Pub Date : 2016-11-28DOI: 10.1017/S0033583516000160
Maya A. Olshina, M. Sharon
Abstract Protein complexes form the critical foundation for a wide range of biological process, however understanding the intricate details of their activities is often challenging. In this review we describe how mass spectrometry (MS) plays a key role in the analysis of protein assemblies and the cellular pathways which they are involved in. Specifically, we discuss how the versatility of mass spectrometric approaches provides unprecedented information on multiple levels. We demonstrate this on the ubiquitin-proteasome proteolytic pathway, a process that is responsible for protein turnover. We follow the various steps of this degradation route and illustrate the different MS workflows that were applied for elucidating molecular information. Overall, this review aims to stimulate the integrated use of multiple mass spectrometry approaches for analyzing complex biological systems.
{"title":"Mass spectrometry: a technique of many faces","authors":"Maya A. Olshina, M. Sharon","doi":"10.1017/S0033583516000160","DOIUrl":"https://doi.org/10.1017/S0033583516000160","url":null,"abstract":"Abstract Protein complexes form the critical foundation for a wide range of biological process, however understanding the intricate details of their activities is often challenging. In this review we describe how mass spectrometry (MS) plays a key role in the analysis of protein assemblies and the cellular pathways which they are involved in. Specifically, we discuss how the versatility of mass spectrometric approaches provides unprecedented information on multiple levels. We demonstrate this on the ubiquitin-proteasome proteolytic pathway, a process that is responsible for protein turnover. We follow the various steps of this degradation route and illustrate the different MS workflows that were applied for elucidating molecular information. Overall, this review aims to stimulate the integrated use of multiple mass spectrometry approaches for analyzing complex biological systems.","PeriodicalId":20828,"journal":{"name":"Quarterly Reviews of Biophysics","volume":"39 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2016-11-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78017017","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-08-31DOI: 10.1017/S0033583516000111
B. Nordén
Abstract Einstein was wrong with his 1927 Solvay Conference claim that quantum mechanics is incomplete and incapable of describing diffraction of single particles. However, the Einstein-Podolsky-Rosen paradox of entangled pairs of particles remains lurking with its ‘spooky action at a distance’. In molecules quantum entanglement can be viewed as basis of both chemical bonding and excitonic states. The latter are important in many biophysical contexts and involve coupling between subsystems in which virtual excitations lead to eigenstates of the total Hamiltonian, but not for the separate subsystems. The author questions whether atomic or photonic systems may be probed to prove that particles or photons may stay entangled over large distances and display the immediate communication with each other that so concerned Einstein. A dissociating hydrogen molecule is taken as a model of a zero-spin entangled system whose angular momenta are in principle possible to probe for this purpose. In practice, however, spins randomize as a result of interactions with surrounding fields and matter. Similarly, no experiment seems yet to provide unambiguous evidence of remaining entanglement between single photons at large separations in absence of mutual interaction, or about immediate (superluminal) communication. This forces us to reflect again on what Einstein really had in mind with the paradox, viz. a probabilistic interpretation of a wave function for an ensemble of identically prepared states, rather than as a statement about single particles. Such a prepared state of many particles would lack properties of quantum entanglement that make it so special, including the uncertainty upon which safe quantum communication is assumed to rest. An example is Zewail's experiment showing visible resonance in the dissociation of a coherently vibrating ensemble of NaI molecules apparently violating the uncertainty principle. Einstein was wrong about diffracting single photons where space-like anti-bunching observations have proven recently their non-local character and how observation in one point can remotely affect the outcome in other points. By contrast, long range photon entanglement with immediate, superluminal response is still an elusive, possibly partly misunderstood issue. The author proposes that photons may entangle over large distances only if some interaction exists via fields that cannot propagate faster than the speed of light. An experiment to settle this ‘interaction hypothesis’ is suggested.
