Navin Pokala - Academia.edu (original) (raw)

Papers by Navin Pokala

Research paper thumbnail of Distinct Circuits for the Formation and Retrieval of an Imprinted Olfactory Memory

Cell, 2016

Memories formed early in life are particularly stable and influential, representing privileged ex... more Memories formed early in life are particularly stable and influential, representing privileged experiences that shape enduring behaviors. We show that exposing newly hatched C. elegans to pathogenic bacteria results in persistent aversion to those bacterial odors, whereas adult exposure generates only transient aversive memory. Long-lasting imprinted aversion has a critical period in the first larval stage and is specific to the experienced pathogen. Distinct groups of neurons are required during formation (AIB, RIM) and retrieval (AIY, RIA) of the imprinted memory. RIM synthesizes the neuromodulator tyramine, which is required in the L1 stage for learning. AIY memory retrieval neurons sense tyramine via the SER-2 receptor, which is essential for imprinted, but not for adult-learned, aversion. Odor responses in several neurons, most notably RIA, are altered in imprinted animals. These findings provide insight into neuronal substrates of different forms of memory, and lay a foundation for further understanding of early learning.

Research paper thumbnail of High Rates of Actin Filament Turnover in Budding Yeast and Roles for Actin in Establishmentand Maintenance of Cell Polarity Revealed Using

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of t... more We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2-5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site.

Research paper thumbnail of High Rates of Actin Filament Turnover in Budding Yeast and Roles for Actin in Establishment and Maintenance of Cell Polarity Revealed Using the Actin Inhibitor Latrunculin-A

The Journal of Cell Biology

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of t... more We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2–5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site. LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actindepende...

Research paper thumbnail of User's Manual for EGAD! a Genetic Algorithm for protein Design

Research paper thumbnail of The structure of an actin-crosslinking domain from human fimbrin

Nature Structural Biology, 1997

Research paper thumbnail of Neural Mechanisms for Evaluating Environmental Variability in Caenorhabditis elegans

Research paper thumbnail of Inducible and titratable silencing of Caenorhabditis elegans neurons in vivo with histamine-gated chloride channels

Proceedings of the National Academy of Sciences of the United States of America, Jan 18, 2014

Recent progress in neuroscience has been facilitated by tools for neuronal activation and inactiv... more Recent progress in neuroscience has been facilitated by tools for neuronal activation and inactivation that are orthogonal to endogenous signaling systems. We describe here a chemical-genetic approach for inducible silencing of Caenorhabditis elegans neurons in intact animals, using the histamine-gated chloride channel HisCl1 from Drosophila and exogenous histamine. Administering histamine to freely moving C. elegans that express HisCl1 transgenes in neurons leads to rapid and potent inhibition of neural activity within minutes, as assessed by behavior, functional calcium imaging, and electrophysiology of neurons expressing HisCl1. C. elegans does not use histamine as an endogenous neurotransmitter, and exogenous histamine has little apparent effect on wild-type C. elegans behavior. HisCl1-histamine silencing of sensory neurons, interneurons, and motor neurons leads to behavioral effects matching their known functions. In addition, the HisCl1-histamine system can be used to titrate ...

Research paper thumbnail of Feedback from Network States Generates Variability in a Probabilistic Olfactory Circuit

Cell, 2015

Variability is a prominent feature of behavior and is an active element of certain behavioral str... more Variability is a prominent feature of behavior and is an active element of certain behavioral strategies. To understand how neuronal circuits control variability, we examined the propagation of sensory information in a chemotaxis circuit of C. elegans where discrete sensory inputs can drive a probabilistic behavioral response. Olfactory neurons respond to odor stimuli with rapid and reliable changes in activity, but downstream AIB interneurons respond with a probabilistic delay. The interneuron response to odor depends on the collective activity of multiple neurons-AIB, RIM, and AVA-when the odor stimulus arrives. Certain activity states of the network correlate with reliable responses to odor stimuli. Artificially generating these activity states by modifying neuronal activity increases the reliability of odor responses in interneurons and the reliability of the behavioral response to odor. The integration of sensory information with network states may represent a general mechanism for generating variability in behavior.

