The Structure of the β-Casein Phosphopeptide Consists of Two Independent Intrinsically Disordered...
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2445-Pos Board B137 Parsing the Contributions of Polypeptide Backbones and Sidechains to Denaturation in Concentrated Aqueous Solutions of Urea and Guanidi- nium Chloride Alex S. Holehouse 1 , Nicholas Lyle 2 , Andreas Vitalis 3 , Devarajan Thirumalai 4 , Rohit V. Pappu 1 . 1 Biomedical Engineering, Washington University in Saint Louis, St Louis, MO, USA, 2 Partek Incorporated, St Louis, MO, USA, 3 Department of Biochemistry, University of Zurich, Zurich, Switzerland, 4 Institute for Physical Sciences and Technology, University of Maryland, College Park, MD, USA. The mechanistic details of protein denaturation are relevant for understanding the nature of the collapse transition that is induced by dilution from denaturing to predominantly aqueous solutions. Concentrated solutions of urea and GdnCl are thought to be good solvents for generic proteins. Here we report results from a series of systematic molecular dynamics simulations. We show that the sol- vent quality of a denaturing solution depends on the chemical details of the polypeptide system. Polyglycine prefers collapsed states in highly concentrated aqueous solutions of GdnCl, implying that these solvents are poor solvents for polypeptide backbones. The induction of chain expansion in 6 M GdnCl requires the addition of specific categories of sidechains including those with aromatic groups, primary amides, and charged groups. Polyglycine expands in highly concentrated aqueous solutions of urea. However, we show that intra-chain and chain-solvent interactions are almost perfectly counterbalanced in 8 M urea, implying that these conditions are theta solvents for generic poly- peptide backbones. The degree of chain expansion can be enhanced in urea by the addition of sidechains that interact favorably with urea molecules. Polypep- tide backbones and sidechains contribute to chain expansion through preferen- tial interactions with denaturant molecules in 6 M GdnCl and 8 M urea, although the contributions from sidechain and backbone specific interactions are dif- ferent in the two milieus. Importantly, we observe that the degree of expansion falls short of the upper limit that is achievable for self-avoiding random walks. This implies that the degree of chain expansion can be sequence-specific and be impacted by residual intra-chain attractions. This should influence the details of the collapse transition upon dilution from denaturants. 2446-Pos Board B138 The Structure of the b-Casein Phosphopeptide Consists of Two Indepen- dent Intrinsically Disordered Domains Muhammad A. Naqvi 1 , Sarah Rauscher 2 , Re ´gis Pome `s 2 , De ´rick Rousseau 3 . 1 Ryerson University, Richmond Hill, ON, Canada, 2 Molecular Structure and Function, Hospital of Sick Children, Toronto, ON, Canada, 3 Ryerson University, Toronto, ON, Canada. The determination of the b-casein phosphopeptide 1-25 (b-CPP) structure has to date remained elusive, yet has important implications for calcium binding and cellular transduction as well as the mechanisms involved with dental remineralization. Though its high net charge of 13e suggests that it is an intrin- sically disordered peptide (IDP), there is significant disagreement regarding its degree of disorder. The structure of b-CPP was examined via molecular dynamics (MD) simulations to investigate its degree of disorder. One hundred independent MD simulations for a cumulative time of 30 ms were conducted in explicit water with 0.1 M sodium chloride. The results showed that b-CPP adopts an ensemble of collapsed conformations (Rg=8.6150.06 A ˚ ) that are stabilized by hydrogen bonding (HB) as well as ionic interactions. The HB contact map showed a lack of interaction between the peptide’s head (RELEELNVPGEIVES x) and tail (S xS xS xEESITR) domains suggesting that they were conformationally independent. Significant backbone HB interactions were observed amongst the amino acids within each domain, further indicating that they were disordered. By determining the conformational ensemble of this peptide, this study has successfully shown that b-CPP is an IDP with two inde- pendent, intrinsically disordered domains. 2447-Pos Board B139 MD Simulation Trajectories of Multiple Intrinsically Disordered Proteins Reveal Order to Disorder Transitions that Bear Functional Significance Tasneem Ali, Mattaparthi Venkata, Satish Kumar, Saurabh Basu, Rajaram Swaminathan. Indian Institute of Technology Guwahati, Guwahati, India. Intrinsically disordered proteins (IDPs) explore a dynamic ensemble of confor- mations and yet retain a functional role inside the cell. However, the correlation of backbone chain dynamics of an IDP correlated with its cellular function is not fully understood. Here in this work we analyze the MD simulation trajec- tories (12500 timesteps of total 10 nanosecond duration) of one ordered protein (catalytic domain of MutY from E. Coli, pdb code: 1MUN), one partially ordered protein (Apoptosis regulator Bcl-xL, 1LXL) and four IDPs (Brak/ CXCL14, 2HDL; sub-domain of staphylococcal nuclease, 2SOB; F6 subunit of ATP synthase, 1VZS and Tyrosyl tRNA synthestase, 1JH3). The analysis included distance variations between chosen multiple points in the protein backbone, probability distributions of the measured distances and their veloc- ities, and full width half maxima of these probability distributions. The results from the analysis yielded a quantitative measure of dynamics at loop regions in comparison to helix and strand regions. In addition the distance probability dis- tributions of loop regions in IDPs specifically displayed multimodal character indicating distinct preferences for certain distances. This was in contrast to velocity probability distributions which were always of unimodal nature for all the six proteins. Further, we attempted to quantify the randomness in disor- dered chain dynamics by computing the Boltzmann entropy for all c-alpha atoms in each of the proteins. This entropy value for a given c-alpha atom correlated with its wandering ability in space. Disorder-order transition regions were spotted from the created protein movies and analyzed quantitatively by constructing Ramachandran plots for those regions across all time steps. Such regions included lysine residues previously speculated to be involved in post-translational modifications in F6 unit of ATP synthase and RNA binding domain in Tyrosyl tRNA synthestase. 2448-Pos Board B140 Physical Bioinformatics Applied to Intrinsically Disordered Nucleoporin Sequences Reveals Universal Functional Features David Ando 1 , Michael Colvin 2 , Michael Rexach 3 , Ajay Gopinathan 1 . 1 Physics, UC Merced, Merced, CA, USA, 2 Chemistry, UC Merced, Merced, CA, USA, 3 Molecular, Cell, and Developmental Biology, UC Santa Cruz, Santa Cruz, CA, USA. Bioinformatics of disordered proteins is especially challenging given high mutation rates for homologous proteins and that functionality may not be strongly related to sequence. We introduce a novel form of bioinformatic anal- ysis, which can be applied to disordered proteins, based on the spatial clustering of physically relevant features such as binding motifs and charges. We apply this technique on thousands of disordered Nuclear Pore Complex (NPC) FG motif containing proteins (FG nups) to elucidate the elusive biophysical mech- anism by which FG nups regulate nucleocytoplasmic transport. Our analysis reveals a set of highly conserved spatial features in the sequence structure of individual FG nups, such as the separation, localization, and ordering of FG motifs and charged residues along the protein chain. These conserved features provide insight into the functioning of the pore and strongly constrain current models. Additionally this method allows us to identify potentially functionally analogous disordered proteins across distantly related species. 2449-Pos Board B141 Extensive use of Host-Mimicking Motifs Supports Complex Regulation of Viral Proteins Tzachi Hagai 1 , Raul Andino 1 , Madan M. Babu 2 , Ariel Azia 3 . 1 University of California, San Francisco, San Francisco, CA, USA, 2 MRC-Laboratory of Molecular Biology, Cambridge, United Kingdom, 3 Bar Ilan University, Ramat Gan, Israel. In order to successfully replicate, viruses engage in complex interactions with their hosts. Small motifs that are used by eukaryotes in cellular regulation are thought to be mimicked by viral proteins to support their interactions with the host. Due to experimental difficulties, comprehensive analyses of which viruses and what proteins use motifs, as well as their role in modulating host-virus interactions, has so far been hampered. Here, we analyze a large set of viral pro- teins representing all viral types and most viral families. We show that the occurrence of motifs varies greatly among viral families and protein types. Some proteins seem to use motifs sparsely whereas others contain numerous motifs that co-occur in a manner that facilitates multiple coordinated interac- tions. Our results reveal a surprising complexity of combinatorial regulation of viral proteins, characteristic of tightly-regulated eukaryotic proteins, to achieve an efficient and finely-tuned infection. Transcription 2450-Pos Board B142 Mechanism of Transcriptional Bursting in Bacteria Shasha Chong 1 , Chongyi Chen 1 , Hao Ge 2 , X. Sunney Xie 1,2 . 1 Harvard University, Cambridge, MA, USA, 2 Peking University, Beijing, China. Many single-cell experiments have shown that transcription of highly ex- pressed genes occurs in stochastic bursts in bacteria. Here we present the mechanism of this ubiquitous phenomenon. We develop a high-throughput in vitro single-molecule assay to observe real-time transcription on individual DNA templates. Using this assay, we demonstrate that positive supercoiling buildup on the DNA by transcription slows down transcription elongation 484a Tuesday, February 18, 2014
Transcript of The Structure of the β-Casein Phosphopeptide Consists of Two Independent Intrinsically Disordered...