{"title":"Quantum entanglement: facts and fiction – how wrong was Einstein after all?","authors":"B. Nordén","doi":"10.1017/S0033583516000111","DOIUrl":"https://doi.org/10.1017/S0033583516000111","url":null,"abstract":"Abstract Einstein was wrong with his 1927 Solvay Conference claim that quantum mechanics is incomplete and incapable of describing diffraction of single particles. However, the Einstein-Podolsky-Rosen paradox of entangled pairs of particles remains lurking with its ‘spooky action at a distance’. In molecules quantum entanglement can be viewed as basis of both chemical bonding and excitonic states. The latter are important in many biophysical contexts and involve coupling between subsystems in which virtual excitations lead to eigenstates of the total Hamiltonian, but not for the separate subsystems. The author questions whether atomic or photonic systems may be probed to prove that particles or photons may stay entangled over large distances and display the immediate communication with each other that so concerned Einstein. A dissociating hydrogen molecule is taken as a model of a zero-spin entangled system whose angular momenta are in principle possible to probe for this purpose. In practice, however, spins randomize as a result of interactions with surrounding fields and matter. Similarly, no experiment seems yet to provide unambiguous evidence of remaining entanglement between single photons at large separations in absence of mutual interaction, or about immediate (superluminal) communication. This forces us to reflect again on what Einstein really had in mind with the paradox, viz. a probabilistic interpretation of a wave function for an ensemble of identically prepared states, rather than as a statement about single particles. Such a prepared state of many particles would lack properties of quantum entanglement that make it so special, including the uncertainty upon which safe quantum communication is assumed to rest. An example is Zewail's experiment showing visible resonance in the dissociation of a coherently vibrating ensemble of NaI molecules apparently violating the uncertainty principle. Einstein was wrong about diffracting single photons where space-like anti-bunching observations have proven recently their non-local character and how observation in one point can remotely affect the outcome in other points. By contrast, long range photon entanglement with immediate, superluminal response is still an elusive, possibly partly misunderstood issue. The author proposes that photons may entangle over large distances only if some interaction exists via fields that cannot propagate faster than the speed of light. An experiment to settle this ‘interaction hypothesis’ is suggested.","PeriodicalId":20828,"journal":{"name":"Quarterly Reviews of Biophysics","volume":"194 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2016-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83094753","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-08-31DOI: 10.1017/S0033583516000123
J. Barber
{"title":"Photosystem II: the water splitting enzyme of photosynthesis and the origin of oxygen in our atmosphere – CORRIGENDUM","authors":"J. Barber","doi":"10.1017/S0033583516000123","DOIUrl":"https://doi.org/10.1017/S0033583516000123","url":null,"abstract":"","PeriodicalId":20828,"journal":{"name":"Quarterly Reviews of Biophysics","volume":"161 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2016-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77095166","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-08-08DOI: 10.1017/S003358351600010X
M. Kaplan, C. Pinto, Klaartje Houben, M. Baldus
Abstract Increasing evidence suggests that most proteins occur and function in complexes rather than as isolated entities when embedded in cellular membranes. Nuclear magnetic resonance (NMR) provides increasing possibilities to study structure, dynamics and assembly of such systems. In our review, we discuss recent methodological progress to study membrane–protein complexes (MPCs) by NMR, starting with expression, isotope-labeling and reconstitution protocols. We review approaches to deal with spectral complexity and limited spectral spectroscopic sensitivity that are usually encountered in NMR-based studies of MPCs. We highlight NMR applications in various classes of MPCs, including G-protein-coupled receptors, ion channels and retinal proteins and extend our discussion to protein–protein complexes that span entire cellular compartments or orchestrate processes such as protein transport across or within membranes. These examples demonstrate the growing potential of NMR-based studies of MPCs to provide critical insight into the energetics of protein–ligand and protein–protein interactions that underlie essential biological functions in cellular membranes.
