Challenges in Large-Scale Molecular Simulations of Intracellular Environments
1
27-Subg Protein Folding in Living Cells Martin Gruebele. U. of Illinois, Urbana, IL, USA. Protein stability, folding and unfolding kinetics are examined in living mamma- lian and bacterial cells. Heterogeneous kinetics are seen in the nucleus, cyto- plasm, ER, as well as among cells in a population. The diffusion coefficient for folding and tuning of the protein free energy landscape by the cellular milieu are quantified. 28-Subg Crowding Induced Conformational Switch Devarajan Thirumalai. University of Maryland, College Park, MD, USA. In this talk I will describe a general theoretical framework for describing con- formational transitions in biomolecules and biomolecular complexes. The the- ory is based on the concept of depletion attraction well known in colloid science. The predictions of theory and simulations using coarse-grained models will be compared to specific experiments. 29-Subg Protein Folding Trends in the Cellular Environment Silvia Cavagnero. University of Wisconsin-Madison, Madison, WI, USA. The pathways of protein folding in vivo are still poorly understood despite the tremendous impact of correct folding for cell function. In the case of small/me- dium-size proteins, kinetic arguments suggest there is ample time for cotransla- tional conformational sampling, while nascent chains are still ribosome-bound and before synthesis of the full length protein is complete. Given the complex- ity of the cellular environment (molecular crowding, chaperones, ribosome), a combination of tailored biological and spectroscopic tools is likely needed to drive progress in this area. This lecture will present our current efforts to un- derstand how proteins acquire the ability to attain their native state as nascent chains elongate from N to C terminus. 30-Subg Challenges in Large-Scale Molecular Simulations of Intracellular Environments Adrian H. Elcock. University of Iowa, Iowa City, IA, USA. Owing to advances in structural biology, proteomics and computational modeling it is now possible to consider constructing working simulation models of certain intra- cellular environments. Using bacterial sys- tems for illustrative purposes, this talk will first briefly describe some promising re- sults obtained with Brownian dynamics simulations methods - such as the ability to capture aspects of protein diffusion and thermodynamic stability in the bacterial cytoplasm. The talk will then discuss re- cent developments and ongoing projects before attempting to highlight a number of significant challenges likely to be faced by simulation models intended to more realistically describe events in vivo. SUBGROUP: Permeation & Transport 31-Subg Hydration Properties of Mechanosensitive Channel Pores: Dewetting and Stabilization of Conductive States Sergei Sukharev. University of Maryland, College Park, MD, USA. MscS is the major osmolyte efflux valve that regulates turgor in many walled organisms from bacteria to higher plants. The crystal structure of WT E. coli MscS revealed a tight packing of seven pore-lining TM3 helices mediated by conserved Ala-Gly motifs. Complementarity of helical interfaces still leaves a 6.5A ˚ unoccluded pore in the hydrophobic constriction (gate) formed by two rings of leucines. Initial computational studies suggested that that the gate is dehydrated and the crystal conformation should be non-conductive. Our further analysis indicated that the ‘outer chamber’ flanking the gate from the periplasmic side may also be dehydrated, producing a ‘vapor plug’ ~20A ˚ long. Experimental analysis of hydrophilic substitutions in the outer chamber revealed decreases in the tension threshold and in-plane protein expansion on opening suggesting that the outer chamber is likely to be dehydrated at rest. The data reveal the principle difference between physical closure of the gate with the backbone-attached side chains and occlusion by vapor: water is mobile and after the breakage of the continuous phase it can freely search for new en- ergy minima setting new boundaries and stabilizing nonconductive states. Un- der membrane tension of ~5-8 mN/m,MscS opens a 16A ˚ pore that is fully hydrated. The opening transition begins with expansion of water-vapor inter- faces, proceeding through wetting of the outer chamber and partial separation of TM3s, which ultimately leads to complete wetting of the hydrophobic pore. We discuss specifically the role of conserved glycines in TM3, which are buried in narrow closed conformations but exposed in the open states that critically changes the wetting characteristics of the pore lining from hydrophobic to hy- drophilic and stabilize the conductive state of the channel. We discuss a similar open-state stabilizing mechanism in MscL. 32-Subg Molecular Mechanisms of Ion Selectivity in Membrane Proteins: Ion Channels and Transporters Sergei Noskov. University of Calgary, Calgary, AB, Canada. The mechanism of ion binding to membrane proteins and subsequent ion reg- ulation of protein function is a subject that has