concentration independent and was subsequently attributed to
a conformational change within the protein (HflX) following
nucleotide binding [5]. As such, pre-steady state kinetics not
only allows for the determination of rate constants but also
provides insight into the kinetic mechanism and structural
dynamics of the protein being studied.
20. Additional reaction components can be added to either reaction solution noting the dilution effect described in Note 14.
21. Titration of nucleotide (or any ligand) for determining the rate
of association yields a graph of the apparent rate at each concentration of ligand tested. The slope of the line is equal to k 1
and the y-intercept is equal to k À1 . The value for k À1 from the
y-intercept can be used to validate the k À1 value determined
from the dissociation experiment (e.g., via nucleotide chase
[5, 6]).
Acknowledgments
This work was supported by H.-J.W.’s Alberta Innovates—Technology Futures (SC60-T2), Canadian Institutes of Health Research
(MOP246392), and Natural Sciences and Engineering Research
Council of Canada (05199-2016). H.E.B. receives funding from
the Natural Sciences and Engineering Research Council of Canada
Postgraduate Scholarship-Doctoral.
References
1. Delgado FF, Cermak N, Hecht VC, Son S,
Li Y, Knudsen SM, Olcum S, Higgins JM,
Chen J, Grover WH, Manalis SR (2013) Intracellular water exchange for measuring the dry
mass, water mass and changes in chemical composition of living cells. PLoS One 8(7):
e67590. https://doi.org/10.1371/journal.
pone.0067590
2. Austin HP, Allen MD, Donohoe BS, Rorrer
NA, Kearns FL, Silveira RL, Pollard BC,
Dominick G, Duman R, El Omari K,
Mykhaylyk V, Wagner A, Michener WE,
Amore A, Skaf MS, Crowley MF, Thorne AW,
Johnson CW, Woodcock HL, McGeehan JE,
Beckham GT (2018) Characterization and
engineering of a plastic-degrading aromatic
polyesterase. Proc Natl Acad Sci U S A 115
(19):E4350–E4357.
https://doi.org/10.
1073/pnas.1718804115
3. Hu JH, Miller SM, Geurts MH, Tang W,
Chen L, Sun N, Zeina CM, Gao X, Rees HA,
Lin Z, Liu DR (2018) Evolved Cas9 variants
with broad PAM compatibility and high DNA
specificity.
Nature
(London)
556
(7699):57–63.
https://doi.org/10.1038/
nature26155
4. Aigner TB, DeSimone E, Scheibel T (2018)
Biomedical applications of recombinant silkbased materials. Adv Mater 30(19):1704636.
https://doi.org/10.1002/adma.201704636
5. Shields MJ, Fischer JJ, Wieden H-J (2009)
Toward understanding the function of the universally conserved GTPase HfIX from Escherichia coli: a kinetic approach. Biochemistry 48
(45):10793–10802.
https://doi.org/10.
1021/bi901074h
6. Fischer JJ, Coatham ML, Eagle Bear S, Brandon HE, De Laurentiis EI, Shields MJ, Wieden
H-J (2012) The ribosome modulates the structural dynamics of the conserved GTPase HflX
and triggers tight nucleotide binding. Biochimie 94(8):1647–1659. https://doi.org/10.
1016/j.biochi.2012.04.016
7. Rosler KS, Mercier E, Andrews IC, Wieden
H-J (2015) Histidine 114 is critical for ATP
hydrolysis by the universally conserved ATPase
286
Harland E. Brandon and Hans-Joachim Wieden
a conformational change within the protein (HflX) following
nucleotide binding [5]. As such, pre-steady state kinetics not
only allows for the determination of rate constants but also
provides insight into the kinetic mechanism and structural
dynamics of the protein being studied.
20. Additional reaction components can be added to either reaction solution noting the dilution effect described in Note 14.
21. Titration of nucleotide (or any ligand) for determining the rate
of association yields a graph of the apparent rate at each concentration of ligand tested. The slope of the line is equal to k 1
and the y-intercept is equal to k À1 . The value for k À1 from the
y-intercept can be used to validate the k À1 value determined
from the dissociation experiment (e.g., via nucleotide chase
[5, 6]).
Acknowledgments
This work was supported by H.-J.W.’s Alberta Innovates—Technology Futures (SC60-T2), Canadian Institutes of Health Research
(MOP246392), and Natural Sciences and Engineering Research
Council of Canada (05199-2016). H.E.B. receives funding from
the Natural Sciences and Engineering Research Council of Canada
Postgraduate Scholarship-Doctoral.
References
1. Delgado FF, Cermak N, Hecht VC, Son S,
Li Y, Knudsen SM, Olcum S, Higgins JM,
Chen J, Grover WH, Manalis SR (2013) Intracellular water exchange for measuring the dry
mass, water mass and changes in chemical composition of living cells. PLoS One 8(7):
e67590. https://doi.org/10.1371/journal.
pone.0067590
2. Austin HP, Allen MD, Donohoe BS, Rorrer
NA, Kearns FL, Silveira RL, Pollard BC,
Dominick G, Duman R, El Omari K,
Mykhaylyk V, Wagner A, Michener WE,
Amore A, Skaf MS, Crowley MF, Thorne AW,
Johnson CW, Woodcock HL, McGeehan JE,
Beckham GT (2018) Characterization and
engineering of a plastic-degrading aromatic
polyesterase. Proc Natl Acad Sci U S A 115
(19):E4350–E4357.
https://doi.org/10.
1073/pnas.1718804115
3. Hu JH, Miller SM, Geurts MH, Tang W,
Chen L, Sun N, Zeina CM, Gao X, Rees HA,
Lin Z, Liu DR (2018) Evolved Cas9 variants
with broad PAM compatibility and high DNA
specificity.
Nature
(London)
556
(7699):57–63.
https://doi.org/10.1038/
nature26155
4. Aigner TB, DeSimone E, Scheibel T (2018)
Biomedical applications of recombinant silkbased materials. Adv Mater 30(19):1704636.
https://doi.org/10.1002/adma.201704636
5. Shields MJ, Fischer JJ, Wieden H-J (2009)
Toward understanding the function of the universally conserved GTPase HfIX from Escherichia coli: a kinetic approach. Biochemistry 48
(45):10793–10802.
https://doi.org/10.
1021/bi901074h
6. Fischer JJ, Coatham ML, Eagle Bear S, Brandon HE, De Laurentiis EI, Shields MJ, Wieden
H-J (2012) The ribosome modulates the structural dynamics of the conserved GTPase HflX
and triggers tight nucleotide binding. Biochimie 94(8):1647–1659. https://doi.org/10.
1016/j.biochi.2012.04.016
7. Rosler KS, Mercier E, Andrews IC, Wieden
H-J (2015) Histidine 114 is critical for ATP
hydrolysis by the universally conserved ATPase
286
Harland E. Brandon and Hans-Joachim Wieden