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Biomedical subjects

David Baker

Publications and source records attributed to David Baker.

At least 109 records · Page 6Linked to original sources
PubMed2004

Computational design of protein-protein interactions.

Computational protein design strategies have been developed to reengineer protein-protein interfaces in an automated, generalizable fashion. In the past two years, these methods have been successfully applied to generate chimeric proteins and protein pairs with specificities different from naturally occurring protein-protein interactions. Although there are shortcomings in current approaches, both in the way conformational space is sampled and in the energy functions used to evaluate designed conformations, the successes suggest we are now entering an era in which computational methods can be used to modulate, reengineer and design protein-protein interaction networks in living cells.

Binding Sites↗
PubMed2004

Exploring folding free energy landscapes using computational protein design.

Recent advances in computational protein design have allowed exciting new insights into the sequence dependence of protein folding free energy landscapes. Whereas most previous studies have examined the sequence dependence of protein stability and folding kinetics by characterizing naturally occurring proteins and variants of these proteins that contain a small number of mutations, it is now possible to generate and characterize computationally designed proteins that differ significantly from naturally occurring proteins in sequence and/or structure. These computer-generated proteins provide insights into the determinants of protein structure, stability and folding, and make it possible to disentangle the properties of proteins that are the consequence of natural selection from those that reflect the fundamental physical chemistry of polypeptide chains.

Computational Biology↗
PubMed2004

Allogeneic bone marrow transplantation in juvenile myelomonocytic leukemia without total body irradiation.

Allogeneic bone marrow transplantation (BMT) without a total body irradiation (TBI) conditioning regimen was investigated in children with juvenile myelomonocytic leukemia (JMML). Eight consecutive patients with JMML (n = 6) or monosomy 7 (n = 2) underwent BMT at a median age of 20 months. Donor source included fully matched related (n = 3), mismatched related (n = 2), or fully matched unrelated (n = 3). The conditioning regimen included busulfan, cyclophosphamide, and etoposide (VP16) (melphalan was substituted for VP16 in one patient). The first patient in the series underwent TBI. Graft-versus-host disease prophylaxis was with cyclosporin and methotrexate and in vivo T-cell depletion (Campath 1 g) for mismatched and unrelated transplants. Seven and two patients, respectively, received chemotherapy and splenectomy before BMT. At a median follow-up of 48 months after BMT, five patients remained in remission. The overall survival rate was 63% at 5 years. All deaths occurred in patients with refractory disease at the time of BMT. Allogeneic BMT without TBI appears to be effective therapy for JMML and avoids some of the potential late sequelae of TBI in preschool children.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2004

Fatigue crack propagation resistance of highly crosslinked polyethylene.

A higher degree of cross-linking has been shown to improve wear properties of ultra-high molecular weight polyethylene in laboratory studies. However, cross-linking can also affect the mechanical properties of ultra-high molecular weight polyethylene. Fatigue crack propagation resistance was determined for electron beam cross-linked ultra-high molecular weight polyethylene and compared with gamma irradiation cross-linked and noncross-linked polyethylene fatigue specimens. Crosslinking was done with different dosages of irradiation followed by melting. For one irradiation dose (50 kGy) extrusion and molding processes were compared. A fracture mechanics approach was used to determine how the degree of cross-linking affects resistance to crack propagation in ultra-high molecular weight polyethylene. Fatigue crack propagation resistance was reduced in proportion to the irradiation dose. The type of irradiation (gamma or electron beam) or manufacturing method (extrusion or molding) did not affect fatigue crack propagation resistance. The reduced fatigue strength of highly cross-linked ultra-high molecular weight polyethylene could lead to mechanical failure in conditions that are associated with cyclic local tensile stresses.

Case-Control Studies↗
PubMed2004

Searching for folded proteins in vitro and in silico.

