Energetics of Substrate Unfolding by ClpA
by
Melva Tonisha James
B.S. Chemistry, University of Mississippi, 2001
Submitted to the Department of Chemistry
in partial fulfillment of the requirements for the degree of
Master of Science in Chemistry
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
September 2007
C Massachusetts Institute of Technology 2007. All rights reserved.
Signature of Author ........................................
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....................................
epartment of Chemistry
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C ertified by. ... ...... ..
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Stuart S. Licht
Samuel A. Goldblith Career Development Professor of Chemistry
Thesis Advisor
Accepted MASSACHUSETTS
by .................................
...................... .. '... ..... .............................
INSTITUTEI·······
Robert W. Field
MASSACHUSETTS INSRTITUTE
OF TECHNOLOGY
Chairman, Department Committee on Graduate Students
SEP 17 2007
LIBRARIES
This is the day which the Lord has made;
Let us rejoice and be glad in it.
-Psalms 118:24
Energetics of Substrate Unfolding by ClpA
by
Melva Tonisha James
Submitted to the Department of Chemistry
on September 5, 2007, in partial fulfillment of the
requirements for the degree of
Master of Science in Chemistry
ABSTRACT
Caseinolytic protease A (ClpA), a member of the Hsp 100 family of heat shock proteins, is the
regulatory subunit of the E. coli protease ClpAP. As such, it recognizes proteins targeted for
degradation by the cell and uses the energy of ATP hydrolysis to unfold substrates and
translocate them to ClpP for proteolysis. ClpA's in vivo contribution to cellular maintenance is
small, but it serves as an ideal model for energy-dependent molecular machines.
The goal of this work is to describe the energetics of ClpA's interactions with both soluble and
membrane-bound protein substrates. Green fluorescent protein modified with an 11-amino acid
tag that facilitates ClpA recognition (GFP-ssrA) is used as a model soluble substrate, and ssrAmodified acetylcholine receptor (AChR-ssrA) is used as a model membrane-bound substrate.
The structural and spectroscopic properties of GFP have been previously described. Here, in
order to determine the minimum amount of energy needed to unfold this protein, we present two
estimates of the free energy of unfolding (AGunfolding) for GFP-ssrA derived from thermal and
chemical denaturation data. We find that GFP-ssrA is highly kinetically stable (tequil > 600 hrs.)
and that AGunfolding = 0-2 kcal/mol. In addition, we show that ClpA retains its enzymatic activity
under conditions which support single-molecule patch-clamp electrophysiology. These
observations made it possible to carry out pilot studies for a novel electrophysiological unfoldase
assay.
ClpA may be able to actively extract protein substrates from the lipid bilayer. We are currently
testing this hypothesis, and we are exploring ways to observe the process of substrate removal in
real-time. The proposed electrophysiological unfoldase assay, once optimized, could be used to
elucidate the mechanistic details of ClpA's interaction with membrane-bound substrates. We are
specifically interested in determining the timescale of substrate removal and determining
whether or not the substrate removal process occurs in one high-energy step or several lowenergy steps.
Thesis Advisor:
Title:
Stuart S. Licht
Samuel A. Goldblith Career Development Professor of Chemistry
ACKNOWLEDGEMENTS
Many people have helped me reach this point. I offer special thanks to my advisor, Stuart Licht,
for his kindness and his patience, the other members of the Licht Group, for their warmth and
hospitality, Hector Hernandez, for his mentorship, Cathy Drennan for her generosity, JoAnne
Stubbe for her insight, and Susan Brighton for being a light in the darkness.
I am tremendously grateful to Dean Blanche Staton, Dean Isaac Colbert, Ed Ballo, and the other
members of the Graduate Students Office staff. Their support and encouragement, throughout
my time at MIT, has been a wonderful gift.
I also thank the staff of the MIT Ombuds Office and the members of the Lutheran Episcopal
Ministry, for their help and good cheer.
Lastly, I acknowledge my family and my friends, all of whom cannot be named. Their love has
sustained me, and I thank them for believing in me when I didn't have the confidence to believe
in myself.
TABLE OF CONTENTS
PAGE
A bstract...................................................................................
...................
3
Acknowledgements ....................................................................
Table of Contents ..............................................................
List of Figures ................................................................
..............
....................5
................
6
List of Tables ..............................................................................
7
Background and Significance...............................................................................8
Chapter 1: Energetics of Unfolding a Soluble Substrate: GFP-ssrA
1.1 Introduction................................................................................
...................................... 13
1.2 Materials and Methods..............................................................................15
1.3 Results...................................................
....................................... 18
1.4 D iscussion..................................................................................
....................................... 29
Chapter 2: Energetics of Unfolding a Membrane-Bound Substrate: AChR-ssrA
2.1 Introduction.................................................................................
..................................... 35
2.2 Materials and Methods..........................................................37
2.3 R esults ........ .............................................................................................
2.4 D iscussion ..................................................................................
39
....................................... 47
References..............................................................................................................52
LIST OF FIGURES
PAGE
Figure 1. ClpA and ClpAP Complex Formation..............................................11
Figure 2. ClpAP-catalyzed Protein Degradation...
............................
....... 12
..............
20
Figure 3. Chemical Denaturation of GFP-ssrA ..................
Figure 4a. Kinetic Stability of GFP-ssrA at Low Denaturant Concentrations ................... 21
Figure 4b. Kinetic Stability of GFP-ssrA at High Denaturant Concentrations ................... 22
Figure 5. GFP-ssrA Rate of Convergence to Equilibrium.......................................23
Figure 6. Thermal Denaturation of GFP-ssrA ...................................
25
Figure 7. Thermal Denaturation Curve for GFP-ssrA at Excitation Maximum.............26
Figure 8. Enzymatic degradation of GFP-ssrA by ClpAP.......................................27
Figure 9. Basal ClpA ATPase Activity at Physiological Temperature .............................. 28
Figure 10. Calculation of GFP-ssrA Stability from Chemical Denaturation Data.............33
Figure 11. Basal ClpA ATPase Activity at Room Temperature.................................40
Figure 12. Mg2+ -dependent degradation of GFP-ssrA by ClpAP .................................... 41
Figure 13. Representative wtAChR patch-clamp data ................................................ 43
Figure 14. Representative Histograms of Open Lifetime as a Function of ATP Concentration.44
Figure 15. Average wtAChR Open Lifetime as a Function of ATP concentration .............45
Figure 16. Average wtAChR Open Probability as a Function of ATP Concentration ...........46
Figure 17. Representative AChR-ssrA patch-clamp data...........................................49
Figure 18. Time-dependence of Open Probability for Electrophysiological Unfoldase Assay
Control Experim ent......
............................................................................... 50
Figure 19. Time-dependence of Open Probability for Electrophysiological Unfoldase Assay...51
LIST OF TABLES
PAGE
Table 1. Thermodynamic Parameters for Thermal Unfolding of GFP-ssrA ...................... 30
Background and Significance
Caseinolytic protease A (ClpA), the ATP-dependent unfoldase that is the subject of this
proposal, is the regulatory subunit of the protease ClpAP (1-3). CIpAP is one of several energydependent proteases in E. coli (4). This enzyme complex was discovered when cell lysates
devoid of Lon, the first known ATP-dependent protease in E. coli, continued to exhibit ATPdependent casein degradation activity (1). ClpAP works, along with other energy-dependent
proteases, to clear the cell of misfolded proteins, overproduced proteins, and some uncomplexed
proteins (4). It is non-essential for cells growing under normal conditions, but the role of the
protease becomes more important when cells are exposed to stresses, such as elevated
temperature (4). The individual contribution of ClpAP to cellular maintenance is small, but its
structure and function have been the targets of intense scrutiny because of its relevance as a
model for energy-dependent molecular machines.
ClpA, a member of the Hsp 100 family of heat shock proteins, forms a hexameric
complex that is structurally and functionally homologous to the 19S regulatory subunit of the
26S proteosome (5). Each subunit is a globular protein of approximately 84 kDa (6). Both the
association of these subunits into the functional hexamer and the binding of ClpA to the
proteolytic component ClpP require the binding of ATP (Figure 1). ATP hydrolysis is not
required (6, 7).
ClpP, though functionally linked to ClpA, has very little structural or sequence homology
to the heat shock proteins and is not included in the same family (5). A tetradecamer in its native
form, ClpP has the ability to cleave short peptides (-5 aa) (8). Degradation of longer peptides
requires the binding of ATP or ATPyS. Proteolysis requires ATP hydrolysis and cannot be
accomplished in the absence of ClpA (6, 8). A possible explanation for these observations is that
the peptidase site in ClpP is inaccessible to large substrates until the energy of ATP hydrolysis
triggers a conformational change that causes the peptidase site to be unmasked or enlarged (7).
ClpP monomers have a molecular weight of approximately 21 kDa (7).
A combination of kinetic and structural (9) work has provided insight into the general
mechanism of the interaction of ClpA with targeted protein substrates. Attachment of a
degradation tag, such as the 11-amino acid ssrA tag (AANDENYALAA), is sufficient, but not
necessary, for recognition by ClpA (10). The mechanism of ClpA's interaction with substrates
consists of three major steps: substrate binding and recognition, substrate unfolding, and,
substrate translocation to ClpP or solution (11-13) (Figure 2).