Research paper thumbnail of The structure of an actin-crosslinking domain from human fimbrin

Natural Structural Biology, 1997

Research paper thumbnail of Neurons Detect Increases and Decreases in Oxygen Levels Using Distinct Guanylate Cyclases

Neuron, 2009

Homeostatic sensory systems detect small deviations in temperature, water balance, pH, and energy... more Homeostatic sensory systems detect small deviations in temperature, water balance, pH, and energy needs to regulate adaptive behavior and physiology. In C. elegans, a homeostatic preference for intermediate oxygen (O2) levels requires cGMP signaling through soluble guanylate cyclases (sGCs), proteins that bind gases through an associated heme group. Here we use behavioral analysis, functional imaging, and genetics to show that reciprocal changes in O2 levels are encoded by sensory neurons that express alternative sets of sGCs. URX sensory neurons are activated by increases in O2 levels, and require the sGCs gcy-35 and gcy-36. BAG sensory neurons are activated by decreases in O2 levels, and require the sGCs gcy-31 and gcy-33. The sGCs are instructive O2 sensors, as forced expression of URX sGC genes causes BAG neurons to detect O2 increases. Both sGC expression and cell-intrinsic dynamics contribute to the differential roles of URX and BAG in O2-dependent behaviors.

Research paper thumbnail of High Rates of Actin Filament Turnover in Budding Yeast and Roles for Actin in Establishment and Maintenance of Cell Polarity Revealed Using the Actin Inhibitor Latrunculin-A

The Journal of Cell Biology, 1997

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of t... more We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2-5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site.

Research paper thumbnail of Oxytocin/Vasopressin-Related Peptides Have an Ancient Role in Reproductive Behavior

Science, 2012

Many biological functions are conserved, but the extent to which conservation applies to integrat... more Many biological functions are conserved, but the extent to which conservation applies to integrative behaviors is unknown. Vasopressin and oxytocin neuropeptides are strongly implicated in mammalian reproductive and social behaviors, yet rodent loss-of-function mutants have relatively subtle behavioral defects. Here we identify an oxytocin/vasopressin-like signaling system in Caenorhabditis elegans, consisting of a peptide and two receptors that are expressed in sexually dimorphic patterns. Males lacking the peptide or its receptors perform poorly in reproductive behaviors, including mate search, mate recognition, and mating, but other sensorimotor behaviors are intact. Quantitative analysis indicates that mating motor patterns are fragmented and inefficient in mutants, suggesting that oxytocin/vasopressin peptides increase the coherence of mating behaviors. These results indicate that conserved molecules coordinate diverse behavioral motifs in reproductive behavior.

Research paper thumbnail of Crystallization and preliminary crystallographic analysis of the N-terminal actin binding domain of human fimbrin

Proteins: Structure, Function, and Genetics, 1997

Research paper thumbnail of Energy functions for protein design I: Efficient and accurate continuum electrostatics and solvation

Protein Science, 2004

Electrostatics and solvation energies are important for defining protein stability, structural sp... more Electrostatics and solvation energies are important for defining protein stability, structural specificity, and molecular recognition. Because these energies are difficult to compute quickly and accurately, they are often ignored or modeled very crudely in computational protein design. To address this problem, we have developed a simple, fast, and accurate approximation for calculating Born radii in the context of protein design calculations. When these approximate Born radii are used with the generalized Born continuum dielectric model, energies calculated by the 10 6 -fold slower finite difference Poisson-Boltzmann model are faithfully reproduced. A similar approach can be used for estimating solvent-accessible surface areas (SASAs). As an independent test, we show that these approximations can be used to accurately predict the experimentally determined pK a s of >200 ionizable groups from 15 proteins.

Research paper thumbnail of Correction: Specific Expression of Channelrhodopsin-2 in Single Neurons of Caenorhabditis elegans

PLoS ONE, 2013

Optogenetic approaches using light-activated proteins like Channelrhodopsin-2 (ChR2) enable inves... more Optogenetic approaches using light-activated proteins like Channelrhodopsin-2 (ChR2) enable investigating the function of populations of neurons in live Caenorhabditis elegans (and other) animals, as ChR2 expression can be targeted to these cells using specific promoters. Sub-populations of these neurons, or even single cells, can be further addressed by restricting the illumination to the cell of interest. However, this is technically demanding, particularly in free moving animals. Thus, it would be helpful if expression of ChR2 could be restricted to single neurons or neuron pairs, as even wide-field illumination would photostimulate only this particular cell. To this end we adopted the use of Cre or FLP recombinases and conditional ChR2 expression at the intersection of two promoter expression domains, i.e. in the cell of interest only. Success of this method depends on precise knowledge of the individual promoters' expression patterns and on relative expression levels of recombinase and ChR2. A bicistronic expression cassette with GFP helps to identify the correct expression pattern. Here we show specific expression in the AVA reverse command neurons and the aversive polymodal sensory ASH neurons. This approach shall enable to generate strains for optogenetic manipulation of each of the 302 C. elegans neurons. This may eventually allow to model the C. elegans nervous system in its entirety, based on functional data for each neuron.

