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1.
Life Sci ; 239: 117060, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31733317

RESUMO

Cancer has become a key healthcare problem worldwide. The background of cancer research has brought the advent of cross-disciplinary collaborations that has enabled us to get an idea of the disease mechanisms at spatial and temporal scales. Understanding the combination of biology and physics of cancer presents a promising field of research with apprehensions in better clarity over both cellular and molecular mechanisms impacting cancer therapy. Investigation of cancer biology has provided a wealth of knowledge on cancer initiation and propagation and has provided newer treatment strategies in the fight against cancer. Understanding the physics of cancer provides wonderful set of equations that take advantage of mechanisms of force production, propagation by the cancer cells and mechanical properties of the tumor tissue. The spatial tissue arrangement in which the tumor growth occurs can be better understood with biophysics. Thus, the combination of biology and physics of cancer contributes crucially in impacting the correct treatment of cancer. The present review is aimed at providing an overview of regulatory networks, regulation of cell division and differentiation, the signal transduction pathways and integration of all sciences including physics, biology, and medicine which is very well needed to tackle the war against cancer and thus influence cancer therapy. These circuits will help us understand whether the therapy will work wonders or cause failure. As cancer is much more than a genetic disease, more insights into the malignancy with physical approaches are designed to use cancer therapy effectively.


Assuntos
Biofísica/métodos , Neoplasias/metabolismo , Neoplasias/patologia , Biologia , Redes Reguladoras de Genes/genética , Humanos , Física , Transdução de Sinais/fisiologia
2.
Nat Commun ; 10(1): 4662, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31604949

RESUMO

While ubiquitous, energy redistribution remains a poorly understood facet of the nonequilibrium thermodynamics of biomolecules. At the molecular level, finite-size effects, pronounced nonlinearities, and ballistic processes produce behavior that diverges from the macroscale. Here, we show that transient thermal transport reflects macromolecular energy landscape architecture through the topological characteristics of molecular contacts and the nonlinear processes that mediate dynamics. While the former determines transport pathways via pairwise interactions, the latter reflects frustration within the landscape for local conformational rearrangements. Unlike transport through small-molecule systems, such as alkanes, nonlinearity dominates over coherent processes at even quite short time- and length-scales. Our exhaustive all-atom simulations and novel local-in-time and space analysis, applicable to both theory and experiment, permit dissection of energy migration in biomolecules. The approach demonstrates that vibrational energy transport can probe otherwise inaccessible aspects of macromolecular dynamics and interactions that underly biological function.


Assuntos
Transferência de Energia , Simulação de Dinâmica Molecular , Biofísica/métodos , Biologia Computacional , Simulação por Computador , Cinética , Termodinâmica
3.
Nat Commun ; 10(1): 4318, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541108

RESUMO

Recent advances in fluorogen-binding "light-up" RNA aptamers have enabled protein-free detection of RNA in cells. Detailed biophysical characterization of folding of G-Quadruplex (GQ)-based light-up aptamers such as Spinach, Mango and Corn is still lacking despite the potential implications on their folding and function. In this work we employ single-molecule fluorescence-force spectroscopy to examine mechanical responses of Spinach2, iMangoIII and MangoIV. Spinach2 unfolds in four discrete steps as force is increased to 7 pN and refolds in reciprocal steps upon force relaxation. In contrast, GQ-core unfolding in iMangoIII and MangoIV occurs in one discrete step at forces >10 pN and refolding occurred at lower forces showing hysteresis. Co-transcriptional folding using superhelicases shows reduced misfolding propensity and allowed a folding pathway different from refolding. Under physiologically relevant pico-Newton levels of force, these aptamers may unfold in vivo and subsequently misfold. Understanding of the dynamics of RNA aptamers will aid engineering of improved fluorogenic modules for cellular applications.


