From the Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500
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ABSTRACT |
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Precursor proteins must be at least partially
unfolded during import into mitochondria, but their actual conformation
during translocation is not known. Are proteins fully unfolded and
threaded through the import machinery amino acid by amino acid, or do
they retain some partial structure? The folding pathway of most
proteins in vitro contains a partially folded intermediate
known as the molten globule state, and it has been suggested that
proteins are in the molten globule state during translocation across
membranes. Here we show that precursors are normally fully unfolded
during import into mitochondria. However, precursors containing
residual structure can be imported, if less efficiently.
Approximately half of all proteins synthesized in a eukaryotic
cell are transported into or across a membrane during their life cycle.
Cells have developed elaborate protein translocation systems, which
share several characteristic features (1). Protein unfolding is
intimately associated with translocation, because proteins do not
maintain their native state during translocation but can be folded both
before and after translocation.
Here we determine the structure of precursor proteins during import
into mitochondria. Precursor proteins are not in their native
conformation during translocation into mitochondria (2), but it is not
known how extensively they unfold. Are precursors fully unfolded and
threaded through the import machinery amino acid by amino acid, or do
they retain some partial structure during transport?
Although precursor proteins can be fully unfolded during translocation
(2), the protein import machinery permits the passage of bulky groups
such as branched polypeptides (3) and precursors with a single- or
double-stranded oligonucleotide attached to the C terminus (4).
Electron micrographs of the protein-conducting channel of the outer
mitochondrial membrane reconstituted in lipid vesicles suggest that it
has an internal diameter of ~20 Å at its entrance (5).
Electrophysiological measurements estimate its internal diameter to be
~22 Å (6). Therefore, the mitochondrial import machinery may
tolerate the passage of proteins that have retained some residual
structure, such as secondary structural elements.
It has been proposed that proteins retain residual structure during
translocation, as in the molten globule state (7, 8). The molten
globule is an in vitro folding intermediate observed on the
refolding pathways of most proteins (8). It is defined as a state in
which the protein lacks defined tertiary structure but retains
secondary structural elements. Many biological membranes contain
negatively charged lipids, and these lipids are expected to increase
the local proton and cation concentrations at the membrane surface (9,
10). The decreased pH at the surface of phospholipid vesicles induces
the molten globule state in the protein toxin colicin A, which allows
the toxin to undergo the conformational changes necessary to insert
into the membrane (11). The outer mitochondrial membrane also contains
negatively charged phospholipids (12), and it has been suggested that
these charges contribute to the unfolding of precursors (13).
Therefore, the surface of mitochondria may induce molten globule
formation in precursors before import. Urea-denatured precursor
proteins refolded in the presence of mitochondria are imported faster
than native precursors, even after long incubations at low temperature
(13). This acceleration of import is not observed when proteins are renatured in the absence of membranes. It appears that in the presence
of mitochondrial membranes denatured precursors do not refold to their
native state but into a translocation-competent folding intermediate,
which may be the molten globule state (8). However, it is not known
whether proteins are transported through membranes as folding
intermediates or whether, before translocation, proteins only
transiently form the intermediate and then unfold completely.
