John Rosenberg - Academia.edu (original) (raw)
Papers by John Rosenberg
Proceedings 2001 IEEE International Conference on Data Mining
Autonomous discovery systems will be able to peruse very large databases more thoroughly than peo... more Autonomous discovery systems will be able to peruse very large databases more thoroughly than people can. In a companion paper [1], we describe a general framework for autonomous systems. We present and evaluate heuristics for use in this framework. Although these heuristics were designed for a prototype system, we believe they provide good initial solutions to problems encountered when implementing fully autonomous discovery systems. As such, these heuristics may be used as the starting point for future research into fully autonomous discovery systems.
Proceedings 2001 IEEE International Conference on Data Mining
We propose and evaluate an agenda-and justificationbased architecture for discovery systems that ... more We propose and evaluate an agenda-and justificationbased architecture for discovery systems that selects the next tasks to perform. This framework has many desirable properties: (1) it facilitates the encoding of general discovery strategies using a variety of background knowledge, (2) it reasons about the appropriateness of the tasks being considered, and (3) it tailors its behavior toward a user's interests. A prototype discovery program called HAMB demonstrates that both reasons and estimates of interestingness contribute to performance in the domains of protein crystallization and patient rehabilitation.
Journal of Biological Chemistry, 1981
The Eco R1 endonuclease and methylase recognize the same hexanucleotide substrate sequence. We ha... more The Eco R1 endonuclease and methylase recognize the same hexanucleotide substrate sequence. We have determined the sequence of a fragment of DNA which encodes these enzymes using the chain-termination method of Sanger (
Journal of Biological Chemistry, 1983
K2P channels regulate nervous, cardiovascular, and immune system functions1,2 through the action ... more K2P channels regulate nervous, cardiovascular, and immune system functions1,2 through the action of their selectivity filter (C-type) gate3-6. Although structural studies show K2P conformations that impact activity7-13, no selectivity filter conformational changes have been observed. Here, combining K2P2.1 (TREK-1) X-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics, and functional studies, we uncover the unprecedented, asymmetric, potassium-dependent conformational changes underlying K2P C-type gating. Low potassium concentrations evoke conformational changes in selectivity filter strand 1 (SF1), selectivity filter strand 2 (SF2), and the SF2-transmembrane helix 4 loop (SF2-M4 loop) that destroy the S1 and S2 ion binding sites and are suppressed by C-type gate activator ML335. Shortening the uniquely long SF2-M4 loop to match the canonical length found in other potassium channels or disrupting the conserved Glu234 hydrogen...
Do complex molecular machines tend to exhibit a single mechanism, as suggested in literature and ... more Do complex molecular machines tend to exhibit a single mechanism, as suggested in literature and textbook cartoons, or is a multiplicity of pathways possible or even prevalent? Motivated by growing evidence for pathway heterogeneity, we develop theoretical and computational approaches for investigating this question, which could have a significant potential impact in both understanding molecular-scale cell biology and in the design of synthetic machines. Focusing on transport in this initial study, we pursue Monte Carlo exploration of 'model space'. Trial moves adjust transition rates among an automatically generated set of conformational and binding states while maintaining fidelity to thermodynamic principles and fitness goal. The simulations yield both single-mechanism and heterogeneous multi-mechanism models for cotransport in a simple environment. In a 'competitive' environment with a decoy substrate, several qualitatively distinct models are found which are cap...
Proceedings of the National Academy of Sciences of the United States of America, Jul 20, 2016
Secondary active transporters, such as those that adopt the leucine-transporter fold, are found i... more Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-faci...
