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Biomedical subjects

M Levitt

Publications and source records attributed to M Levitt.

At least 19 recordsLinked to original sources
PubMed2003

Protein decoy assembly using short fragments under geometric constraints.

A small set of protein fragments can represent adequately all known local protein structure. This set of fragments, along with a construction scheme that assembles these fragments into structures, defines a discrete (relatively small) conformation space, which approximates protein structures accurately. We generate protein decoys by sampling geometrically valid structures from this conformation space, biased by the secondary structure prediction for the protein. Unlike other methods, secondary structure prediction is the only protein-specific information used for generating the decoys. Nevertheless, these decoys are qualitatively similar to those found by others. The method works well for all-alpha proteins, and shows promising results for alpha and beta proteins.

Databases, Protein↗
PubMed2001

Quantification of the hydrophobic interaction by simulations of the aggregation of small hydrophobic solutes in water.

The hydrophobic interaction, the tendency for nonpolar molecules to aggregate in solution, is a major driving force in biology. In a direct approach to the physical basis of the hydrophobic effect, nanosecond molecular dynamics simulations were performed on increasing numbers of hydrocarbon solute molecules in water-filled boxes of different sizes. The intermittent formation of solute clusters gives a free energy that is proportional to the loss in exposed molecular surface area with a constant of proportionality of 45 +/- 6 cal/mol A(2). The molecular surface area is the envelope of the solute cluster that is impenetrable by solvent and is somewhat smaller than the more traditional solvent-accessible surface area, which is the area transcribed by the radius of a solvent molecule rolled over the surface of the cluster. When we apply a factor relating molecular surface area to solvent-accessible surface area, we obtain 24 cal/mol A(2). Ours is the first direct calculation, to our knowledge, of the hydrophobic interaction from molecular dynamics simulations; the excellent qualitative and quantitative agreement with experiment proves that simple van der Waals interactions and atomic point-charge electrostatics account for the most important driving force in biology.

Computer Simulation↗
PubMed2001

A novel method for sampling alpha-helical protein backbones.

We present a novel technique of sampling the configurations of helical proteins. Assuming knowledge of native secondary structure, we employ assembly rules gathered from a database of existing structures to enumerate the geometrically possible three-dimensional arrangements of the constituent helices. We produce a library of possible folds for 25 helical protein cores. In each case, our method finds significant numbers of conformations close to the native structure. In addition, we assign coordinates to all atoms for four of the 25 proteins and show that this has a small effect on the number of near-native conformations. In the context of database driven exhaustive enumeration our method performs extremely well, yielding significant percentages of conformations (between 0.02% and 82%) within 6 A of the native structure. The method's speed and efficiency make it a valuable tool for predicting protein structure.

Amino Acid Sequence↗
PubMed2001

Euroscreen 2: towards community policy on insurance, commercialization and public awareness.

The project Euroscreen 2 has examined genetic screening and testing with particular reference to implications for insurance, commercialization through marketing of genetic tests direct to the public, and issues surrounding raising public awareness of these and other developments in genetics, including the practical experiment of a Gene Shop. This paper provides a snapshot of the three year project. The study group's work included monitoring developments in different European countries and exploring possibilities for regulation in insurance and commercialization together with public attitudes to regulation. The success or failure of different strategies is not independent of public awareness. Exploration of policy, however, also requires examination of fundamental concepts such as solidarity and geneticization.

Attitude to Health↗
PubMed2001

'Not so wrong that we are prepared to threaten the entire service': the regulation of reproductive technologies in the UK.

In the UK human embryo research and infertility treatments are regulated by the Human Fertilisation and Embryology Authority (HFEA), a national statutory body set up in 1991. Clinics are required by law to be licensed by the HFEA and open to inspection. In 1999 there were 107 clinics licensed to carry out IVF and/or donor insemination. There are regional variations in National Health Service funding and seventy-five percent of those treated pay for most of their treatment, usually to avoid long waiting lists or because they do not meet the eligibility criteria set by their local health authority. Having described the code of practice laid down by the HFEA the paper discusses the implications of new reproductive technologies from a social science perspective, focusing on three topics which continue to be debated by the media, the regulatory authorities, health professionals and 'lay' people. First, age limits and other criteria for assisted reproduction which raise the question 'who should be allowed to reproduce?' Second, donor anonymity and the question of payment for donors. Finally, the screening and selection of embryos for implantation and the recurring theme in the media of 'designer babies'. The HFEA can be seen to fulfill its intended role, mediating between the infertility professionals, patients and the wider general public. It seeks to allay public fears while supporting professionals by avoiding restrictions on research and treatment and by consulting with the public as to when extensions to research will be acceptable.

Confidentiality↗
PubMed2000

Ab initio construction of protein tertiary structures using a hierarchical approach.

