Quantification of functional dynamics of membrane proteins reconstituted in nanodiscs membranes by single turnover functional readout

TY - CHAP

T1 - Quantification of functional dynamics of membrane proteins reconstituted in nanodiscs membranes by single turnover functional readout

AU - Moses, Matias Emil

AU - Hedegård, Per

AU - Hatzakis, Nikos

PY - 2016

Y1 - 2016

N2 - Single-molecule measurements are emerging as a powerful tool to study the individual behavior of biomolecules, revolutionizing our understanding of biological processes. Their ability to measure the distribution of behaviors, instead of the average behavior, allows the direct observation and quantification of the activity, abundance, and lifetime of multiple states and transient intermediates in the energy landscape that are typically averaged out in nonsynchronized ensemble measurements. Studying the function of membrane proteins at the single-molecule level remains a formidable challenge, and to date there is limited number of available functional assays. In this chapter, we describe in detail our recently developed methodology to reconstitute membrane proteins such as the integral membrane protein cytochrome P450 oxidoreductase on membrane systems such as Nanodiscs and study their functional dynamics by recordings at the fundamental resolution of individual catalytic turnovers using prefluorescent substrate analogues. We initially describe the methodology for reconstitution, surface immobilization, and data acquisition of individual enzyme catalytic turnovers. We then explain in detail the statistical analysis, with an emphasis on the model development, the potential pitfalls for correctly identifying the abundance, lifetime, and likelihood of sampling protein functional states. This methodology may enable studies of functional dynamics and their role in biology for a spectrum of membrane proteins.

AB - Single-molecule measurements are emerging as a powerful tool to study the individual behavior of biomolecules, revolutionizing our understanding of biological processes. Their ability to measure the distribution of behaviors, instead of the average behavior, allows the direct observation and quantification of the activity, abundance, and lifetime of multiple states and transient intermediates in the energy landscape that are typically averaged out in nonsynchronized ensemble measurements. Studying the function of membrane proteins at the single-molecule level remains a formidable challenge, and to date there is limited number of available functional assays. In this chapter, we describe in detail our recently developed methodology to reconstitute membrane proteins such as the integral membrane protein cytochrome P450 oxidoreductase on membrane systems such as Nanodiscs and study their functional dynamics by recordings at the fundamental resolution of individual catalytic turnovers using prefluorescent substrate analogues. We initially describe the methodology for reconstitution, surface immobilization, and data acquisition of individual enzyme catalytic turnovers. We then explain in detail the statistical analysis, with an emphasis on the model development, the potential pitfalls for correctly identifying the abundance, lifetime, and likelihood of sampling protein functional states. This methodology may enable studies of functional dynamics and their role in biology for a spectrum of membrane proteins.

KW - Allosteric regulation

KW - Fluorescent microscopy

KW - Functional dynamics

KW - Nanodiscs

KW - Single enzyme

U2 - 10.1016/bs.mie.2016.08.026

DO - 10.1016/bs.mie.2016.08.026

M3 - Book chapter

C2 - 27793281

AN - SCOPUS:84994267485

T3 - Methods in Enzymology

SP - 227

EP - 256

BT - Single-molecule enzymology

A2 - Spies, Maria

A2 - Chemla, Yann R.

PB - Academic Press

ER -