In this study we investigate whether precursor proteins retain residual
structure during translocation into mitochondria by determining import
kinetics for a series of precursors into which we introduced disulfide
bridges that covalently cross-link two, three, four, or five of the
strands of a Precursor Proteins and Mitochondria--
Mitochondrial precursor
proteins, consisting of a presequence fused to the N terminus of a
passenger protein, were constructed using standard molecular biology
techniques in pGEM-3Zf(+) vectors (Promega, Madison, WI) and verified
by DNA sequencing. The presequence was derived from the first 95 amino
acids of yeast cytochrome b2 (15), starting at
the initiator methionine. The presequence contained a Cys14
Radioactive precursors were expressed from a T7 promoter by in
vitro transcription and translation in rabbit reticulocyte lysate
(Promega) supplemented with [35S]methionine and partially
purified by high speed centrifugation and ammonium sulfate
precipitation as described (15). Mitochondria were isolated from
Saccharomyces cerevisiae strain D273-10B (MAT Selection of Sites for Cysteine Mutation--
Three single
disulfide bridge mutants and one double disulfide bridge mutant were
described previously (22). To link Disulfide Bridge Formation--
To induce disulfide bridge
formation between Cys residues, precursors were oxidized with 10 mM K3Fe(CN)6 for 2 min at room temperature before precipitation with ammonium sulfate. In other experiments disulfide bridge formation was prevented by resuspending ammonium sulfate-precipitated precursors in import buffer containing 10 mM dithiothreitol
(DTT).1
To test for disulfide bridge formation, unreacted cysteine residues
were modified with 4-acetamido-4'-maleididylstilbene-2,2' disulfonic
acid (stilbene disulfonate maleimide (SDSM), Molecular Probes) and
detected through a change in mobility of the modified proteins in
SDS-PAGE. Precursors were oxidized with
K3Fe(CN)6, partially purified by precipitation
with ammonium sulfate, and reacted with 0.1 mM SDSM at
25 °C for 2 h in the dark. SDSM modification was detected
through gel shift after autoradiography and
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine-SDS-PAGE (26).
Import Assays--
Import kinetics of precursor proteins into
purified yeast mitochondria were performed at 20 °C essentially as
described previously (15). Briefly, radiolabeled precursors were
treated with K3Fe(CN)6 or DTT to form or break
disulfide bridges. 30 µl of precursor were prewarmed and then
incubated with 570 µl of mitochondrial suspension at 0.5 mg of
mitochondrial protein/ml in import buffer (0.6 M sorbitol,
20 mM HEPES-KOH, pH 7.4, 1 mg/ml fatty acid-free bovine
serum albumin) containing 4 mM ATP, 10 mM
creatine phosphate, and 0.15 mg/ml creatine kinase. For the import of
precursors with reduced disulfides, the import reaction contained 10 mM DTT. At indicated time points, 50-µl samples were
transferred to 100 µl of ice-cold stop buffer (0.6 M
sorbitol, 20 mM HEPES-KOH, pH 7.4, 2 µM
valinomycin, 0.2 mg/ml proteinase K). After 10 min, proteinase K was
inhibited with 1 mM phenylmethylsulfonyl fluoride. The
mitochondria were then reisolated by centrifugation at 7000 × g and resuspended in SDS-PAGE sample buffer containing 2 mM phenylmethylsulfonyl fluoride. Samples were analyzed by
SDS-PAGE, and the amount of imported protein was quantified by
electronic autoradiography. Analysis with reducing and nonreducing
SDS-PAGE yields the same kinetic parameters. The extent of import was
plotted as a percentage of the total amount of precursor in the import
reaction, and import kinetics were analyzed using the software package
Kaleidagraph (Abelbeck Software) by assuming a simple first-order
process and fitting to the equation:
Import specifically into the matrix space was measured by rupturing the
outer mitochondrial membrane by hypo-osmotic shock (mitoplasting; Ref.
27) before assaying for import. The efficiency of mitoplasting was
determined by quantifying the amount of the intermembrane space protein
cytochrome b2 and the matrix protein Precursor Proteins--
We constructed a series of mitochondrial
precursor proteins in which specific parts of the structure were
prevented from unfolding by covalent cross-links. All precursor
proteins consisted of a 95-amino acid mitochondrial presequence derived
from cytochrome b2 fused to the ribonuclease
barnase (15, 29). Cross-links were introduced into barnase by mutating
pairs of residues to cysteine at positions that allow disulfide bridge
formation upon oxidation (Fig. 1). The
disulfide bonds covalently link neighboring
Three single disulfide bridge mutants were described previously (22)
and cross-link Disulfide Bridges Are Formed by Oxidation--
Neighboring Cys
residues were induced to form disulfide bridges by oxidation with
ferricyanide (K3Fe(CN)6). Disulfide bond formation was assessed by modification of free thiol groups with SDSM.