Biophysical Journal, 2014
Background and purpose: Amphetamines bind to the plasmalemmal transporters for the monoamines dop... more Background and purpose: Amphetamines bind to the plasmalemmal transporters for the monoamines dopamine (DAT), norepinephrine (NET) and serotonin (SERT); influx of amphetamine leads to efflux of substrates. Various models have been put forth to account for this amphetamine-induced reverse transport in mechanistic terms. A most notable example is the molecular stent hypothesis, which posits a special amphetamine-induced conformation that is not foreseen in alternate access models of transport. The current study was designed to evaluate the explanatory power of these models and the molecular stent hypothesis. Experimental approach: Xenopus laevis oocytes and HEK293 cells expressing human (h)SERT were voltage clamped and exposed to serotonin (5-HT), p-chloroamphetamine (pCA) or methylenedioxyamphetamine (MDMA). Key results: In contrast to currents induced by 5-HT, pCA-triggered currents through SERT decayed slowly (i.e., with a half-life of 20 s at 3 micromolar) in Xenopus laevis oocytes once the agonist was removed (consistent with the molecular stent hypothesis). However, when SERT was expressed in HEK293 cells, currents induced by 3 micromolar or 100 micromolar pCA decayed 10 or 100 times faster, respectively, after pCA removal. Conclusions and implications: This discrepancy in decay rates is inconsistent with the molecular stent hypothesis. In contrast, a multi-state version of the alternate access model accounts for all the observations and reproduces the kinetic parameters extracted from the electrophysiological recordings. A crucial feature that explains the action of amphetamines is their lipophilic nature, which allows for rapid diffusion through the membrane.
Springer Proceedings in Physics, 2004
We introduce an adaptive Monte Carlo method to calculate free energy differences for continuous s... more We introduce an adaptive Monte Carlo method to calculate free energy differences for continuous systems. The method uses a biasing potential based on integrating the derivative of the Hamiltonian with respect to parameters of interest. Tests on the two-dimensional Lennard-Jones fluid are used to demonstrate the efficiency of the method.
Science, 1990
The original model of Eco RI endonuclease (1) proved refractory to crystallographic refinement, w... more The original model of Eco RI endonuclease (1) proved refractory to crystallographic refinement, whereby model coordinates were adjusted to optimize the fit to the observed diffraction data (the best R factor for the original model is 0.25). We have now obtained five additional isomorphous derivatives by cocrystallizing the protein with the halogenated oligonucleotides: U'CGC-GAATTCGCG, TCGCGAAU'TCGCG, TC-GCGAATU'CGCG, TCGCGAATICGC'G
Proceedings of the National Academy of Sciences, 1989
The arginine at position 200 of EcoRI endonuclease is thought to make two hydrogen bonds to the g... more The arginine at position 200 of EcoRI endonuclease is thought to make two hydrogen bonds to the guanine of the sequence GAATTC and thus be an important determinant of sequence discrimination. Arg-200 was replaced by each of the other 19 naturally occurring amino acids, and the mutant endonucleases were assessed for activities in vivo and in vitro. The mutant endonuclease with lysine at position 200 exhibits the most in vivo activity of all the position 200 mutants, although the in vitro activity is less than 1/100th of wild-type activity. Five other mutants show more drastically reduced levels of in vivo activity (Cys, Pro, Val, Ser, and Trp). The Cys, Val, and Ser mutant enzymes appear to have in vivo activity which is specific for the wild-type canonical site despite the loss of hydrogen bonding potential at position 200. The Pro and Trp mutants retain in vivo activity which is independent of the presence of the EcoRI methylase. In crude cell lysates, only the Cys mutant shows a v...
Proceedings of the National Academy of Sciences, 1973
The sodium salt of guanylyl-3′,5′-cytidine crystallizes in a monoclinic unit cell with one molecu... more The sodium salt of guanylyl-3′,5′-cytidine crystallizes in a monoclinic unit cell with one molecule in the asymmetric unit. Each molecule is related to another molecule by a 2-fold rotation axis which results in the formation of an antiparallel, right-handed double helix with complementary hydrogen bonding between the guanine and cytosine residues. The crystal is heavily hydrated with 36 water molecules in the unit cell. The geometry of this crystalline double helix is very similar to those which have been derived from studies of fiber x-ray diffraction patterns of double-stranded RNA, even though the latter do not yield data at atomic resolution.
Nucleic Acids Research, 1977
A procedure has been developed which eliminates the commonly observed inactivation of the DNA bin... more A procedure has been developed which eliminates the commonly observed inactivation of the DNA binding activity of the lac repressor during purification. The operator binding activity of the repressor obtained by this method is 100 ±10%. The repressor can be stored frozen indefinitely without losing its affinity for DNA.
