Biomedical subjects

David Baker

Publications and source records attributed to David Baker.

At least 91 records · Page 5Linked to original sources

PubMed2004

Improvement of comparative model accuracy by free-energy optimization along principal components of natural structural variation.

Accurate high-resolution refinement of protein structure models is a formidable challenge because of the delicate balance of forces in the native state, the difficulty in sampling the very large number of alternative tightly packed conformations, and the inaccuracies in current force fields. Indeed, energy-based refinement of comparative models generally leads to degradation rather than improvement in model quality, and, hence, most current comparative modeling procedures omit physically based refinement. However, despite their inaccuracies, current force fields do contain information that is orthogonal to the evolutionary information on which comparative models are based, and, hence, refinement might be able to improve comparative models if the space that is sampled is restricted sufficiently so that false attractors are avoided. Here, we use the principal components of the variation of backbone structures within a homologous family to define a small number of evolutionarily favored sampling directions and show that model quality can be improved by energy-based optimization along these directions.

Models, Molecular↗

PubMed2004

A new hydrogen-bonding potential for the design of protein-RNA interactions predicts specific contacts and discriminates decoys.

RNA-binding proteins play many essential roles in the regulation of gene expression in the cell. Despite the significant increase in the number of structures for RNA-protein complexes in the last few years, the molecular basis of specificity remains unclear even for the best-studied protein families. We have developed a distance and orientation-dependent hydrogen-bonding potential based on the statistical analysis of hydrogen-bonding geometries that are observed in high-resolution crystal structures of protein-DNA and protein-RNA complexes. We observe very strong geometrical preferences that reflect significant energetic constraints on the relative placement of hydrogen-bonding atom pairs at protein-nucleic acid interfaces. A scoring function based on the hydrogen-bonding potential discriminates native protein-RNA structures from incorrectly docked decoys with remarkable predictive power. By incorporating the new hydrogen-bonding potential into a physical model of protein-RNA interfaces with full atom representation, we were able to recover native amino acids at protein-RNA interfaces.

Amino Acid Sequence↗

PubMed2004

Analysis of anisotropic side-chain packing in proteins and application to high-resolution structure prediction.

pi-pi, Cation-pi, and hydrophobic packing interactions contribute specificity to protein folding and stability to the native state. As a step towards developing improved models of these interactions in proteins, we compare the side-chain packing arrangements in native proteins to those found in compact decoys produced by the Rosetta de novo structure prediction method. We find enrichments in the native distributions for T-shaped and parallel offset arrangements of aromatic residue pairs, in parallel stacked arrangements of cation-aromatic pairs, in parallel stacked pairs involving proline residues, and in parallel offset arrangements for aliphatic residue pairs. We then investigate the extent to which the distinctive features of native packing can be explained using Lennard-Jones and electrostatics models. Finally, we derive orientation-dependent pi-pi, cation-pi and hydrophobic interaction potentials based on the differences between the native and compact decoy distributions and investigate their efficacy for high-resolution protein structure prediction. Surprisingly, the orientation-dependent potential derived from the packing arrangements of aliphatic side-chain pairs distinguishes the native structure from compact decoys better than the orientation-dependent potentials describing pi-pi and cation-pi interactions.

Amino Acids↗

PubMed2004

Protein structure prediction and analysis using the Robetta server.

The Robetta server (http://robetta.bakerlab.org) provides automated tools for protein structure prediction and analysis. For structure prediction, sequences submitted to the server are parsed into putative domains and structural models are generated using either comparative modeling or de novo structure prediction methods. If a confident match to a protein of known structure is found using BLAST, PSI-BLAST, FFAS03 or 3D-Jury, it is used as a template for comparative modeling. If no match is found, structure predictions are made using the de novo Rosetta fragment insertion method. Experimental nuclear magnetic resonance (NMR) constraints data can also be submitted with a query sequence for RosettaNMR de novo structure determination. Other current capabilities include the prediction of the effects of mutations on protein-protein interactions using computational interface alanine scanning. The Rosetta protein design and protein-protein docking methodologies will soon be available through the server as well.