After oxidation with ferricyanide and purification, none of the
disulfide mutants could be modified with SDSM, whereas cysteine mutants
that were not oxidized with ferricyanide before treatment with SDSM run
slower in SDS-PAGE than ferricyanide-treated precursors (data not
shown). Thus, all disulfide bonds form quantitatively after oxidation.
Nonreducing SDS-PAGE after oxidation showed that <2% of precursors
formed dimers or higher order multimers (data not shown).
Two results suggest that precursor proteins are in their native
conformation after disulfide bridge formation. First, oxidized precursors are resistant to proteinase K to similar extents as wild-type protein (data not shown). Second, even precursors with eight
Cys residues do not form dimers or multimers after oxidation, as judged
by nonreducing SDS-PAGE, yet all Cys residues are involved in disulfide
bridges. It is unlikely that the Cys residues could efficiently form
non-native disulfide bridge pairs within a properly folded precursor protein.
Residual Structure in Precursors Inhibits Import--
We measured
the effect of increasing amounts of residual structure in barnase
precursor proteins on import into purified yeast mitochondria under
conditions where precursor unfolding is not rate limiting for import
(15). Radiolabeled precursors were synthesized in vitro by
coupled transcription and translation, treated with
K3Fe(CN)6 or DTT to form or break disulfide
bridges, partially purified, and incubated with purified yeast
mitochondria in import buffer. At designated time points samples were
removed and analyzed for imported protein (Fig.
2A). The extent of import was
plotted as a percentage of the total amount of precursor presented to
the mitochondria (Fig. 2, B and C).
As precursors were forced to retain an increasing amount of residual
structure during import by the cross-linking of
The effect of oxidation on import was not attributable to the formation
of precursor dimers or other multimers, because import kinetics
analyzed by reducing or nonreducing SDS-PAGE yield identical kinetic
parameters. Import of the cross-linked precursors was not attributable
to slow reduction of the disulfide bridges, because disulfide bonds
remained formed for at least 1 h when oxidized precursors were
incubated with de-energized mitochondria in import buffer (data not
shown). Import experiments conducted at 30 °C yielded import
inhibitions equivalent to those performed at 20 °C.
The import inhibition of the oxidized precursors containing Cys
residues is caused by the formation of disulfide bridges and not the
presence of Cys residues themselves. 10 mM DTT fully
reduces the previously characterized single disulfide barnase mutants (22). When import experiments were conducted in the presence of 10 mM DTT, all of the disulfide mutants showed import rates approximately equal to those of the wild-type precursor (Table I and
Fig. 2C). The nondisulfide bridge containing wild-type precursor was imported at identical rates under oxidizing or reducing conditions (Table I and Fig. 2C).
Precursor Unfolding Is Not Rate Limiting for Import under the
Chosen Conditions--
Disulfide bridges may affect the stability of
the fully folded precursor protein against global unfolding (22), and
therefore, their effect on import rates could be caused by an effect on
the stability of the native precursor rather than residual structure induced in the unfolded precursor. We have shown previously that import
of barnase and dihydrofolate reductase precursors with the 95-amino
acid presequence described here is not limited by unfolding of the
passenger protein (15). When the length of the targeting sequence is
reduced to 35 amino acids, unfolding of the passenger protein at the
mitochondrial surface becomes rate limiting for import (15), and
destabilizing mutations in barnase, such as the mutation of the Ile at
position 25 in barnase to Ala, greatly accelerate import (>170-fold
rate enhancement of a pseudo-wild-type barnase
precursor).2 The
Ile25 Import Across the Inner Mitochondrial Membrane--
Import across
both mitochondrial membranes into the matrix can be differentiated from
import across only the outer membrane into the intermembrane space by
rupturing the outer membrane by hypo-osmotic shock (mitoplasting)
before assaying for import. When import of the precursors described
here was analyzed by mitoplasting, no significant amounts of precursor
could be detected between the two membranes, indicting that all of the
precursors were imported completely into the matrix. Import rate
constants for wild-type precursor and all the disulfide bridge mutants
are shown in Table III together with the
relative inhibition of import rates by the disulfide bridges. As for
import across the outer membrane, introducing an additional
Ile25 To determine the structure of proteins during import, we forced
increasing amounts of residual structure into a translocating precursor
protein by cross-linking parts of the passenger protein barnase with
disulfide bridges. The disulfide bridges cross-linked pairs of
neighboring The largest amount of structure that any one disulfide bridge will