We present a hierarchical method to predict protein tertiary structure models from sequence. We start with complete enumeration of conformations using a simple tetrahedral lattice model. We then build conformations with increasing detail, and at each step select a subset of conformations using empirical energy functions with increasing complexity. After enumeration on lattice, we select a subset of low energy conformations using a statistical residue-residue contact energy function, and generate all-atom models using predicted secondary structure. A combined knowledge-based atomic level energy function is then used to select subsets of the all-atom models. The final predictions are generated using a consensus distance geometry procedure. We test the feasibility of the procedure on a set of 12 small proteins covering a wide range of protein topologies. A rigorous double-blind test of our method was made under the auspices of the CASP3 experiment, where we did ab initio structure predictions for 12 proteins using this approach. The performance of our methodology at CASP3 is reasonably good and completely consistent with our initial tests.

Algorithms↗
PubMed2000

The ASTRAL compendium for protein structure and sequence analysis.

The ASTRAL compendium provides several databases and tools to aid in the analysis of protein structures, particularly through the use of their sequences. The SPACI scores included in the system summarize the overall characteristics of a protein structure. A structural alignments database indicates residue equivalencies in superimposed protein domain structures. The PDB sequence-map files provide a linkage between the amino acid sequence of the molecule studied (SEQRES records in a database entry) and the sequence of the atoms experimentally observed in the structure (ATOM records). These maps are combined with information in the SCOPdatabase to provide sequences of protein domains. Selected subsets of the domain database, with varying degrees of similarity measured in several different ways, are also available. ASTRALmay be accessed at http://astral.stanford.edu/

Amino Acid Sequence↗
PubMed2000

Colonic J pouch reconstruction of the radiation-damaged neorectum.

BACKGROUND: Postoperative radiotherapy after restorative resection for rectal carcinoma can cause fibrosis, stricture and proctitis. There are few accounts in the literature of the surgical approach to the radiation-damaged neorectum. METHODS: Reported are four patients who underwent resection of the radiation-damaged neorectum with colonic J pouch reconstruction between September 1996 and July 1997. The operative and postoperative course is described. Parameters of bowel function were assessed by standardized interview 6 months after ileostomy reversal. Ongoing follow up has occurred to the present time. RESULTS: There was no mortality. Complications were splenic injury requiring splenectomy and a pelvic collection requiring percutaneous drainage. At 6 months after ileostomy reversal all patients had ongoing disturbance of bowel function, and two patients described some degree of incontinence. The mean frequency of bowel actions was four per day. Two patients have subsequently undergone further resection and formation of an end stoma. CONCLUSIONS: Resection of the radiation-damaged neorectum with colonic J pouch reconstruction is technically feasible. Functional results in the present series were disappointing and half the patients went on to formation of a permanent stoma.

Adult↗
PubMed2000

Constructing side chains on near-native main chains for ab initio protein structure prediction.

Is there value in constructing side chains while searching protein conformational space during an ab initio simulation? If so, what is the most computationally efficient method for constructing these side chains? To answer these questions, four published approaches were used to construct side chain conformations on a range of near-native main chains generated by ab initio protein structure prediction methods. The accuracy of these approaches was compared with a naive approach that selects the most frequently observed rotamer for a given amino acid to construct side chains. An all-atom conditional probability discriminatory function is useful at selecting conformations with overall low all-atom root mean square deviation (r.m.s.d.) and the discrimination improves on sets that are closer to the native conformation. In addition, the naive approach performs as well as more sophisticated methods in terms of the percentage of chi(1) angles built accurately and the all-atom r. m.s.d., between the native and near-native conformations. The results suggest that the naive method would be extremely useful for fast and efficient side chain construction on vast numbers of conformations for ab initio prediction of protein structure.

Amino Acids↗
PubMed2000

Expectations from structural genomics.

Structural genomics projects aim to provide an experimental structure or a good model for every protein in all completed genomes. Most of the experimental work for these projects will be directed toward proteins whose fold cannot be readily recognized by simple sequence comparison with proteins of known structure. Based on the history of proteins classified in the SCOP structure database, we expect that only about a quarter of the early structural genomics targets will have a new fold. Among the remaining ones, about half are likely to be evolutionarily related to proteins of known structure, even though the homology could not be readily detected by sequence analysis.

Databases, Factual↗
PubMed2000

Decoys 'R' Us: a database of incorrect conformations to improve protein structure prediction.

The development of an energy or scoring function for protein structure prediction is greatly enhanced by testing the function on a set of computer-generated conformations (decoys) to determine whether it can readily distinguish native-like conformations from nonnative ones. We have created "Decoys 'R' Us," a database containing many such sets of conformations, to provide a resource that allows scoring functions to be improved.

Algorithms↗
PubMed2000

Probing structure-function relationships of the DNA polymerase alpha-associated zinc-finger protein using computational approaches.

We present the application of a method for protein structure prediction to aid the determination of structure-function relationships by experiment. The structure prediction method was rigourously tested by making blind predictions at the third meeting on the Critical Assessment of Protein Structure methods (CASP3). The method is a combined hierarchical approach involving exhaustive enumeration of all possible folds of a small protein sequence on a tetrahedral lattice. A set of filters, primarily in the form of discriminatory functions, are applied to these conformations. As the filters are applied, greater detail is added to the models resulting in a handful of all-atom "final" conformations. Encouraged by the results at CASP3, we used our approach to help solve a practical biological problem: the prediction of the structure and function of the 67-residue C-terminal zinc-finger region of the DNA polymerase alpha-associated zinc-finger (PAZ) protein. We discuss how the prediction points to a novel function relative to the sequence homologs, in conjunction with evidence from experiment, and how the predicted structure is guiding further experimental studies. This work represents a move from the theoretical realm to actual application of structure prediction methods for gaining unique insight to guide experimental biologists.