fascinated scientists for a long time. Although the implications and applications of selective ion binding are many, ranging from management of cell volume to proper electric signaling upon subsequent transport across cellular membranes to being integral part of new nano-materials, our understanding of the underlying complex thermo- dynamics of ion binding and subsequent permeation across cellular mem- branes is very limited. Recent progress in structural studies of secondary amino-acid transporters provides us with a unique opportunity to address mo- lecular mechanism of the cation selectivity in the protein with available high- resolution crystal structure and confirmed Naþ/Kþ or Kþ/Naþ selectivity. Combined results of our studies on K-selective channels (KcsA and MthK), non-selective channels (NaK) and Naþ-coupled secondary transporters will be presented. A combination of the free energy simulations with classical and polarizable force-fields as well as recently developed QM/MM FEP pro- tocol was used for evaluation of different factors in the observed selectivity in protein sites. Atomistics simulations blazed the trail for the development of an analytical theory than allows quantification of ion selectivity within a reduced framework. In conclusion, I will present a theory to clarify the molecular de- terminants of ion selectivity in protein binding sites developed recently (Yu et al., PNAS 2011) that integrates our current knowledge on monovalent cation selectivity. 33-Subg Ion Selectivity and Permeation in a Potassium Ion Transporter Ming Zhou. Columbia University, New York, NY, USA. The TrkH/TrkG/KtrB family of proteins mediate K þ uptake in bacteria and likely evolved from simple K þ channels by multiple gene duplications or fusions. We have solved the crystal structure of a TrkH from Vibrio para- haemolyticus to 3.5 A ˚ . TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to that in K þ channels but significantly shorter, is lined by backbone and side chain oxygen atoms. Functional studies showed that TrkH allows permeation of K þ and Rb þ but not smaller ions such as Na þ or Li þ . Immediately intracel- lular to the selectivity filter is an intramembrane loop and an arginine residue, both highly conserved, that constrict an otherwise open permeation pathway. Functions of the loop and the positive charge (Arg) will be addressed. 6a Saturday, March 5, 2011
Transcript of Challenges in Large-Scale Molecular Simulations of Intracellular Environments
6a Saturday, March 5, 2011
27-SubgProtein Folding in Living CellsMartin Gruebele.U. of Illinois, Urbana, IL, USA.Protein stability, folding and unfolding kinetics are examined in living mamma-lian and bacterial cells. Heterogeneous kinetics are seen in the nucleus, cyto-plasm, ER, as well as among cells in a population. The diffusion coefficientfor folding and tuning of the protein free energy landscape by the cellularmilieu are quantified.
28-SubgCrowding Induced Conformational SwitchDevarajan Thirumalai.University of Maryland, College Park, MD, USA.In this talk I will describe a general theoretical framework for describing con-formational transitions in biomolecules and biomolecular complexes. The the-ory is based on the concept of depletion attraction well known in colloidscience. The predictions of theory and simulations using coarse-grained modelswill be compared to specific experiments.
29-SubgProtein Folding Trends in the Cellular EnvironmentSilvia Cavagnero.University of Wisconsin-Madison, Madison, WI, USA.The pathways of protein folding in vivo are still poorly understood despite thetremendous impact of correct folding for cell function. In the case of small/me-dium-size proteins, kinetic arguments suggest there is ample time for cotransla-tional conformational sampling, while nascent chains are still ribosome-boundand before synthesis of the full length protein is complete. Given the complex-ity of the cellular environment (molecular crowding, chaperones, ribosome),a combination of tailored biological and spectroscopic tools is likely neededto drive progress in this area. This lecture will present our current efforts to un-derstand how proteins acquire the ability to attain their native state as nascentchains elongate from N to C terminus.
30-SubgChallenges in Large-Scale Molecular Simulations of IntracellularEnvironmentsAdrian H. Elcock.University of Iowa, Iowa City, IA, USA.
Owing to advances in structural biology,proteomics and computational modeling itis now possible to consider constructingworking simulation models of certain intra-cellular environments. Using bacterial sys-tems for illustrative purposes, this talk willfirst briefly describe some promising re-sults obtained with Brownian dynamicssimulations methods - such as the abilityto capture aspects of protein diffusion andthermodynamic stability in the bacterialcytoplasm. The talk will then discuss re-cent developments and ongoing projects
before attempting to highlight a number of significant challenges likely to befaced by simulation models intended to more realistically describe eventsin vivo.