Research paper thumbnail of A hub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans

Nature, 2009

Innate social behaviors emerge from neuronal circuits that interpret sensory information based on... more Innate social behaviors emerge from neuronal circuits that interpret sensory information based on an individual's own genotype, sex, and experience. The regulated aggregation behavior of C. elegans, a simple animal with only 302 neurons, is an attractive system to analyze these circuits. Wild social strains of the nematode Caenorhabditis elegans aggregate in the presence of specific sensory cues, but solitary strains do not1 , 2 , 3 , 4. Here we identify the RMG inter/motor neuron as the hub of a regulated circuit that controls aggregation and related behaviors. RMG is the central site of action of the neuropeptide receptor gene npr-1, which distinguishes solitary strains (high npr-1 activity) from wild social strains (low npr-1 activity); high RMG activity is essential for all aspects of social behavior. Anatomical gap junctions connect RMG to multiple classes of sensory neurons known to promote aggregation, and to ASK sensory neurons, which are implicated in male attraction to hermaphrodite pheromones5. We find that ASK neurons respond directly to pheromones, and that high RMG activity enhances ASK responses in social strains, causing hermaphrodite attraction to pheromones at concentrations that repel solitary hermaphrodites. The coordination of social behaviors by RMG suggests an anatomical hub-and-spoke model for sensory integration in aggregation, and points to functions for related circuit motifs in the C. elegans wiring diagram.

Research paper thumbnail of Review: Protein Design—Where We Were, Where We Are, Where We're Going

Journal of Structural Biology, 2001

Protein design has become a powerful approach for understanding the relationship between amino ac... more Protein design has become a powerful approach for understanding the relationship between amino acid sequence and 3-dimensional structure. In the past 5 years, there have been many breakthroughs in the development of computational methods that allow the selection of novel sequences given the structure of a protein backbone. Successful design of protein scaffolds has now paved the way for new endeavors to design function. The ability to design sequences compatible with a fold may also be useful in structural and functional genomics by expanding the range of proteins used for fold recognition and for the identification of functionally important domains from multiple sequence alignments.

Research paper thumbnail of Energy Functions for Protein Design: Adjustment with Protein–Protein Complex Affinities, Models for the Unfolded State, and Negative Design of Solubility and Specificity

Journal of Molecular Biology, 2005

The development of the EGAD program and energy function for protein design is described. In contr... more The development of the EGAD program and energy function for protein design is described. In contrast to most protein design methods, which require several empirical parameters or heuristics such as patterning of residues or rotamers, EGAD has a minimalist philosophy; it uses very few empirical factors to account for inaccuracies resulting from the use of fixed backbones and discrete rotamers in protein design calculations, and describes the unfolded state, aggregates, and alternative conformers explicitly with physical models instead of fitted parameters. This approach unveils important issues in protein design that are often camouflaged by heuristic-emphasizing methods. Inter-atom energies are modeled with the OPLS-AA all-atom forcefield, electrostatics with the generalized Born continuum model, and the hydrophobic effect with a solvent-accessible surface area-dependent term. Experimental characterization of proteins designed with an unmodified version of the energy function revealed problems with under-packing, stability, aggregation, and structural specificity. Under-packing was addressed by modifying the van der Waals function. By optimizing only three parameters, the effects of O400 mutations on protein-protein complex formation were predicted to within 1.0 kcal mol K1 . As an independent test, this modified energy function was used to predict the stabilities of O1500 mutants to within 1.0 kcal mol K1 ; this required a physical model of the unfolded state that includes more interactions than traditional tripeptide-based models. Solubility and structural specificity were addressed with simple physical approximations of aggregation and conformational equilibria. The complete energy function can design protein sequences that have high levels of identity with their natural counterparts, and have predicted structural properties more consistent with soluble and uniquely folded proteins than the initial designs.

