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M Levitt

Publications and source records attributed to M Levitt.

At least 73 records · Page 4Linked to original sources
PubMed1996

Using a hydrophobic contact potential to evaluate native and near-native folds generated by molecular dynamics simulations.

There are several knowledge-based energy functions that can distinguish the native fold from a pool of grossly misfolded decoys for a given sequence of amino acids. These decoys, which are typically generated by mounting, or "threading", the sequence onto the backbones of unrelated protein structures, tend to be non-compact and quite different from the native structure: the root-mean-squared (RMS) deviations from the native are commonly in the range of 15 to 20 angstroms. Effective energy functions should also demonstrate a similar recognition capability when presented with compact decoys that depart only slightly in conformation from the correct structure (i.e. those with RMS deviations of approximately 5 angstroms or less). Recently, we developed a simple yet powerful method for native fold recognition based on the tendency for native folds to form hydrophobic cores. Our energy measure, which we call the hydrophobic fitness score, is challenged to recognize the native fold from 2000 near-native structures generated for each of five small monomeric proteins. First, 1000 conformations for each protein were generated by molecular dynamics simulation at room temperature. The average RMS deviation of this set of 5000 was 1.5 angstroms. A total of 323 decoys had energies lower than native; however, none of these had RMS deviations greater than 2 angstroms. Another 1000 structures were generated for each at high temperature, in which a greater range of conformational space was explored (4.3 angstroms RMS deviation). Out of this set, only seven decoys were misrecognized. The hydrophobic fitness energy of a conformation is strongly dependent upon the RMS deviation. On average our potential yields energy values which are lowest for the population of structures generated at room temperature, intermediate for those produced at high temperature and highest for those constructed by threading methods. In general, the lowest energy decoy conformations have backbones very close to native structure. The possible utility of our method for screening backbone candidates for the purpose of modelling by side-chain packing optimization is discussed.

Protein Folding↗
PubMed1996

Using iterative dynamic programming to obtain accurate pairwise and multiple alignments of protein structures.

We show how a basic pairwise alignment procedure can be improved to more accurately align conserved structural regions, by using variable, position-dependent gap penalties that depend on secondary structure and by taking the consensus of a number of suboptimal alignments. These improvements, which are novel for structural alignment, are direct analogs of what is possible with normal sequences alignment. They are feasible for us since our basic structural alignment procedure, unlike others, is so similar to normal sequence alignment. We further present preliminary results that show how our procedure can be generalized to produce a multiple alignment of a family of structures. Our approach is based on finding a "median" structure from doing all possible pairwise alignments and then aligning everything to it.

Amino Acid Sequence↗
PubMed1995

Recognizing native folds by the arrangement of hydrophobic and polar residues.

Central to the ab initio protein folding problem is the development of an energy function for which the correct native structure has a lower energy than all other conformations. Existing potentials of mean force typically rely extensively on database-derived contact frequencies or knowledge of three-dimensional structural information in order to be successful in the problem of recognizing the native fold for a given sequence from a set of decoy backbone conformations. Is the detailed statistical information or sophisticated analysis used by these knowledge-based potentials needed to achieve the observed degree of success in fold recognition? Here we introduce a novel pairwise energy function that enumerates contacts between hydrophobic residues while weighting their sum by the total number of residues surrounding these hydrophobic residues. Thus it effectively selects compact folds with the desired structural feature of a buried, intact core. This approach represents an advance over using pairwise terms whose energies of interaction that are independent of the position in the protein and greatly improves the discrimination capability of an energy function. Our results show that 85% of a set of 195 representative native folds were recognized correctly. The 29 exceptions were lipophilic proteins, small proteins with prosthetic groups or disulfide bonds, and oligomeric proteins. Overall, our method separates the native fold from incorrect folds by a larger margin (measured in standard deviation units) than has been previously demonstrated by more sophisticated methods. The arrangement of hydrophobic and polar residues alone as evaluated by our novel scoring scheme, is unexpectedly effective at recognizing native folds in general. It is surprising that a simple binary pattern of hydrophobic and polar residues apparently selects a give unique fold topology.

Amino Acids↗
PubMed1995

The volume of atoms on the protein surface: calculated from simulation, using Voronoi polyhedra.