{"title":"Nuclear magnetic resonance (NMR) applied to membrane–protein complexes","authors":"M. Kaplan, C. Pinto, Klaartje Houben, M. Baldus","doi":"10.1017/S003358351600010X","DOIUrl":"https://doi.org/10.1017/S003358351600010X","url":null,"abstract":"Abstract Increasing evidence suggests that most proteins occur and function in complexes rather than as isolated entities when embedded in cellular membranes. Nuclear magnetic resonance (NMR) provides increasing possibilities to study structure, dynamics and assembly of such systems. In our review, we discuss recent methodological progress to study membrane–protein complexes (MPCs) by NMR, starting with expression, isotope-labeling and reconstitution protocols. We review approaches to deal with spectral complexity and limited spectral spectroscopic sensitivity that are usually encountered in NMR-based studies of MPCs. We highlight NMR applications in various classes of MPCs, including G-protein-coupled receptors, ion channels and retinal proteins and extend our discussion to protein–protein complexes that span entire cellular compartments or orchestrate processes such as protein transport across or within membranes. These examples demonstrate the growing potential of NMR-based studies of MPCs to provide critical insight into the energetics of protein–ligand and protein–protein interactions that underlie essential biological functions in cellular membranes.","PeriodicalId":20828,"journal":{"name":"Quarterly Reviews of Biophysics","volume":"40 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2016-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86864550","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-08-01DOI: 10.1017/S0033583516000093
J. Barber
Abstract About 3 billion years ago an enzyme emerged which would dramatically change the chemical composition of our planet and set in motion an unprecedented explosion in biological activity. This enzyme used solar energy to power the thermodynamically and chemically demanding reaction of water splitting. In so doing it provided biology with an unlimited supply of reducing equivalents needed to convert carbon dioxide into the organic molecules of life while at the same time produced oxygen to transform our planetary atmosphere from an anaerobic to an aerobic state. The enzyme which facilitates this reaction and therefore underpins virtually all life on our planet is known as Photosystem II (PSII). It is a pigment-binding, multisubunit protein complex embedded in the lipid environment of the thylakoid membranes of plants, algae and cyanobacteria. Today we have detailed understanding of the structure and functioning of this key and unique enzyme. The journey to this level of knowledge can be traced back to the discovery of oxygen itself in the 18th-century. Since then there has been a sequence of mile stone discoveries which makes a fascinating story, stretching over 200 years. But it is the last few years that have provided the level of detail necessary to reveal the chemistry of water oxidation and O–O bond formation. In particular, the crystal structure of the isolated PSII enzyme has been reported with ever increasing improvement in resolution. Thus the organisational and structural details of its many subunits and cofactors are now well understood. The water splitting site was revealed as a cluster of four Mn ions and a Ca ion surrounded by amino-acid side chains, of which seven provide direct ligands to the metals. The metal cluster is organised as a cubane structure composed of three Mn ions and a Ca2+ linked by oxo-bonds with the fourth Mn ion attached to the cubane. This structure has now been synthesised in a non-protein environment suggesting that it is a totally inorganic precursor for the evolution of the photosynthetic oxygen-evolving complex. In summary, the overall structure of the catalytic site has given a framework on which to build a mechanistic scheme for photosynthetic dioxygen generation and at the same time provide a blue-print and incentive to develop catalysts for artificial photo-electrochemical systems to split water and generate renewable solar fuels.
{"title":"‘Photosystem II: the water splitting enzyme of photosynthesis and the origin of oxygen in our atmosphere’","authors":"J. Barber","doi":"10.1017/S0033583516000093","DOIUrl":"https://doi.org/10.1017/S0033583516000093","url":null,"abstract":"Abstract About 3 billion years ago an enzyme emerged which would dramatically change the chemical composition of our planet and set in motion an unprecedented explosion in biological activity. This enzyme used solar energy to power the thermodynamically and chemically demanding reaction of water splitting. In so doing it provided biology with an unlimited supply of reducing equivalents needed to convert carbon dioxide into the organic molecules of life while at the same time produced oxygen to transform our planetary atmosphere from an anaerobic to an aerobic state. The enzyme which facilitates this reaction and therefore underpins virtually all life on our planet is known as Photosystem II (PSII). It is a pigment-binding, multisubunit protein complex embedded in the lipid environment of the thylakoid membranes of plants, algae and cyanobacteria. Today we have detailed understanding of the structure and functioning of this key and unique enzyme. The journey to this level of knowledge can be traced back to the discovery of oxygen itself in the 18th-century. Since then there has been a sequence of mile stone discoveries which makes a fascinating story, stretching over 200 years. But it is the last few years that have provided the level of detail necessary to reveal the chemistry of water oxidation and O–O bond formation. In particular, the crystal structure of the isolated PSII enzyme has been reported with ever increasing improvement in resolution. Thus the organisational and structural details of its many subunits and cofactors are now well understood. The water splitting site was revealed as a cluster of four Mn ions and a Ca ion surrounded by amino-acid side chains, of which seven provide direct ligands to the metals. The metal cluster is organised as a cubane structure composed of three Mn ions and a Ca2+ linked by oxo-bonds with the fourth Mn ion attached to the cubane. This structure has now been synthesised in a non-protein environment suggesting that it is a totally inorganic precursor for the evolution of the photosynthetic oxygen-evolving complex. In summary, the overall structure of the catalytic site has given a framework on which to build a mechanistic scheme for photosynthetic dioxygen generation and at the same time provide a blue-print and incentive to develop catalysts for artificial photo-electrochemical systems to split water and generate renewable solar fuels.","PeriodicalId":20828,"journal":{"name":"Quarterly Reviews of Biophysics","volume":"1 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2016-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76748561","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-07-22DOI: 10.1017/S0033583516000068