Research paper thumbnail of An object-oriented library for computational protein design

Journal of Computational Chemistry, 2007

Recent advances in computational protein design have established it as a viable technique for the... more Recent advances in computational protein design have established it as a viable technique for the rational generation of stable protein sequences, novel protein folds, and even enzymatic activity. We present a new and object-oriented library of code, written specifically for protein design applications in C(++), called EGAD Library. The modular fashion in which this library is written allows developers to tailor various energy functions and minimizers for a specific purpose. It also allows for the generation of novel protein design applications with a minimal amount of code investment. It is our hope that this will permit labs that have not considered protein design to apply it to their own systems, thereby increasing its potential as a tool in biology. We also present various uses of EGAD Library: in the development of Interaction Viewer, a PyMOL plug-in for viewing interactions between protein residues; in the repacking of protein cores; and in the prediction of protein-protein complex stabilities.

Research paper thumbnail of Structure and in Vitro Molecular Chaperone Activity of Cytosolic Small Heat Shock Proteins from Pea

Journal of Biological Chemistry, 1995

Plants synthesize several classes of small heat shock proteins ranging in size from 15 to 30 kDa.... more Plants synthesize several classes of small heat shock proteins ranging in size from 15 to 30 kDa. Two conserved classes, designated class I and class II, are localized to the cytosol. Recombinant HSP18.1 and HSP17.7, representing class I and class II proteins from pea, respectively, were expressed in Escherichia coli and purified. Non-denaturing polyacrylamide gel electrophoresis and electron microscopy demonstrated that the purified proteins formed discretely sized, high molecular weight complexes. Sedimentation equilibrium analytical ultracentrifugation revealed that the HSP18.1 and HSP17.7 complexes were composed of approximately 12 subunits. Both proteins were able to enhance the refolding of chemically denatured citrate synthase and lactate dehydrogenase at stoichiometric levels in an ATP-independent manner. Furthermore, HSP18.1 and HSP17.7 prevented aggregation of citrate synthase at 45 degrees C and irreversible inactivation of citrate synthase at 38 degrees C. HSP18.1 also suppressed aggregation of lactate dehydrogenase at 55 degrees C. These findings demonstrate that HSP18.1 and HSP17.7 can function as molecular chaperones in vitro.

Research paper thumbnail of Distinct Circuits for the Formation and Retrieval of an Imprinted Olfactory Memory

Cell, 2016

Memories formed early in life are particularly stable and influential, representing privileged ex... more Memories formed early in life are particularly stable and influential, representing privileged experiences that shape enduring behaviors. We show that exposing newly hatched C. elegans to pathogenic bacteria results in persistent aversion to those bacterial odors, whereas adult exposure generates only transient aversive memory. Long-lasting imprinted aversion has a critical period in the first larval stage and is specific to the experienced pathogen. Distinct groups of neurons are required during formation (AIB, RIM) and retrieval (AIY, RIA) of the imprinted memory. RIM synthesizes the neuromodulator tyramine, which is required in the L1 stage for learning. AIY memory retrieval neurons sense tyramine via the SER-2 receptor, which is essential for imprinted, but not for adult-learned, aversion. Odor responses in several neurons, most notably RIA, are altered in imprinted animals. These findings provide insight into neuronal substrates of different forms of memory, and lay a foundation for further understanding of early learning.

Research paper thumbnail of High Rates of Actin Filament Turnover in Budding Yeast and Roles for Actin in Establishmentand Maintenance of Cell Polarity Revealed Using

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of t... more We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2-5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site.

Research paper thumbnail of High Rates of Actin Filament Turnover in Budding Yeast and Roles for Actin in Establishment and Maintenance of Cell Polarity Revealed Using the Actin Inhibitor Latrunculin-A

The Journal of Cell Biology

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of t... more We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2–5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site. LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actindepende...