Data from steady-state experiments carried out on ClpAP (14-17) and the homologous
protease system ClpXP (18-20) have given basic information, such as V.ma, keat, and KM for ATP
hydrolysis. Some have also gone further, addressing the modulation of these parameters in
response to various substrates. Specifically, data have been presented that suggest that local
stability near the degradation tag is more important than global stability in determining the
ability of a protein to be degraded (16, 20), that the overall rate of ATP hydrolysis is the same for
proteins of various thermodynamic stabilities (19), and that the partitioning of the observed rate
of ATP hydrolysis between unfolding and translocation shifts with substrate stability (20).
Despite this wealth of information, little is known about details of individual chemical
steps in ClpA-catalyzed protein unfolding. Even less is known about the role of subunit
interactions in this process. The experiments described below are designed to address how ClpA
uses the energy of ATP hydrolysis to do work on substrate proteins. Topics of particular interest
include how much energy is needed to fully process a targeted substrate, how efficient ClpA is as
a molecular machine, and whether soluble and membrane-bound substrates are processed by the
same mechanism.
CIpP14
*
U
,of7
ATP
ATP
CIpAs
CIpA
CIpAP
Figure 1. ClpA and ClpAP Complex Formation. Binding, but not hydrolysis, of ATP (or
ATPyS) is required for ClpA hexamerization and ClpAP complex formation. ClpA is only
active in its hexameric form. Adapted from Gottesman et al.(l1).
,
NDENYALAA
e~r
41
ATP
I
ATP
ADP + PPi
ClpAP
CIpAP
Figure 2. ClpAP-catalyzed Protein Degradation. ClpA recognizes all protein substrates that
carry the 11-aa SsrA degradation tag. ClpA uses the energy of ATP hydrolysis to unfold and
translocate these substrates to ClpP for proteolysis. Proteolysis is not energy-dependent.
Adapted from Gottesman et al. (11).
Chapter 1
Energetics of Unfolding a Soluble Substrate: GFP-ssrA
1.1 Introduction
ClpA, the regulatory portion of the E. coli proteolytic complex ClpAP, assists the cell in
managing damaged proteins and short-lived regulatory proteins (5, 21). Protein substrates
targeted to ClpA are processed by a mechanism that consists of three major steps: substrate
binding and recognition, substrate unfolding, and substrate translocation (11-13). ClpA requires
ATP to perform the work of unfolding and transporting targeted substrates. The details of the
mechanism by which ClpA uses the free energy of ATP hydrolysis, however, have yet to be
elucidated.
Experiments designed to provide insight into energy use by ClpA are described below,
with modified green fluorescent protein (GFP) used as a model soluble substrate. GFP, while
not an in vivo substrate of ClpA, is a convenient substitute for use in vitro assays because its
concentration can be monitored by several analytical methods, including fluorescence.
In its native state, GFP contains a chromophore, formed by residues Ser 65, Tyr 66, and
Gly 67, which requires only oxygen to fluoresce (22). This unique spectroscopic handle gives
GFP its characteristic bright green appearance and facilitates detection of the protein in solution.
It also allows one to monitor structural changes in GFP. As GFP is unfolded, the local
environment of the chromophore becomes destabilized, and fluorescence is quenched. This fact
is exploited in the experiments described below.
GFP-ssrA, the variant of GFP used experimentally, is modified by the addition of 11
amino acids to the C-terminus of the protein. These 11 amino acids, AANDENYALLA, are
referred to as the ssrA sequence. The ssrA modification can be used to make both endogenous
and exogenous proteins substrates for ClpA (23, 24).
The ultimate goal of this project is to understand ClpA. To facilitate accomplishment of
this goal, the thermodynamic and kinetic stabilities of GFP-ssrA have been measured. These
values will be used compare the parameters of spontaneous unfolding of GFP-ssrA with those of
ClpA-catalyzed unfolding of the same substrate.
1.2 Materials and Methods
1.2.1 Overexpression and Purification of ClpA
The pET9a overexpression vector carrying clpA was a generous gift from Profs. Robert
Sauer and Tania Baker (MIT). The M169T mutation, which provides both enhanced solubility
and enhanced levels of full-length protein expression (25, 26), was introduced into the plasmid
using the QuikChange Site-Directed Mutagenesis Kit protocol (Stratagene), and the sequence of
the modified plasmid was confirmed.
ClpA M169T, henceforth referred to as ClpA, was expressed in E. coli strain
BL21/DE3/pLysS. Aliquots of starter culture derived from a single colony were used to seed
liter-scale growth in LB medium containing 30 gg/mL kanamycin. Cells were grown at 37 'C
until OD 600 = 0.6. Each volume of cells was then induced with isopropyl-P-Dthiogalactopyranoside (IPTG) to a final concentration of 1 mM. Cells were grown at 25 'C for 3
hours post-induction and harvested by centrifugation (10 min @ 6,000 x g, 4 'C). Cell pellets
were frozen in liquid nitrogen, and stored at -80 OC.
ClpA was purified in the method described by Maurizi et al.(8) with the following
modifications. Cells were lysed by sonication. After ammonium sulfate precipitation, ClpAcontaining pellets were resuspended in buffer and loaded onto a Macro-Prep High S support
(Bio-Rad) cation exchange column. A 50 mL linear gradient from 0.1 to 1 M KCl was applied.
ClpA was eluted at approximately 0.6 M KC1.
Throughout the purification, the concentration of protein was monitored using
absorbance at 280 nm. Enzymatic activity was determined using the NADH-coupled ATPase
assay (27). Briefly, this assay permits the indirect measurement of ATP hydrolysis by linking
the reaction to NADH oxidation via the glycolytic enzymes pyruvate kinase and lactate
dehydrogenase. The coupled reaction is monitored by absorbance (@ 340 nm), which decreases
as NAD+ is produced.
ATPase assays were performed at 37 'C with 0.2 lgM ClpA 6 in 50 mM HEPES, pH 7.5,
300 mM NaC1, 30 mM MgCI 2 , 0.5 mM DTT, 10% glycerol, 2.5 mM ATP, 0.23 mM NADH, 7.5
mM phosphoenolpyruvate, 19 U/mL pyruvate kinase (from rabbit muscle, Sigma), and 21 U/mL
lactate dehydrogenase (from rabbit muscle, Sigma), in the presence or absence of 0.1 liM ClpP1 4.
The specific activity of the prepared ClpA is -170 gLmol/hr/mg ClpA. This value is significantly
higher than the activity of -60 glmol/hr/mg ClpA reported in the literature (8, 28), but it is
comparable to the specific activity obtained by other members of the Licht Group (Laura
Jennings, Mary Farbman, Amy Weeks; unpublished data).
1.2.2 Overexpression and Purification of ClpP
A plasmid for expression of ClpP with a C-terminal His 6 tag was kindly provided by
Profs. Robert Sauer and Tania Baker (MIT). ClpP-His 6, henceforth referred to as ClpP, was
expressed in E. coli strain DH5a/QE704/KI175. Aliquots of starter culture derived from a single
colony were used to seed liter-scale growth in 2YT medium containing 100 gg/mL ampicillin.
Cells were grown at 370 C until OD 600 = 0.5. Each volume of cells was then induced with IPTG
to a final concentration of 1 mM. Cells were grown at 25 'C for 3 hours post-induction and
harvested by centrifugation (10 min @ 6,000 x g, 4 °C). Cell pellets were frozen in liquid
nitrogen, and stored at -80 'C.
ClpP was purified in the method described by Kim et al. (18) and stored concentrated at 80 'C. Concentrations were monitored throughout the purification using absorbance at 280 nm.
Enzymatic activity was determined using the Suc-Leu-Tyr-(7-amino-4-methyl)coumarin (AMC)
peptide degradation assay (8, 29-31). Hydrolysis of Suc-Leu-Tyr-AMC, a fluorogenic substrate,
generates AMC, an amine product whose absorption and emission spectra exhibit longer
wavelengths than the uncleaved peptide (Ex. = 346 nm; Em = 442 nm @ pH 7.0) (32). The
specific activity of the prepared ClpP is 7 ± 0.5 [Lmol/min/•pmol ClpP. This value is comparable
to the activity of 3 ± 0.5 p.mol/min/gpmol ClpP reported in the literature (28).
1.2.3 Overexpression and Purification of GFP-ssrA
A plasmid expressing GFP containing both the S65G and S72A mutations that enhance
the intensity of green fluorescence (33) and a C-terminal ssrA tag was kindly provided by Prof.
Soren Molin (34). This protein (GFP-ssrA) was expressed in E. coli strain JB401. Aliquots of
starter culture derived from a single colony were used to seed liter-scale growth in 2YT medium
containing 100 [tg/mL ampicillin. Cells were grown at 37 'C until OD 600 = 0.5. Each volume of
cells was then induced with IPTG to a final concentration of 1 mM. Cells were grown at 25 oC
for 3 hours post-induction and harvested by centrifugation (10 min @ 6,000 x g, 4 °C). Cell
pellets were frozen in liquid nitrogen, and stored at -80 'C.