K. Vinothkumar, R. Henderson
Abstract There has been enormous progress during the last few years in the determination of three-dimensional biological structures by single particle electron cryomicroscopy (cryoEM), allowing maps to be obtained with higher resolution and from fewer images than required previously. This is due principally to the introduction of a new type of direct electron detector that has 2- to 3-fold higher detective quantum efficiency than available previously, and to the improvement of the computational algorithms for image processing. In spite of the great strides that have been made, quantitative analysis shows that there are still significant gains to be made provided that the problems associated with image degradation can be solved, possibly by minimising beam-induced specimen movement and charge build up during imaging. If this can be achieved, it should be possible to obtain near atomic resolution structures of smaller single particles, using fewer images and resolving more conformational states than at present, thus realising the full potential of the method. The recent popularity of cryoEM for molecular structure determination also highlights the need for lower cost microscopes, so we encourage development of an inexpensive, 100 keV electron cryomicroscope with a high-brightness field emission gun to make the method accessible to individual groups or institutions that cannot afford the investment and running costs of a state-of-the-art 300 keV installation. A key requisite for successful high-resolution structure determination by cryoEM includes interpretation of images and optimising the biochemistry and grid preparation to obtain nicely distributed macromolecules of interest. We thus include in this review a gallery of cryoEM micrographs that shows illustrative examples of single particle images of large and small macromolecular complexes.
{"title":"Single particle electron cryomicroscopy: trends, issues and future perspective","authors":"K. Vinothkumar, R. Henderson","doi":"10.1017/S0033583516000068","DOIUrl":"https://doi.org/10.1017/S0033583516000068","url":null,"abstract":"Abstract There has been enormous progress during the last few years in the determination of three-dimensional biological structures by single particle electron cryomicroscopy (cryoEM), allowing maps to be obtained with higher resolution and from fewer images than required previously. This is due principally to the introduction of a new type of direct electron detector that has 2- to 3-fold higher detective quantum efficiency than available previously, and to the improvement of the computational algorithms for image processing. In spite of the great strides that have been made, quantitative analysis shows that there are still significant gains to be made provided that the problems associated with image degradation can be solved, possibly by minimising beam-induced specimen movement and charge build up during imaging. If this can be achieved, it should be possible to obtain near atomic resolution structures of smaller single particles, using fewer images and resolving more conformational states than at present, thus realising the full potential of the method. The recent popularity of cryoEM for molecular structure determination also highlights the need for lower cost microscopes, so we encourage development of an inexpensive, 100 keV electron cryomicroscope with a high-brightness field emission gun to make the method accessible to individual groups or institutions that cannot afford the investment and running costs of a state-of-the-art 300 keV installation. A key requisite for successful high-resolution structure determination by cryoEM includes interpretation of images and optimising the biochemistry and grid preparation to obtain nicely distributed macromolecules of interest. We thus include in this review a gallery of cryoEM micrographs that shows illustrative examples of single particle images of large and small macromolecular complexes.","PeriodicalId":20828,"journal":{"name":"Quarterly Reviews of Biophysics","volume":"295 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2016-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73479439","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-07-18DOI: 10.1017/S0033583516000081
Lin Ren, Lisa J. Mellander, Jacqueline D. Keighron, A. Cans, M. Kurczy, I. Svir, A. Oleinick, C. Amatore, A. Ewing
Abstract Exocytosis is the fundamental process by which cells communicate with each other. The events that lead up to the fusion of a vesicle loaded with chemical messenger with the cell membrane were the subject of a Nobel Prize in 2013. However, the processes occurring after the initial formation of a fusion pore are very much still in debate. The release of chemical messenger has traditionally been thought to occur through full distention of the vesicle membrane, hence assuming exocytosis to be all or none. In contrast to the all or none hypothesis, here we discuss the evidence that during exocytosis the vesicle-membrane pore opens to release only a portion of the transmitter content during exocytosis and then close again. This open and closed exocytosis is distinct from kiss-and-run exocytosis, in that it appears to be the main content released during regular exocytosis. The evidence for this partial release via open and closed exocytosis is presented considering primarily the quantitative evidence obtained with amperometry.
