Identification of Multiple Kinetic Populations of DNA-Binding Proteins in Live Cells.
Biophys J
; 117(5): 950-961, 2019 09 03.
Article
en En
| MEDLINE
| ID: mdl-31383358
ABSTRACT
Understanding how multiprotein complexes function in cells requires detailed quantitative understanding of their association and dissociation kinetics. Analysis of the heterogeneity of binding lifetimes enables the interrogation of the various intermediate states formed during the reaction. Single-molecule fluorescence imaging permits the measurement of reaction kinetics inside living organisms with minimal perturbation. However, poor photophysical properties of fluorescent probes limit the dynamic range and accuracy of measurements of off rates in live cells. Time-lapse single-molecule fluorescence imaging can partially overcome the limits of photobleaching; however, limitations of this technique remain uncharacterized. Here, we present a structured analysis of which timescales are most accessible using the time-lapse imaging approach and explore uncertainties in determining kinetic subpopulations. We demonstrate the effect of shot noise on the precision of the measurements as well as the resolution and dynamic range limits that are inherent to the method. Our work provides a convenient implementation to determine theoretical errors from measurements and to support interpretation of experimental data.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas de Unión al ADN
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Biophys J
Año:
2019
Tipo del documento:
Article
País de afiliación:
Australia