta-turn and neighboring 3(10)-helix of DTX(K) are critical for its interaction with neuronal K+ channels.

Animals↗

PubMed1997

Ricin A-chain structural determinant for binding substrate analogues: a molecular dynamics simulation analysis.

Ricin A-chain is a cytotoxic protein that attacks ribosomes by hydrolyzing a specific adenine base from a highly conserved, single-stranded rRNA hairpin containing the tetraloop sequence GAGA. Molecular-dynamics simulation methods are used to analyze the structural determinant for three substrate analogues bound to the ricin A-chain molecule. Simulations were applied to the binding of the dinucleotide adenyl-3',5'-guanosine employing the x-ray crystal structure of the ricin complex and a modeled CGAGAG hexanucleotide loop taken from the NMR solution structure of a 29-mer oligonucleotide hairpin. A third simulation model is also presented describing a conformational search of the docked 29-mer structure by using a simulated-annealing method. Analysis of the structural interaction energies for each model shows the overall binding dominated by nonspecific interactions, which are mediated by specific arginine contracts from the highly basic region on the protein surface. The tetraloop conformation of the 29-mer was found to make specific interactions with conserved protein residues, in a manner that favored the GAGA sequence. A comparison of the two docked loop conformations with the NMR structure revealed significant positional deviations, suggesting that ricin may use an induced fit mechanism to recognize and bind the rRNA substrate. The conserved Tyr-80 may play an important conformational entropic role in the binding and release of the target adenine in the active site.

Binding Sites↗

PubMed1997

Predicting differential antigen-antibody contact regions based on solvent accessibility.

A novel computational approach was examined for predicting epitopes from primary structures of the seven immunologically distinct botulinum neurotoxins (BoNT/A-G) and tetanus toxin (TeTX). An artificial neural network [Rost and Sander (1994), Proteins 20, 216] was used to estimate residue solvent accessibilities in multiple aligned sequences. A similar network trained to predict secondary structures was also used to examine this protein family, whose tertiary fold is presently unknown. The algorithm was validated by showing that it was 80% accurate in determining the secondary structure of avian egg-white lysozyme and that it correctly identified highly solvent-exposed residues that correspond to the major contact regions of lysozyme-antibody cocrystals. When sequences of the heavy (H) chains of TeTX and BoNT/A-G were analyzed, this algorithm predicted that the most highly exposed regions were clustered at the sequentially nonconserved N- and C-termini [Lebeda and Olson (1994), Proteins 20, 293]. The secondary structures and the remaining highly solvent-accessible regions were, in contrast, predicted to be conserved. In experiments reported by others, H-chain fragments that induced immunological protection against BoNT/A overlap with these predicted most highly exposed regions. It is also known that the C-terminal halves of the TeTX and BoNT/A H-chains interfere with holotoxin binding to ectoacceptors on nerve endings. Thus, the present results provide a theoretical framework for predicting the sites that could assist in the development of genetically engineered vaccines and that could interact with neurally located toxin ectoacceptors. Finally, because the most highly solvent-exposed regions were not well conserved, it is hypothesized that nonconserved, potential contact sites partially account for the existence of different dominant binding regions for type-specific neutralizing antibodies.

Algorithms↗

PubMed1997

Quantitative research on therapeutic touch. An integrative review of the literature 1985-1995.

Quantitative research on Therapeutic Touch (TT), published in referred nursing journals from 1985 to 1995, is reviewed. Therapeutic Touch is defined by Dolores Krieger, the founder of this nursing intervention. The authors of this Integrative Review examine what is known and not known to date in order to facilitate appropriate application of this modality in practice, and to offer recommendations for future research. Critical characteristics of eleven quantitative studies are identified and analyzed. These characteristics include: author/year/journal/title; study purpose (hypotheses); background/literature review/conceptual citations; sample selection method; study design/random assignment; independent variable/length of treatment/control and confounders; dependent variables/measurements; outcomes; study limitations; and implications for future research. After reviewing the studies, it is concluded that there is evidence to support the practice of Therapeutic Touch for the reduction of pain or anxiety. There is clearly a lack of congruity between the research statement, conceptual framework, operational definition of TT and the findings. This incongruity is discussed and incorporated in the recommendations for future research including outcome, theory-generating and theory-testing research.

Confounding Factors, Epidemiologic↗

PubMed1995

Simulation analysis of formycin 5'-monophosphate analog substrates in the ricin A-chain active site.

Ricin is an RNA N-glycosidase that hydrolyzes a single adenine base from a conserved loop of 28S ribosomal RNA, thus inactivating protein synthesis. Molecular-dynamics simulation methods are used to analyze the structural interactions and thermodynamics that govern the binding of formycin 5'-monophosphate (FMP) and several of its analogs to the active site of ricin A-chain. Simulations are carried out initiated from the X-ray crystal structure of the ricin-FMP complex with the ligand modeled as a dianion, monoanion and zwitterion. Relative changes in binding free energies are estimated for FMP analogs constructed from amino substitutions at the 2- and 2'-positions, and from hydroxyl substitution at the 2'-position.

Binding Sites↗

PubMed1995

Structural predictions of the channel-forming region of botulinum neurotoxin heavy chain.

A novel combination of theoretical approaches was exploited to predict which amino acid residues of various botulinum neurotoxin serotypes participate in forming ion channels. Estimates of sequence hydrophobic moments were used initially to identify residues within amphipathic regions in the N-terminal half of the heavy chain. A neural network algorithm was then used to make additional secondary structural predictions for these regions. Together, these approaches predicted a complimentary pattern of four, adjacent amphipathic, possibly transmembrane, regions that may be separated by solvent-exposed loops. Both the hydrophobic moment and the neural net analyses predicted that at least one of these amphipathic segments may be in an extended conformation. These theoretical results are discussed in view of our current knowledge of other transmembrane structures.

Algorithms↗

PubMed1995

Cloning and expression of mamba toxins.

Mamba venoms contain pharmacologically active proteins that interfere with neuromuscular transmission by binding to and altering the normal functioning of neuronal proteins involved, directly or indirectly, with regulating nerve transmission. Of the mamba toxins studied to date, many act on voltage-sensitive K+ channels, nicotinic or muscarinic acetylcholine receptors, or acetylcholinesterase. In an attempt to clone, characterize, and express the genes encoding these toxins, as well as other genes specifying activities not completely elucidated as yet, a cDNA library was constructed from mRNA isolated from the glands of the black mamba. Clones from the library harboring sequences encoding 14 different mamba toxins were isolated and characterized by nucleotide sequence analysis. Genes coding for three proteins, dendrotoxins (DTX) K, I, and E, were expressed as maltose-binding (MBP) fusion proteins in the periplasmic space of Escherichia coli. The DTXK-MBP fusion protein was affinity purified, cleaved from its chaperon, and the recombinant DTXK purified from MBP. Recombinant DTXK was shown to be identical to native DTXK in its N-terminal sequence, chromatographic behavior, convulsion-inducing activity, and binding to voltage-activated K+ channels in bovine synaptic membranes. Computer modeling was employed to create three-dimensional structures of DTXK and DTX1 from the X-ray crystal structure of alpha-DTX utilizing both structural and sequence homologies. Comparisons were made between the three toxins, providing a framework for site-directed mutagenesis.

Amino Acid Sequence↗