{"title":"The evidence for open and closed exocytosis as the primary release mechanism","authors":"Lin Ren, Lisa J. Mellander, Jacqueline D. Keighron, A. Cans, M. Kurczy, I. Svir, A. Oleinick, C. Amatore, A. Ewing","doi":"10.1017/S0033583516000081","DOIUrl":"https://doi.org/10.1017/S0033583516000081","url":null,"abstract":"Abstract Exocytosis is the fundamental process by which cells communicate with each other. The events that lead up to the fusion of a vesicle loaded with chemical messenger with the cell membrane were the subject of a Nobel Prize in 2013. However, the processes occurring after the initial formation of a fusion pore are very much still in debate. The release of chemical messenger has traditionally been thought to occur through full distention of the vesicle membrane, hence assuming exocytosis to be all or none. In contrast to the all or none hypothesis, here we discuss the evidence that during exocytosis the vesicle-membrane pore opens to release only a portion of the transmitter content during exocytosis and then close again. This open and closed exocytosis is distinct from kiss-and-run exocytosis, in that it appears to be the main content released during regular exocytosis. The evidence for this partial release via open and closed exocytosis is presented considering primarily the quantitative evidence obtained with amperometry.","PeriodicalId":20828,"journal":{"name":"Quarterly Reviews of Biophysics","volume":"3 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2016-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74185291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-06-24DOI: 10.1017/S003358351600007X
Kathleen A. Leamy, S. Assmann, D. Mathews, P. Bevilacqua
Abstract Deciphering the folding pathways and predicting the structures of complex three-dimensional biomolecules is central to elucidating biological function. RNA is single-stranded, which gives it the freedom to fold into complex secondary and tertiary structures. These structures endow RNA with the ability to perform complex chemistries and functions ranging from enzymatic activity to gene regulation. Given that RNA is involved in many essential cellular processes, it is critical to understand how it folds and functions in vivo. Within the last few years, methods have been developed to probe RNA structures in vivo and genome-wide. These studies reveal that RNA often adopts very different structures in vivo and in vitro, and provide profound insights into RNA biology. Nonetheless, both in vitro and in vivo approaches have limitations: studies in the complex and uncontrolled cellular environment make it difficult to obtain insight into RNA folding pathways and thermodynamics, and studies in vitro often lack direct cellular relevance, leaving a gap in our knowledge of RNA folding in vivo. This gap is being bridged by biophysical and mechanistic studies of RNA structure and function under conditions that mimic the cellular environment. To date, most artificial cytoplasms have used various polymers as molecular crowding agents and a series of small molecules as cosolutes. Studies under such in vivo-like conditions are yielding fresh insights, such as cooperative folding of functional RNAs and increased activity of ribozymes. These observations are accounted for in part by molecular crowding effects and interactions with other molecules. In this review, we report milestones in RNA folding in vitro and in vivo and discuss ongoing experimental and computational efforts to bridge the gap between these two conditions in order to understand how RNA folds in the cell.