Journal of Computational Chemistry, 1992
The Weighted Histogram Analysis Method (WHAM), an extension of Ferrenberg and Swendsen's Mult... more The Weighted Histogram Analysis Method (WHAM), an extension of Ferrenberg and Swendsen's Multiple Histogram Technique, has been applied for the first time on complex biomolecular Hamiltonians. The method is presented here as an extension of the Umbrella Sampling method for free‐energy and Potential of Mean Force calculations. This algorithm possesses the following advantages over methods that are currently employed: (1) It provides a built‐in estimate of sampling errors thereby yielding objective estimates of the optimal location and length of additional simulations needed to achieve a desired level of precision; (2) it yields the “best” value of free energies by taking into account all the simulations so as to minimize the statistical errors; (3) in addition to optimizing the links between simulations, it also allows multiple overlaps of probability distributions for obtaining better estimates of the free‐energy differences. By recasting the Ferrenberg–Swendsen Multiple Histogr...
Journal of Biological Chemistry, 2003
Biophysical Journal, 2012
able to (I) find evidence that the key residue-encompassing core region in the AcrB trans-membran... more able to (I) find evidence that the key residue-encompassing core region in the AcrB trans-membrane domain is monomer-specifically connected to bulk water by up to 3 periplasmic and 1 cytoplasmic water channels, suggesting three alternative routes of proton transfer; (II) provide evidence that contrary to the available crystal structures the outer membrane efflux duct TolC, while freely accessible from the extracellular medium, is locked only on periplasmic side in a sodium-dependent manner; (III) show that the spontaneous binding of the isolated AcrB docking domain to TolC does not induce an opening of the efflux duct within 1ms simulation time, suggesting that either a longer response time or an additional key is required to unlock the channel; (IV) provide evidence that the currently proposed model of the assembled AcrAB-TolC complex is most likely not correct proposing an alternative model that is consistent with all experimental data currently available including the crystal structure of the membrane fusion protein / inner membrane translocase complex of the homologue copper transporter CusBA.
Proceedings 2001 IEEE International Conference on Data Mining
Autonomous discovery systems will be able to peruse very large databases more thoroughly than peo... more Autonomous discovery systems will be able to peruse very large databases more thoroughly than people can. In a companion paper [1], we describe a general framework for autonomous systems. We present and evaluate heuristics for use in this framework. Although these heuristics were designed for a prototype system, we believe they provide good initial solutions to problems encountered when implementing fully autonomous discovery systems. As such, these heuristics may be used as the starting point for future research into fully autonomous discovery systems.
Proceedings 2001 IEEE International Conference on Data Mining
We propose and evaluate an agenda-and justificationbased architecture for discovery systems that ... more We propose and evaluate an agenda-and justificationbased architecture for discovery systems that selects the next tasks to perform. This framework has many desirable properties: (1) it facilitates the encoding of general discovery strategies using a variety of background knowledge, (2) it reasons about the appropriateness of the tasks being considered, and (3) it tailors its behavior toward a user's interests. A prototype discovery program called HAMB demonstrates that both reasons and estimates of interestingness contribute to performance in the domains of protein crystallization and patient rehabilitation.
Journal of Biological Chemistry, 1981
The Eco R1 endonuclease and methylase recognize the same hexanucleotide substrate sequence. We ha... more The Eco R1 endonuclease and methylase recognize the same hexanucleotide substrate sequence. We have determined the sequence of a fragment of DNA which encodes these enzymes using the chain-termination method of Sanger (
Journal of Biological Chemistry, 1983
K2P channels regulate nervous, cardiovascular, and immune system functions1,2 through the action ... more K2P channels regulate nervous, cardiovascular, and immune system functions1,2 through the action of their selectivity filter (C-type) gate3-6. Although structural studies show K2P conformations that impact activity7-13, no selectivity filter conformational changes have been observed. Here, combining K2P2.1 (TREK-1) X-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics, and functional studies, we uncover the unprecedented, asymmetric, potassium-dependent conformational changes underlying K2P C-type gating. Low potassium concentrations evoke conformational changes in selectivity filter strand 1 (SF1), selectivity filter strand 2 (SF2), and the SF2-transmembrane helix 4 loop (SF2-M4 loop) that destroy the S1 and S2 ion binding sites and are suppressed by C-type gate activator ML335. Shortening the uniquely long SF2-M4 loop to match the canonical length found in other potassium channels or disrupting the conserved Glu234 hydrogen...
