From the
SoxR protein governs the soxRS (superoxide response)
regulon of Escherichia coli by becoming a transcriptional
activator when the cells are exposed to compounds that mediate
univalent redox reactions, many of which produce superoxide as a
by-product. SoxR was overproduced and purified to near homogeneity from
a strain bearing an expression vector. It could bind specifically to
the soxS operator even in the absence of RNA polymerase. The
aerobically purified protein, which is readily autooxidized, could
activate the transcription of soxS DNA even without exposure
to known inducing agents. SoxR is a globular homodimer. It contains one
[2Fe-2S] cluster per polypeptide chain, as demonstrated by
optical and EPR spectroscopy combined with stoichiometric analysis of
iron content, unpaired-electron-spin density, and reduction by
dithionite. The protein is active in its oxidized
([2Fe-2S]
The transcriptional regulators, SoxR and SoxS, govern a regulon
of Escherichia coli that responds to superoxide generators
such as paraquat (methyl viologen)
(1, 2) . The
soxRS regulon and the oxyR regulon (which protects
against H
The induction of the
soxRS regulon occurs in two steps
(6, 7) . SoxR
responds to an oxidative signal and activates the transcription of
soxS. The overproduced SoxS then activates the transcription
of the target genes of the regulon. This cascade increases sensitivity
through signal amplification. Although SoxS is a member of the AraC
family of transcriptional activators, it lacks a region corresponding
to the sensor domains of its relatives
(8, 9) . The
following findings suggested that SoxR provides this sensor function.
The action of SoxR precedes that of SoxS, some soxR mutants
have a regulon-constitutive phenotype, and SoxR has a cluster of four
cysteines that may be part of a redox-sensing center
(6, 7, 8, 9) .
In preliminary
experiments
(10) , we obtained active SoxR with an average of
1.0 iron atom per chain and the optical spectrum of an iron-sulfur
protein. A recent report
(11) described the purification of
SoxR that had 1.6 iron atoms per polypeptide and a similar spectrum.
The low iron content was consistent with the well known instability of
many Fe-S clusters. The number of iron atoms per Fe-S center and the
number of clusters per polypeptide were not determined. In this study,
we report the large scale production and purification of SoxR that
contains a full complement of Fe-S clusters, a prerequisite for
crystallographic and other physical studies. We determine some of its
molecular parameters, and we show that the active protein is a globular
homodimer containing one [2Fe-2S] center per polypeptide
chain. Thus, SoxR is related to a class of well characterized electron
transport proteins, the [2Fe-2S] ferredoxins. This
identification of the redox center provides an important insight into
the mechanism of activation of the soxRS regulon by oxidative
stress.
Overproduction was also achieved in
500-ml flasks on a water bath shaker in room atmosphere. One ml of a
saturated culture was added to 100 ml of medium in each flask. The
temperature shift was performed at a cell density of 5
Whatman P-11 phosphocellulose
was equilibrated with the loading buffer and packed into a column (7.6
cm
The first five N-terminal amino acids of the
purified protein matched those predicted by the DNA sequence of
soxR (8) , indicating that there was no amino-terminal
processing. A secondary band detected by SDS-PAGE
(Fig. 2 B, IV), had the mobility and N-terminal
sequence expected of undissociated SoxR dimer.
Active SoxR is a homodimer. Because each chain contains a
DNA-binding (helix-turn-helix) motif
(8, 9) , the
protein is likely to recognize a repeated sequence in DNA. Indeed, the
soxS operator, which covers most of the promoter region
(11) , contains an 18-nucleotide palindrome
(8) . The
Fe-S clusters in SoxR are probably coordinated to all four of its
cysteines, as is the case for most [2Fe-2S] proteins of known
structure. However, there are exceptions
(31, 36) , and
the unusual spacing of the cysteines in SoxR adds to our uncertainty.
It is also possible that Fe-S clusters might bridge the two polypeptide
chains.
The superoxide response ( soxRS) regulon was so
named because it is induced by superoxide generators like paraquat. It
was therefore unexpected that SoxR, when purified from cells that were
not exposed to these agents, was already active
(11) (this
work) and that this activity was not enhanced in vitro by
superoxide generators. However, recent evidence (reviewed in Liochev
and Fridovich
(4) ) suggested that SoxR is likely to be
activated in vivo through an oxidative chain rather than by
superoxide directly.
