2,851 research outputs found
Entropy-driven genome organization
DNA and RNA polymerases active on bacterial and human genomes in the crowded environment of a cell are modeled as beads spaced along a string. Aggregation of the large polymerizing complexes increases the entropy of the system through an increase in entropy of the many small crowding molecules; this occurs despite the entropic costs of looping the intervening DNA. Results of a quantitative cost/benefit analysis are consistent with observations that active polymerases cluster into replication and transcription âfactoriesâ in both pro- and eukaryotes. We conclude that the second law of thermodynamics acts through nonspecific entropic forces between engaged polymerases to drive the self-organization of genomes into loops containing several thousands (and sometimes millions) of basepairs
Machine learning applied to enzyme turnover numbers reveals protein structural correlates and improves metabolic models.
Knowing the catalytic turnover numbers of enzymes is essential for understanding the growth rate, proteome composition, and physiology of organisms, but experimental data on enzyme turnover numbers is sparse and noisy. Here, we demonstrate that machine learning can successfully predict catalytic turnover numbers in Escherichia coli based on integrated data on enzyme biochemistry, protein structure, and network context. We identify a diverse set of features that are consistently predictive for both in vivo and in vitro enzyme turnover rates, revealing novel protein structural correlates of catalytic turnover. We use our predictions to parameterize two mechanistic genome-scale modelling frameworks for proteome-limited metabolism, leading to significantly higher accuracy in the prediction of quantitative proteome data than previous approaches. The presented machine learning models thus provide a valuable tool for understanding metabolism and the proteome at the genome scale, and elucidate structural, biochemical, and network properties that underlie enzyme kinetics
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Adaptations of Escherichia coli strains to oxidative stress are reflected in properties of their structural proteomes.
BACKGROUND:The reconstruction of metabolic networks and the three-dimensional coverage of protein structures have reached the genome-scale in the widely studied Escherichia coli K-12 MG1655 strain. The combination of the two leads to the formation of a structural systems biology framework, which we have used to analyze differences between the reactive oxygen species (ROS) sensitivity of the proteomes of sequenced strains of E. coli. As proteins are one of the main targets of oxidative damage, understanding how the genetic changes of different strains of a species relates to its oxidative environment can reveal hypotheses as to why these variations arise and suggest directions of future experimental work. RESULTS:Creating a reference structural proteome for E. coli allows us to comprehensively map genetic changes in 1764 different strains to their locations on 4118 3D protein structures. We use metabolic modeling to predict basal ROS production levels (ROStype) for 695 of these strains, finding that strains with both higher and lower basal levels tend to enrich their proteomes with antioxidative properties, and speculate as to why that is. We computationally assess a strain's sensitivity to an oxidative environment, based on known chemical mechanisms of oxidative damage to protein groups, defined by their localization and functionality. Two general groups - metalloproteins and periplasmic proteins - show enrichment of their antioxidative properties between the 695 strains with a predicted ROStype as well as 116 strains with an assigned pathotype. Specifically, proteins that a) utilize a molybdenum ion as a cofactor and b) are involved in the biogenesis of fimbriae show intriguing protective properties to resist oxidative damage. Overall, these findings indicate that a strain's sensitivity to oxidative damage can be elucidated from the structural proteome, though future experimental work is needed to validate our model assumptions and findings. CONCLUSION:We thus demonstrate that structural systems biology enables a proteome-wide, computational assessment of changes to atomic-level physicochemical properties and of oxidative damage mechanisms for multiple strains in a species. This integrative approach opens new avenues to study adaptation to a particular environment based on physiological properties predicted from sequence alone
Cryo-EM structures of herpes simplex virus type 1 portal vertex and packaged genome.