Assuntos
Biofísica/métodos , Mangifera/metabolismo , Dobramento de Proteína , Spinacia oleracea/metabolismo , Aptâmeros de Nucleotídeos , Compostos de Benzil , Quadruplex G , Imidazolinas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
4.
Eur Biophys J ; 48(5): 413-424, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30972433

RESUMO

Recent decades brought a revolution to biology, driven mainly by exponentially increasing amounts of data coming from "'omics" sciences. To handle these data, bioinformatics often has to combine biologically heterogeneous signals, for which methods from statistics and engineering (e.g. machine learning) are often used. While such an approach is sometimes necessary, it effectively treats the underlying biological processes as a black box. Similarly, systems biology deals with inherently complex systems, characterized by a large number of degrees of freedom, and interactions that are highly non-linear. To deal with this complexity, the underlying physical interactions are often (over)simplified, such as in Boolean modelling of network dynamics. In this review, we argue for the utility of applying a biophysical approach in bioinformatics and systems biology, including discussion of two examples from our research which address sequence analysis and understanding intracellular gene expression dynamics.


Assuntos
Biofísica/métodos , Proteômica/métodos , Biologia de Sistemas/métodos , Regulação da Expressão Gênica , Análise de Sequência de DNA
5.
Radiat Prot Dosimetry ; 183(1-2): 116-120, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30668835

RESUMO

The mechanobiology is providing novel perspectives in the study of cancer and is contributing to evaluate the cancer responses, from a biophysical point of view, to classical therapeutic approaches- radiotherapy and chemotherapy. Here we have explored the effects of two doses (4 and 8 Gy) of 6 MeV photons on spreading, focal adhesions, migration and mechanical properties of BALB/c 3T3 and their SV40 transformed equivalent, SVT2. Cell biophysical responses to 4 and 8 Gy were analysed and compared with those reported in previous published work when lower doses (1 and 2 Gy) were administered Panzetta et al. (Effects of high energy X-rays on cell morphology and functions. Proc. Book 2017;16:116). We observed that the range of sensitivity to ionising radiations profoundly changes depending on the patho-physiological state of cells. In particular, we found that X-rays induce morphological and functional variations in both cell lines (decreased motility, increased adhesion and increased cytoskeleton stiffness). These changes were slightly dependent on doses in the case of SVT2 cells and may indicate a possible mechanical normalisation in their phenotype. Nevertheless, the responses of BALB/c 3T3 were negligible only for the low dose of 1 Gy and increased significantly in a dose-dependent manner with higher doses. We believe that the characterisation of X-rays effects on the cell mechanobiology could shed new light in the design and customisation of radiotherapy treatments.


Assuntos
Biofísica/métodos , Células Cultivadas/efeitos da radiação , Linhagem Celular , Citoesqueleto/efeitos da radiação , Relação Dose-Resposta à Radiação , Fótons , Eficiência Biológica Relativa , Raios X
6.
Biosystems ; 177: 5-8, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30610909

RESUMO

We present a model of diffusion in a system consisting of a medium A, in which normal diffusion occurs, connected to a subdiffusive medium B in which absorption of diffusing particles may take place. Diffusion in B is described by the subdiffusion-absorption equation with a fractional time derivative. Solutions to equations are obtained for a specific boundary condition at the border between media. Based on the obtained results we briefly discuss the opportunity to experimentally determine whether absorption is taking place in the medium B if measurement of particles' concentration is possible in the medium A only.


Assuntos
Algoritmos , Biofísica/métodos , Modelos Teóricos , Difusão
7.
Trends Biotechnol ; 37(4): 358-372, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30343948

RESUMO

Two-photon lithography is a laser writing technique that can produce 3D microstructures with resolutions below the diffraction limit. This review focuses on its applications to study mechanical properties of cells, an emerging field known as mechanobiology. We review 3D structural designs and materials in the context of new experimental designs, including estimating forces exerted by single cells, studying selective adhesion on substrates, and creating 3D networks of cells. We then focus on emerging applications, including structures for assessing cancer cell invasiveness, whose migration properties depend on the cell mechanical response to the environment, and 3D architectures and materials to study stem cell differentiation, as 3D structure shape and patterning play a key role in defining cell fates.