Research paper thumbnail of User's Manual for EGAD! a Genetic Algorithm for protein Design

Research paper thumbnail of The structure of an actin-crosslinking domain from human fimbrin

Nature Structural Biology, 1997

Research paper thumbnail of Neural Mechanisms for Evaluating Environmental Variability in Caenorhabditis elegans

Research paper thumbnail of Inducible and titratable silencing of Caenorhabditis elegans neurons in vivo with histamine-gated chloride channels

Proceedings of the National Academy of Sciences of the United States of America, Jan 18, 2014

Recent progress in neuroscience has been facilitated by tools for neuronal activation and inactiv... more Recent progress in neuroscience has been facilitated by tools for neuronal activation and inactivation that are orthogonal to endogenous signaling systems. We describe here a chemical-genetic approach for inducible silencing of Caenorhabditis elegans neurons in intact animals, using the histamine-gated chloride channel HisCl1 from Drosophila and exogenous histamine. Administering histamine to freely moving C. elegans that express HisCl1 transgenes in neurons leads to rapid and potent inhibition of neural activity within minutes, as assessed by behavior, functional calcium imaging, and electrophysiology of neurons expressing HisCl1. C. elegans does not use histamine as an endogenous neurotransmitter, and exogenous histamine has little apparent effect on wild-type C. elegans behavior. HisCl1-histamine silencing of sensory neurons, interneurons, and motor neurons leads to behavioral effects matching their known functions. In addition, the HisCl1-histamine system can be used to titrate ...

Research paper thumbnail of Feedback from Network States Generates Variability in a Probabilistic Olfactory Circuit

Cell, 2015

Variability is a prominent feature of behavior and is an active element of certain behavioral str... more Variability is a prominent feature of behavior and is an active element of certain behavioral strategies. To understand how neuronal circuits control variability, we examined the propagation of sensory information in a chemotaxis circuit of C. elegans where discrete sensory inputs can drive a probabilistic behavioral response. Olfactory neurons respond to odor stimuli with rapid and reliable changes in activity, but downstream AIB interneurons respond with a probabilistic delay. The interneuron response to odor depends on the collective activity of multiple neurons-AIB, RIM, and AVA-when the odor stimulus arrives. Certain activity states of the network correlate with reliable responses to odor stimuli. Artificially generating these activity states by modifying neuronal activity increases the reliability of odor responses in interneurons and the reliability of the behavioral response to odor. The integration of sensory information with network states may represent a general mechanism for generating variability in behavior.

Research paper thumbnail of The structure of an actin-crosslinking domain from human fimbrin

Natural Structural Biology, 1997

Research paper thumbnail of Neurons Detect Increases and Decreases in Oxygen Levels Using Distinct Guanylate Cyclases

Neuron, 2009

Homeostatic sensory systems detect small deviations in temperature, water balance, pH, and energy... more Homeostatic sensory systems detect small deviations in temperature, water balance, pH, and energy needs to regulate adaptive behavior and physiology. In C. elegans, a homeostatic preference for intermediate oxygen (O2) levels requires cGMP signaling through soluble guanylate cyclases (sGCs), proteins that bind gases through an associated heme group. Here we use behavioral analysis, functional imaging, and genetics to show that reciprocal changes in O2 levels are encoded by sensory neurons that express alternative sets of sGCs. URX sensory neurons are activated by increases in O2 levels, and require the sGCs gcy-35 and gcy-36. BAG sensory neurons are activated by decreases in O2 levels, and require the sGCs gcy-31 and gcy-33. The sGCs are instructive O2 sensors, as forced expression of URX sGC genes causes BAG neurons to detect O2 increases. Both sGC expression and cell-intrinsic dynamics contribute to the differential roles of URX and BAG in O2-dependent behaviors.

Research paper thumbnail of High Rates of Actin Filament Turnover in Budding Yeast and Roles for Actin in Establishment and Maintenance of Cell Polarity Revealed Using the Actin Inhibitor Latrunculin-A

The Journal of Cell Biology, 1997

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of t... more We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2-5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site.

Research paper thumbnail of Oxytocin/Vasopressin-Related Peptides Have an Ancient Role in Reproductive Behavior

Science, 2012

Many biological functions are conserved, but the extent to which conservation applies to integrat... more Many biological functions are conserved, but the extent to which conservation applies to integrative behaviors is unknown. Vasopressin and oxytocin neuropeptides are strongly implicated in mammalian reproductive and social behaviors, yet rodent loss-of-function mutants have relatively subtle behavioral defects. Here we identify an oxytocin/vasopressin-like signaling system in Caenorhabditis elegans, consisting of a peptide and two receptors that are expressed in sexually dimorphic patterns. Males lacking the peptide or its receptors perform poorly in reproductive behaviors, including mate search, mate recognition, and mating, but other sensorimotor behaviors are intact. Quantitative analysis indicates that mating motor patterns are fragmented and inefficient in mutants, suggesting that oxytocin/vasopressin peptides increase the coherence of mating behaviors. These results indicate that conserved molecules coordinate diverse behavioral motifs in reproductive behavior.