{"title":"Bridging the gap between in vitro and in vivo RNA folding","authors":"Kathleen A. Leamy, S. Assmann, D. Mathews, P. Bevilacqua","doi":"10.1017/S003358351600007X","DOIUrl":"https://doi.org/10.1017/S003358351600007X","url":null,"abstract":"Abstract Deciphering the folding pathways and predicting the structures of complex three-dimensional biomolecules is central to elucidating biological function. RNA is single-stranded, which gives it the freedom to fold into complex secondary and tertiary structures. These structures endow RNA with the ability to perform complex chemistries and functions ranging from enzymatic activity to gene regulation. Given that RNA is involved in many essential cellular processes, it is critical to understand how it folds and functions in vivo. Within the last few years, methods have been developed to probe RNA structures in vivo and genome-wide. These studies reveal that RNA often adopts very different structures in vivo and in vitro, and provide profound insights into RNA biology. Nonetheless, both in vitro and in vivo approaches have limitations: studies in the complex and uncontrolled cellular environment make it difficult to obtain insight into RNA folding pathways and thermodynamics, and studies in vitro often lack direct cellular relevance, leaving a gap in our knowledge of RNA folding in vivo. This gap is being bridged by biophysical and mechanistic studies of RNA structure and function under conditions that mimic the cellular environment. To date, most artificial cytoplasms have used various polymers as molecular crowding agents and a series of small molecules as cosolutes. Studies under such in vivo-like conditions are yielding fresh insights, such as cooperative folding of functional RNAs and increased activity of ribozymes. These observations are accounted for in part by molecular crowding effects and interactions with other molecules. In this review, we report milestones in RNA folding in vitro and in vivo and discuss ongoing experimental and computational efforts to bridge the gap between these two conditions in order to understand how RNA folds in the cell.","PeriodicalId":20828,"journal":{"name":"Quarterly Reviews of Biophysics","volume":"98 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2016-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85330825","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-06-24DOI: 10.1017/S0033583516000056
Jin Chen, Junhong Choi, S. O’Leary, Arjun Prabhakar, A. Petrov, Rosslyn Grosely, E. V. Puglisi, J. Puglisi
Abstract Translation of proteins by the ribosome regulates gene expression, with recent results underscoring the importance of translational control. Misregulation of translation underlies many diseases, including cancer and many genetic diseases. Decades of biochemical and structural studies have delineated many of the mechanistic details in prokaryotic translation, and sketched the outlines of eukaryotic translation. However, translation may not proceed linearly through a single mechanistic pathway, but likely involves multiple pathways and branchpoints. The stochastic nature of biological processes would allow different pathways to occur during translation that are biased by the interaction of the ribosome with other translation factors, with many of the steps kinetically controlled. These multiple pathways and branchpoints are potential regulatory nexus, allowing gene expression to be tuned at the translational level. As research focus shifts toward eukaryotic translation, certain themes will be echoed from studies on prokaryotic translation. This review provides a general overview of the dynamic data related to prokaryotic and eukaryotic translation, in particular recent findings with single-molecule methods, complemented by biochemical, kinetic, and structural findings. We will underscore the importance of viewing the process through the viewpoints of regulation, translational control, and heterogeneous pathways.
{"title":"The molecular choreography of protein synthesis: translational control, regulation, and pathways","authors":"Jin Chen, Junhong Choi, S. O’Leary, Arjun Prabhakar, A. Petrov, Rosslyn Grosely, E. V. Puglisi, J. Puglisi","doi":"10.1017/S0033583516000056","DOIUrl":"https://doi.org/10.1017/S0033583516000056","url":null,"abstract":"Abstract Translation of proteins by the ribosome regulates gene expression, with recent results underscoring the importance of translational control. Misregulation of translation underlies many diseases, including cancer and many genetic diseases. Decades of biochemical and structural studies have delineated many of the mechanistic details in prokaryotic translation, and sketched the outlines of eukaryotic translation. However, translation may not proceed linearly through a single mechanistic pathway, but likely involves multiple pathways and branchpoints. The stochastic nature of biological processes would allow different pathways to occur during translation that are biased by the interaction of the ribosome with other translation factors, with many of the steps kinetically controlled. These multiple pathways and branchpoints are potential regulatory nexus, allowing gene expression to be tuned at the translational level. As research focus shifts toward eukaryotic translation, certain themes will be echoed from studies on prokaryotic translation. This review provides a general overview of the dynamic data related to prokaryotic and eukaryotic translation, in particular recent findings with single-molecule methods, complemented by biochemical, kinetic, and structural findings. We will underscore the importance of viewing the process through the viewpoints of regulation, translational control, and heterogeneous pathways.","PeriodicalId":20828,"journal":{"name":"Quarterly Reviews of Biophysics","volume":"1 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2016-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90879468","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-06-09DOI: 10.1017/S0033583516000044