Alanine↗

PubMed2004

Maternal toxicity with continuous nevirapine in pregnancy: results from PACTG 1022.

OBJECTIVE: To compare the safety of nelfinavir and nevirapine-based antiretroviral treatment in HIV-1-infected pregnant women. METHODS: In Pediatric AIDS Clinical Trials Group Protocol 1022, 38 antiretroviral-naive pregnant women at 10-30 weeks' gestation were randomized to nelfinavir or nevirapine with zidovudine plus lamivudine. The study was suspended because of greater than expected toxicity and changes in nevirapine prescribing information. The incidence of treatment-limiting hepatic or cutaneous toxicity was compared between groups for all subjects and for the subset with CD4 cell counts greater than 250 cells/microL at study entry. RESULTS: Toxicity was seen in 1 (5%) of 21 subjects randomized to nelfinavir and 5 (29%) of 17 subjects randomized to nevirapine (P = 0.07). Within the nevirapine group, 1 subject developed fulminant hepatic failure and died, and another developed Stevens-Johnson syndrome. The one adverse event associated with nelfinavir occurred in a subject with a CD4 cell count less than 250 cells/microL. All 5 events among subjects with a CD4 cell count greater than 250 cells/microL were associated with nevirapine (P = 0.04). CONCLUSIONS: Continuous nevirapine may be associated with increased toxicity among HIV-1-infected pregnant women with CD4 cell counts greater than 250 cells/microL, as has been observed in non-pregnant women.

Adult↗

PubMed2004

Modeling structurally variable regions in homologous proteins with rosetta.

A major limitation of current comparative modeling methods is the accuracy with which regions that are structurally divergent from homologues of known structure can be modeled. Because structural differences between homologous proteins are responsible for variations in protein function and specificity, the ability to model these differences has important functional consequences. Although existing methods can provide reasonably accurate models of short loop regions, modeling longer structurally divergent regions is an unsolved problem. Here we describe a method based on the de novo structure prediction algorithm, Rosetta, for predicting conformations of structurally divergent regions in comparative models. Initial conformations for short segments are selected from the protein structure database, whereas longer segments are built up by using three- and nine-residue fragments drawn from the database and combined by using the Rosetta algorithm. A gap closure term in the potential in combination with modified Newton's method for gradient descent minimization is used to ensure continuity of the peptide backbone. Conformations of variable regions are refined in the context of a fixed template structure using Monte Carlo minimization together with rapid repacking of side-chains to iteratively optimize backbone torsion angles and side-chain rotamers. For short loops, mean accuracies of 0.69, 1.45, and 3.62 A are obtained for 4, 8, and 12 residue loops, respectively. In addition, the method can provide reasonable models of conformations of longer protein segments: predicted conformations of 3A root-mean-square deviation or better were obtained for 5 of 10 examples of segments ranging from 13 to 34 residues. In combination with a sequence alignment algorithm, this method generates complete, ungapped models of protein structures, including regions both similar to and divergent from a homologous structure. This combined method was used to make predictions for 28 protein domains in the Critical Assessment of Protein Structure 4 (CASP 4) and 59 domains in CASP 5, where the method ranked highly among comparative modeling and fold recognition methods. Model accuracy in these blind predictions is dominated by alignment quality, but in the context of accurate alignments, long protein segments can be accurately modeled. Notably, the method correctly predicted the local structure of a 39-residue insertion into a TIM barrel in CASP 5 target T0186.

Algorithms↗

PubMed2004

Realistic protein-protein association rates from a simple diffusional model neglecting long-range interactions, free energy barriers, and landscape ruggedness.