Although the regulon is induced by univalent
redox compounds, it is not induced by H
We gratefully acknowledge the helpful advice of Dr.
David P. Ballou.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) state. The presence of a
prosthetic group capable of univalent redox reactions may help to
explain the activation of the regulon in vivo by compounds
that can mediate such reactions.
O
) are the two best studied bacterial
regulatory systems for adaptive defenses against oxidants
(3) .
The soxRS regulon includes the following genes of known
function: sodA, encoding Mn
-superoxide
dismutase; nfo, encoding endonuclease IV, a DNA repair enzyme;
zwf, encoding glucose-6-phosphate dehydrogenase, which
regenerates the reducing equivalents (in the form of NADPH) that are
consumed during redox cycling of the inducers; micF, whose
mRNA inhibits the porin gene, ompF; fpr, encoding
ferredoxin (flavodoxin):NADPH oxidoreductase, which assists in the
reduction of Fe-S proteins
(4) ; and fumC, encoding an
oxidatively stable fumarase
(5) .
Strains and Plasmids
E. coli strain
BL21(DE3) and plasmid pET-11
(12) were obtained from
Novagen. Plasmid pHE6:: soxR, a derivative of pHE6
(13) , will be separately described.
Molecular Biological Methods
General methods for
cloning and for electrophoretic analysis of polynucleotides and
proteins were as described
(14, 15) . DNA concentrations
were estimated by staining with ethidium bromide
(14) or by
fluorimetry with Hoechst 33258 dye
(16) .
Overproduction of SoxR
High density bacterial
growth was performed as described
(17) , with the following
modifications. A 60-ml saturated culture of E. coli BL21(pET11K- soxR, DE3) was inoculated into 3 liters
of medium containing kanamycin (50 µg/ml) and incubated at 37
°C under oxygen. At an OD
of 21 (11 h), the culture
was rapidly cooled to 15 °C, and 2 ml of a 20% IPTG
(
)
solution were added, followed 6 h later by 600 mg of
rifampicin. The cells were harvested 23 h after induction (OD
= 32, pH 6.6). The cell paste (130 g) was washed and
resuspended in an equal volume of 10% sucrose, 10 mM
Tris
HCl buffer (pH 8.0), dripped into liquid N
, and
stored at
80 °C.
10
ml
. Rifampicin was omitted. The yield was 1.7
g of cell paste per flask.
Purification of SoxR
All operations were performed
at 0 to 4 °C in a standard buffer containing 20 mM
MOPS/KOH (pH 7.6), 1 mM dithiothreitol, and 10% glycerol.
Precipitates were collected by centrifugation at 27,000 g for 10 min after 20 min of stirring. Twenty grams of the frozen
cell suspension were sonicated in 40 ml of the standard buffer
containing 0.2 M KCl and 0.0025% phenylmethylsulfonyl
fluoride, and cell debris was removed by centrifugation at 33,000
g for 15 min. NaCl (5 M) and
polyethyleneimine-HCl (5%, pH 7.0) were added to final concentrations
of 0.1 M and 0.225%, respectively, and the precipitate was
removed by centrifugation. An equal volume of a saturated solution of
(NH
)
SO
was slowly added. The
precipitate was dissolved in 40 ml of standard buffer, precipitated
again with 60 ml of a saturated (NH
)
SO
solution, and redissolved in 200 ml of standard buffer containing
0.2 M KCl (loading buffer).
1 cm) to which the protein solution was applied followed by
12 ml of the loading buffer. Elution was performed with 60 ml of the
loading buffer containing a linear gradient of from 0.2 to 1.0
M KCl. SoxR was eluted in a single peak at 0.5 to 0.6
M KCl. It was detected by its reddish brown color and
electrophoretic mobility.