Amino Acid Sequence↗
PubMed2000

Towards a complete map of the protein space based on a unified sequence and structure analysis of all known proteins.

In search for global principles that may explain the organization of the space of all possible proteins, we study all known protein sequences and structures. In this paper we present a global map of the protein space based on our analysis. Our protein space contains all protein sequences in a non-redundant (NR) database, which includes all major sequence databases. Using the PSI-BLAST procedure we defined 4,670 clusters of related sequences in this space. Of these clusters, 1,421 are centered on a sequence of known structure. All 4,670 clusters were then compared using either a structure metric (when 3D structures are known) or a novel sequence profile metric. These scores were used to define a unified and consistent metric between all clusters. Two schemes were employed to organize these clusters in a meta-organization. The first uses a graph theory method and cluster the clusters in an hierarchical organization. This organization extends our ability to predict the structure and function of many proteins beyond what is possible with existing tools for sequence analysis. The second uses a variation on a multidimensional scaling technique to embed the clusters in a low dimensional real space. This last approach resulted in a projection of the protein space onto a 2D plane that provides us with a bird's eye view of the protein space. Based on this map we suggest a list of possible target sequences with unknown structure that are likely to adopt new, unknown folds.

Algorithms↗
PubMed1999

Oxidative stress increases ubiquitin--protein conjugates in synaptosomes.

Synaptosomes were incubated in the presence of FeSO4 to test the hypothesis that iron-catalyzed oxidative damage causes an increase in the ubiquitination of synaptosomal proteins. Incubation with 10 or 50 microM FeSO4 caused concentration-dependent increases in carbonyl groups (an indication of protein oxidation) and ubiquitinated proteins (determined by probing Western blots with a monoclonal antibody to ubiquitin). Differences in protein ubiquitination occurred within 5 min of incubation, indicating a rapid response to oxidative stress. Results of experiments with MG-132, an inhibitor of the degradation of ubiquitinated proteins, suggested that oxidative damage stimulated ubiquitination rather than inhibited degradation of ubiquitinated proteins. The data are consistent with the hypothesis that synaptic terminals utilize the ubiquitin/proteasome proteolytic pathway to degrade oxidatively damaged proteins.

Animals↗
PubMed1999

De novo protein design. I. In search of stability and specificity.

We have developed a fully automated protein design strategy that works on the entire sequence of the protein and uses a full atom representation. At each step of the procedure, an all-atom model of the protein is built using the template protein structure and the current designed sequence. The energy of the model is used to drive a Monte Carlo optimization in sequence space: random moves are either accepted or rejected based on the Metropolis criterion. We rely on the physical forces that stabilize native protein structures to choose the optimum sequence. Our energy function includes van der Waals interactions, electrostatics and an environment free energy. Successful protein design should be specific and generate a sequence compatible with the template fold and incompatible with competing folds. We impose specificity by maintaining the amino acid composition constant, based on the random energy model. The specificity of the optimized sequence is tested by fold recognition techniques. Successful sequence designs for the B1 domain of protein G, for the lambda repressor and for sperm whale myoglobin are presented. We show that each additional term of the energy function improves the performance of our design procedure: the van der Waals term ensures correct packing, the electrostatics term increases the specificity for the correct native fold, and the environment solvation term ensures a correct pattern of buried hydrophobic and exposed hydrophilic residues. For the globin family, we show that we can design a protein sequence that is stable in the myoglobin fold, yet incompatible with the very similar hemoglobin fold.

Amino Acid Sequence↗
PubMed1999

De novo protein design. II. Plasticity in sequence space.

It is generally accepted that many different protein sequences have similar folded structures, and that there is a relatively high probability that a new sequence possesses a previously observed fold. An indirect consequence of this is that protein design should define the sequence space accessible to a given structure, rather than providing a single optimized sequence. We have recently developed a new approach for protein sequence design, which optimizes the complete sequence of a protein based on the knowledge of its backbone structure, its amino acid composition and a physical energy function including van der Waals interactions, electrostatics, and environment free energy. The specificity of the designed sequence for its template backbone is imposed by keeping the amino acid composition fixed. Here, we show that our procedure converges in sequence space, albeit not to the native sequence of the protein. We observe that while polar residues are well conserved in our designed sequences, non-polar amino acids at the surface of a protein are often replaced by polar residues. The designed sequences provide a multiple alignment of sequences that all adopt the same three-dimensional fold. This alignment is used to derive a profile matrix for chicken triose phosphate isomerase, TIM. The matrix is found to recognize significantly the native sequence for TIM, as well as closely related sequences. Possible application of this approach to protein fold recognition is discussed.

Amino Acid Sequence↗