We develop a simple but rigorous model of protein-protein association kinetics based on diffusional association on free energy landscapes obtained by sampling configurations within and surrounding the native complex binding funnels. Guided by results obtained on exactly solvable model problems, we transform the problem of diffusion in a potential into free diffusion in the presence of an absorbing zone spanning the entrance to the binding funnel. The free diffusion problem is solved using a recently derived analytic expression for the rate of association of asymmetrically oriented molecules. Despite the required high steric specificity and the absence of long-range attractive interactions, the computed rates are typically on the order of 10(4)-10(6) M(-1) sec(-1), several orders of magnitude higher than rates obtained using a purely probabilistic model in which the association rate for free diffusion of uniformly reactive molecules is multiplied by the probability of a correct alignment of the two partners in a random collision. As the association rates of many protein-protein complexes are also in the 10(5)-10(6) M(-1) sec(-1) range, our results suggest that free energy barriers arising from desolvation and/or side-chain freezing during complex formation or increased ruggedness within the binding funnel, which are completely neglected in our simple diffusional model, do not contribute significantly to the dynamics of protein-protein association. The transparent physical interpretation of our approach that computes association rates directly from the size and geometry of protein-protein binding funnels makes it a useful complement to Brownian dynamics simulations.

Computer Simulation↗

PubMed2004

Characterization of the folding energy landscapes of computer generated proteins suggests high folding free energy barriers and cooperativity may be consequences of natural selection.

To determine the extent to which protein folding rates and free energy landscapes have been shaped by natural selection, we have examined the folding kinetics of five proteins generated using computational design methods and, hence, never exposed to natural selection. Four of these proteins are complete computer-generated redesigns of naturally occurring structures and the fifth protein, called Top7, has a computer-generated fold not yet observed in nature. We find that three of the four redesigned proteins fold much faster than their naturally occurring counterparts. While natural selection thus does not appear to operate on protein folding rates, the majority of the designed proteins unfold considerably faster than their naturally occurring counterparts, suggesting possible selection for a high free energy barrier to unfolding. In contrast to almost all naturally occurring proteins of less than 100 residues but consistent with simple computational models, the folding energy landscape for Top7 appears to be quite complex, suggesting the smooth energy landscapes and highly cooperative folding transitions observed for small naturally occurring proteins may also reflect the workings of natural selection.

Computational Biology↗

PubMed2004

Close agreement between the orientation dependence of hydrogen bonds observed in protein structures and quantum mechanical calculations.

Hydrogen bonding is a key contributor to the exquisite specificity of the interactions within and between biological macromolecules, and hence accurate modeling of such interactions requires an accurate description of hydrogen bonding energetics. Here we investigate the orientation and distance dependence of hydrogen bonding energetics by combining two quite disparate but complementary approaches: quantum mechanical electronic structure calculations and protein structural analysis. We find a remarkable agreement between the energy landscapes obtained from the electronic structure calculations and the distributions of hydrogen bond geometries observed in protein structures. In contrast, molecular mechanics force fields commonly used for biomolecular simulations do not consistently exhibit close correspondence to either quantum mechanical calculations or experimentally observed hydrogen bonding geometries. These results suggest a route to improved energy functions for biological macromolecules that combines the generality of quantum mechanical electronic structure calculations with the accurate context dependence implicit in protein structural analysis.

Amino Acids↗

PubMed2004

Computational redesign of protein-protein interaction specificity.

We developed a 'computational second-site suppressor' strategy to redesign specificity at a protein-protein interface and applied it to create new specifically interacting DNase-inhibitor protein pairs. We demonstrate that the designed switch in specificity holds in in vitro binding and functional assays. We also show that the designed interfaces are specific in the natural functional context in living cells, and present the first high-resolution X-ray crystallographic analysis of a computer-redesigned functional protein-protein interface with altered specificity. The approach should be applicable to the design of interacting protein pairs with novel specificities for delineating and re-engineering protein interaction networks in living cells.

Bacterial Proteins↗

PubMed2004

Contributions of amino acid side chains to the kinetics and thermodynamics of the bivalent binding of protein L to Ig kappa light chain.

