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1.
Stud Health Technol Inform ; 242: 64-71, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28873778

RESUMO

This paper presents the theoretical and methodological framework underpinning the advancement of new technology enabling seniors domicile in residential homes to live with independence, quality of life and dignity. In addition, it presents the preliminary findings of this research including the emerging user interface design solution.


Assuntos
Vida Independente , Qualidade de Vida , Equipamentos de Autoajuda , Idoso , Humanos , Tecnologia
2.
J Am Chem Soc ; 139(29): 9937-9948, 2017 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-28658579

RESUMO

Specific binding between biomolecules, i.e., molecular recognition, controls virtually all biological processes including the interactions between cells and biointerfaces, both natural and synthetic. Such binding often relies on the conformation of biomacromolecules, which can be highly heterogeneous and sensitive to environmental perturbations, and therefore difficult to characterize and control. An approach is demonstrated here that directly connects the binding kinetics and stability of the protein receptor integrin αvß3 to the conformation of the ligand fibronectin (FN), which are believed to control cellular mechanosensing. Specifically, we investigated the influence of surface-adsorbed FN structure and dynamics on αvß3 binding using high-throughput single-molecule three-color Förster resonance energy transfer (FRET) tracking methods. By controlling FN structure and dynamics through tuning surface chemistry, we found that as the conformational and translational dynamics of FN increased, the rate of binding, particularly to folded FN, and stability of the bound FN-αvß3 complex decreased significantly. These findings highlight the importance of the conformational plasticity and accessibility of the arginine-glycine-aspartic acid (RGD) binding site in FN, which, in turn, mediates cell signaling in physiological and synthetic environments.


Assuntos
Cor , Fibronectinas/química , Transferência Ressonante de Energia de Fluorescência , Integrina alfaVbeta3/química , Termodinâmica , Sítios de Ligação , Ensaios de Triagem em Larga Escala , Humanos , Integrina alfaVbeta3/isolamento & purificação , Ligantes , Conformação Proteica , Propriedades de Superfície
6.
J Bacteriol ; 198(20): 2853-63, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27501982

RESUMO

UNLABELLED: When microbes are faced with an environmental challenge or opportunity, preexisting enzymes with promiscuous secondary activities can be recruited to provide newly important functions. Mutations that increase the efficiency of a new activity often compromise the original activity, resulting in an inefficient bifunctional enzyme. We have investigated the mechanisms by which growth of Escherichia coli can be improved when fitness is limited by such an enzyme, E383A ProA (ProA*). ProA* can serve the functions of both ProA (required for synthesis of proline) and ArgC (required for synthesis of arginine), albeit poorly. We identified four genetic changes that improve the growth rate by up to 6.2-fold. Two point mutations in the promoter of the proBA* operon increase expression of the entire operon. Massive amplification of a genomic segment around the proBA* operon also increases expression of the entire operon. Finally, a synonymous point mutation in the coding region of proB creates a new promoter for proA* This synonymous mutation increases the level of ProA* by 2-fold but increases the growth rate by 5-fold, an ultrasensitive response likely arising from competition between two substrates for the active site of the inefficient bifunctional ProA*. IMPORTANCE: The high-impact synonymous mutation we discovered in proB is remarkable for two reasons. First, most polar effects documented in the literature are detrimental. This finding demonstrates that polar effect mutations can have strongly beneficial effects, especially when an organism is facing a difficult environmental challenge for which it is poorly adapted. Furthermore, the consequence of the synonymous mutation in proB is a 2-fold increase in the level of ProA* but a disproportionately large 5.1-fold increase in growth rate. While ultrasensitive responses are often found in signaling networks and genetic circuits, an ultrasensitive response to an adaptive mutation has not been previously reported.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Glutamato-5-Semialdeído Desidrogenase/genética , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Glutamato-5-Semialdeído Desidrogenase/metabolismo , Cinética , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Óperon , Fosfotransferases (Aceptor do Grupo Carboxila)/metabolismo , Mutação Puntual , Regiões Promotoras Genéticas
12.
Mol Biol Evol ; 32(1): 100-8, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25246702

RESUMO

Neutral drift occurring over millions or billions of years results in substantial sequence divergence among enzymes that catalyze the same reaction. Although natural selection maintains the primary activity of orthologous enzymes, there is, by definition, no selective pressure to maintain physiologically irrelevant promiscuous activities. Thus, the levels and the evolvabilities of promiscuous activities may vary among orthologous enzymes. Consistent with this expectation, we have found that the levels of a promiscuous activity in nine gamma-glutamyl phosphate reductase (ProA) orthologs vary by about 50-fold. Remarkably, a single amino acid change from Glu to Ala near the active site appeared to be critical for improvement of the promiscuous activity in every ortholog. The effects of this change varied dramatically. The improvement in the promiscuous activity varied from 50- to 770-fold, and, importantly, was not correlated with the initial level of the promiscuous activity. The decrease in the original activity varied from 190- to 2,100-fold. These results suggest that evolution of a novel enzyme may be possible in some microbes, but not in others. Further, these results underscore the importance of using multiple orthologs as starting points for directed evolution of novel enzyme activities.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/genética , Evolução Molecular , Oxirredutases/genética , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Evolução Molecular Direcionada , Deriva Genética , Glutamina/análogos & derivados , Glutamina/metabolismo , Modelos Moleculares , Mutação , Oxirredutases/química , Oxirredutases/metabolismo , Filogenia
14.
Biomed Instrum Technol ; 48(2): 74, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24712348
17.
Proc Natl Acad Sci U S A ; 110(48): 19396-401, 2013 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-24235137

RESUMO

A method was developed to monitor dynamic changes in protein structure and interfacial behavior on surfaces by single-molecule Förster resonance energy transfer. This method entails the incorporation of unnatural amino acids to site-specifically label proteins with single-molecule Förster resonance energy transfer probes for high-throughput dynamic fluorescence tracking microscopy on surfaces. Structural changes in the enzyme organophosphorus hydrolase (OPH) were monitored upon adsorption to fused silica (FS) surfaces in the presence of BSA on a molecule-by-molecule basis. Analysis of >30,000 individual trajectories enabled the observation of heterogeneities in the kinetics of surface-induced OPH unfolding with unprecedented resolution. In particular, two distinct pathways were observed: a majority population (∼ 85%) unfolded with a characteristic time scale of 0.10 s, and the remainder unfolded more slowly with a time scale of 0.7 s. Importantly, even after unfolding, OPH readily desorbed from FS surfaces, challenging the common notion that surface-induced unfolding leads to irreversible protein binding. This suggests that protein fouling of surfaces is a highly dynamic process because of subtle differences in the adsorption/desorption rates of folded and unfolded species. Moreover, such observations imply that surfaces may act as a source of unfolded (i.e., aggregation-prone) protein back into solution. Continuing study of other proteins and surfaces will examine whether these conclusions are general or specific to OPH in contact with FS. Ultimately, this method, which is widely applicable to virtually any protein, provides the framework to develop surfaces and surface modifications with improved biocompatibility.


Assuntos
Arildialquilfosfatase/química , Materiais Biocompatíveis/química , Caulobacteraceae/enzimologia , Microscopia de Fluorescência/métodos , Modelos Moleculares , Conformação Proteica , Adsorção , Arildialquilfosfatase/metabolismo , Materiais Biocompatíveis/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cinética , Dióxido de Silício/química , Dióxido de Silício/metabolismo
19.
Biomed Instrum Technol ; 47(4): 278, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23919779
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