Quantification of functional dynamics of membrane proteins reconstituted in nanodiscs membranes by single turnover functional readout
Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review
Standard
Quantification of functional dynamics of membrane proteins reconstituted in nanodiscs membranes by single turnover functional readout. / Moses, Matias Emil; Hedegård, Per; Hatzakis, Nikos.
Single-molecule enzymology: fluorescence-based and high-throughput methods. ed. / Maria Spies; Yann R. Chemla. Academic Press, 2016. p. 227-256 (Methods in Enzymology, Vol. 581).Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review
Harvard
APA
Vancouver
Author
Bibtex
}
RIS
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 -
ID: 170765659