Herpesviruses are enveloped viruses that are prevalent in the human population and are responsible for diverse pathologies, including cold sores, birth defects and cancers. They are characterized by a highly pressurized pseudo-icosahedral capsid-with triangulation number (T) equal to 16-encapsidating a tightly packed double-stranded DNA (dsDNA) genome1-3. A key process in the herpesvirus life cycle involves the recruitment of an ATP-driven terminase to a unique portal vertex to recognize, package and cleave concatemeric dsDNA, ultimately giving rise to a pressurized, genome-containing virion4,5. Although this process has been studied in dsDNA phages6-9-with which herpesviruses bear some similarities-a lack of high-resolution in situ structures of genome-packaging machinery has prevented the elucidation of how these multi-step reactions, which require close coordination among multiple actors, occur in an integrated environment. To better define the structural basis of genome packaging and organization in herpes simplex virus type 1 (HSV-1), we developed sequential localized classification and symmetry relaxation methods to process cryo-electron microscopy (cryo-EM) images of HSV-1 virions, which enabled us to decouple and reconstruct hetero-symmetric and asymmetric elements within the pseudo-icosahedral capsid. Here we present in situ structures of the unique portal vertex, genomic termini and ordered dsDNA coils in the capsid spooled around a disordered dsDNA core. We identify tentacle-like helices and a globular complex capping the portal vertex that is not observed in phages, indicative of herpesvirus-specific adaptations in the DNA-packaging process. Finally, our atomic models of portal vertex elements reveal how the fivefold-related capsid accommodates symmetry mismatch imparted by the dodecameric portal-a longstanding mystery in icosahedral viruses-and inform possible DNA-sequence recognition and headful-sensing pathways involved in genome packaging. This work showcases how to resolve symmetry-mismatched elements in a large eukaryotic virus and provides insights into the mechanisms of herpesvirus genome packaging
Structural Prediction of ProteinâProtein Interactions by Docking: Application to Biomedical Problems
A huge amount of genetic information is available thanks to the recent advances in sequencing technologies and the larger computational capabilities, but the interpretation of such genetic data at phenotypic level remains elusive. One of the reasons is that proteins are not acting alone, but are specifically interacting with other proteins and biomolecules, forming intricate interaction networks that are essential for the majority of cell processes and pathological conditions. Thus, characterizing such interaction networks is an important step in understanding how information flows from gene to phenotype. Indeed, structural characterization of proteinâprotein interactions at atomic resolution has many applications in biomedicine, from diagnosis and vaccine design, to drug discovery. However, despite the advances of experimental structural determination, the number of interactions for which there is available structural data is still very small. In this context, a complementary approach is computational modeling of protein interactions by docking, which is usually composed of two major phases: (i) sampling of the possible binding modes between the interacting molecules and (ii) scoring for the identification of the correct orientations. In addition, prediction of interface and hot-spot residues is very useful in order to guide and interpret mutagenesis experiments, as well as to understand functional and mechanistic aspects of the interaction. Computational docking is already being applied to specific biomedical problems within the context of personalized medicine, for instance, helping to interpret pathological mutations involved in proteinâprotein interactions, or providing modeled structural data for drug discovery targeting proteinâprotein interactions.Spanish Ministry of Economy grant number BIO2016-79960-R; D.B.B. is supported by a
predoctoral fellowship from CONACyT; M.R. is supported by an FPI fellowship from the
Severo Ochoa program. We are grateful to the Joint BSC-CRG-IRB Programme in
Computational Biology.Peer ReviewedPostprint (author's final draft
Nanoporous silica-based protocells at multiple scales for designs of life and nanomedicine.