J. Seelig, H. Schönfeld
Abstract Thermally-induced protein unfolding is commonly described with the two-state model. This model assumes only two types of protein molecules in solution, the native (N) and the denatured, unfolded (U) protein. In reality, protein unfolding is a multistep process, even if intermediate states are only sparsely populated. As an alternative approach we explore the Zimm–Bragg theory, originally developed for the α-helix-to-random coil transition of synthetic polypeptides. The theory includes intermediate structures with concentrations determined by the cooperativity of the unfolding reaction. We illustrate the differences between the two-state model and the Zimm–Bragg theory with measurements of apolipoprotein A-1 and lysozyme by differential scanning calorimetry (DSC) and CD spectroscopy. Nine further protein examples are taken from the literature. The Zimm–Bragg theory provides a perfect fit of the calorimetric unfolding transitions for all proteins investigated. In contrast, the transition curves and enthalpies predicted by the two-state model differ considerably from the experimental results. Apolipoprotein A-1 is ~50% α-helical at ambient temperature and its unfolding follows the classical α-helix-to-random coil equilibrium. The unfolding of proteins with little α-helix content, such as lysozyme, can also be analyzed with the Zimm–Bragg theory by introducing the concept of ‘folded’ and ‘unfolded’ peptide units assuming an average unfolding enthalpy per peptide unit. DSC is the method of choice to measure the unfolding enthalpy, $Delta H_{rm exp} ^0 $, but CD spectroscopy in combination with the two-state model is often used to deduce the unfolding enthalpy. This can lead to erroneous result. Not only are different enthalpies required to describe the CD and DSC transition curves but these values deviate distinctly from the experimental result. In contrast, the Zimm–Bragg theory predicts the DSC and CD unfolding transitions with the same set of parameters.
{"title":"Thermal protein unfolding by differential scanning calorimetry and circular dichroism spectroscopy Two-state model versus sequential unfolding","authors":"J. Seelig, H. Schönfeld","doi":"10.1017/S0033583516000044","DOIUrl":"https://doi.org/10.1017/S0033583516000044","url":null,"abstract":"Abstract Thermally-induced protein unfolding is commonly described with the two-state model. This model assumes only two types of protein molecules in solution, the native (N) and the denatured, unfolded (U) protein. In reality, protein unfolding is a multistep process, even if intermediate states are only sparsely populated. As an alternative approach we explore the Zimm–Bragg theory, originally developed for the α-helix-to-random coil transition of synthetic polypeptides. The theory includes intermediate structures with concentrations determined by the cooperativity of the unfolding reaction. We illustrate the differences between the two-state model and the Zimm–Bragg theory with measurements of apolipoprotein A-1 and lysozyme by differential scanning calorimetry (DSC) and CD spectroscopy. Nine further protein examples are taken from the literature. The Zimm–Bragg theory provides a perfect fit of the calorimetric unfolding transitions for all proteins investigated. In contrast, the transition curves and enthalpies predicted by the two-state model differ considerably from the experimental results. Apolipoprotein A-1 is ~50% α-helical at ambient temperature and its unfolding follows the classical α-helix-to-random coil equilibrium. The unfolding of proteins with little α-helix content, such as lysozyme, can also be analyzed with the Zimm–Bragg theory by introducing the concept of ‘folded’ and ‘unfolded’ peptide units assuming an average unfolding enthalpy per peptide unit. DSC is the method of choice to measure the unfolding enthalpy, $Delta H_{rm exp} ^0 $, but CD spectroscopy in combination with the two-state model is often used to deduce the unfolding enthalpy. This can lead to erroneous result. Not only are different enthalpies required to describe the CD and DSC transition curves but these values deviate distinctly from the experimental result. In contrast, the Zimm–Bragg theory predicts the DSC and CD unfolding transitions with the same set of parameters.","PeriodicalId":20828,"journal":{"name":"Quarterly Reviews of Biophysics","volume":"33 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2016-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81472058","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：