Assuntos
Biofísica/métodos , Imagem Tridimensional/métodos , Fenômenos Mecânicos , Imagem Óptica/métodos , Células-Tronco/fisiologia , Células Tumorais Cultivadas/fisiologia , Animais , Biofísica/instrumentação , Diferenciação Celular , Movimento Celular , Humanos , Imagem Tridimensional/instrumentação , Imagem Óptica/instrumentação
8.
Biochem Soc Trans ; 47(1): 63-76, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30578343

RESUMO

There are numerous methods for studying molecular interactions. However, each method gives rise to false negative- or false positive binding results, stemming from artifacts of the scientific equipment or from shortcomings of the experimental format. To validate an initial positive binding result, additional methods need to be applied to cover the shortcomings of the primary experiment. The aim of such a validation procedure is to exclude as many artifacts as possible to confirm that there is a true molecular interaction that meets the standards for publishing or is worth investing considerable resources for follow-up activities in a drug discovery project. To simplify this validation process, a graphical scheme - the validation cross - can be used. This simple graphic is a powerful tool for identifying blind spots of a binding hypothesis, for selecting the most informative combination of methods to reveal artifacts and, in general, for understanding more thoroughly the nature of a validation process. The concept of the validation cross was originally introduced for the validation of protein-ligand interactions by NMR in drug discovery. Here, an attempt is made to expand the concept to further biophysical methods and to generalize it for binary molecular interactions.


Assuntos
Biofísica/métodos , Animais , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Ligação Proteica
10.
Orthod Fr ; 89(4): 343-353, 2018 12.
Artigo em Francês | MEDLINE | ID: mdl-30565553

RESUMO

INTRODUCTION: Mechanobiology, at the interface between biology and biophysics, studies the impact of mechanical forces on tissues, cells and biomolecules. The application of orthodontic forces, followed by induced tooth displacement, is a striking example of its clinical application. OBJECTIVE: The purpose of this article was to compile a review of the literature on the subject of mechanobiology; from its detection at bone level to the presentation of stimulated intracellular pathways. MATERIALS AND METHODS: The literature search was conducted on the Pubmed database in April 2018, with associations of the terms "mechanobiology", "orthodontics", "cell culture", "physiopathology". RESULTS: Three major areas of research were selected: highlighting of the phenomenon and its application in the field of bone biology; the cellular effectors of mechanobiology and its clinical applications. The use of mechanobiology in dentofacial orthopedics opens up a new field of reflection for clinicians regarding future advances in orthodontics.


Assuntos
Fenômenos Biomecânicos/fisiologia , Biofísica/tendências , Osso e Ossos/fisiologia , Ortodontia/tendências , Biofísica/métodos , Humanos , Ortodontia/métodos , Osteoblastos/fisiologia , Estresse Mecânico
11.
Sci Rep ; 8(1): 15599, 2018 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-30348953

RESUMO

Cartilage provides low-friction properties and plays an essential role in diarthrodial joints. A hydrated ground substance composed mainly of proteoglycans (PGs) and a fibrillar collagen network are the main constituents of cartilage. Unfortunately, traumatic joint loading can destroy this complex structure and produce lesions in tissue, leading later to changes in tissue composition and, ultimately, to post-traumatic osteoarthritis (PTOA). Consequently, the fixed charge density (FCD) of PGs may decrease near the lesion. However, the underlying mechanisms leading to these tissue changes are unknown. Here, knee cartilage disks from bovine calves were injuriously compressed, followed by a physiologically relevant dynamic compression for twelve days. FCD content at different follow-up time points was assessed using digital densitometry. A novel cartilage degeneration model was developed by implementing deviatoric and maximum shear strain, as well as fluid velocity controlled algorithms to simulate the FCD loss as a function of time. Predicted loss of FCD was quite uniform around the cartilage lesions when the degeneration algorithm was driven by the fluid velocity, while the deviatoric and shear strain driven mechanisms exhibited slightly discontinuous FCD loss around cracks. Our degeneration algorithm predictions fitted well with the FCD content measured from the experiments. The developed model could subsequently be applied for prediction of FCD depletion around different cartilage lesions and for suggesting optimal rehabilitation protocols.