Protein L is a bacterial surface protein with 4-5 immunoglobulin (Ig)-binding domains (B1-B5), each of which appears to have two binding sites for Ig, corresponding to the two edges of its beta-sheet. To verify these sites biochemically and to probe their relative contributions to the protein L-Ig kappa light chain (kappa) interaction, we compared the binding of PLW (the Y47W mutant of the B1 domain) to that of mutants designed to disrupt binding to sites 1 and 2, using gel filtration, BIAcore surface plasmon resonance, fluorescence titration, and solid-phase radioimmunoassays. Gel filtration experiments show that PLW binds kappa both in 1:1 complexes and multivalently, consistent with two binding sites. Covalent dimers of the A20C and V51C mutants of PLW were prepared to eliminate site 1 and site 2 binding, respectively; both the A20C and V51C dimers bind kappa in 1:1 complexes and multivalently, indicating that neither site 1 nor site 2 is solely responsible for kappa binding. The A20R mutant was designed computationally to eliminate site 1 binding while preserving site 2 binding; consistent with this design, the A20R mutant binds kappa in 1:1 complexes but not multivalently. To probe the contributions of amino acid side chains to binding, we prepared 75 point mutants spanning nearly every residue of PLW; BIAcore studies of these mutants revealed two binding-energy "hot spots" consistent with sites 1 and 2. These data indicate that PLW binds kappa at both sites with similar affinities (high nanomolar), with the strongest contributions to the binding energy from Tyr34 (site 2) and Tyr36 (site 1). Compared to other protein-protein complexes, the binding is insensitive to amino acid substitutions at these sites, consistent with the large number of main chain interactions relative to side chain interactions. The strong binding of protein L to Ig kappa light chains of various species may result from the ambidextrous binding of the B1-B5 domains and the unimportance of specific side chain interactions.

Alanine↗

PubMed2004

Protein structure prediction for the male-specific region of the human Y chromosome.

The complete sequence of the male-specific region of the human Y chromosome (MSY) has been determined recently; however, detailed characterization for many of its encoded proteins still remains to be done. We applied state-of-the-art protein structure prediction methods to all 27 distinct MSY-encoded proteins to provide better understanding of their biological functions and their mechanisms of action at the molecular level. The results of such large-scale structure-functional annotation provide a comprehensive view of the MSY proteome, shedding light on MSY-related processes. We found that, in total, at least 60 domains are encoded by 27 distinct MSY genes, of which 42 (70%) were reliably mapped to currently known structures. The most challenging predictions include the unexpected but confident 3D structure assignments for three domains identified here encoded by the USP9Y, UTY, and BPY2 genes. The domains with unknown 3D structures that are not predictable with currently available theoretical methods are established as primary targets for crystallographic or NMR studies. The data presented here set up the basis for additional scientific discoveries in human biology of the Y chromosome, which plays a fundamental role in sex determination.

Amino Acid Sequence↗

PubMed2004

No effect of rosiglitazone for treatment of HIV-1 lipoatrophy: randomised, double-blind, placebo-controlled trial.

BACKGROUND: Lipodystrophy commonly complicates antiretroviral therapy of HIV-1 infection. Thiazolidinediones such as rosiglitazone promote subcutaneous fat growth in type 2 diabetics and adults with congenital lipodystrophy, and can prevent HIV-1 protease inhibitor toxicity to adipocytes in vitro. We postulated that rosiglitazone would improve HIV lipoatrophy. METHODS: 108 HIV-1-infected lipoatrophic adults on antiretroviral therapy were randomised to rosiglitazone 4 mg twice daily (n=53) or matching placebo (n=55) for 48 weeks. The study had 80% power to detect a 0.5 kg difference in changes in limb fat (using dual-energy X-ray absorptiometry) between groups at week 48 by intention-to-treat analysis, and a 0.7 kg difference within each protease inhibitor stratum. FINDINGS: Limb fat increased by 0.14 kg in the rosiglitazone group and 0.18 kg in the placebo group (mean difference -0.04 kg [95%CI -0.29 to 0.21]; p=0.74 by t test), with three participants (one on rosiglitazone and two controls), lost to follow-up. Rosiglitazone had no significant benefit on any other measure of lipodystrophy, despite large relative increases in plasma adiponectin (4.2 mmol/L [102%]; p<0.0001) and in three markers of insulin sensitivity (p=0.01 to 0.02). Six participants ceased study drug in each group, four participants (three on rosiglitazone and one control) for related adverse events. The main adverse effects, which seem to be almost unique to this population, were asymptomatic hypertriglyceridaemia (mean relative increase 0.9 mmol/L at week 48; p=0.04) and hypercholesterolaemia (1.5 mmol/L; p=0.001). INTERPRETATION: Rosiglitazone for 48 weeks did not improve lipoatrophy in HIV-1-infected adults receiving antiretroviral therapy. Use of less toxic antiretroviral treatment is necessary to prevent lipoatrophy.