stabilize is a pair of neighboring The differential effects of the three combinations of two disulfide
bridges we examined can be understood if one considers that precursors
are imported from the N terminus to the C terminus. A single disulfide
bridge in a precursor will introduce a loop and thus change the
direction of the peptide chain. Therefore, if the directionality of
import from N to C terminus is to be maintained, three strands of the
precursor must pass through the import machinery at the same time. In
the mutants
Cys43-Cys80/Cys70-Cys92
and
Cys70-Cys92/Cys85-Cys102
the second disulfide bridge cross-links the neighboring strand to the
loop induced by the first disulfide bridge (Fig. 1). This second
cross-link restricts the conformations allowed to all three neighboring
strands and leads to a small increase in inhibition of import over that
induced by a single disulfide bridge. In contrast, in the mutant
Cys43-Cys80/Cys85-Cys102
two pairs of two neighboring The structure of proteins during import will be influenced by the
internal diameter of the mitochondrial protein import channel. Small
amounts of residual structure in precursor proteins, as induced by
single disulfide bridges, slow import, but even precursors with large
amounts of residual structure, as induced by several disulfide bridges,
are imported. Import is progressively inhibited as the residual
structure in precursors increases. Thus, the import channels do not
show a clear size cutoff. Rather, they appear to be flexible and can
accommodate structures considerably larger than a single strand of
protein when required. However, the alternative explanation that
mitochondria contain a range of import channels of different sizes
cannot be ruled out.
Because import across the inner membrane is slower than across the
outer membrane, and disulfide bridges inhibit import across the inner
membrane more strongly than across the outer membrane, the import
channel in the inner membrane appears to be tighter than that in the
outer membrane.
The flexibility of the import channel may be biologically important.
Mitochondria establish an electrochemical gradient across their inner
membrane. Channels with large openings would make maintaining an
electrochemical potential difficult. At the same time, the import
channel may need to accommodate large structures. For example, some
mitochondrial tRNAs are imported into mitochondria from the cytosol by
the protein import machinery, and it has been suggested that they may
be imported in association with their cognate aminoacyl tRNA
synthetases (31). The task of maintaining ion gradients across the
membrane would be facilitated by the import channel in the inner
membrane having a small diameter and adjusting its size to expand
around large structures that pass through it.
How do our conclusions apply to proteins other than barnase?
Conclusions about the import channel itself, such as its size and
flexibility, can be expected to be valid generally, because barnase was
used simply as a probe to determine properties intrinsic to the
channel. Conclusions about the structure of precursor proteins during
import may or may not be general, depending on how good a model barnase
is for the behavior of other proteins. The folding and unfolding of
barnase have been studied extensively in vitro and appear
typical when compared with the behavior of other proteins. Our results
suggest that the structure of proteins during import is influenced by
the properties of the channel. Size limitations imposed by the internal
diameter of the import channel appear to favor minimal residual
structure in precursors. Could
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-sheet. Because mitochondria have no mechanism for
reducing disulfides at their surface (14), disulfide bridges make it
impossible for the import machinery to separate the cross-linked
strands. If the
-sheet persisted during translocation, locking the
structure with disulfide bridges would have no effect on import. On the
other hand, if preventing
-strands from separating completely during
import inhibits translocation relative to translocation of wild-type
protein, the
-sheet is not normally present during import. We show
that proteins are normally fully unfolded during import and not in the
molten globule state. However, precursors containing residual structure
can be imported, if less efficiently.
EXPERIMENTAL PROCEDURES
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Ser mutation to prevent disulfide bond formation between targeting sequences, an Arg30
Gly mutation to prevent processing
by the mitochondrial matrix processing protease (16), and a
Leu62
Pro mutation to target the precursor to the
mitochondrial matrix (17). The passenger protein was barnase, a
ribonuclease from Bacillus amyloliquefaciens (18). In
addition to the introduction of cysteine residues, barnase contained
two further mutations: one changing His102 of the authentic
barnase sequence to Ala to inactivate barnase (19) and one mutating
Gln2 of barnase to Met to allow radioactive labeling.