484a Tuesday, February 18, 2014
2445-Pos Board B137Parsing the Contributions of Polypeptide Backbones and Sidechains toDenaturation in Concentrated Aqueous Solutions of Urea and Guanidi-nium ChlorideAlex S. Holehouse1, Nicholas Lyle2, Andreas Vitalis3,Devarajan Thirumalai4, Rohit V. Pappu1.1Biomedical Engineering, Washington University in Saint Louis, St Louis,MO, USA, 2Partek Incorporated, St Louis, MO, USA, 3Department ofBiochemistry, University of Zurich, Zurich, Switzerland, 4Institute forPhysical Sciences and Technology, University of Maryland, College Park,MD, USA.The mechanistic details of protein denaturation are relevant for understandingthe nature of the collapse transition that is induced by dilution from denaturingto predominantly aqueous solutions. Concentrated solutions of urea and GdnClare thought to be good solvents for generic proteins. Here we report results froma series of systematic molecular dynamics simulations. We show that the sol-vent quality of a denaturing solution depends on the chemical details of thepolypeptide system. Polyglycine prefers collapsed states in highly concentratedaqueous solutions of GdnCl, implying that these solvents are poor solvents forpolypeptide backbones. The induction of chain expansion in 6 M GdnClrequires the addition of specific categories of sidechains including those witharomatic groups, primary amides, and charged groups. Polyglycine expandsin highly concentrated aqueous solutions of urea. However, we show thatintra-chain and chain-solvent interactions are almost perfectly counterbalancedin 8 M urea, implying that these conditions are theta solvents for generic poly-peptide backbones. The degree of chain expansion can be enhanced in urea bythe addition of sidechains that interact favorably with urea molecules. Polypep-tide backbones and sidechains contribute to chain expansion through preferen-tial interactions with denaturant molecules in 6MGdnCl and 8M urea, althoughthe contributions from sidechain and backbone specific interactions are dif-ferent in the two milieus. Importantly, we observe that the degree of expansionfalls short of the upper limit that is achievable for self-avoiding random walks.This implies that the degree of chain expansion can be sequence-specific and beimpacted by residual intra-chain attractions. This should influence the details ofthe collapse transition upon dilution from denaturants.
2446-Pos Board B138The Structure of the b-Casein Phosphopeptide Consists of Two Indepen-dent Intrinsically Disordered DomainsMuhammad A. Naqvi1, Sarah Rauscher2, Regis Pomes2, Derick Rousseau3.1Ryerson University, Richmond Hill, ON, Canada, 2Molecular Structure andFunction, Hospital of Sick Children, Toronto, ON, Canada, 3RyersonUniversity, Toronto, ON, Canada.The determination of the b-casein phosphopeptide 1-25 (b-CPP) structure hasto date remained elusive, yet has important implications for calcium bindingand cellular transduction as well as the mechanisms involved with dentalremineralization. Though its high net charge of 13e suggests that it is an intrin-sically disordered peptide (IDP), there is significant disagreement regarding itsdegree of disorder. The structure of b-CPP was examined via moleculardynamics (MD) simulations to investigate its degree of disorder. One hundredindependent MD simulations for a cumulative time of 30 ms were conductedin explicit water with 0.1 M sodium chloride. The results showed thatb-CPP adopts an ensemble of collapsed conformations (Rg=8.6150.06 A)that are stabilized by hydrogen bonding (HB) as well as ionic interactions.The HB contact map showed a lack of interaction between the peptide’shead (RELEELNVPGEIVESx) and tail (SxSxSxEESITR) domains suggesting thatthey were conformationally independent. Significant backbone HB interactionswere observed amongst the amino acids within each domain, further indicatingthat they were disordered. By determining the conformational ensemble of thispeptide, this study has successfully shown that b-CPP is an IDP with two inde-pendent, intrinsically disordered domains.