Anemia↗

PubMed2004

Computational alanine scanning of protein-protein interfaces.

Protein-protein interactions are key components of all signal transduction processes, so methods to alter these interactions promise to become important tools in dissecting function of connectivities in these networks. We have developed a fast computational approach for the prediction of energetically important amino acid residues in protein-protein interfaces (available at http://robetta.bakerlab.org/alaninescan), which we, following Peter Kollman, have termed "computational alanine scanning." The input consists of a three-dimensional structure of a protein-protein complex; output is a list of "hot spots," or amino acid side chains that are predicted to significantly destabilize the interface when mutated to alanine, analogous to the results of experimental alanine-scanning mutagenesis. 79% of hot spots and 68% of neutral residues were correctly predicted in a test of 233 mutations in 19 protein-protein complexes. A single interface can be analyzed in minutes. The computational methodology has been validated by the successful design of protein interfaces with new specificity and activity, and has yielded new insights into the mechanisms of receptor specificity and promiscuity in biological systems.

Alanine↗

PubMed2004

Strand-loop-strand motifs: prediction of hairpins and diverging turns in proteins.

Beta-sheet proteins have been particularly challenging for de novo structure prediction methods, which tend to pair adjacent beta-strands into beta-hairpins and produce overly local topologies. To remedy this problem and facilitate de novo prediction of beta-sheet protein structures, we have developed a neural network that classifies strand-loop-strand motifs by local hairpins and nonlocal diverging turns by using the amino acid sequence as input. The neural network is trained with a representative subset of the Protein Data Bank and achieves a prediction accuracy of 75.9 +/- 4.4% compared to a baseline prediction rate of 59.1%. Hairpins are predicted with an accuracy of 77.3 +/- 6.1%, diverging turns with an accuracy of 73.9 +/- 6.0%. Incorporation of the beta-hairpin/diverging turn classification into the ROSETTA de novo structure prediction method led to higher contact order models and somewhat improved tertiary structure predictions for a test set of 11 all-beta-proteins and 3 alphabeta-proteins. The beta-hairpin/diverging turn classification from amino acid sequences is available online for academic use (Meiler and Kuhn, 2003; www.jens-meiler.de/turnpred.html).

Algorithms↗

PubMed2004

In vivo pharmacological actions of two novel inhibitors of anandamide cellular uptake.