We analyze the volume of atoms on the protein surface during a molecular-dynamics simulation of a small protein (pancreatic trypsin inhibitor). To calculate volumes, we use a particular geometric construction, called Voronoi polyhedra, that divides the total volume of the simulation box amongst the atoms, rendering them relatively larger or smaller depending on how tightly they are packed. We find that most of the atoms on the protein surface are larger than those buried in the core (by approximately 6%), except for the charged atoms, which decrease in size, presumably due to electroconstriction. We also find that water molecules are larger near apolar atoms on the protein surface and smaller near charged atoms, in comparison to "bulk" water molecules far from the protein. Taken together, these findings necessarily imply that apolar atoms on the protein surface and their associated water molecules are less tightly packed (than corresponding atoms in the protein core and bulk water) and the opposite is the case for charged atoms. This looser apolar packing and tighter charged packing fundamentally reflects protein-water distances that are larger or smaller than those expected from van der Waals radii. In addition to the calculation of mean volumes, simulations allow us to investigate the volume fluctuations and hence compressibilities of the protein and solvent atoms. The relatively large volume fluctuations of atoms at the protein-water interface indicates that they have a more variable packing than corresponding atoms in the protein core or in bulk water. We try to adhere to traditional conventions throughout our calculations. Nevertheless, we are aware of and discuss three complexities that significantly qualify our calculations: the positioning of the dividing plane between atoms, the problem of vertex error, and the choice of atom radii. In particular, our results highlight how poor a "compromise" the commonly accepted value of 1.4 A is for the radius of a water molecule.

Animals↗
PubMed1995

The complexity and accuracy of discrete state models of protein structure.

The prediction of protein structure depends on the quality of the models used. In this paper, we examine the relationship between the complexity and accuracy of representation of various models of protein alpha-carbon backbone structure. First, we develop an efficient algorithm for the near optimal fitting of arbitrary lattice and off-lattice models of polypeptide chains to their true X-ray structures. Using this, we show that the relationship between the complexity of a model, taken as the number of possible conformational states per residue, and the simplest measure of accuracy, the root-mean-square deviation from the X-ray structure, is approximately (Accuracy) varies; is directly proportional to (Complexity)-1/2. This relationship is insensitive to the particularities of individual models, i.e. lattice and off-lattice models of the same complexity tend to have similar average root-mean-square deviations, and this also implies that improvements in model accuracy with increasing complexity are very small. However, other measures of model accuracy, such as the preservation of X-ray residue-residue contacts and the alpha-helix, do distinguish among models. In addition, we show that low complexity models, which take into account the uneven distribution of residue conformations in real proteins, can represent X-ray structures as accurately as more complex models, which do not: a selected 6-state model can represent protein structures almost as accurately (1.7 A root-mean-square) as a 17-state lattice model (1.6 A root-mean-square). Finally, we use a novel optimization procedure to generate eight 4-state models, which fit native proteins to an average of 2.4 A, and preserve 85% of native residue-residue contacts. We discuss the implications of these findings for protein folding and the prediction of protein conformation.

Algorithms↗
PubMed1995

Structural diversity in a conserved cholera toxin epitope involved in ganglioside binding.

Cholera is a widespread disease for which there is no efficient vaccine. A better understanding of the conformational rearrangements at the epitope might be very helpful for the development of a good vaccine. Cholera toxin (CT) as well as the closely related heat-labile toxin from Escherichia coli (LT) are composed of two subunits, A and B, which form an oligomeric assembly AB5. Residues 50-64 on the surface of the B subunits comprise a conserved loop (CTP3), which is involved in saccharide binding to the receptor on epithelial cells. This loop exhibits remarkable conformational plasticity induced by environmental constraints. The crystal structure of this loop is compared in the free and receptor-bound toxins as well as in the crystal and solution structures of a complex with TE33, a monoclonal antibody elicited against CTP3. In the toxins this loop forms an irregular structure connecting a beta-strand to the central alpha-helix. Ser 55 and Gln 56 exhibit considerable conformational variability in the five subunits of the unliganded toxins. Saccharide binding induces a change primarily in Ser 55 and Gln 56 to a conformation identical in all five copies. Thus, saccharide binding confers rigidity upon the loop. The conformation of CTP3 in complex with TE33 is quite different. The amino-terminal part of CTP3 forms a beta-turn that fits snugly into a deep binding pocket on TE33, in both the crystal and NMR-derived solution structure. Only 8 and 12 residues out of 15 are seen in the NMR and crystal structures, respectively. Despite these conformational differences, TE33 is cross-reactive with intact CT, albeit with a thousandfold decrease in affinity. This suggests a different interaction of TE33 with intact CT.

Amino Acid Sequence↗
PubMed1995

Activity of melphalan in combination with the glutathione transferase inhibitor sulfasalazine.