SUBGROUP: Permeation & Transport
31-SubgHydration Properties of Mechanosensitive Channel Pores:Dewetting and Stabilization of Conductive StatesSergei Sukharev.University of Maryland, College Park, MD, USA.MscS is the major osmolyte efflux valve that regulates turgor in many walledorganisms from bacteria to higher plants. The crystal structure of WT E. coliMscS revealed a tight packing of seven pore-lining TM3 helices mediated by
conserved Ala-Gly motifs. Complementarity of helical interfaces still leavesa 6.5A unoccluded pore in the hydrophobic constriction (gate) formed bytwo rings of leucines. Initial computational studies suggested that that thegate is dehydrated and the crystal conformation should be non-conductive.Our further analysis indicated that the ‘outer chamber’ flanking the gate fromthe periplasmic side may also be dehydrated, producing a ‘vapor plug’ ~20Along. Experimental analysis of hydrophilic substitutions in the outer chamberrevealed decreases in the tension threshold and in-plane protein expansion onopening suggesting that the outer chamber is likely to be dehydrated at rest.The data reveal the principle difference between physical closure of the gatewith the backbone-attached side chains and occlusion by vapor: water is mobileand after the breakage of the continuous phase it can freely search for new en-ergy minima setting new boundaries and stabilizing nonconductive states. Un-der membrane tension of ~5-8 mN/m,MscS opens a 16A pore that is fullyhydrated. The opening transition begins with expansion of water-vapor inter-faces, proceeding through wetting of the outer chamber and partial separationof TM3s, which ultimately leads to complete wetting of the hydrophobic pore.We discuss specifically the role of conserved glycines in TM3, which are buriedin narrow closed conformations but exposed in the open states that criticallychanges the wetting characteristics of the pore lining from hydrophobic to hy-drophilic and stabilize the conductive state of the channel. We discuss a similaropen-state stabilizing mechanism in MscL.
32-SubgMolecular Mechanisms of Ion Selectivity in Membrane Proteins:Ion Channels and TransportersSergei Noskov.University of Calgary, Calgary, AB, Canada.The mechanism of ion binding to membrane proteins and subsequent ion reg-ulation of protein function is a subject that has fascinated scientists for a longtime. Although the implications and applications of selective ion binding aremany, ranging from management of cell volume to proper electric signalingupon subsequent transport across cellular membranes to being integral partof new nano-materials, our understanding of the underlying complex thermo-dynamics of ion binding and subsequent permeation across cellular mem-branes is very limited. Recent progress in structural studies of secondaryamino-acid transporters provides us with a unique opportunity to address mo-lecular mechanism of the cation selectivity in the protein with available high-resolution crystal structure and confirmed Naþ/Kþ or Kþ/Naþ selectivity.Combined results of our studies on K-selective channels (KcsA and MthK),non-selective channels (NaK) and Naþ-coupled secondary transporters willbe presented. A combination of the free energy simulations with classicaland polarizable force-fields as well as recently developed QM/MM FEP pro-tocol was used for evaluation of different factors in the observed selectivity inprotein sites. Atomistics simulations blazed the trail for the development of ananalytical theory than allows quantification of ion selectivity within a reducedframework. In conclusion, I will present a theory to clarify the molecular de-terminants of ion selectivity in protein binding sites developed recently(Yu et al., PNAS 2011) that integrates our current knowledge on monovalentcation selectivity.
33-SubgIon Selectivity and Permeation in a Potassium Ion TransporterMing Zhou.Columbia University, New York, NY, USA.The TrkH/TrkG/KtrB family of proteins mediate Kþ uptake in bacteria andlikely evolved from simple Kþ channels by multiple gene duplications orfusions. We have solved the crystal structure of a TrkH from Vibrio para-haemolyticus to 3.5 A. TrkH is a homodimer, and each protomer contains anion permeation pathway. A selectivity filter, similar in architecture to that inKþ channels but significantly shorter, is lined by backbone and side chainoxygen atoms. Functional studies showed that TrkH allows permeation ofKþ and Rbþ but not smaller ions such as Naþ or Liþ. Immediately intracel-lular to the selectivity filter is an intramembrane loop and an arginineresidue, both highly conserved, that constrict an otherwise open permeationpathway. Functions of the loop and the positive charge (Arg) will beaddressed.
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