Various protocell models have been constructed de novo with the bottom-up approach. Here we describe a silica-based protocell composed of a nanoporous amorphous silica core encapsulated within a lipid bilayer built by self-assembly that provides for independent definition of cell interior and the surface membrane. In this review, we will first describe the essential features of this architecture and then summarize the current development of silica-based protocells at both micro- and nanoscale with diverse functionalities. As the structure of the silica is relatively static, silica-core protocells do not have the ability to change shape, but their interior structure provides a highly crowded and, in some cases, authentic scaffold upon which biomolecular components and systems could be reconstituted. In basic research, the larger protocells based on precise silica replicas of cells could be developed into geometrically realistic bioreactor platforms to enable cellular functions like coupled biochemical reactions, while in translational research smaller protocells based on mesoporous silica nanoparticles are being developed for targeted nanomedicine. Ultimately we see two different motivations for protocell research and development: (1) to emulate life in order to understand it; and (2) to use biomimicry to engineer desired cellular interactions
ModĂ©lisation mĂ©tabolique Ă lâĂ©chelle du gĂ©nome de la bactĂ©rie quasi-minimale Mesoplasma florum
Des avancĂ©es significatives au niveau de la synthĂšse et de lâassemblage de fragments dâacide dĂ©soxyribonuclĂ©ique (ADN), le support physique des fonctions cellulaires encodĂ©es dans une cellule vivante, permettent maintenant la construction de gĂ©nomes entiers. Ce progrĂšs permet dâimaginer que la conception dâorganismes synthĂ©tiques deviendra routiniĂšre au cours des prochaines annĂ©es. Cette capacitĂ© promet de transformer radicalement le domaine de la biologie en formant une nouvelle discipline dâingĂ©nierie biologique. Parmi les retombĂ©es anticipĂ©es, on note le remplacement de synthĂšses chimiques par des procĂ©dĂ©s biologiques renouvelables tels que la production de biocarburants, la synthĂšse de mĂ©dicaments microbiens, ou des approches alternatives pour le traitement des maladies.
Dans ce contexte, il devient particuliĂšrement important dâarriver Ă prĂ©dire correctement le phĂ©notype rĂ©sultant des gĂ©nomes qui seront gĂ©nĂ©rĂ©s. Pour y arriver, il convient de rĂ©duire la complexitĂ© biologique en travaillant dâabord avec les cellules les plus simples possibles. Ce type dâorganisme ayant subi un processus de rĂ©duction de gĂ©nome et dont la majoritĂ© des gĂšnes sont essentiels afin de survivre en conditions dĂ©finies se nomme une cellule minimale. Le groupe phylogĂ©nĂ©tique des mollicutes, bactĂ©ries dĂ©pourvues de paroi cellulaire, contient les espĂšces vivant avec les plus petits gĂ©nomes connus Ă ce jour. Membre de ce groupe, le pathogĂšne humain Mycoplasma genitalium possĂšde le plus petit gĂ©nome capable de croissance autonome (560kbp codant pour 482 protĂ©ines. Cependant, sa pathogĂ©nicitĂ© et sa vitesse de croissance rĂ©duite (~24h) limitent lâapplicabilitĂ© de M. genitalium en biologie synthĂ©tique.
Pour remĂ©dier Ă ce problĂšme, notre laboratoire a choisi de travailler avec Mesoplasma florum dont le temps de doublement est trĂšs rapide (~32 min) et qui ne cause pas de maladies chez lâhumain. Les travaux effectuĂ©s chez M. florum permettent maintenant le clonage et la transplantation de son gĂ©nome et des travaux rĂ©cents ont permis de caractĂ©riser les propriĂ©tĂ©s physico-chimiques de sa cellule ainsi que plusieurs paramĂštres biologiques. Afin de permettre la conception de gĂ©nomes synthĂ©tiques basĂ©s sur M. florum, il convient dâintĂ©grer un maximum de connaissances dans un cadre informatique structurĂ© capable de gĂ©nĂ©rer des prĂ©dictions phĂ©notypiques. Un modĂšle mĂ©tabolique Ă lâĂ©chelle du gĂ©nome (GEM) reposant sur la mĂ©thode dâanalyse des flux Ă lâĂ©quilibre (FBA) reprĂ©sente un format particuliĂšrement intĂ©ressant pour initier ces travaux de biologie des systĂšmes.