Glutathione (GSH) transferases (GST), a family of detoxification enzyme proteins, are suggested to play an important role in tumor cell resistance to melphalan. The GST-activity inhibitor ethacrynic acid has been shown to increase the antitumor activity of melphalan in vitro as well as in vivo. In this study we determined the activity and toxicity of melphalan in combination with another GST-activity inhibitor, sulfasalazine, an agent used to treat ulcerative colitis. We entered 37 previously treated patients with advanced cancer of different histologies on sulfasalazine given at the individually calculated maximum tolerated dose (MTD) and melphalan given at doses beginning at 20 mg/m2. The main toxicity arising from this combination was nausea and vomiting, whereas increased myelosuppression was not observed. A partial response was seen in 2/4 of the ovarian cancer patients only. Plasma sulfasalazine levels varied between 2.5 and 47.1 micrograms/ml. Although reductions in GSH/GST levels were observed in peripheral mononuclear cells of certain patients following sulfasalazine treatment, there was no correlation between the extent of reduction and the plasma sulfasalazine level. A larger patient population must be studied to determine the usefulness of this combination.

Drug Administration Schedule↗
PubMed1995

Zonal differences in adrenocortical lipid peroxidation: role of alpha-tocopherol.

Studies were done to evaluate the relationship between alpha-tocopherol (alpha-T) concentrations and lipid peroxidation (LP) in vitro in microsomal preparations from the inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Microsomes were incubated with ferrous ion (Fe2+) to promote free radical production, and alpha-T levels and LP were monitored after various incubation times. alpha-T concentrations were far lower in inner than outer zone preparations and were rapidly depleted from inner zone microsomes by incubation with Fe2+. Coinciding with alpha-T depletion was a large and rapid increase in LP. With outer zone microsomes, alpha-T depletion required more than 30 min, and very little LP was demonstrable during this period. However, once alpha-T depletion occurred, LP was rapidly initiated and reached levels similar to those obtained with inner zone preparations. Inhibition of LP by MnCl2 prevented the Fe(2+)-induced declines in alpha-T in both zones. The results demonstrate the importance of alpha-T as a modulator of adrenal LP and indicate that the zonal differences in LP are largely attributable to the differences in alpha-T concentrations.

Adrenal Cortex↗
PubMed1995

Desire for death in the terminally ill.

OBJECTIVE: Euthanasia and physician-assisted suicide have become prominent medical and social issues. This study investigated the prevalence of the desire for death in terminally ill patients, the stability of this desire over time, and its association with psychiatric disorders. METHOD: Two hundred terminally ill inpatients were given semistructured interviews that assessed their desire for death and evaluated them for major and minor depressive episodes according to the Research Diagnostic Criteria. Each patient also completed a short form of the Beck Depression Inventory and provided ratings of pain and social support. When possible, patients who expressed a desire for death received a follow-up interview after a 2-week interval. RESULTS: Although occasional wishes that death would come soon were common (reported by 44.5% of the patients), only 17 (8.5%) of these individuals acknowledged a serious and pervasive desire to die. The desire for death was correlated with ratings of pain and low family support but most significantly with measures of depression. The prevalence of diagnosed depressive syndromes was 58.8% among patients with a desire to die and 7.7% among patients without such a desire. Follow-up interviews were conducted with six patients; in four cases, the desire to die had decreased during the 2-week interval. CONCLUSIONS: The desire for death in terminally ill patients is closely associated with clinical depression--a potentially treatable condition--and can also decrease over time. Informed debate about euthanasia should recognize the importance of psychiatric considerations, as well as the inherent transience of many patients' expressed desire to die.

Adult↗
PubMed1995

When drug treatment in the elderly is not cost effective. An ethical dilemma in an environment of healthcare rationing.

In light of moves towards the view that rationing of healthcare resources is inevitable, the question arises as to whether age is a relevant consideration. To hold age as relevant in this context attracts the charge of agism; however, age may be related to some other characteristic(s) that are pertinent. In the context of drug treatment of the elderly, there is evidence to suggest that there are important differences between older and younger patients in terms of adverse responses to drugs because of factors such as physiological changes and multiple health problems, which occur with aging. On the other hand, some studies show beneficial effects of drug therapy. There is a need for more empirical data on cost effectiveness. Ethics arguably have to take into account factors other than cost effectiveness, such as equity. A central question is whether what is required is a set of criteria in addition to cost effectiveness, or an interpretation of cost effectiveness sensitive to broader considerations, such as quality of life (QOL). In relation to the latter, the issue of age-specific measurements of QOL remains problematic.

Aged↗
PubMed1994

Exploring conformational space with a simple lattice model for protein structure.