Do complex molecular machines tend to exhibit a single mechanism, as suggested in literature and ... more Do complex molecular machines tend to exhibit a single mechanism, as suggested in literature and textbook cartoons, or is a multiplicity of pathways possible or even prevalent? Motivated by growing evidence for pathway heterogeneity, we develop theoretical and computational approaches for investigating this question, which could have a significant potential impact in both understanding molecular-scale cell biology and in the design of synthetic machines. Focusing on transport in this initial study, we pursue Monte Carlo exploration of 'model space'. Trial moves adjust transition rates among an automatically generated set of conformational and binding states while maintaining fidelity to thermodynamic principles and fitness goal. The simulations yield both single-mechanism and heterogeneous multi-mechanism models for cotransport in a simple environment. In a 'competitive' environment with a decoy substrate, several qualitatively distinct models are found which are cap...
Proceedings of the National Academy of Sciences of the United States of America, Jul 20, 2016
Secondary active transporters, such as those that adopt the leucine-transporter fold, are found i... more Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-faci...
Biophysical Journal, 2014
Background and purpose: Amphetamines bind to the plasmalemmal transporters for the monoamines dop... more Background and purpose: Amphetamines bind to the plasmalemmal transporters for the monoamines dopamine (DAT), norepinephrine (NET) and serotonin (SERT); influx of amphetamine leads to efflux of substrates. Various models have been put forth to account for this amphetamine-induced reverse transport in mechanistic terms. A most notable example is the molecular stent hypothesis, which posits a special amphetamine-induced conformation that is not foreseen in alternate access models of transport. The current study was designed to evaluate the explanatory power of these models and the molecular stent hypothesis. Experimental approach: Xenopus laevis oocytes and HEK293 cells expressing human (h)SERT were voltage clamped and exposed to serotonin (5-HT), p-chloroamphetamine (pCA) or methylenedioxyamphetamine (MDMA). Key results: In contrast to currents induced by 5-HT, pCA-triggered currents through SERT decayed slowly (i.e., with a half-life of 20 s at 3 micromolar) in Xenopus laevis oocytes once the agonist was removed (consistent with the molecular stent hypothesis). However, when SERT was expressed in HEK293 cells, currents induced by 3 micromolar or 100 micromolar pCA decayed 10 or 100 times faster, respectively, after pCA removal. Conclusions and implications: This discrepancy in decay rates is inconsistent with the molecular stent hypothesis. In contrast, a multi-state version of the alternate access model accounts for all the observations and reproduces the kinetic parameters extracted from the electrophysiological recordings. A crucial feature that explains the action of amphetamines is their lipophilic nature, which allows for rapid diffusion through the membrane.
Springer Proceedings in Physics, 2004
We introduce an adaptive Monte Carlo method to calculate free energy differences for continuous s... more We introduce an adaptive Monte Carlo method to calculate free energy differences for continuous systems. The method uses a biasing potential based on integrating the derivative of the Hamiltonian with respect to parameters of interest. Tests on the two-dimensional Lennard-Jones fluid are used to demonstrate the efficiency of the method.