Assuntos
Cartilagem Articular/lesões , Cartilagem Articular/patologia , Proteoglicanas/análise , Estresse Mecânico , Animais , Biofísica/métodos , Bovinos , Modelos Biológicos , Fatores de Tempo
12.
Phys Rev Lett ; 121(7): 078102, 2018 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-30169087

RESUMO

We present an ultrafast single submicron particle detection method based on a half-bowtie coplanar waveguide. The method is capable of resolving the translocation of these particles at a bandwidth greater than 30 MHz. We compare experimentally the simultaneous use of our radio-frequency technique with conventional dc based resistive pulse recordings and find that our method has a throughput that is enhanced by 2 orders of magnitude. The technique incorporates a microfluidic circuit and has the potential to be employed for screening microparticles and biological cells at frequencies in excess of 1 GHz.


Assuntos
Dispositivos Lab-On-A-Chip , Biofísica/instrumentação , Biofísica/métodos , DNA/análise , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Tamanho da Partícula
13.
Sci Rep ; 8(1): 13501, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30201976

RESUMO

Incorporating membrane proteins into membrane mimicking systems is an essential process for biophysical studies and structure determination. Monodisperse lipid nanodiscs have been found to be a suitable tool, as they provide a near-native lipid bilayer environment. Recently, a covalently circularized nanodisc (cND) assembled with a membrane scaffold protein (MSP) in circular form, instead of conventional linear form, has emerged. Covalently circularized nanodiscs have been shown to have improved stability, however the optimal strategies for the incorporation of membrane proteins, as well as the physicochemical properties of the membrane protein embedded in the cND, have not been studied. Bacteriorhodopsin (bR) is a seven-transmembrane helix (7TM) membrane protein, and it forms a two dimensional crystal consisting of trimeric bR on the purple membrane of halophilic archea. Here it is reported that the bR trimer in its active form can be directly incorporated into a cND from its native purple membrane. Furthermore, the assembly conditions of the native purple membrane nanodisc (PMND) were optimized to achieve homogeneity and high yield using a high sodium chloride concentration. Additionally, the native PMND was demonstrated to have the ability to assemble over a range of different pHs, suggesting flexibility in the preparation conditions. The native PMND was then found to not only preserve the trimeric structure of bR and most of the native lipids in the PM, but also maintained the photocycle function of bR. This suggests a promising potential for assembling a cND with a 7TM membrane protein, extracted directly from its native membrane environment, while preserving the protein conformation and lipid composition.


Assuntos
Bacteriorodopsinas/química , Bicamadas Lipídicas/química , Nanoestruturas/química , Membrana Purpúrea/química , Bacteriorodopsinas/metabolismo , Biofísica/métodos , Halobacterium salinarum/química , Halobacterium salinarum/metabolismo , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/metabolismo , Multimerização Proteica , Membrana Purpúrea/metabolismo
14.
Nat Protoc ; 13(10): 2200-2216, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30218102

RESUMO

The goal of mechanobiology is to understand the links between changes in the physical properties of living cells and normal physiology and disease. This requires mechanical measurements that have appropriate spatial and temporal resolution within a single cell. Conventional atomic force microscopy (AFM) methods that acquire force curves pointwise are used to map the heterogeneous mechanical properties of cells. However, the resulting map acquisition time is much longer than that required to study many dynamic cellular processes. Dynamic AFM (dAFM) methods using resonant microcantilevers are compatible with higher-speed, high-resolution scanning; however, they do not directly acquire force curves and they require the conversion of a limited number of instrument observables to local mechanical property maps. We have recently developed a technique that allows commercial AFM systems equipped with direct cantilever excitation to quantitatively map the viscoelastic properties of live cells. The properties can be obtained at several widely spaced frequencies with nanometer-range spatial resolution and with fast image acquisition times (tens of seconds). Here, we describe detailed procedures for quantitative mapping, including sample preparation, AFM calibration, and data analysis. The protocol can be applied to different biological samples, including cells and viruses. The transition from dAFM imaging to quantitative mapping should be easily achievable for experienced AFM users, who will be able to set up the protocol in <30 min.


