RESUMEN
The binding constant is an important characteristic of a DNA-binding protein. A large number of methods exist to measure the binding constant, but many of those methods have intrinsic flaws that influence the outcome of the characterization. Tethered particle motion (TPM) is a simple, cheap, and high-throughput single-molecule method that can be used to measure binding constants of proteins binding to DNA reliably, provided that they distort DNA. In TPM, the motion of a bead tethered to a surface by DNA is tracked using light microscopy. A protein binding to the DNA will alter bead motion. This change in bead motion makes it possible to measure the DNA-binding properties of proteins. We use the bacterial protein integration host factor (IHF) and the archaeal histone HMfA as examples to show how specific binding to DNA can be measured. Moreover, we show how the end-to-end distance can provide structural insights into protein-DNA binding.
Asunto(s)
ADN , Unión Proteica , ADN/metabolismo , ADN/química , Imagen Individual de Molécula/métodos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/química , Factores de Integración del Huésped/metabolismo , Factores de Integración del Huésped/química , Histonas/metabolismo , Histonas/química , Movimiento (Física)RESUMEN
The binding constant is an important characteristic of a DNA-binding protein. A large number of methods exist to measure the binding constant, but many of those methods have intrinsic flaws that influence the outcome of the characterization. Tethered Particle Motion (TPM) is a simple, cheap, and high-throughput single-molecule method that can be used to reliably measure binding constants of proteins binding to DNA, provided that they distort DNA. In TPM, the motion of a bead tethered to a surface by DNA is tracked using light microscopy. A protein binding to the DNA will alter bead motion. This makes it possible to measure binding properties. We use the bacterial protein Integration Host Factor (IHF) as an example to show how specific binding to DNA can be measured. Moreover, we show a new intuitive quantitative approach to displaying data obtained via TPM.