Science, 1990
The original model of Eco RI endonuclease (1) proved refractory to crystallographic refinement, w... more The original model of Eco RI endonuclease (1) proved refractory to crystallographic refinement, whereby model coordinates were adjusted to optimize the fit to the observed diffraction data (the best R factor for the original model is 0.25). We have now obtained five additional isomorphous derivatives by cocrystallizing the protein with the halogenated oligonucleotides: U'CGC-GAATTCGCG, TCGCGAAU'TCGCG, TC-GCGAATU'CGCG, TCGCGAATICGC'G
Proceedings of the National Academy of Sciences, 1989
The arginine at position 200 of EcoRI endonuclease is thought to make two hydrogen bonds to the g... more The arginine at position 200 of EcoRI endonuclease is thought to make two hydrogen bonds to the guanine of the sequence GAATTC and thus be an important determinant of sequence discrimination. Arg-200 was replaced by each of the other 19 naturally occurring amino acids, and the mutant endonucleases were assessed for activities in vivo and in vitro. The mutant endonuclease with lysine at position 200 exhibits the most in vivo activity of all the position 200 mutants, although the in vitro activity is less than 1/100th of wild-type activity. Five other mutants show more drastically reduced levels of in vivo activity (Cys, Pro, Val, Ser, and Trp). The Cys, Val, and Ser mutant enzymes appear to have in vivo activity which is specific for the wild-type canonical site despite the loss of hydrogen bonding potential at position 200. The Pro and Trp mutants retain in vivo activity which is independent of the presence of the EcoRI methylase. In crude cell lysates, only the Cys mutant shows a v...
Proceedings of the National Academy of Sciences, 1973
The sodium salt of guanylyl-3′,5′-cytidine crystallizes in a monoclinic unit cell with one molecu... more The sodium salt of guanylyl-3′,5′-cytidine crystallizes in a monoclinic unit cell with one molecule in the asymmetric unit. Each molecule is related to another molecule by a 2-fold rotation axis which results in the formation of an antiparallel, right-handed double helix with complementary hydrogen bonding between the guanine and cytosine residues. The crystal is heavily hydrated with 36 water molecules in the unit cell. The geometry of this crystalline double helix is very similar to those which have been derived from studies of fiber x-ray diffraction patterns of double-stranded RNA, even though the latter do not yield data at atomic resolution.
Nucleic Acids Research, 1977
A procedure has been developed which eliminates the commonly observed inactivation of the DNA bin... more A procedure has been developed which eliminates the commonly observed inactivation of the DNA binding activity of the lac repressor during purification. The operator binding activity of the repressor obtained by this method is 100 ±10%. The repressor can be stored frozen indefinitely without losing its affinity for DNA.
Journal of Computational Chemistry, 1992
The Weighted Histogram Analysis Method (WHAM), an extension of Ferrenberg and Swendsen's Mult... more The Weighted Histogram Analysis Method (WHAM), an extension of Ferrenberg and Swendsen's Multiple Histogram Technique, has been applied for the first time on complex biomolecular Hamiltonians. The method is presented here as an extension of the Umbrella Sampling method for free‐energy and Potential of Mean Force calculations. This algorithm possesses the following advantages over methods that are currently employed: (1) It provides a built‐in estimate of sampling errors thereby yielding objective estimates of the optimal location and length of additional simulations needed to achieve a desired level of precision; (2) it yields the “best” value of free energies by taking into account all the simulations so as to minimize the statistical errors; (3) in addition to optimizing the links between simulations, it also allows multiple overlaps of probability distributions for obtaining better estimates of the free‐energy differences. By recasting the Ferrenberg–Swendsen Multiple Histogr...
Journal of Biological Chemistry, 2003
Biophysical Journal, 2012
able to (I) find evidence that the key residue-encompassing core region in the AcrB trans-membran... more able to (I) find evidence that the key residue-encompassing core region in the AcrB trans-membrane domain is monomer-specifically connected to bulk water by up to 3 periplasmic and 1 cytoplasmic water channels, suggesting three alternative routes of proton transfer; (II) provide evidence that contrary to the available crystal structures the outer membrane efflux duct TolC, while freely accessible from the extracellular medium, is locked only on periplasmic side in a sodium-dependent manner; (III) show that the spontaneous binding of the isolated AcrB docking domain to TolC does not induce an opening of the efflux duct within 1ms simulation time, suggesting that either a longer response time or an additional key is required to unlock the channel; (IV) provide evidence that the currently proposed model of the assembled AcrAB-TolC complex is most likely not correct proposing an alternative model that is consistent with all experimental data currently available including the crystal structure of the membrane fusion protein / inner membrane translocase complex of the homologue copper transporter CusBA.