GFP-ssrA was purified in the method described by Yakhnin et al. (35) and stored
concentrated at 4 "C. Total protein concentration was monitored throughout the purification
using the Bradford assay (36), and the concentration of the final fraction was verified by
absorbance at 280 nm. In addition, the relative amount of native GFP-ssrA in each fraction was
determined by fluorescence (Ex. = 460 nm, Em. = 540 nm; 25 °C). The specific activity of the
prepared GFP-ssrA is approximately 600,000 relative fluorescence units (RFU) /mg protein.
1.2.4 Chemical Denaturation of GFP-ssrA
In a micro-well plate, a series of 100 giL-samples were prepared by diluting a 10X
concentrated protein solution into seventeen (17) different guanidinium hydrochloride (GdnHC1)
concentrations (5.4 jtg GFP-ssrA in 20 mM Tris-HC1, 1 mM EDTA, 0 - 8 M GdnHC1, pH 8.0).
After preparation, samples were incubated at 25 'C, and fluorescence data (Ex. = 460 nm, Em. =
540 nm) were taken. Samples were covered and stored in darkness between timepoints to
minimize buffer evaporation and to reduce quenching of the fluorophore by ambient conditions.
1.2.5 Thermal Denaturation of GFP-ssrA
In a 500 g.L quartz cuvette, a single protein solution (2.12 PM GFP-ssrA in 20 mM TrisHC1, 1 mM EDTA, pH 8.0) was incubated for 3 min. at 20, 40, 60, 65, 70, 72, 74, 76, 78, and 80
'C, respectively. Absorbance spectra (250 - 600 nm) were collected at each temperature.
1.2.6 Enzymatic Degradation of GFP-ssrA
Degradation assays were performed at 37 'C with 10 nM ClpA6 , 5 nM ClpP14 , and 0-10
jiM GFP-ssrA 50 mM HEPES, pH 7.5, 450 mM KC1, 20 mM MgC12 , 0.5 mM DTT, 10%
glycerol, 10 mM ATP. Fluorescence loss (Ex. = 467 nm, Em. = 511 nm) due to GFP-ssrA
unfolding was monitored as a function of time. Data were analyzed to extract initial rates.
1.3 Results
1.3.2 Chemical Denaturation of GFP-ssrA
Monitoring the rate of approach to equilibrium under denaturing conditions is one way to
assess the kinetic stability of a protein. A slow rate of approach to equilibrium is consistent with
existence of a high transition-state energy barrier (AGt), or activation energy (Ea), for the
reaction, while a rapid approach to equilibrium indicates a low-energy barrier to state change. It
is important to note that the activation energy of a reaction is an intrinsic property of the highest-
energy transition state (i.e., rate-limiting step) along the reaction coordinate. Its value remains
constant unless access to the transition state is facilitated by a catalyst, such as an enzyme.
Here, fluorescence endpoint values were collected over several days and used to create a
series of curves that illustrates how the environment of GFP-ssrA's fluorophore changes in
response to the concentration of the denaturant GdnHCI (Figure 3). These data suggest that
GFP-ssrA is highly kinetically stable (Figures 4a-4b). Indeed, even at GdnHCI concentrations
above 4M, the approach to equilibrium takes place on the timescale of tens of hours (Figure 4b;
Figure 5).
The kinetic data shown could also be analyzed, via a chevron plot, to give information
about the thermodynamic stability of GFP-ssrA. A chevron plot is the biphasic graph of a
protein's approach to equilibrium, also known as the observed relaxation rate (k o bs), as a function
of the concentration of disruptive reagent (37). One can get an estimate of a protein's stability in
water by linearly extrapolating kobs to a denaturant concentration of zero. This method is
commonly used to extract information, such as the unfolding midpoint, from denaturation data
(38).
1.3.2 Thermal Stability of GFP-ssrA
Thermal denaturation was used as an alternate method of obtaining information about the
thermodynamic stability of the model substrate. GFP-ssrA was incubated at a range of
temperatures (20 - 80 oC), and absorbance spectra were collected at each temperature. The
resultant series of spectra show, qualitatively, how the environment of GFP-ssrA's fluorophore
changes in response to increasing temperature. They also, when plotted as a function of
wavelength, reveal an isosbestic point at 381 nm (Figure 6). The convergence of multiple
absorbance spectra at a single wavelength is indicative of a two-state transition.
GFP-ssrA Fluorescence vs. Denaturant
Concentration
.3UU
-o- T=0 hrs.
2500
2000
= 1500
1000
-
500
0
0
2
-a
---*-
T=24 hrs.
T=53 hrs.
T=100 hrs.
T=352 hrs.
T=594 hrs.
-
T=762 hrs.
(M)
lGdnHCll
[GdnHC1] (M)
Figure 3. Chemical Denaturation of GFP-ssrA. GFP-ssrA unfolding was measured, as a
function of GdnHCl concentration, under the following conditions (2 gLM GFP-ssrA in 20 mM
Tris-HC1, 1 mM EDTA, 0 - 8 M GdnHC1, pH 8.0). After preparation, samples were incubated
at 25 'C, and fluorescence data (Ex. = 460 nm, Em. = 540 nm) were collected at the times
shown. Each data point represents the average value from three repetitions. Error bars represent
the standard deviation of the measurements.
-F`P F~lnore~cence! vie Time!
[0 - 3 M GdnHCI]
S2500
2000
OM GdnHC1
-
0.5M Gdn HCI
-a- IM GdnHCI
1500
o
-
1000
500
0
0
200
400
rlrqe
•
miT
(U
e
600
800
-
1.5M GdnHCI
a
2M GdnHCI
-
2.5M GdnHCI
-i-
3M GdnHCI
.L_.
(hrs)
Figure 4a. Kinetic Stability of GFP-ssrA at Low Denaturant Concentrations. GFP-ssrA
unfolding was measured, as a function of GdnHCl concentration, under the following conditions
(2 giM GFP-ssrA in 20 mM Tris-HC1, 1 mM EDTA, 0 - 8 M GdnHC1, pH 8.0). After
preparation, samples were incubated at 25 °C, and fluorescence data (Ex. = 460 nm, Em. = 540
nm) were collected at the times shown. Each data point represents the average value from three
repetitions. Error bars represent the standard deviation of the measurements. Data are the same
as in Figure 3.
GFP Fluorescence vs. Time
[3.5- 8 M GdnHCl]
-
3.5M GdnHCI
-
ILUU
4M GdnHCI
S1000
800
-- 4.5M GdnHCI
600
400
200
0
-- 5.5M GdnHCI
:s
5M GdnHCI
0
10
20
30
Time (hrs)
40
50
-.-
6M GdnHCI
---
6.5M GdnHCI
--
7M GdnHCI
+
---
7.5M GdnHCI
8M GdnHCI
Figure 4b. Kinetic Stability of GFP-ssrA at High Denaturant Concentrations. GFP-ssrA
unfolding was measured, as a function of GdnHCl concentration, under the following conditions
(2 gLM GFP-ssrA in 20 mM Tris-HCl, 1 mM EDTA, 0 - 8 M GdnHC1, pH 8.0). After
preparation, samples were incubated at 25 'C, and fluorescence data (Ex. = 460 nm, Em. = 540
nm) were collected at the times shown. Each data point represents the average value from three
repetitions. Error bars represent the standard deviation of the measurements. Data are the same
as in Figure 3.
GFP-ssrA Convergence to Equilibrium
4.0
S3.5
U 3.0
S2.5
S2,.0
"• 1.5
*- 1.0
0.5
0
200
400
600
800
Time (hrs)
Figure 5. GFP-ssrA Rate of Convergence to Equilibrium. Using the data from Figure 3, a
midpoint values for each time series was calculated. Each data point represents the average
mean GdHCl concentration of three repetitions. Error bars represent the standard deviation of the
means. The data were fit to a double exponential using Prism GraphPad. The half-lives are t/2 =
14 hrs. and tl/2 = 440 hrs. for the fast and the slow components, respectively.
Further evidence of a two-state transition is shown in Figure 7. A subset of the
absorbance data was used to generate and unfolding curve for GFP-ssrA. This unfolding curve
clearly shows a single, major transition in absorbance at 345 K (-72 'C). It also demonstrates
that GFP-ssrA is highly resistant to thermal unfolding, as expected (22, 39).
These data, because the protein sample is already at equilibrium at the point of data
collection, cannot be used to determine kinetic parameters. They can, however be used to
calculate the free energy (AG) of the unfolding reaction, a thermodynamic property. AG, unlike
AG + , varies with changes in reaction conditions, such as increased temperature or denaturant
concentration. This is because AG, the difference in energy between the folded and unfolded
states, is directly related to the equilibrium constant (Keq) of the reaction. Detailed analysis and
interpretation of the temperature denaturation curve is presented in the Discussion section.
1.3.3 Kinetics of GFP-ssrA Degradation by CIpAP (Fluorescence)
A steady-state experiment yielding the kinetic parameters of GFP-ssrA degradation by
ClpAP was conducted. As expected, ClpAP catalysis accelerated the rate of GFP-ssrA
fluorescence loss by several orders of magnitude (Figure 4a). A nonlinear least-squares fit of the
data reveals a sigmoidal dependence of the unfolding rate on GFP-ssrA concentration, with a
Hill coefficient of 2.0 ± 0.5, a Vmax of 13 ± 2 ýpM GFP/min/iM ClpA 6P1 4), and a KM of - 3 gtM
GFP (Figure 8). The values for KM and Vmax are comparable to those reported in the literature
(17, 40). However, GFP-ssrA-dependent cooperativity is not observed in those cases.