, ATCC 25657) (20) and purified by centrifugation through a Nycodenz gradient (21).
-strands 4 and 5 covalently,
residues 96 and 110 of barnase were mutated to Cys. The positions of
the cysteine mutations were selected by using the program EDPDB (23)
and visual inspection of the barnase structure (24) using the program
Xtalview (25).
where A is the extent of import at any given time
(t), A0 is the extent of import at
infinite time, k is the import rate constant, and
C is a constant offset attributable to background.
-ketoglutarate dehydrogenase present before and after mitoplasting by quantitative Western blotting (28). Mitoplasting ruptured the outer
membrane in 95-99% of the sample, whereas the inner mitochondrial
membrane remains intact in ~90% of the sample (data not shown).
RESULTS
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-strands within the
five-stranded antiparallel
-sheet of barnase and prevent their
complete separation during import, because mitochondria have no
mechanism for reducing disulfide bridges at their surface (see below
and Ref. 14).
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Fig. 1.
Wire diagram of barnase. -Helices are
shown as cylinders,
-strands as arrows.
Disulfide bridges are drawn in gray and labeled with the
positions of the participating cysteine residues.
-strands 1 and 2 (Cys43-Cys80),
-strands 2 and 3 (Cys70-Cys92), and
-strands 3 and 4 (Cys85-Cys102) (Fig. 1). The structure of these
proteins was determined by x-ray crystallography and found to be almost
identical to that of wild-type barnase (30). To covalently link
-strands 4 and 5, we introduced an additional disulfide bridge by
mutating residues 96 and 110 of barnase to cysteines. We constructed
precursor proteins containing all single disulfide bridges and all
combinations of two and three disulfide bridges between the first four
-strands, as well as a precursor with four disulfide bridges
cross-linking all five
-strands.
View larger version (27K):
[in a new window]
Fig. 2.
Import kinetics of oxidized and reduced
cytochrome b2-barnase precursor proteins.
A, autoradiograms of SDS-PAGE analysis of import experiments
with wild-type and
Cys43-Cys80/Cys70-Cys92/Cys85-Cys102
precursors after oxidation with ferricyanide. Bands represent the
amount of precursor associated with mitochondria after protease
treatment and reisolation of mitochondria at the indicated times;
T, total amount of precursor presented to mitochondria.
Top, wild type; bottom,
43-80/70-92/85-102. B, import of precursor proteins with no,
one, two, three, or four disulfide bridges after oxidation with
ferricyanide. C, import of oxidized and reduced barnase
precursors lacking disulfide bonds and precursors containing three
disulfide bonds. The oxidized precursors were treated with
ferricyanide, the reduced precursors were treated with DTT.
y-axis, amount of precursor imported as a percentage of the
total amount of precursor presented to the mitochondria.
-strands, both the
import rate constants and the extent of import decreased (Fig.
2B and Table I, oxidized).
Import rates of precursors containing a single disulfide bridge were
inhibited by a small but significant amount compared with import rates
of precursors lacking disulfide bridges. With introduction of an
additional disulfide bridge, import rates were inhibited further.
Precursors containing three disulfide bridges (four cross-linked
-strands) were imported approximately four times more slowly than
the wild-type precursor. In addition, the extent of import was reduced
to ~50% of that of the precursor lacking disulfide bridges. Finally,
precursors containing four disulfide bridges (five cross-linked
-strands) were imported eight to nine times more slowly than the
wild-type precursor, and only ~30% of the cross-linked precursor
could be imported. It is not immediately clear why residual structure
in a precursor protein reduces the extent of import as well as the rates of import. However, it is commonly observed that the efficiency of import decreases with decreasing import rates. Two possible explanations are that aggregation of partially imported precursors competes with import, and that mitochondrial preparations deteriorate throughout import reactions.