Understanding the sequence determinants of protein structure, stability and folding is critical for understanding how natural proteins have evolved and how proteins can be engineered to perform novel functions. The complexity of the protein folding problem requires the ability to search large volumes of sequence space for proteins with specific structural or functional characteristics. Here we describe our efforts to identify novel proteins using a phage-display selection strategy from a 'mini-exon' shuffling library generated from the yeast genome and from completely random sequence libraries, and compare the results to recent successes in generating novel proteins using in silico protein design.

Evolution, Molecular↗
PubMed2004

Cannabinoid-mediated neuroprotection following interferon-gamma treatment in a three-dimensional mouse brain aggregate cell culture.

Multiple sclerosis is increasingly recognized as a neurodegenerative disease which is triggered by inflammation in the central nervous system (CNS). Demyelination-associated axonal or neuronal damage is a primary cause of disability and has thus far not been successfully targeted by available drug therapies. The neuroprotective properties of both endogenous and administered cannabinoids have been shown in in vivo and in vitro models of CNS damage following excitotoxic, oxidative, traumatic and ischaemic insults, with a predominantly apoptotic effector mechanism. In this study a foetal mouse telencephalon aggregate cell culture system was developed to compare tissue from cannabinoid receptor 1 knockout mice with wildtype counterparts. Aggregate formation and neurofilament/myelin basic protein accumulation were dependent on the age of foetal dissection and species used. Following treatment with interferon-gamma, levels of myelin basic protein, neurofilament, neuronal dephosphorylation and caspase 3 activation were assessed in telencephalon tissue in vitro. Cytokine treatment resulted in significant loss of the neuronal marker neurofilament-H in cannabinoid receptor 1 knockout cultures but not in wildtypes, indicating that presence of the cannabinoid receptor 1 gene can be neuroprotective. Caspase 3 activation was higher in cultures from knockout animals, indicating an apoptotic mechanism of cell death. Dephosphorylated neurofilament levels were significantly elevated in knockout mice, lending support to the premise that neurofilament dephosphorylation is a marker for neuronal damage. Taken together, these results indicate that neuroprotection could be elicited through the cannabinoid receptor 1, and point towards a potential therapeutic role for cannabinoid compounds in demyelinating conditions such as multiple sclerosis.

Animals↗
PubMed2003

Rapid protein fold determination using unassigned NMR data.

Experimental structure determination by x-ray crystallography and NMR spectroscopy is slow and time-consuming compared with the rate at which new protein sequences are being identified. NMR spectroscopy has the advantage of rapidly providing the structurally relevant information in the form of unassigned chemical shifts (CSs), intensities of NOESY crosspeaks [nuclear Overhauser effects (NOEs)], and residual dipolar couplings (RDCs), but use of these data are limited by the time and effort needed to assign individual resonances to specific atoms. Here, we develop a method for generating low-resolution protein structures by using unassigned NMR data that relies on the de novo protein structure prediction algorithm, rosetta [Simons, K. T., Kooperberg, C., Huang, E. & Baker, D. (1997) J. Mol. Biol. 268, 209-225] and a Monte Carlo procedure that searches for the assignment of resonances to atoms that produces the best fit of the experimental NMR data to a candidate 3D structure. A large ensemble of models is generated from sequence information alone by using rosetta, an optimal assignment is identified for each model, and the models are then ranked based on their fit with the NMR data assuming the identified assignments. The method was tested on nine protein sequences between 56 and 140 amino acids and published CS, NOE, and RDC data. The procedure yielded models with rms deviations between 3 and 6 A, and, in four of the nine cases, the partial assignments obtained by the method could be used to refine the structures to high resolution (0.6-1.8 A) by repeated cycles of structure generation guided by the partial assignments, followed by reassignment using the newly generated models.

Algorithms↗
PubMed2003

Design of a novel globular protein fold with atomic-level accuracy.