Research paper thumbnail of Crystallization and preliminary crystallographic analysis of the N-terminal actin binding domain of human fimbrin

Proteins: Structure, Function, and Genetics, 1997

Research paper thumbnail of Energy functions for protein design I: Efficient and accurate continuum electrostatics and solvation

Protein Science, 2004

Electrostatics and solvation energies are important for defining protein stability, structural sp... more Electrostatics and solvation energies are important for defining protein stability, structural specificity, and molecular recognition. Because these energies are difficult to compute quickly and accurately, they are often ignored or modeled very crudely in computational protein design. To address this problem, we have developed a simple, fast, and accurate approximation for calculating Born radii in the context of protein design calculations. When these approximate Born radii are used with the generalized Born continuum dielectric model, energies calculated by the 10 6 -fold slower finite difference Poisson-Boltzmann model are faithfully reproduced. A similar approach can be used for estimating solvent-accessible surface areas (SASAs). As an independent test, we show that these approximations can be used to accurately predict the experimentally determined pK a s of >200 ionizable groups from 15 proteins.

Research paper thumbnail of Correction: Specific Expression of Channelrhodopsin-2 in Single Neurons of Caenorhabditis elegans

PLoS ONE, 2013

Optogenetic approaches using light-activated proteins like Channelrhodopsin-2 (ChR2) enable inves... more Optogenetic approaches using light-activated proteins like Channelrhodopsin-2 (ChR2) enable investigating the function of populations of neurons in live Caenorhabditis elegans (and other) animals, as ChR2 expression can be targeted to these cells using specific promoters. Sub-populations of these neurons, or even single cells, can be further addressed by restricting the illumination to the cell of interest. However, this is technically demanding, particularly in free moving animals. Thus, it would be helpful if expression of ChR2 could be restricted to single neurons or neuron pairs, as even wide-field illumination would photostimulate only this particular cell. To this end we adopted the use of Cre or FLP recombinases and conditional ChR2 expression at the intersection of two promoter expression domains, i.e. in the cell of interest only. Success of this method depends on precise knowledge of the individual promoters' expression patterns and on relative expression levels of recombinase and ChR2. A bicistronic expression cassette with GFP helps to identify the correct expression pattern. Here we show specific expression in the AVA reverse command neurons and the aversive polymodal sensory ASH neurons. This approach shall enable to generate strains for optogenetic manipulation of each of the 302 C. elegans neurons. This may eventually allow to model the C. elegans nervous system in its entirety, based on functional data for each neuron.

Research paper thumbnail of A hub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans

Nature, 2009

Innate social behaviors emerge from neuronal circuits that interpret sensory information based on... more Innate social behaviors emerge from neuronal circuits that interpret sensory information based on an individual's own genotype, sex, and experience. The regulated aggregation behavior of C. elegans, a simple animal with only 302 neurons, is an attractive system to analyze these circuits. Wild social strains of the nematode Caenorhabditis elegans aggregate in the presence of specific sensory cues, but solitary strains do not1 , 2 , 3 , 4. Here we identify the RMG inter/motor neuron as the hub of a regulated circuit that controls aggregation and related behaviors. RMG is the central site of action of the neuropeptide receptor gene npr-1, which distinguishes solitary strains (high npr-1 activity) from wild social strains (low npr-1 activity); high RMG activity is essential for all aspects of social behavior. Anatomical gap junctions connect RMG to multiple classes of sensory neurons known to promote aggregation, and to ASK sensory neurons, which are implicated in male attraction to hermaphrodite pheromones5. We find that ASK neurons respond directly to pheromones, and that high RMG activity enhances ASK responses in social strains, causing hermaphrodite attraction to pheromones at concentrations that repel solitary hermaphrodites. The coordination of social behaviors by RMG suggests an anatomical hub-and-spoke model for sensory integration in aggregation, and points to functions for related circuit motifs in the C. elegans wiring diagram.