Assuntos
Biofísica/métodos , Microscopia de Força Atômica/métodos , Animais , Aplysia/citologia , Fenômenos Biomecânicos , Biofísica/instrumentação , Células Cultivadas , Elasticidade , Fibroblastos/citologia , Camundongos , Microscopia de Força Atômica/instrumentação , Células NIH 3T3 , Neurônios/citologia , Imagem Óptica , Viscosidade
15.
Yakugaku Zasshi ; 138(8): 1033-1041, 2018.
Artigo em Japonês | MEDLINE | ID: mdl-30068844

RESUMO

In small molecule drug discovery, researchers must find specific binders that interact with a target protein and inhibit its function in connection with human diseases. It is of critical importance to know the binding mode of compounds interacting with a target protein to assure hit validation and optimization. Biophysical analysis is a powerful quantitative approach to evaluate the binding modes of such candidates. Since the level of sensitivity of biophysical analysis is suitable to quantitatively detect the binding of fragment compounds, and because of the remarkable success of compound libraries of small molecules, the development and adaptation of biophysical analysis for these applications is in great demand. Herein, we describe the technical developments of biophysical methods, especially thermodynamic and kinetic analysis, for the purpose of screenings which employ small molecules. In addition, we discuss the interaction mechanisms of small molecules to find hit compounds based on these biophysical analyses.


Assuntos
Biofísica/métodos , Calorimetria , Descoberta de Drogas , Humanos , Cinética , Tamanho da Partícula , Ressonância de Plasmônio de Superfície , Termodinâmica
16.
Artigo em Inglês | MEDLINE | ID: mdl-30093344

RESUMO

Noninvasive neuroimaging has revolutionized the study of the organization of the human brain and how its structure and function are altered in psychiatric disorders. A critical explanatory gap lies in our mechanistic understanding of how systems-level neuroimaging biomarkers emerge from underlying synaptic-level perturbations associated with a disease state. We describe an emerging computational psychiatry approach leveraging biophysically based computational models of large-scale brain dynamics and their potential integration with clinical and pharmacological neuroimaging. In particular, we focus on neural circuit models, which describe how patterns of functional connectivity observed in resting-state functional magnetic resonance imaging emerge from neural dynamics shaped by inter-areal interactions through underlying structural connectivity defining long-range projections. We highlight the importance of local circuit physiological dynamics, in combination with structural connectivity, in shaping the emergent functional connectivity. Furthermore, heterogeneity of local circuit properties across brain areas, which impacts large-scale dynamics, may be critical for modeling whole-brain phenomena and alterations in psychiatric disorders and pharmacological manipulation. Finally, we discuss important directions for future model development and biophysical extensions, which will expand their utility to link clinical neuroimaging to neurobiological mechanisms.


Assuntos
Biofísica/métodos , Encéfalo , Conectoma/métodos , Transtornos Mentais , Modelos Teóricos , Neuroimagem/métodos , Psiquiatria/métodos , Transcriptoma , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Encéfalo/fisiopatologia , Humanos , Transtornos Mentais/diagnóstico por imagem , Transtornos Mentais/patologia , Transtornos Mentais/fisiopatologia
17.
Methods Mol Biol ; 1813: 65-73, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30097861

RESUMO

The poly(ADP-ribose) polymerase (PARP) family of proteins utilize NAD+ as the substrate to modify protein acceptors with either mono(ADP-ribose) (MAR) or poly(ADP-ribose) (PAR). MAR and PAR have been shown to regulate distinct cellular processes. Iso-ADP-ribose (iso-ADPr) is the smallest internal PAR structural unit containing the characteristic ribose-ribose glycosidic bond formed during poly(ADP-ribosyl)ation. The WWE domain of RNF146 specifically recognizes the iso-ADPr moiety in PAR but does not interact with MAR. This provides a way to distinguish PAR from MAR modification and to isolate PARylated proteins. Iso-ADPr can be used to detect the PAR-specific binding properties of interested proteins. Here we describe the detailed method to generate and purify iso-ADPr and its use in PAR-binding analysis through isothermal titration calorimetry (ITC) analysis.