1.3.4 Energetics of Nucleotide Degradation by ClpA
A steady-state experiment yielding the kinetic parameters of ClpA's basal ATPase
activity was conducted (Figure 9). A nonlinear least-squares fit of the data reveals a sigmoidal
GFP-ssrA Absorbance vs. Temperature
A
10
200C
V.10
0.16
A
S0.14
0.12
0.10
.o 0.08
0.06
1
0.04
800C
0.02
250
300
350
400
450
500
550
600
Wavelength (nm)
Figure 6. Thermal Denaturation of GFP-ssrA. A single protein solution (2.12 RLM GFP-ssrA in
20 mM Tris-HC1, 1 mM EDTA, pH 8.0) was incubated for 3 min. at one of ten temperature
points (20-80 'C). Absorbance spectra (250 - 600 nm) were collected at each temperature.
GFP-ssrA Absorbance vs. Temperature
U.2.,I-
g 0.150.10
0.05
0.00280
I
I
I
I
300
320
340
360
Temperature (K)
Figure 7. Thermal Denaturation Curve for GFP-ssrA. A thermal denaturation curve for GFPssrA was constructed using data from Figure 5. Each point corresponds to the absorbance value
of the protein solution at each temperature (,ax = 498 nm).
CIpAP Degradation of GFP-ssrA
I
=LL
aPo
0
2
10
[GFPI (uM)
Figure 8. Enzymatic degradation of GFP-ssrA by ClpAP. The rate of GFP-ssrA degradation by
ClpAP was measured, as a function of GFP-ssrA concentration, under the following conditions
(10 nM ClpA 6, 5 nM ClpPl4, and 0-10 ýpM GFP-ssrA in 50 mM HEPES, pH 7.5, 450 mM KC1,
20 mM MgC12 , 0.5 mM DTT, 10% glycerol, 10 mM ATP, 37 'C). Loss of fluorescence was
monitored with excitation @ 467 nm and emission @ 511 nm.
ClpA Basal ATPase Activity (370C)
A
,<
E
P04
0
v
1
m
m
[ATPI (mM)
[ATP] (mM)
Figure 9. Basal ClpA ATPase Activity at Physiological Temperature. The rate of ATP
hydrolysis by CIpA was measured, as a function of ATP concentration, under the following
conditions (0.2 giM ClpA 6 in 50 mM HEPES, pH 7.5, 300 mM NaCI, 30 mM MgC12, 0.5 mM
DTT, 10% glycerol, 2.5 mM ATP, 0.23 mM NADH, 7.5 mM phosphoenolpyruvate, 19 U/mL
pyruvate kinase, and 21 U/mL lactate dehydrogenase, 37 'C). Absorbance was monitored @
340 nm. Each data point represents the average value from three repetitions. Error bars
represent the standard deviation of the measurements.
dependence of the ATPase rate on ATP concentration, with a Hill coefficient of 2.4 ± 0.2, a Vmax
of 2.7 ± 0.1 9Imol ATP/min/mg ClpA, and a Km of 1.3 ± 0.1 mM ATP. The observed pattern of
ATP-dependence is consistent with cooperative behavior between the two distinct sites of ATP
hydrolysis within ClpA (8, 41).
The complementary experiment exploring how ClpA's rate of ATP hydrolysis changes in
the presence of saturating amounts of GFP-ssrA was not conducted. Comparison of these data
with results from other members of the Licht Group, however, suggest that ClpA consumes
approximately two-fold less ATP independently as it does when in complex with ClpP and in the
presence of GFP-ssrA (17). This observation is consistent with other reports that suggest the rate
of ATP hydrolysis by ClpA increases in the presence of protein substrates (8, 9).
1.4 Discussion
We have shown preliminary data regarding the stability of GFP-ssrA. These data, when
analyzed, give us insight into the kinetic and the thermodynamic properties this model soluble
substrate. The chemical denaturation and thermal denaturation data collected, more specifically,
were used to make empirical determinations of the unfolding free energy (AG) for GFP-ssrA.
The process for determining this thermodynamic parameter is described below.
Chemical denaturation data are often conveniently analyzed to reveal accurate
quantitative descriptions of both the kinetic and the thermodynamic stabilities of a protein of
interest. GFP-ssrA, however, is very stable, and its unfolding kinetics are extremely slow
(Figures 4a-4b; Figure 5). Equilibrium, for all concentrations of denaturant tested, is approached
at t > 700 hrs. (Figure 5). Fluorescence data from t = 752 hrs. were used to calculate the change
in free energy of unfolding for GFP-ssrA in water (AG = 2.2 ± 0.1 kcal/mol; Figure 10). This
estimate is comparable to values of AG(37 'C) reported in the literature (AG = 2.6 ± 0.2 kcal/mol
(42, 43)).
Thermal denaturation was used in order to make a second estimate of the thermodynamic
stability of GFP-ssrA. These experiments took advantage of the observation that denaturation of
GFP-ssrA leads to a loss of absorbance in the blue region of the spectrum (22). The absorbance
at 498 nm of a dilute solution of GFP-ssrA in aqueous buffer was monitored over a wide
temperature range, and the change in absorbance was plotted as a function of temperature to give
a temperature denaturation curve (Figure 6). This plot, which is roughly sigmoidal in shape, was
analyzed in Origin (OriginLab, Northhampton, MA, USA) using non-linear least squares
analysis. The equation used to fit the data is shown below (44, 45):
y = {bf + mfT + (b, + mrnT) exp [(-AHm(l - T/Tm))/RT]}/ {I +exp [(-AHm(I - T/Tm))/RT]} Eq.1
Values for the midpoint of the transition from the native to denatured states, or melting
temperature (Tm), and the change in enthalpy at the melting temperature (AHm) were determined,
and the calculated parameters are shown in Table 1. The melting temperature (Tm) converted to
Table 1. Thermodynamic Parameters for Thermal Unfolding of GFP-ssrA
Parameter Symbol
P5
AHm
P6
T,
I
Definition
Calculated Value
change in enthalpy for unfolding at T,
166 ± 14.0 (kcal/mol)
melting point
345 ± 0.300 (K)
Celsius is approximately 72 'C. This value is consistent with a highly stable protein, but it is
significantly less than the reported value of 76 'C for wtGFP (42).
Analysis of thermal denaturation data is straightforward, however, one must have
accurate values for the change in heat capacity (ACp) in order to calculate an accurate estimate of
AG. In the limit that the change in heat capacity upon unfolding is negligible (ACp=0), one
obtains a maximum theoretical value for the unfolding free energy. This often, however, is not a
reasonable assumption because the folded and denatured states of a protein are expected to have
significantly different heat capacities. Using the empirically-determined values of AHm and Tm
(Table 1), estimates of AG for GFP-ssrA unfolding were calculated according to Equation 2.
AG(T) = AHm (1 - T/Tm) - ACp[(Tm _T) + (TIn(T/Tm)]
Eq. 2
When ACp is estimated as 0 kcal/mol-K, the calculated value of AG(298 K) is 21 - 24 kcal/mol.
Alternately, when ACp is estimated as 30 kJ/mol-K (46) (- 7 kcal/mol-K), the calculated value of
AGunfold is approximately 0 kcal/mol. This estimate of the free energy of GFP-ssrA unfolding is
qualitatively consistent with
the AG calculated using GdnHCI denaturation. It is also comparable to recently reported values
of this thermodynamic parameter (42, 43).
Future Work
In addition to obtaining information about the model substrate GFP-ssrA, we have also
gathered preliminary data on the energetics of ATP hydrolysis by ClpA. Analysis of these data
show that ClpA expressed and purified in the Licht Group exhibits high ATPase activity and that
the rate of ATP hydrolysis likely increases to compensate for the additional energy required to
process GFP-ssrA. Additional experiments are required, however, in order to make an accurate
determination of ClpA's efficiency as an unfoldase (i.e., the ratio of the substrate unfolding free
energy versus the amount of energy provided by ClpA via ATP hydrolysis). Such experiments
include determining ClpA's rate of ATP hydrolysis in the presence of GFP-ssrA, determining the
rate of GFP-ssrA unfolding by ClpA in the absence of ClpP. One might also consider improving
the accuracy of the experimentally determined value of the unfolding free energy for GFP-ssrA
by using differential scanning calorimetry to make a direct measurement of heat capacity (AC,)
(45, 47).
Change in Free Energy as a Function of
Denaturant Concentration
2.5
2.0
y = -2.2585x + 2.0527
S1.5
R2 = 0.9997
" 0.5
-1.5
-2.0
[GdnHC1] (M)
Figure 10. Calculation of the Thermodynamic Stability of GFP-ssrA from Chemical
Denaturation Data. Using data from Figure 3 ([GdnHCl] = 0.5 M-1.5 M; t = 752 hrs.), a series
of free energies were calculated from the mean fluorescence signal at each denaturant
concentration (solid line). Each data point represents the average value from three repetitions.
Error bars represent the standard deviation of the measurements. The equation for the fit of these
values is shown. An estimate of the unfolding free energy of GFP-ssrA was found by linear
extrapolation to ([GdnHCl] = 0 M).