2447-Pos Board B139MD Simulation Trajectories of Multiple Intrinsically Disordered ProteinsReveal Order to Disorder Transitions that Bear Functional SignificanceTasneem Ali, Mattaparthi Venkata, Satish Kumar, Saurabh Basu,Rajaram Swaminathan.Indian Institute of Technology Guwahati, Guwahati, India.Intrinsically disordered proteins (IDPs) explore a dynamic ensemble of confor-mations and yet retain a functional role inside the cell. However, the correlationof backbone chain dynamics of an IDP correlated with its cellular function isnot fully understood. Here in this work we analyze the MD simulation trajec-tories (12500 timesteps of total 10 nanosecond duration) of one ordered protein(catalytic domain of MutY from E. Coli, pdb code: 1MUN), one partiallyordered protein (Apoptosis regulator Bcl-xL, 1LXL) and four IDPs (Brak/CXCL14, 2HDL; sub-domain of staphylococcal nuclease, 2SOB; F6 subunit
of ATP synthase, 1VZS and Tyrosyl tRNA synthestase, 1JH3). The analysisincluded distance variations between chosen multiple points in the proteinbackbone, probability distributions of the measured distances and their veloc-ities, and full width half maxima of these probability distributions. The resultsfrom the analysis yielded a quantitative measure of dynamics at loop regions incomparison to helix and strand regions. In addition the distance probability dis-tributions of loop regions in IDPs specifically displayed multimodal characterindicating distinct preferences for certain distances. This was in contrast tovelocity probability distributions which were always of unimodal nature forall the six proteins. Further, we attempted to quantify the randomness in disor-dered chain dynamics by computing the Boltzmann entropy for all c-alphaatoms in each of the proteins. This entropy value for a given c-alpha atomcorrelated with its wandering ability in space. Disorder-order transition regionswere spotted from the created protein movies and analyzed quantitatively byconstructing Ramachandran plots for those regions across all time steps.Such regions included lysine residues previously speculated to be involved inpost-translational modifications in F6 unit of ATP synthase and RNA bindingdomain in Tyrosyl tRNA synthestase.
2448-Pos Board B140Physical Bioinformatics Applied to Intrinsically Disordered NucleoporinSequences Reveals Universal Functional FeaturesDavid Ando1, Michael Colvin2, Michael Rexach3, Ajay Gopinathan1.1Physics, UC Merced, Merced, CA, USA, 2Chemistry, UC Merced, Merced,CA, USA, 3Molecular, Cell, and Developmental Biology, UC Santa Cruz,Santa Cruz, CA, USA.Bioinformatics of disordered proteins is especially challenging given highmutation rates for homologous proteins and that functionality may not bestrongly related to sequence. We introduce a novel form of bioinformatic anal-ysis, which can be applied to disordered proteins, based on the spatial clusteringof physically relevant features such as binding motifs and charges. We applythis technique on thousands of disordered Nuclear Pore Complex (NPC) FGmotif containing proteins (FG nups) to elucidate the elusive biophysical mech-anism by which FG nups regulate nucleocytoplasmic transport. Our analysisreveals a set of highly conserved spatial features in the sequence structure ofindividual FG nups, such as the separation, localization, and ordering of FGmotifs and charged residues along the protein chain. These conserved featuresprovide insight into the functioning of the pore and strongly constrain currentmodels. Additionally this method allows us to identify potentially functionallyanalogous disordered proteins across distantly related species.
2449-Pos Board B141Extensive use of Host-Mimicking Motifs Supports Complex Regulation ofViral ProteinsTzachi Hagai1, Raul Andino1, Madan M. Babu2, Ariel Azia3.1University of California, San Francisco, San Francisco, CA, USA,2MRC-Laboratory of Molecular Biology, Cambridge, United Kingdom,3Bar Ilan University, Ramat Gan, Israel.In order to successfully replicate, viruses engage in complex interactions withtheir hosts. Small motifs that are used by eukaryotes in cellular regulation arethought to be mimicked by viral proteins to support their interactions with thehost. Due to experimental difficulties, comprehensive analyses of which virusesand what proteins use motifs, as well as their role in modulating host-virusinteractions, has so far been hampered. Here, we analyze a large set of viral pro-teins representing all viral types and most viral families. We show that theoccurrence of motifs varies greatly among viral families and protein types.Some proteins seem to use motifs sparsely whereas others contain numerousmotifs that co-occur in a manner that facilitates multiple coordinated interac-tions. Our results reveal a surprising complexity of combinatorial regulationof viral proteins, characteristic of tightly-regulated eukaryotic proteins, toachieve an efficient and finely-tuned infection.
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2450-Pos Board B142Mechanism of Transcriptional Bursting in BacteriaShasha Chong1, Chongyi Chen1, Hao Ge2, X. Sunney Xie1,2.1Harvard University, Cambridge, MA, USA, 2Peking University, Beijing,China.Many single-cell experiments have shown that transcription of highly ex-pressed genes occurs in stochastic bursts in bacteria. Here we present themechanism of this ubiquitous phenomenon. We develop a high-throughput invitro single-molecule assay to observe real-time transcription on individualDNA templates. Using this assay, we demonstrate that positive supercoilingbuildup on the DNA by transcription slows down transcription elongation