Rate constants for import into mitochondria and import inhibitions for
oxidized and reduced barnase precursor proteins
Ala mutation did not accelerate import of any of
the disulfide-containing precursors described here (Table
II). This demonstrates that, under the
conditions tested, global unfolding of the native precursor was never
rate limiting for import. Therefore, the observed import rate
inhibition is the result of the residual structure induced in the
unfolded precursor protein by the disulfide bonds rather than any
effect on the stability of the native conformation of barnase.
Rate constants for import into mitochondria and accelerations through
destabilization for oxidized barnase precursors
Ala mutation in barnase did not affect inhibition
by the disulfide bridges (Table III). Comparison of Tables I and III
shows that import across the inner membrane appears somewhat slower
than import across the outer membrane. Residual structure in the
precursor protein has a stronger effect on import across the
inner membrane than on import across the outer membrane.
Rate constants for import across the inner mitochondrial membrane,
inhibitions by disulfide bridges, and accelerations by destabilization
Ala of barnase.
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-strands in the single antiparallel
-sheet of
barnase. When two disulfide bridges were introduced simultaneously, they cross-linked either three neighboring
-strands, as in the mutants
Cys43-Cys80/Cys70-Cys92
and
Cys70-Cys92/Cys85-Cys102,
or two sets of two
-strands, as in the mutant
Cys43-Cys80/Cys85-Cys102
(Fig. 1). When three disulfide bridges were introduced simultaneously, they cross-linked four neighboring
-strands, and four disulfide bridges cross-linked all five
-strands of the
-sheet.
-strands. However, the
cross-linked parts of the protein need not remain in a
-sheet conformation during translocation but could rearrange to minimize steric bulk if the import channel imposed size constraints on the
precursor. Tables I and III show that even single disulfide bridges
inhibited import and that the inhibition becomes stronger as more
-strands are cross-linked to each other. Therefore, the entire
-sheet in barnase is clearly not normally maintained during import
and barnase precursors are not in the molten globule state. Furthermore, because even one and two disulfide bridges inhibited import, we conclude that barnase is normally fully unfolded
during import into mitochondria.
-strands are cross-linked, presumably forcing four or five strands of protein to be imported next to each
other simultaneously. As expected, this double disulfide bridge mutant
shows a larger inhibition of import than the two double disulfide
bridge mutants with cross-links between three neighboring
-strands.
In the triple and quadruple disulfide bridge mutants, the conformation
of the five protein strands during translocation is increasingly
restricted, which leads to a correspondingly stronger inhibition of import.
-helical structures persist in a
precursor protein during import? The anhydrous diameter of an
-helix
is 10-12 Å, and that of three extended protein strands is between 10 and 20 Å, depending on their arrangement. Therefore, our data cannot
rule out that
-helices are maintained in a protein during import.
However, this appears unlikely, because
-helices are unstable
outside the context of other structural elements, and our results
suggest that the structure of the import channel minimizes residual
structure in precursors.
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ACKNOWLEDGEMENTS |
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We acknowledge the use of instruments in the Keck Biophysics Facility at Northwestern University (http://x.biochem.nwu.edu/Keck/keckmain.html). We thank Karl Schmid (Biozentrum, University of Basel, Basel, Switzerland) for excellent technical assistance in the initial stages of the project.
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FOOTNOTES |
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* This work was supported in part by Basil O'Connor Starter Scholar Research Award 5-FY97-0666 from the March of Dimes Birth Defects Foundation and by an individual allocation from American Cancer Society institutional research Grant IRG-93-037-04 (Robert H. Lurie Cancer Center).
Recipient of a Gramm travel fellowship award from the Lurie Cancer
Center of Northwestern University.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2153 Sheridan Rd., Evanston, IL 60208-3500. Tel.: 847-467-3570; Fax 847-467-6489; E-mail: matouschek{at}nwu.edu.
2 M. P. Schwartz, S. Huang, and A. Matouschek, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: DTT, dithiothreitol; SDSM, stilbene disulfonate maleimide; PAGE, polyacrylamide gel electrophoresis.
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