A major challenge of computational protein design is the creation of novel proteins with arbitrarily chosen three-dimensional structures. Here, we used a general computational strategy that iterates between sequence design and structure prediction to design a 93-residue alpha/beta protein called Top7 with a novel sequence and topology. Top7 was found experimentally to be folded and extremely stable, and the x-ray crystal structure of Top7 is similar (root mean square deviation equals 1.2 angstroms) to the design model. The ability to design a new protein fold makes possible the exploration of the large regions of the protein universe not yet observed in nature.

Algorithms↗
PubMed2003

Efficient minimization of angle-dependent potentials for polypeptides in internal coordinates.

Angular potentials play an important role in the refinement of protein structures through angle-dependent restraints (e.g., those determined by cross-correlated relaxations, residual dipolar couplings, and hydrogen bonds). Analytic derivatives of such angular potentials with respect to the dihedral angles of proteins would be useful for optimizing such restraints and other types of angular potentials (i.e., such as we are now introducing into protein structure prediction) but have not been described. In this article, analytic derivatives are calculated for four types of angular potentials and integrated with the efficient recursive derivative calculation methods of Gō and coworkers. The formulas are implemented in publicly available software and illustrated by refining a low-resolution protein structure with idealized vector-angle, dipolar-coupling, and hydrogen-bond restraints. The method is now being used routinely to optimize hydrogen-bonding potentials in ROSETTA.

Hydrogen Bonding↗
PubMed2003

Coupled prediction of protein secondary and tertiary structure.

The strong coupling between secondary and tertiary structure formation in protein folding is neglected in most structure prediction methods. In this work we investigate the extent to which nonlocal interactions in predicted tertiary structures can be used to improve secondary structure prediction. The architecture of a neural network for secondary structure prediction that utilizes multiple sequence alignments was extended to accept low-resolution nonlocal tertiary structure information as an additional input. By using this modified network, together with tertiary structure information from native structures, the Q3-prediction accuracy is increased by 7-10% on average and by up to 35% in individual cases for independent test data. By using tertiary structure information from models generated with the ROSETTA de novo tertiary structure prediction method, the Q3-prediction accuracy is improved by 4-5% on average for small and medium-sized single-domain proteins. Analysis of proteins with particularly large improvements in secondary structure prediction using tertiary structure information provides insight into the feedback from tertiary to secondary structure.

Computer Simulation↗
PubMed2003

An improved protein decoy set for testing energy functions for protein structure prediction.

We have improved the original Rosetta centroid/backbone decoy set by increasing the number of proteins and frequency of near native models and by building on sidechains and minimizing clashes. The new set consists of 1,400 model structures for 78 different and diverse protein targets and provides a challenging set for the testing and evaluation of scoring functions. We evaluated the extent to which a variety of all-atom energy functions could identify the native and close-to-native structures in the new decoy sets. Of various implicit solvent models, we found that a solvent-accessible surface area-based solvation provided the best enrichment and discrimination of close-to-native decoys. The combination of this solvation treatment with Lennard Jones terms and the original Rosetta energy provided better enrichment and discrimination than any of the individual terms. The results also highlight the differences in accuracy of NMR and X-ray crystal structures: a large energy gap was observed between native and non-native conformations for X-ray structures but not for NMR structures.

Algorithms↗
PubMed2003

A large scale test of computational protein design: folding and stability of nine completely redesigned globular proteins.

A previously developed computer program for protein design, RosettaDesign, was used to predict low free energy sequences for nine naturally occurring protein backbones. RosettaDesign had no knowledge of the naturally occurring sequences and on average 65% of the residues in the designed sequences differ from wild-type. Synthetic genes for ten completely redesigned proteins were generated, and the proteins were expressed, purified, and then characterized using circular dichroism, chemical and temperature denaturation and NMR experiments. Although high-resolution structures have not yet been determined, eight of these proteins appear to be folded and their circular dichroism spectra are similar to those of their wild-type counterparts. Six of the proteins have stabilities equal to or up to 7kcal/mol greater than their wild-type counterparts, and four of the proteins have NMR spectra consistent with a well-packed, rigid structure. These encouraging results indicate that the computational protein design methods can, with significant reliability, identify amino acid sequences compatible with a target protein backbone.