Assuntos
Adenosina Difosfato Ribose/química , Biofísica/métodos , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerases/química , Adenosina Difosfato Ribose/genética , Animais , Reparo do DNA/genética , Humanos , Poli(ADP-Ribose) Polimerase-1/genética , Poli Adenosina Difosfato Ribose/química , Poli Adenosina Difosfato Ribose/genética , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica
18.
Physiol Rev ; 98(4): 2097-2131, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30067160

RESUMO

Voltage-sensing phosphatase (VSP) contains a voltage sensor domain (VSD) similar to that in voltage-gated ion channels, and a phosphoinositide phosphatase region similar to phosphatase and tensin homolog deleted on chromosome 10 (PTEN). The VSP gene is conserved from unicellular organisms to higher vertebrates. Membrane depolarization induces electrical driven conformational rearrangement in the VSD, which is translated into catalytic enzyme activity. Biophysical and structural characterization has revealed details of the mechanisms underlying the molecular functions of VSP. Coupling between the VSD and the enzyme is tight, such that enzyme activity is tuned in a graded fashion to the membrane voltage. Upon VSP activation, multiple species of phosphoinositides are simultaneously altered, and the profile of enzyme activity depends on the history of the membrane potential. VSPs have been the obvious candidate link between membrane potential and phosphoinositide regulation. However, patterns of voltage change regulating VSP in native cells remain largely unknown. This review addresses the current understanding of the biophysical biochemical properties of VSP and provides new insight into the proposed functions of VSP.


Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Biofísica/métodos , Humanos , Canais Iônicos/metabolismo , Potenciais da Membrana/fisiologia , Fosfatidilinositóis/metabolismo
19.
Exp Dermatol ; 27(8): 867-875, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30019358

RESUMO

In this review, we aim to give a concise and selective overview of noninvasive biophysical analysis techniques for skin barrier analysis (transepidermal water loss, electrical methods, confocal Raman microspectroscopy, sebumeter, reflectance spectrophotometry, tristimulus colorimetry, diffuse reflectance spectroscopy and reflectance confocal microscopy), including advantages and limitations. Rather than giving an exhaustive description of the many techniques currently available, we show the usefulness of a representative selection of techniques in the functional and morphological evaluation of the skin barrier. Furthermore, we introduce human minimally invasive skin challenging models as a means to study the mechanisms regulating skin homoeostasis and disease and subsequently show how biophysical analysis techniques can be combined with these in vivo skin challenging models in the functional and morphological evaluation of the skin barrier in healthy human skin. We are convinced that the widespread application of biophysical analysis techniques in dermatological practice and in cosmetic sciences will prove invaluable in offering personalized and noninvasive skin treatment solutions. Furthermore, combining the human in vivo challenging models with these novel noninvasive techniques will provide valuable methodology and tools for detailed characterization of the skin barrier in health and disease.


Assuntos
Biofísica/métodos , Fenômenos Fisiológicos da Pele , Pele/metabolismo , Animais , Colorimetria , Cosméticos , Humanos , Leucotrieno B4/química , Microscopia Confocal , Análise Espectral Raman , Água/química
20.
Sci Rep ; 8(1): 9892, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29967470

RESUMO

Bacterial global post-transcriptional regulators execute hundreds of interactions with targets that display varying molecular features while retaining specificity. Herein, we develop, validate, and apply a biophysical, statistical thermodynamic model of canonical target mRNA interactions with the CsrA global post-transcriptional regulator to understand the molecular features that contribute to target regulation. Altogether, we model interactions of CsrA with a pool of 236 mRNA: 107 are experimentally regulated by CsrA and 129 are suspected interaction partners. Guided by current understanding of CsrA-mRNA interactions, we incorporate (i) mRNA nucleotide sequence, (ii) cooperativity of CsrA-mRNA binding, and (iii) minimization of mRNA structural changes to identify an ensemble of likely binding sites and their free energies. The regulatory impact of bound CsrA on mRNA translation is determined with the RBS calculator. Predicted regulation of 66 experimentally regulated mRNAs adheres to the principles of canonical CsrA-mRNA interactions; the remainder implies that other, diverse mechanisms may underlie CsrA-mRNA interaction and regulation. Importantly, results suggest that this global regulator may bind targets in multiple conformations, via flexible stretches of overlapping predicted binding sites. This novel observation expands the notion that CsrA always binds to its targets at specific consensus sequences.


Assuntos
Proteínas de Escherichia coli/metabolismo , Modelos Teóricos , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Regiões 5' não Traduzidas , Sítios de Ligação , Biofísica/métodos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Reprodutibilidade dos Testes , Termodinâmica
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