Chapter 2
Energetics of Unfolding a Membrane-Bound Substrate: AChR-ssrA
2.1 Introduction
ClpA, like the related protein FtsH (48, 49), may be able to actively extract protein
substrates from the lipid bilayer. To test this hypothesis, an assay using single-molecule patchclamp electrophysiology to detect substrate degradation, was designed, and mouse nicotinic
acetylcholine receptor (AChR) was selected as a model membrane-bound substrate for this
assay.
Wild-type (wt) AChR, like GFP, is not an in vivo substrate of ClpA, but it is a reasonable
choice for monitoring the unfoldase activity of ClpA because its structure is known (50, 51), and
its electrophysiological properties are well-defined (52). Originally discovered as the target of
the paralyzing neurotoxin curare, AChR is now known as a post-synaptic receptor of the
neurotransmitter acetylcholine (53, 54). As such, it plays a major role in the propagation of
electrical signals in the body (53).
Functional adult AChR is pentameric and is composed of four distinct polypeptide
subunits (a2:j3:8:) (50, 51, 53-55). Each subunit is structurally important, but a-AChR has the
additional distinction of containing both of the two known ACh binding sites on AChR (50, 51,
53-55). Since AChR will not open in the absence of bound ligand, one would expect the
channel's open probability to decrease in the event of a-AChR removal by ClpA. For this
reason, the ssrA tag, which promotes ClpA recognition, was added to the C-terminus of the alpha
subunit. C-terminal, rather than N-terminal, tag placement was necessary because the Cterminus alone points outward into the extracellular medium, allowing interaction with the
unfoldase (50, 51, 53).
ClpA and AChR reside in different environments, thus, the conditions for optimal AChR
activity and optimal ClpA activity are different. The first step in the development of the
electrophysiological unfoldase assay, therefore, was to assess the levels of ClpA's ATPase and
unfoldase activities under conditions known to support single-molecule patch-clamp
electrophysiology. The second step in assay development was to verify that AChR-ssrA could
be successfully expressed in heterologous cells and that the addition of the ssrA tag to the asubunit would not disrupt the channel's function. Finally, correct interpretation of data
generated from the unfoldase experiments requires that we know whether or not ATP elicits any
enzyme-independent changes in the system. Therefore, the responses of both wtAChR and
AChR-ssrA to various concentrations of ATP were measured.
Characterization of the channels' behavior in the presence of ATP is an especially
important control because earlier work suggests that extracellular ATP may activate AChR (5659). Igusa et al. report that the frequency of single-channel currents increases in a concentrationdependent fashion when cultured Xenopus laevis muscle cells are exposed to 0.1-10 AiM ATP in
the presence or absence of 0.1 nM ACh (56). The same group reports that the frequency greatly
decreases at nucleotide concentrations greater than 10 jiM ATP (56). In a later study, Eterovi6 et
al. report that current amplitudes from whole-cell recordings of AChR derived from mouse and
Torpedo californicaincrease in a concentration-dependent fashion when transiently transfected
Xenopus oocytes are exposed to 10-1,000 AiM ATP in the presence of 0.6 AM ACh (57). The
same study reports that ATP alone elicits no measurable effect on current amplitude (57). Lu et
al.subsequently reported that the frequency of agonist-independent events increases in the
presence 10 jiM ATP and that the open probability of single-channel currents increases in a
concentration-dependent fashion when rat skeletal muscle cells are exposed to 0-500 AiM ATP in
the presence of 0.1 jIM ACh (58). The same study reports neither the mean open time nor the
single-channel conductance is altered by the presence of 500 jiM ATP (58). Finally, in a 1992
study, Mozrzymas et al. report that ATP-activated single-channel currents exhibit a shorter mean
open time than ACh (200 nM)-activated single-channel currents from freshly dissociated rat
skeletal muscle fibers (59).
The fact that several research groups give differing accounts of the specific effects
reported for ATP modulation of AChR gating properties is consistent with the fact that the
environment of ion channels in primary cells is extremely complex. We, therefore, needed to
conduct independent experiments to establish the phenotype of ATP's effect on AChR gating
when expressed in heterologous cells
2.2 Materials and Methods
2.2.1 Characterization of ClpA Under Unfoldase Assay Conditions
ClpA was overexpressed and purified as described in Section 1.2.1. Basal ATPase
activity at was determined using the NADH-coupled assay (27). The conditions for this assay
were the same as described in Section 1.2.1, with the exception of temperature (25 °C).
Magnesium (Mg2+)-dependent degradation assays were performed at room temperature
(~26 °C) with 0.2 giM ClpA 6, 0.1 9tM ClpP1 4, and 7.4 jgM GFP-ssrA in phosphate-buffered saline
(PBS), pH 7.5, 0.9 mM CaC12, 0 - 20 mM MgCl 2, and 2.5 mM ATP. Fluorescence loss due to
GFP-ssrA (Ex. = 467 nm, Em. = 511 nm) unfolding was monitored as a function of time. Assays
were performed in triplicate and then analyzed to extract initial rates.
2.2.2 Cloning and Overexpression of AChR and AChR-ssrA
Four pRBG4 expression vectors, each carrying the cDNA for one of the four mouse
muscle AChR subunits, were the generous gift of Prof. Anthony Auerbach (SUNY Buffalo).
The AChR-ssrA plasmid was a gift of Dina Volfson (Licht Group).
Both wtAChR and AChR-ssrA were overexpressed in the method described by Salamone
et al. (52), with the following modifications. Human embryonic kidney (HEK 293) cells, after
reaching -50% confluency, were transiently transfected by calcium phosphate precipitation (60).
For every dish of cells used, 3.6 glg cDNA, in the ratio 2:1:1:1 (a:#:&e), was added to an ice-cold
CaCl 2 solution (final concentration: 250 mM). This mixture was then carefully layered onto an
equal volume of cold buffer (50 mM HEPES, 280 NaC1, 1.5 Na2HPO 4, pH 7.0) and allowed to
stand at room temperature (24-26 "C) for 10-15 min. After this incubation period, the solution
was mixed and allowed to stand for an additional 15 min. Equal volumes of this suspension
were added to each 35-mm culture dish. Approximately 24 hours after transfection, the cell
medium was changed. Electrophysiological recordings were performed 24-48 hours later. Cells
were washed twice with equal volumes of bath solution immediately before use in patch-clamp
experiments.
2.2.3 Electrophysiology
Single-channel recordings were obtained using the cell-attached patch configuration
(61). Patch pipettes were made from borosilicate glass capillaries (World Precision Instruments,
Sarasota, FL, USA) and pulled to a resistance of about 10 MO using an upright puller (Narishige
PP-830; Narishige Group, Tokyo, Japan). After pulling, pipettes were coated with Sylgard (Dow
Coming, Midland, MI, USA) and polished in a microforge (Narishige MF-830). Pipettes were
used within 12 hours of pulling.
The bath solution (DPBS, 0.9 mM CaCl 2, 0.5 mM MgC12, pH 7.2) and pipette solutions
were the same except pipette solutions contained 1-2 jiM acetylcholine (ACh) with varying
concentrations of ATP (0-2 mM) and ClpA 6 (0 uM or 0.13 uM). After loading, patch pipettes
were lowered into the bath under positive pressure. Negative pressure was applied upon contact
with the cell membrane, and the membrane potential was held at -70 mV. Currents were
amplified with an Axopatch 200B (Axon Instrument Corporation, USA) and saved directly to
hard disk in digital format. Data from gigaohm seals were analyzed using the QUB
electrophysiology suite [www.qub.buffalo.edu].
2.3 Results
2.3.1 ClpA Activity Under Unfoldase Assay Conditions
The steady-state rate of ATP hydrolysis ClpA at 25 °C was measured (Figure 11).
Analysis of the data generated from these experiments yields a Vmax of 1.1 + 0.2 p.mol
ATP/min/mg ClpA, a KM of 0.7 ± 0.01 mM ATP, and a Hill coefficient of 2.2 ± 0.1.
Comparison of these values with those determined at 37 'C (Section 1.3.4.) shows that ClpA's
rate of ATP hydrolysis at 25 0 C (kcat - 600 min-') is approximately two-fold lower than the rate of
ATP hydrolysis at 37 'C (klat -1,400 min-'). Despite this reduction in activity, the ATPase rate
of ClpA at 25 OC was still found to be much higher than the uncoupled rate of NADH hydrolysis
at 37 'C (kcat - 4 min').
The steady-state rate of GFP-ssrA degradation by ClpAP at room temperature (-26 °C)
was measured. Here, the standard HEPES-based buffer system was replaced by the DPBS
solution used for patch-clamp experiments. MgCl 2 was varied in order to determine the optimal
Mg2+ concentration to use in the patch-clamp assay. A steady-state experiment yielding the
kinetic parameters of GFP-ssrA degradation by ClpAP was conducted. A nonlinear least-squares
fit of the data reveals a sigmoidal dependence of the unfolding rate on magnesium concentration,
with a Hill coefficient of 1.7 ± 0.3, a Vmax of 3.1 ± 0.2 gtM GFP/min/PtM ClpA 6P14), and a KM of
-4 mM MgC12 (Figure 12). Maximum enzymatic activity is observed at 20 mM MgCl 2 (Figure
12). This result is consistent with data shown by Maurizi et al. (8).