Amino Acid Sequence↗
PubMed2003

Deciphering a novel thioredoxin-like fold family.

Sequence--and structure-based searching strategies have proven useful in the identification of remote homologs and have facilitated both structural and functional predictions of many uncharacterized protein families. We implement these strategies to predict the structure of and to classify a previously uncharacterized cluster of orthologs (COG3019) in the thioredoxin-like fold superfamily. The results of each searching method indicate that thioltransferases are the closest structural family to COG3019. We substantiate this conclusion using the ab initio structure prediction method rosetta, which generates a thioredoxin-like fold similar to that of the glutaredoxin-like thioltransferase (NrdH) for a COG3019 target sequence. This structural model contains the thiol-redox functional motif CYS-X-X-CYS in close proximity to other absolutely conserved COG3019 residues, defining a novel thioredoxin-like active site that potentially binds metal ions. Finally, the rosetta-derived model structure assists us in assembling a global multiple-sequence alignment of COG3019 with two other thioredoxin-like fold families, the thioltransferases and the bacterial arsenate reductases (ArsC).

Amino Acid Sequence↗
PubMed2003

Conserved residue clustering and protein structure prediction.

Protein residues that are critical for structure and function are expected to be conserved throughout evolution. Here, we investigate the extent to which these conserved residues are clustered in three-dimensional protein structures. In 92% of the proteins in a data set of 79 proteins, the most conserved positions in multiple sequence alignments are significantly more clustered than randomly selected sets of positions. The comparison to random subsets is not necessarily appropriate, however, because the signal could be the result of differences in the amino acid composition of sets of conserved residues compared to random subsets (hydrophobic residues tend to be close together in the protein core), or differences in sequence separation of the residues in the different sets. In order to overcome these limits, we compare the degree of clustering of the conserved positions on the native structure and on alternative conformations generated by the de novo structure prediction method Rosetta. For 65% of the 79 proteins, the conserved residues are significantly more clustered in the native structure than in the alternative conformations, indicating that the clustering of conserved residues in protein structures goes beyond that expected purely from sequence locality and composition effects. The differences in the spatial distribution of conserved residues can be utilized in de novo protein structure prediction: We find that for 79% of the proteins, selection of the Rosetta generated conformations with the greatest clustering of the conserved residues significantly enriches the fraction of close-to-native structures.

Acyl Coenzyme A↗
PubMed2003

Protein-protein docking with simultaneous optimization of rigid-body displacement and side-chain conformations.

Protein-protein docking algorithms provide a means to elucidate structural details for presently unknown complexes. Here, we present and evaluate a new method to predict protein-protein complexes from the coordinates of the unbound monomer components. The method employs a low-resolution, rigid-body, Monte Carlo search followed by simultaneous optimization of backbone displacement and side-chain conformations using Monte Carlo minimization. Up to 10(5) independent simulations are carried out, and the resulting "decoys" are ranked using an energy function dominated by van der Waals interactions, an implicit solvation model, and an orientation-dependent hydrogen bonding potential. Top-ranking decoys are clustered to select the final predictions. Small-perturbation studies reveal the formation of binding funnels in 42 of 54 cases using coordinates derived from the bound complexes and in 32 of 54 cases using independently determined coordinates of one or both monomers. Experimental binding affinities correlate with the calculated score function and explain the predictive success or failure of many targets. Global searches using one or both unbound components predict at least 25% of the native residue-residue contacts in 28 of the 32 cases where binding funnels exist. The results suggest that the method may soon be useful for generating models of biologically important complexes from the structures of the isolated components, but they also highlight the challenges that must be met to achieve consistent and accurate prediction of protein-protein interactions.

Algorithms↗