Two inhibitors of the cellular uptake of the endocannabinoid anandamide, (R)-N-oleoyl-(1'-hydroxybenzyl)-2'-ethanolamine and (S)-N-oleoyl-(1'-hydroxybenzyl)-2'-ethanolamine (OMDM-1 and OMDM-2, respectively), were recently synthesized, and their in vitro pharmacological activity described. Here we have assessed their activity in two typical pharmacological responses of cannabimimetic compounds. We first examined whether these compounds exert any effect per se on locomotion and pain perception in rats, and/or enhance the effects of anandamide on these two processes. We compared the effects of the novel compounds with those produced by a previously developed selective inhibitor, N-arachidonoyl-(2-methyl-4-hydroxyphenyl)amine (VDM-11). When assayed alone, OMDM-1 and OMDM-2 (1-10 mg/kg, i.p.) did not affect any of the five motor parameters under investigation, although the former compound exhibited a trend for the inhibition of ambulation, fast movements, and speed in rats. OMDM-2 and, to a lesser extent, VDM-11 (5 mg/kg, i.p.) enhanced the motor-inhibitory effects of a noneffective dose (2 mg/kg, i.p.) of anandamide, while OMDM-1 did not. In a typical test of acute analgesia, OMDM-2 and VDM-11 (1-10 mg/kg, i.p.), but not OMDM-1, significantly enhanced the time spent by rats on a "hot plate." However, the same compounds (5 mg/kg, i.p.) did not enhance the analgesic effect of a subeffective dose (2 mg/kg, i.p.) of anandamide, whereas OMDM-1 exerted a strong trend towards potentiation (P=0.06). We next explored the possible use of the two novel compounds in a pathological condition. Thus, we determined if, like other previously developed anandamide reuptake inhibitors, OMDM-1 and OMDM-2 inhibit spasticity in an animal model of multiple sclerosis-the chronic relapsing experimental allergic encephalomyelitis in mice. As previously shown with a higher dose of VDM-11, both novel compounds (5 mg/kg, i.v.) significantly reduced spasticity of the hindlimb in mice with chronic relapsing experimental allergic encephalomyelitis. We suggest that OMDM-1 and, particularly, OMDM-2 are useful pharmacological tools for the study of the (patho)physiological role of the anandamide cellular uptake process, and represent unique templates for the development of new antispastic drugs.

Animals↗

PubMed2004

Disease-related epitope spread in a humanized T cell receptor transgenic model of multiple sclerosis.

While EAE has been an invaluable model for the immunopathogenesis of multiple sclerosis, it has sometimes been difficult to bridge the gap between findings and therapies in the rodent models and the cellular and molecular interactions that can be studied in the human disease. Humanized transgenic models offer a means of achieving this, through the expression of disease-implicated HLA class II molecules, co-expressed with a cognate HLA-class II-restricted, myelin-specific TCR derived from a human T cell clone implicated in disease. We have generated such a transgenic line, called line 8, that co-expresses a high level of HLA-DR15 and a human TCR specific for HLA-DR15/MBP 85-99. T cells from the transgenic line are skewed to the CD4 single-positive compartment and produce IFN-gamma in response to peptide from mylein basic protein. Mice develop a spontaneous disease phenotype, showing poverty of movement, although this rarely develops into paralysis except following immunization with peptide. On induction of paralysis by immunization with peptide, disease correlates with epitope spread to a number of additional, HLA-DR15-restricted myelin epitopes. This model should be valuable for analyzing epitope spread in a humanized immunogenetic environment and for the testing of specific immunotherapies.

Animals↗

PubMed2004

Cost-effectiveness of herpes simplex virus type 2 serologic testing and antiviral therapy in pregnancy.

OBJECTIVE: The purpose of this study was to determine whether serologic testing for herpes simplex virus type 2 (HSV-2) in pregnant women and their partners is cost-effective. STUDY DESIGN: A decision analysis model was developed to investigate the cost-effectiveness of providing type-specific serologic testing at week 15 of pregnancy for all women unaware of their HSV-2 status, and offering antiviral suppressive therapy from week 36 until delivery to all seropositive women. This scenario was compared with current care, in which only a minority of women diagnosed with genital herpes (GH) receives antiviral suppressive therapy (AST). In a third scenario, testing is offered to partners of pregnant women who test seronegative, and antiviral suppressive therapy is offered to the partners who test seropositive. RESULTS: Compared with current care, offering testing and antiviral suppressive therapy to 100,000 pregnant women resulted in an incremental cost of $3.1 million, 15.7 fewer cases of neonatal herpes, 186 fewer cesarean deliveries, and an incremental cost per quality-adjusted life- year gained (QALY) of $18,680. Offering testing and suppressive therapy to both the pregnant women and their partners resulted in an increased cost of $8.6 million, 16.8 fewer cases of neonatal herpes, 192 fewer cesarean deliveries, and an incremental cost per QALY of $48,946 compared with no testing. CONCLUSION: Compared with commonly accepted benchmarks for cost-effectiveness (<$50,000/QALY), type-specific HSV-2 serologic testing of pregnant women may be a cost-effective strategy.

Adolescent↗