Research paper thumbnail of Review: Protein Design—Where We Were, Where We Are, Where We're Going

Journal of Structural Biology, 2001

Protein design has become a powerful approach for understanding the relationship between amino ac... more Protein design has become a powerful approach for understanding the relationship between amino acid sequence and 3-dimensional structure. In the past 5 years, there have been many breakthroughs in the development of computational methods that allow the selection of novel sequences given the structure of a protein backbone. Successful design of protein scaffolds has now paved the way for new endeavors to design function. The ability to design sequences compatible with a fold may also be useful in structural and functional genomics by expanding the range of proteins used for fold recognition and for the identification of functionally important domains from multiple sequence alignments.

Research paper thumbnail of Energy Functions for Protein Design: Adjustment with Protein–Protein Complex Affinities, Models for the Unfolded State, and Negative Design of Solubility and Specificity

Journal of Molecular Biology, 2005

The development of the EGAD program and energy function for protein design is described. In contr... more The development of the EGAD program and energy function for protein design is described. In contrast to most protein design methods, which require several empirical parameters or heuristics such as patterning of residues or rotamers, EGAD has a minimalist philosophy; it uses very few empirical factors to account for inaccuracies resulting from the use of fixed backbones and discrete rotamers in protein design calculations, and describes the unfolded state, aggregates, and alternative conformers explicitly with physical models instead of fitted parameters. This approach unveils important issues in protein design that are often camouflaged by heuristic-emphasizing methods. Inter-atom energies are modeled with the OPLS-AA all-atom forcefield, electrostatics with the generalized Born continuum model, and the hydrophobic effect with a solvent-accessible surface area-dependent term. Experimental characterization of proteins designed with an unmodified version of the energy function revealed problems with under-packing, stability, aggregation, and structural specificity. Under-packing was addressed by modifying the van der Waals function. By optimizing only three parameters, the effects of O400 mutations on protein-protein complex formation were predicted to within 1.0 kcal mol K1 . As an independent test, this modified energy function was used to predict the stabilities of O1500 mutants to within 1.0 kcal mol K1 ; this required a physical model of the unfolded state that includes more interactions than traditional tripeptide-based models. Solubility and structural specificity were addressed with simple physical approximations of aggregation and conformational equilibria. The complete energy function can design protein sequences that have high levels of identity with their natural counterparts, and have predicted structural properties more consistent with soluble and uniquely folded proteins than the initial designs.

Research paper thumbnail of An object-oriented library for computational protein design

Journal of Computational Chemistry, 2007

Recent advances in computational protein design have established it as a viable technique for the... more Recent advances in computational protein design have established it as a viable technique for the rational generation of stable protein sequences, novel protein folds, and even enzymatic activity. We present a new and object-oriented library of code, written specifically for protein design applications in C(++), called EGAD Library. The modular fashion in which this library is written allows developers to tailor various energy functions and minimizers for a specific purpose. It also allows for the generation of novel protein design applications with a minimal amount of code investment. It is our hope that this will permit labs that have not considered protein design to apply it to their own systems, thereby increasing its potential as a tool in biology. We also present various uses of EGAD Library: in the development of Interaction Viewer, a PyMOL plug-in for viewing interactions between protein residues; in the repacking of protein cores; and in the prediction of protein-protein complex stabilities.

Research paper thumbnail of Structure and in Vitro Molecular Chaperone Activity of Cytosolic Small Heat Shock Proteins from Pea

Journal of Biological Chemistry, 1995

Plants synthesize several classes of small heat shock proteins ranging in size from 15 to 30 kDa.... more Plants synthesize several classes of small heat shock proteins ranging in size from 15 to 30 kDa. Two conserved classes, designated class I and class II, are localized to the cytosol. Recombinant HSP18.1 and HSP17.7, representing class I and class II proteins from pea, respectively, were expressed in Escherichia coli and purified. Non-denaturing polyacrylamide gel electrophoresis and electron microscopy demonstrated that the purified proteins formed discretely sized, high molecular weight complexes. Sedimentation equilibrium analytical ultracentrifugation revealed that the HSP18.1 and HSP17.7 complexes were composed of approximately 12 subunits. Both proteins were able to enhance the refolding of chemically denatured citrate synthase and lactate dehydrogenase at stoichiometric levels in an ATP-independent manner. Furthermore, HSP18.1 and HSP17.7 prevented aggregation of citrate synthase at 45 degrees C and irreversible inactivation of citrate synthase at 38 degrees C. HSP18.1 also suppressed aggregation of lactate dehydrogenase at 55 degrees C. These findings demonstrate that HSP18.1 and HSP17.7 can function as molecular chaperones in vitro.