ClpA Basal ATPase Activity (250C)
0o
q4i
=1.
eE
0rOC
v
0
[ATP] (mM)
[ATP] (mM)
Figure 11. Basal ClpA ATPase Activity at Room Temperature. The rate of ATP hydrolysis by
ClpA was measured, as a function of ATP concentration, under the following conditions (0.2 igM
ClpA6 in 50 mM HEPES, pH 7.5, 300 mM NaCl, 30 mM MgC12, 0.5 mM DTT, 10% glycerol,
2.5 mM ATP, 0.23 mM NADH, 7.5 mM phosphoenolpyruvate, 19 U/mL pyruvate kinase, and
21 U/mL lactate dehydrogenase, 25 'C). Absorbance was monitored @ 340 nm. Each data
point represents the average value from three repetitions. Error bars represent the standard
deviation of the measurements.
Mg2-Dependence of GFP-ssrA Degradation
3
2
0 'C1
_
0~
=L
0
5
(mM)
[MgCI2I
20
[MgC1 2 ] (mM)
Figure 12. Mg2+-dependent degradation of GFP-ssrA by ClpAP. The rate of GFP-ssrA
degradation by ClpAP was measured, as a function of MgCl 2 concentration, under the conditions
that simulated the proposed patch-clamp assay (DPBS, 26 'C). Loss of fluorescence was
monitored with excitation @ 467 nm and emission @ 511 nm. Each data point represents the
average value from three repetitions.
2.3.2 AChR Activity Under Unfoldase Assay Conditions
AChR was expressed in the immortalized human kidney cell line, HEK-293, to separate
the activity of wtAChR from that of other cholinergic channels found in primary cells (62).
Single-molecule data were generated using the cell-attached patch method (Figure 13), and
analysis of these data suggests that, within experimental error, there is no significant change in
either the mean open lifetime or the open probability of the wild-type channel over the range of
ATP concentrations tested (100 - 2,000 giM ATP; Figures 13-16). In addition, no channel
activity was observed in control experiments where ACh was excluded from the patch pipette
solution (data not shown). Our data, in other words, do not support the idea of independent
activation of wtAChR by ATP.
It is important to note that the single-molecule patch-clamp experiments, as well as all
single-molecule experiments, due to their sensitivity, generate data that are noisy. Interpretation
of these data requires an understanding of likely sources of noise (63-68). The data below
exhibit large errors for several potential reasons: 1) interruptions in current flow due to protein
conformational changes (Figures 13-16), 2) heterogeneity in channel microenvironments
(Figures 13-16), 3) post-translational modifications, and 4) the influence of an unknown number
of silent channels (Figure 16) (69). The significance of the difference between the average values
obtained with each ATP concentration was verified via t-test.
2VI
20ms
roko011---
..IN-4op
11,L
L ij
ow
Figure 13. Representative patch-clamp data. HEK-293 cells were transiently transfected with
wtAChR. The bath solution was DPBS + 0.9 mM CaC12 + 0.5 mM MgCl 2. The pipette solution
was the same buffer + 2 pM ACh + 500 jiM ATP. The data shown are from a continuous 30 s
segment excerpted from a 10 min. recording. All data were collected at 25 'C.
0 pM ATP
100 pM ATP
S0.10 0.05
0.05
-
S0.00
......
0.00
Duration [IloglO ms]
Duration IloglO ms]
C.
D.
500 giM ATP
200 gM ATP
0.10 -
0o.10
2 0.05
2
U 0.00
U 0.00 -
0.05 -
M
Duration [loglO ms]
E.
1000 iM ATP
0.05
6
Duration [log10 ms]
F.
2000 gIM ATP
D
0.00
Duration [loglO msj
Duration [logl ms]
Figure 14. Representative Histograms of Open Lifetime as a Function of ATP Concentration.
All experiments (A-F) were conducted with wtAChR at 250 C in the presence of 2 gM ACh. All
data are included in the open lifetimes averaged in Figure 15 (dead time = 0.5 ms).
Average wtAChR Single Opening Lifetime vs. [ATP]
e
2
2
Co 2
ME2
1
0
C
01
,o
4-0
>0
0
0
200 400 600 800 1000 1200 1400 1600 1800 2000 2200
[ATP] (pM)
Figure 15. Average open lifetime for a single opening as a function of ATP concentration. All
experiments were conducted at 250C in the presence of 2 AiM ACh. The number of gigaohm
seals analyzed at each condition is indicated beneath the corresponding data point. Each data
point represents the average value of all repetitions. Error bars show the standard deviation from
the mean.
Average wtAChR Open Probability vs. [ATP]
0.75 0.70 0.65 0.60 -
0.55 0.50 0.45 c
0.40 -
4)
0.35 -
z*
0.30 -
2
0.25 0.20 -
0.15-
f
0.10-
9
4
L--
0.05 -
0.00 -0.05-
_
500
500
'
I
1000
1500
I2000
2000
[ATP] (pM)
Figure 16. Average wtAChR product of the number of channels (N) and open probability (Popen)
as a function of ATP concentration. Data and symbols are the same as Figure 15.
2.3.3 Characterization of AChR-ssrA Activity
The functionality of the AChR-ssrA construct was tested under conditions previously
shown to support wild-type acetylcholine receptor activity (DPBS, 0.9 mM CaC12 , 0.5 mM
MgCl 2, 1 gtM ACh, pH 7.2, 25 'C. Preliminary data suggest that the AChR-ssrA construct can
be used to generate functional mutant ion channels (Figure 17). These results are in agreement
with previous studies reporting that epitope tags can be introduced at the C-terminus of neuronal
AChR without damaging channel activity (70).
2.3.3 Preliminary Unfoldase Assays
The functionality of the AChR-ssrA construct was also tested under conditions optimized
to support to support both acetylcholine receptor activity and ClpA ATPase activity (DPBS, 0.9
mM CaC12, 0.5 - 30 mM MgC12 , 0 - 20mM ATP, 1 iM ACh, pH 7.2, 25 0 C). Preliminary data
suggest that ClpA may be able to remove tagged ion channels, as indicated by the reduction in
open probability (N*Popen) between a ClpA-minus control (Figure 18) and a ClpA-containing
experimental counterpart (Figure 19).
2.4 Discussion
Preliminary data for the controls of the electrophysiological unfoldase assay are
presented. We show that AChR-ssrA can be expressed in the heterologous cell line, HEK-293,
that the C-terminal ssrA tag does not inhibit channel activity, and that the presence of
extracellular ATP alters neither the open time nor the open probability of AChR.
Preliminary data for the unfoldase assay is also shown. These data, although initially
promising, must be reinterpreted in light of results from more recent experiments (Licht Group,
unpublished results). Kee-Hyun Choi (Licht Group), upon repeating the electrophysiological
unfoldase assay, saw no evidence that the presence of ClpA decreased the open probability of
AChR-ssrA over time, and Mathew Tantama (Licht Group), who performed western blotting as
an independent means of verifying the ClpA-AChR-ssrA interaction, saw no evidence that aAChR-ssrA had been removed from the membrane by ClpA and trapped inside the cavity of the
inactivated protease, ClpP S65G (18), (71).
Based on these results, the conditions of the electrophysiological unfoldase assay must be
further adjusted to optimize the probability of selective ion channel removal by ClpA. One such
modification could be to create C-terminal linkers of varying length so that the ssrA can extend
further into the bath. Alternately, the unfoldase experiment could be conducted at higher
temperature in order to maximize ClpA's catalytic activity.
IVI
20 ms
Figure 17. Representative AChR-ssrA patch-clamp data. HEK-293 cells were transiently
transfected with AChR-ssrA. The bath solution was DPBS + 0.9 mM CaC12 + 0.5 mM MgCl 2.
The pipette solution was the same buffer + 1 jgM ACh. The data shown are from a continuous 3
s segment excerpted from an 11 min. recording. All data were collected at 25 'C.
N*Popen vs. Segment-i mM ACh (+CIpA)
U.4U
0.35
0.30
0.25
a
0.20
0.15
0.10
0.05
0.00
0
100
200
300
400
500
Segment Number (5s)
Figure 18. Time-dependence of N-Pope for representative electrophysiological unfoldase assay
control experiment. HEK-293 cells were transiently transfected with AChR-ssrA. The bath
solution was DPBS + 0.9 mM CaC12 + 0.5 mM MgCl 2. The pipette solution was DPBS + 0.9
mM CaC12 + 10 mM MgC12 + 20 mM ATP + 1 jgM ACh. Data shown are from a 40 min
recording idealized in 5 s segments.
Figure 19. Time-dependence of NPopen for representative electrophysiological unfoldase assay.
Data shown are from a 40 min recording idealized in 5 s segments. Conditions are the same as in
Figure 18, except pipette solution also contains 0.13 gM ClpA6 .
References
1.
2.
3.
Katayama-Fujimura, Y., Gottesman, S., and Maurizi, M. R. (1987) A multiplecomponent, ATP-dependent protease from Escherichia coli. JBiol Chem 262, 4477-85.
Hwang, B. J., Woo, K. M., Goldberg, A. L., and Chung, C. H. (1988) Protease Ti, a new
ATP-dependent protease in Escherichia coli, contains protein-activated ATPase and
proteolytic functions in distinct subunits. JBiol Chem 263, 8727-34.