We present a low resolution lattice model for which we can exhaustively generate all possible compact backbone conformations for small proteins. Using simple structural and energetic criteria, for a variety of proteins, we can select for lattice structures that have significant similarities with their known native structures. Our energetic parameters are based on pairwise amino acid contact frequencies in a database of experimentally determined structures. A key step in our method involves the threading of a sequence onto every lattice model, such that a locally optimal pattern of tertiary interactions is formed. We evaluate our results against statistics collected for structures covering all of conformational space, and against statistics collected for permuted sequences. Despite the low resolution of the model, our low energy structures contain many native features. These results indicate that the overall pattern of hydrophobicity of a sequence significantly constrains the range of folds that sequence is likely to adopt.

Algorithms↗
PubMed1994

Induction of squamous differentiation by interferon beta in a human non-small-cell lung cancer cell line.

BACKGROUND: More than 95% of lung cancers occur in the bronchi, appearing as adenocarcinoma, squamous carcinoma, large-cell and small-cell carcinoma, or mixed types. Generally, the least aggressive form is squamous cell lung cancer, suggesting the possibility that promotion of squamous cell differentiation may have therapeutic potential for non-small-cell lung cancer, a disease having no effective systemic therapy. Interferons are a group of glycoproteins with known antiproliferative effects, including the ability to induce differentiation in certain cases. PURPOSE: These studies were conducted to determine whether interferon beta induces squamous cell differentiation in non-small-cell lung cancer in vitro. METHODS: NCI-H596 adenosquamous cells were grown to confluence to maximize their differentiation potential. Growth and parameters for squamous differentiation (cross-linked envelope competence, transglutaminase activity, and relative involucrin expression) were then measured when the cells were exposed to various concentrations of interferon beta. RESULTS: Interferon beta inhibited growth of the NCI-H596 cell line and stimulated envelope competence, involucrin expression, and type 2 transglutaminase activity. Alterations in transglutaminase activity and involucrin expression preceded induction of envelope competence and growth suppression. CONCLUSIONS: Interferon beta suppresses the growth and stimulates markers of squamous differentiation in NCI-H596. While the mechanism(s) for such effects are unknown, the sequence of effects suggests a causal relationship between differentiation induction and subsequent growth suppression. IMPLICATIONS: Interferon beta may have clinical usefulness in squamous differentiation strategies for the treatment of non-small-cell lung cancer. More must be learned about the mechanisms whereby interferons and other biologic agents induce differentiation, and clinical trials will be needed to determine whether in vitro results are pertinent in vivo.

Apoptosis↗
PubMed1994

Different protein sequences can give rise to highly similar folds through different stabilizing interactions.

We report an interesting case of structural similarity between 2 small, nonhomologous proteins, the third domain of ovomucoid (ovomucoid) and the C-terminal fragment of ribosomal L7/L12 protein (CTF). The region of similarity consists of a 3-stranded beta-sheet and an alpha-helix. This region is highly similar; the corresponding elements of secondary structure share a common topology, and the RMS difference for "equivalent" C alpha atoms is 1.6 A. Surprisingly, this common structure arises from completely different sequences. For the common core, the sequence identity is less than 3%, and there is neither significant sequence similarity nor similarity in the position or orientation of conserved hydrophobic residues. This superposition raises the question of how 2 entirely different sequences can produce an identical structure. Analyzing this common region in ovomucoid revealed that it is stabilized by disulfide bonds. In contrast, the corresponding structure in CTF is stabilized in the alpha-helix by a composition of residues with high helix-forming propensities. This result suggests that different sequences and different stabilizing interactions can produce an identical structure.

Amino Acid Sequence↗
PubMed1993

Protein unfolding pathways explored through molecular dynamics simulations.

Herein we describe the results of molecular dynamics simulations of the bovine pancreatic trypsin inhibitor (BPTI) in solution at a variety of temperatures both with and without disulfide bonds. The reduced form of the protein unfolded at high temperature to an ensemble of conformations with all the properties of the molten globule state. In this account we outline the structural details of the actual unfolding process between the native and molten globule states. The first steps of unfolding involved expansion of the protein, which disrupted packing interactions. The solvent-accessible surface area also quickly increased. The unfolding was localized mostly to the turn and loop regions of the molecule, while leaving the secondary structure intact. Then, there was more gradual unfolding of the secondary structure and non-native turns became prevalent. This same trajectory was continued and more drastic unfolding occurred that resulted in a relatively compact state devoid of stable secondary structure.

Aprotinin↗