La qualitĂ© des prĂ©dictions gĂ©nĂ©rĂ©es par ce type de modĂšle est dĂ©pendante de la prĂ©cision de lâobjectif Ă atteindre. Pour simuler la croissance, les GEMs doivent satisfaire un objectif nommĂ© âfonction objective de biomasseâ (BOF) qui contient lâensemble des mĂ©tabolites nĂ©cessaires Ă la production dâune nouvelle cellule avec des coefficients stĆchiomĂ©triques reprĂ©sentatifs de lâabondance de ces composantes dans la cellule. Pendant mon parcours de doctorat, jâai dĂ©veloppĂ© le logiciel BOFdat qui permet la dĂ©finition dâune BOF reprĂ©sentative de la composition cellulaire spĂ©cifique Ă une espĂšce avec les donnĂ©es expĂ©rimentales associĂ©es. Les deux premiĂšres des trois Ă©tapes de BOFdat dĂ©terminent les coefficients stoechiomĂ©triques de molĂ©cules connues pour faire partie de la composition cellulaire telles que les macromolĂ©cules principales (Ă©tape 1, ADN, ARN et protĂ©ines) et les coenzymes essentiels (Ă©tape 2). LâĂ©tape 3 de BOFdat propose une mĂ©thode non-biaisĂ©e pour dĂ©terminer les mĂ©tabolites susceptibles dâamĂ©liorer la prĂ©diction dâessentialitĂ© des gĂšnes formulĂ©e par le modĂšle. Pour ce faire, un algorithme gĂ©nĂ©tique maximise la composition de la biomasse en fonction des donnĂ©es dâessentialitĂ© expĂ©rimentales Ă lâĂ©chelle du gĂ©nome. BOFdat a Ă©tĂ© validĂ© en reconstruisant la BOF du modĂšle iML1515 de la bactĂ©rie modĂšle Escherichia coli. Lâutilisation de BOFdat a permis de rĂ©capituler le taux de croissance prĂ©dit avec la BOF originale tout en amĂ©liorant la qualitĂ© des prĂ©dictions dâessentialitĂ© de gĂšnes de iML1515. BOFdat est disponible en libre accĂšs pour quiconque dĂ©sire construire une BOF pour un modĂšle mĂ©tabolique.
Ensuite, un GEM nommĂ© iJL208 a Ă©tĂ© produit et contient 208 des 676 protĂ©ines reprĂ©sentant lâensemble du mĂ©tabolisme de M. florum. La qualitĂ© de lâannotation du gĂ©nome a dâabord Ă©tĂ© Ă©valuĂ©e en intĂ©grant lâinformation obtenue par trois approches bio-informatiques, rĂ©vĂ©lant que la majoritĂ© des protĂ©ines (418/676) ont une qualitĂ© suffisante pour ĂȘtre incorporĂ©es dans le modĂšle. Ensuite, les rĂ©actions ont Ă©tĂ© identifiĂ©es et rigoureusement incorporĂ©es une Ă la fois afin de construire le rĂ©seau mĂ©tabolique de cette bactĂ©rie quasi-minimale. LâĂ©tude de la carte mĂ©tabolique reconstruite rĂ©vĂšle une dĂ©pendance prononcĂ©e pour lâimport de composantes Ă partir du milieu de culture ainsi que lâimportance des mĂ©canismes de recyclage des mĂ©tabolites. Pour sa production dâĂ©nergie, M. florum est entiĂšrement dĂ©pendante de la glycolyse et ne possĂšde pas la machinerie nĂ©cessaire Ă la respiration cellulaire. LâĂ©laboration dâun milieu de culture semi-dĂ©fini a rĂ©duit la prĂ©sence de sucres contaminants dans le milieu de culture initial et ainsi de distinguer la croissance avec ou sans supplĂ©mentation de sucrose. Cette avancĂ©e importante a permis de mesurer les taux dâassimilation de sucrose et de production des dĂ©chets mĂ©taboliques lactate et acĂ©tate. Ces paramĂštres ont Ă©tĂ© utilisĂ©s afin de contraindre le modĂšle et de mieux comprendre la sensibilitĂ© du modĂšle Ă une variĂ©tĂ© de paramĂštres. Aussi, la croissance de M. florum a pu ĂȘtre validĂ©e expĂ©rimentalement avec diffĂ©rents sucres. Lâinformation contextuelle obtenue, combinĂ©e Ă une analyse de structures tridimensionnelles de protĂ©ines clĂ©s, a permis de suggĂ©rer des hypothĂšses crĂ©dibles supportant lâassimilation de ces sucres par M. florum.