Katayama, Y., Gottesman, S., Pumphrey, J., Rudikoff, S., Clark, W. P., and Maurizi, M.
R. (1988) The two-component, ATP-dependent Clp protease of Escherichia coli.
Purification, cloning, and mutational analysis of the ATP-binding component. JBiol
Chem 263, 15226-36.
4.
5.
6.
Gottesman, S. (1996) Proteases and their targets in Escherichia coli. Annu Rev Genet 30,
465-506.
Schirmer, E. C., Glover, J. R., Singer, M. A., and Lindquist, S. (1996) HSP100/Clp
proteins: a common mechanism explains diverse functions. Trends Biochem Sci 21, 28996.
Maurizi, M. R., Singh, S. K., Thompson, M. W., Kessel, M., and Ginsburg, A. (1998)
Molecular properties of ClpAP protease of Escherichia coli: ATP-dependent association
of ClpA and ClpP. Biochemistry 37, 7778-7786.
7.
Chung, C. H. (1993) Proteases in Escherichiacoli. Science 262, 372-374.
8.
Maurizi, M. R., Thompson, M. W., Singh, S. K., and Kim, S. H. (1994) Endopeptidase
Clp: ATP-dependent Clp protease from Escherichia coli. Methods Enzymol 244, 314-31.
Seol, J. H., Baek, S. H., Kang, M. S., Ha, D. B., and Chung, C. H. (1995) Distinctive
roles of the two ATP-binding sites in ClpA, the ATPase component of protease Ti in
Escherichia coli. JBiol Chem 270, 8087-92.
Hoskins, J. R., Pak, M., Maurizi, M. R., and Wickner, S. (1998) The role of the ClpA
9.
10.
chaperone in proteolysis by ClpAP. Proceedingsof the NationalAcademy of Sciences of
the United States ofAmerica 95, 12135-12140.
11.
12.
13.
Gottesman, S., Maurizi, M. R., and Wickner, S. (1997) Regulatory subunits of energydependent proteases. Cell 91, 435-438.
Weber-Ban, E. U., Reid, B. G., Miranker, A. D., and Horwich, A. L. (1999) Global
unfolding of a substrate protein by the Hsp 100 chaperone ClpA. Nature 401, 90-93.
Hoskins, J. R., Kim, S. Y., and Wickner, S. (2000) Substrate recognition by the ClpA
chaperone component of ClpAP protease. Journalof Biological Chemistry 275, 35361-
14.
35367.
Singh, S. K., Guo, F., and Maurizi, M. R. (1999) ClpA and ClpP remain associated
during multiple rounds of ATP-dependent protein degradation by ClpAP protease.
Biochemistry 38, 14906-15.
15.
Singh, S. K., Grimaud, R., Hoskins, J.R., Wickner, S., and Maurizi, M. R. (2000)
Unfolding and internalization of proteins by the ATP-dependent proteases ClpXP and
ClpAP. Proceedingsof the NationalAcademy of Sciences of the United States ofAmerica
16.
97, 8898-8903.
Lee, C., Schwartz, M. P., Prakash, S., Iwakura, M., and Matouschek, A. (2001) ATPdependent proteases degrade their substrates by processively unraveling them from the
degradation signal. Mol Cell 7, 627-37.
17.
18.
19.
Choi, K. H., and Licht, S. (2005) Control of peptide product sizes by the energydependent protease ClpAP. Biochemistry 44, 13921-31.
Kim, Y. I., Burton, R. E., Burton, B. M., Sauer, R. T., and Baker, T. A. (2000) Dynamics
of substrate denaturation and translocation by the ClpXP degradation machine. Mol Cell
5, 639-48.
Burton, B. M., Williams, T. L., and Baker, T. A. (2001) ClpX-mediated remodeling of
Mu transpososomes: Selective unfolding of subunits destabilizes the entire complex.
Molecular Cell 8, 449-454.
20.
21.
Kenniston, J. A., Baker, T. A., Fernandez, J. M., and Sauer, R. T. (2003) Linkage
between ATP consumption and mechanical unfolding during the protein processing
reactions of an AAA+ degradation machine. Cell 114, 511-20.
Maurizi, M. R., Clark, W. P., Katayama, Y., Rudikoff, S., Pumphrey, J., Bowers, B., and
Gottesman, S. (1990) Sequence and Structure of Clp-P, the Proteolytic Component of the
Atp-Dependent Clp Protease of Escherichia-Coli. JournalofBiological Chemistry 265,
22.
23.
24.
12536-12545.
Tsien, R. Y. (1998) The green fluorescent protein. Annu Rev Biochem 67, 509-44.
Hayes, C. S., Bose, B., and Sauer, R. T. (2002) Proline residues at the C terminus of
nascent chains induce SsrA tagging during translation termination. JBiol Chem 277,
33825-32.
Farrell, C. M., Grossman, A. D., and Sauer, R. T. (2005) Cytoplasmic degradation of
ssrA-tagged proteins. MolecularMicrobiology 57, 1750-1761.
25.
26.
Seol, J. H., Yoo, S. J., Kim, K. I., Kang, M. S., Ha, D. B., and Chung, C. H. (1994) The
65-kDa protein derived from the internal translational initiation site of the clpA gene
inhibits the ATP-dependent protease Ti in Escherichia coli. JBiol Chem 269, 29468-73.
Seol, J. H., Yoo, S. J., Kang, M. S., Ha, D. B., and Chung, C. H. (1995) The 65-kDa
protein derived from the internal translational start site of the clpA gene blocks
autodegradation of ClpA by the ATP-dependent protease Ti in Escherichia coli. Febs
Letters 377, 41-43.
27.
Lo, J. H., Baker, T. A., and Sauer, R. T. (2001) Characterization of the N-terminal repeat
domain of Escherichia coli ClpA - A class I Clp/HSP 100 ATPase. ProteinScience 10,
551-559.
28.
29.
Thompson, M. W., and Maurizi, M. R. (1994) Activity and specificity of Escherichia coli
ClpAP protease in cleaving model peptide substrates. JBiol Chem 269, 18201-8.
Zimmerman, M., Yurewicz, E., and Patel, G. (1976) A new fluorogenic substrate for
chymotrypsin. Anal Biochem 70, 258-62.
30.
31.
32.
33.
Woo, K. M., Chung, W. J., Ha, D. B., Goldberg, A. L., and Chung, C. H. (1989) Protease
Ti from Escherichia coli requires ATP hydrolysis for protein breakdown but not for
hydrolysis of small peptides. JBiol Chem 264, 2088-91.
Thompson, M. W., and Maurizi, M. R. (1994) Activity and Specificity of EscherichiaColi Clpap Protease in Cleaving Model Peptide-Substrates. JournalofBiological
Chemistry 269, 18201-18208.
The Handbook - A Guide to FluorescentProbes andLabeling Technologies, 10th ed.,
Invitrogen, Carlsbad, California.
Cormack, B. P., Valdivia, R. H., and Falkow, S. (1996) FACS-optimized mutants of the
green fluorescent protein (GFP). Gene 173, 33-8.
34.
Andersen, J. B., Sternberg, C., Poulsen, L. K., Bjorn, S. P., Givskov, M., and Molin, S.
(1998) New unstable variants of green fluorescent protein for studies of transient gene
expression in bacteria. Appl Environ Microbiol64, 2240-6.
35.
36.
Yakhnin, A. V., Vinokurov, L. M., Surin, A. K., and Alakhov, Y. B. (1998) Green
fluorescent protein purification by organic extraction. ProteinExpr Purif14, 382-6.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 24854.
37.
Pace, C. N., and Shaw, K. L. (2000) Linear extrapolation method of analyzing solvent
denaturation curves. ProteinsSuppl 4, 1-7.
38.
39.
Maxwell, K. L., Wildes, D., Zarrine-Afsar, A., De Los Rios, M. A., Brown, A. G., Friel,
C. T., Hedberg, L., Horng, J. C., Bona, D., Miller, E. J., Vallee-Belisle, A., Main, E. R.,
Bemporad, F., Qiu, L., Teilum, K., Vu, N. D., Edwards, A. M., Ruczinski, I., Poulsen, F.
M., Kragelund, B. B., Michnick, S. W., Chiti, F., Bai, Y., Hagen, S. J., Serrano, L.,
Oliveberg, M., Raleigh, D. P., Wittung-Stafshede, P., Radford, S. E., Jackson, S. E.,
Sosnick, T. R., Marqusee, S., Davidson, A. R., and Plaxco, K. W. (2005) Protein folding:
defining a "standard" set of experimental conditions and a preliminary kinetic data set of
two-state proteins. Protein Sci 14, 602-16.
Bokman, S. H., and Ward, W. W. (1981) Renaturation ofAequorea gree-fluorescent
protein. Biochem Biophys Res Commun 101, 1372-80.
40.
Xia, D., Esser, L., Singh, S. K., Guo, F., and Maurizi, M. R. (2004) Crystallographic
investigation of peptide binding sites in the N-domain of the ClpA chaperone. JStruct
Biol 146, 166-79.
41.
Seol, J.H., Baek, S. H., Kang, M. S., Ha, D. B., and Chung, C. H. (1995) Distinctive
Roles of the 2 Atp-Binding Sites in Clpa, the Atpase Component of Protease Ti in
Escherichia-Coli. Journalof Biological Chemistry 270, 8087-8092.
42.
43.
Herberhold, H., Marchal, S., Lange, R., Scheyhing, C. H., Vogel, R. F., and Winter, R.
(2003) Characterization of the pressure-induced intermediate and unfolded state of redshifted green fluorescent protein--a static and kinetic FTIR, UVNIS and fluorescence
spectroscopy study. J Mol Biol 330, 1153-64.
Campanini, B., Bologna, S., Cannone, F., Chirico, G., Mozzarelli, A., and Bettati, S.
(2005) Unfolding of Green Fluorescent Protein mut2 in wet nanoporous silica gels.
Protein Sci 14, 1125-33.
44.
45.
Santoro, M. M., and Bolen, D. W. (1988) Unfolding free energy changes determined by
the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alphachymotrypsin using different denaturants. Biochemistry 27, 8063-8.
Pace, C. N., and Schultz, J. M. (1997) Measuring the conformational stability of a
protein., in ProteinStructure:A PracticalApproach (Creighton, T. E., Ed.) pp 299-321,
46.
47.
Oxford University Press.
Krasnenko, V., Tkaczyk, A. H., Tkaczyk, E. R., Farkas, 0., and Mauring, K. (2005)
Vibrations-determined properties of green fluorescent protein. Biopolymers 78, 140-6.
Pace, C. N., Hebert, E. J., Shaw, K. L., Schell, D., Both, V., Krajcikova, D., Sevcik, J.,
Wilson, K. S., Dauter, Z., Hartley, R. W., and Grimsley, G. R. (1998) Conformational
stability and thermodynamics of folding of ribonucleases Sa, Sa2 and Sa3. JMol Biol
279, 271-86.
48.
Chiba, S., Akiyama, Y., and Ito, K. (2002) Membrane protein degradation by FtsH can be
initiated from either end. JBacteriol184, 4775-82.
49.
50.
51.
52.
53.
Kihara, A., Akiyama, Y., and Ito, K. (1999) Dislocation of membrane proteins in FtsHmediated proteolysis. Embo J 18, 2970-81.
Unwin, N. (1993) Nicotinic acetylcholine receptor at 9 A resolution. JMol Biol 229,
1101-24.
Unwin, N. (1995) Acetylcholine receptor channel imaged in the open state. Nature 373,
37-43.
Salamone, F. N., Zhou, M., and Auerbach, A. (1999) A re-examination of adult mouse
nicotinic acetylcholine receptor channel activation kinetics. JPhysiol 516 (Pt 2), 315-30.
Unwin, N. (2000) The Croonian Lecture 2000. Nicotinic acetylcholine receptor and the
structural basis of fast synaptic transmission. Philos Trans R Soc Lond B Biol Sci 355,
54.
1813-29.
Karlin, A. (2002) Emerging structure of the nicotinic acetylcholine receptors. Nat Rev
Neurosci 3, 102-14.
55.
Galzi, J. L., and Changeux, J. P. (1995) Neuronal nicotinic receptors: molecular
organization and regulations. Neuropharmacology34, 563-82.
56.
57.
58.
59.
60.
Igusa, Y. (1988) Adenosine 5'-triphosphate activates acetylcholine receptor channels in
cultured Xenopus myotomal muscle cells. JPhysiol 405, 169-85.
Eterovic, V. A., Li, L., Palma, A., and McNamee, M. G. (1990) Regulation of nicotinic
acetylcholine receptor function by adenine nucleotides. Cell Mol Neurobiol 10, 423-33.
Lu, Z., and Smith, D. O. (1991) Adenosine 5'-triphosphate increases acetylcholine
channel opening frequency in rat skeletal muscle. JPhysiol436, 45-56.
Mozrzymas, J. W., and Ruzzier, F. (1992) ATP activates junctional and extrajunctional
acetylcholine receptor channels in isolated adult rat muscle fibres. Neurosci Lett 139,
217-20.
Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J., and Struhl, K.
(1995) Short Protocols in Molecular Biology, John Wiley & Sons, New York City.
61.
Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981) Improved
patch-clamp techniques for high-resolution current recording from cells and cell-free
membrane patches. PflugersArch 391, 85-100.
62.
Tsim, K. W., Choi, R. C., Siow, N. L., Cheng, A. W., Ling, K. K., Jiang, J. X., Tung, E.
K., Lee, H. H., Xie, Q. H., Simon, J., and Barnard, E. A. (2003) ATP induces postsynaptic gene expressions in vertebrate skeletal neuromuscular junctions. JNeurocytol
32, 603-17.
63.
64.
65.
66.
67.
Heinemann, S. H., and Sigworth, F. J. (1988) Open channel noise. IV. Estimation of
rapid kinetics of formamide block in gramicidin A channels. Biophys J 54, 757-64.
Heinemann, S. H., and Sigworth, F. J. (1990) Open channel noise. V. Fluctuating barriers
to ion entry in gramicidin A channels. Biophys J 57, 499-514.
Heinemann, S. H., and Sigworth, F. J. (1991) Open channel noise. VI. Analysis of
amplitude histograms to determine rapid kinetic parameters. Biophys J 60, 577-87.
Sigworth, F. J. (1985) Open channel noise. I. Noise in acetylcholine receptor currents
suggests conformational fluctuations. Biophys J47, 709-20.
Sigworth, F. J. (1986) Open channel noise. II. A test for coupling between current
fluctuations and conformational transitions in the acetylcholine receptor. Biophys J 49,
1041-6.
68.
Sigworth, F. J., Urry, D. W., and Prasad, K. U. (1987) Open channel noise. III. Highresolution recordings show rapid current fluctuations in gramicidin A and four chemical
analogues. Biophys J 52, 1055-64.
69.
70.
Alvarez, O., Gonzalez, C., and Latorre, R. (2002) Counting channels: a tutorial guide on
ion channel fluctuation analysis. Adv Physiol Educ 26, 327-41.
Anand, R., Bason, L., Saedi, M. S., Gerzanich, V., Peng, X., and Lindstrom, J. (1993)
Reporter epitopes: a novel approach to examine transmembrane topology of integral
membrane proteins applied to the alpha 1 subunit of the nicotinic acetylcholine receptor.
Biochemistry 32, 9975-84.
71.
Flynn, J. M., Neher, S. B., Kim, Y. I., Sauer, R. T., and Baker, T. A. (2003) Proteomic
discovery of cellular substrates of the ClpXP protease reveals five classes of ClpXrecognition signals. Mol Cell 11, 671-83.
Melva Tonisha James
Secondary Address:
77 Massachusetts Avenue
Room 56-522
Cambridge, MA 02139
(617) 258-6268 (lab)
Primary Address:
70 Pacific Street
Room 298
Cambridge, MA 02139
(617) 452-4252 (home)
melva@mit.edu
Education
Awards
University of Mississippi
Cum laude; B.S. Chemistry, 2001.
University, MS
Robert M. Callaway High School
Salutatorian, 1997. Hall of Fame, 1997.
Jackson, MS
NIH Ruth L. Kirschstein Predoctoral Fellow (2004-); Lester Wolfe Predoctoral Fellow (20032004); MIT Provost Fellow (2002-2003); Phi Beta Kappa, Beta of Mississippi Charter Member
(2001); Who's Who Among Students at American Colleges and Universities (2001); Mortar
Board (2000-2001); NASA Space Grant (3 consecutive semesters, 1999-2000); Phi Kappa Phi
(2000); Sagner Fellow (1999); All-American Scholar (1999); Golden Key National Honor
Society (1999); 2nd-year German Government Prize (1999); Alpha Lambda Delta (1998);
McDonnell-Barksdale Honors College (1997-2001); Mississippi Eminent Scholar (1997);
National Achievement Award (1997).
Activities
Resident Advisor (July 2005-), Kappa Alpha Theta; Coordinator (2004-2005; 2005-2006),
Women in Chemistry (MIT); Member (2005-), Lutheran Episcopal Ministry; Volunteer
(2004, 2005), CONVERGE Minority Recruitment Program (MIT); Hall Councilor (20032004), Sidney-Pacific Graduate Residence (MIT); Treasurer (2003-2004), Black Graduate
Students Association (MIT); Secretary (2002), National Organization for the Professional
Advancement of Black Chemists and Chemical Engineers (UM). Officer (1998-2000),
Student Affiliates of the American Chemical Society (UM). Mentor (Summer 1998),
Member (1997-2000), Mississippi Alliance for Minority Participation. Chief organizer
(2000), Students Envisioning Equality through Diversity (UM). Student organizer (1999),
Statewide Student Summit on Race (UM); Member (1998-2000), Sigma Alpha Iota Music
Fraternity for Women; Member (1997-2001), UM Orchestra.
References
Prof. Stuart S. Licht
MIT Department of
Chemistry
77 Massachusetts Ave.
16-573B
(617) 452-3525
lichts@mit.edu
Prof. Catherine L. Drennan
MIT Department of
Chemistry
77 Massachusetts Ave.
16-573A
(617) 253-5622
cdrennan@mit.edu
Prof. Maurice R. Eftink
Department of Chemistry
& Biochemistry
University of Mississippi
P.O. Box 1848
University, MS 38677
(662) 915-5330
chmre(cotton.vislab.
olemiss.edu