Finalement, iJL208 a Ă©tĂ© utilisĂ© afin de formuler une prĂ©diction de gĂ©nome minimal pour M. florum en simulant itĂ©rativement de larges dĂ©lĂ©tions dans son gĂ©nome. Combiner lâintĂ©gration de donnĂ©es expĂ©rimentales avec les prĂ©dictions du modĂšle constitue une voie dâavenir pour la conception de gĂ©nomes synthĂ©tiques qui rejoint les capacitĂ©s techniques dâassemblage de chromosomes en biologie synthĂ©tique. Globalement, les projets rĂ©alisĂ©s au cours de mon doctorat contribuent Ă lâavancement de la biologie des systĂšmes chez M. florum dans le but de prĂ©dire efficacement les phĂ©notypes de la souche naturelle et de variants synthĂ©tiques qui pourront ĂȘtre produits au cours des prochaines annĂ©es
Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB
This is the peer reviewed version of the following article: Kemege, K. E., Hickey, J. M., Barta, M. L., Wickstrum, J., Balwalli, N., Lovell, S., Battaile, K. P. and Hefty, P. S. (2015), Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB. Molecular Microbiology, 95: 365â382. doi:10.1111/mmi.12855, which has been published in final form at http://doi.org/10.1111/mmi.12855. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.Cell division in Chlamydiae is poorly understood as apparent homologs to most conserved bacterial cell division proteins are lacking and presence of elongation (rod shape) associated proteins indicate non-canonical mechanisms may be employed. The rod-shape determining protein MreB has been proposed as playing a unique role in chlamydial cell division. In other organisms, MreB is part of an elongation complex that requires RodZ for proper function. A recent study reported that the protein encoded by ORF CT009 interacts with MreB despite low sequence similarity to RodZ. The studies herein expand on those observations through protein structure, mutagenesis, and cellular localization analyses. Structural analysis indicated that CT009 shares high level of structural similarity to RodZ, revealing the conserved orientation of two residues critical for MreB interaction. Substitutions eliminated MreB protein interaction and partial complementation provided by CT009 in RodZ deficient E. coli. Cellular localization analysis of CT009 showed uniform membrane staining in Chlamydia. This was in contrast to the localization of MreB, which was restricted to predicted septal planes. MreB localization to septal planes provides direct experimental observation for the role of MreB in cell division and supports the hypothesis that it serves as a functional replacement for FtsZ in Chlamydia
Chromatography-free purification strategies for large biological macromolecular complexes involving fractionated PEG precipitation and density gradients
A complex interplay between several biological macromolecules maintains cellular homeostasis. Generally, the demanding chemical reactions which sustain life are not performed by individual macromolecules, but rather by several proteins that together form a macromolecular complex. Understanding the functional interactions amongst subunits of these macromolecular machines is fundamental to elucidate mechanisms by which they maintain homeostasis. As the faithful function of macromolecular complexes is essential for cell survival, their mis-function leads to the development of human diseases. Furthermore, detailed mechanistic nterrogation of the function of macromolecular machines can be exploited to develop and optimize biotechnological processes. The purification of intact macromolecular complexes is an essential prerequisite for this; however, chromatographic purification schemes can induce the dissociation of subunits or the disintegration of the whole complex. Here, we discuss the development and application of chromatography-free purification strategies based on fractionated PEG precipitation and orthogonal density gradient centrifugation that overcomes existing limitations of established chromatographic purification protocols. The presented case studies illustrate the capabilities of these procedures for the purification of macromolecular complexes
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