DNA Substrates
The soxS promoter segment
used for electrophoretic mobility shift assays was an
EcoRI- BstYI fragment of an M13mp19:: soxRS plasmid that had a terminal deletion of most of the soxR gene
(8) ; it contained nucleotides 459-586 of the
published sequence
(8) . The control ( soxR) DNA was an
EcoRI- SmaI fragment of a similar plasmid that had an
intact soxR gene; it contained nucleotides 977-1099. The
soxS promoter DNA used for transcription assays extended from
nucleotides 421-594. It was generated by a polymerase chain
reaction
(15) that employed a fragment of plasmid
pUC18:: soxS (6) as a template and the following
oligonucleotides as primers: TAAGCGGCTGGTCAATATGC and
GCGGGGTAATTTCTTTTCCA.
Electrophoretic Band Shift Assays
Mobility shift
DNA binding assays using low ionic strength PAGE were performed as
described elsewhere
(15) . Prior to electrophoresis, the
P-labeled DNA fragments (7500 cpm each) were incubated
with from 0 to 200 ng of SoxR protein for 20 min at 30 °C in 15
µl of a solution containing 133 µg/ml poly(dI-dC), 300
µg/ml bovine serum albumin, 12% glycerol, 10 mM KCl, 1
mM dithiothreitol, and 12 mM HEPES/NaOH buffer (pH
7.9).
Transcription Assays
SoxR was diluted in a
solution containing 20 mM TrisHCl buffer (pH 8.0), 100
mM KCl, 1 mM dithiothreitol, and 50 µg/ml bovine
serum albumin. SoxR (5-50 ng in 3 µl of diluent) was added to
18 µl of a solution containing 15 nmol of DNA template and 0.16
mM each of ATP, GTP, and CTP, 40 mM Tris
HCl
buffer (pH 8.0), 10 mM MgCl
, 50 mM KCl, 1
mM dithiothreitol, and 1 mg/ml bovine serum albumin. After the
mixture was incubated for 10 min at 37 °C, 0.15 unit of E. coli RNA polymerase-
holoenzyme (Epicentre
Technologies) was added in 3 µl. After an additional 10 min at 37
°C, 3 µl of heparin sulfate (1 mg/ml) and 3 µl of 80
µM
-[
P]UTP (3-30
Bq/mmol) were added simultaneously. The mixtures were incubated at 37
°C for 15 min and treated with phenol. The RNA products were
precipitated with ethanol and analyzed by electrophoresis in an 8%
polyacrylamide, 7.7 M urea gel
(15) . Radioactivity was
measured with the Ambis Corp. (San Diego, CA) radioanalytic imaging
system. One standard unit of activity is defined as the incorporation
of 1 fmol of UTP under these conditions. The activity was proportional
(±5%) to the concentration of SoxR in the stated range.
Protein Analyses
Protein concentrations were
measured with the Pierce Coomassie Blue dye reagent, using bovine serum
albumin as a standard
(18) . Values for SoxR were multiplied by
0.323 to agree with those obtained with a Beckman 7300 Amino Acid
Analyzer at the W. M. Keck Foundation, Yale University. N-terminal
sequences were determined by Edman degradation, using an Applied
Biosystems model 470 sequencer at the University of Michigan Protein
and Carbohydrate Structure Facility.
Gel Permeation Chromatography
A column (1-cm
diameter, 35-ml volume) of Bio-Gel P-60 (Bio-Rad) was equilibrated and
operated at 4 °C with a solution containing 0.25 M KCl, 10
mM dithiothreitol, 20 mM MOPS/NaOH buffer (pH 7.6),
and 10% glycerol. SoxR (45 µg) or other proteins (1 mg each) were
applied in a volume of 0.1 ml. The void volume was determined with
bovine catalase (240 kDa). Of the material applied, 37% of the SoxR
protein and 24% of its original activity were recovered in the combined
peak fractions. The markers used were ovalbumin, pancreatic DNase,
bovine chymotrypsinogen, and horse heart cytochrome c.
Sedimentation Analysis
Band sedimentation was
performed in 4.5-ml solutions containing 0.25 M KCl, 10
mM dithiothreitol, 20 mM MOPS/NaOH buffer (pH 7.6),
and a linear gradient of from 10 to 30% glycerol. The protein samples
were 0.3 to 0.5 mg. Ovalbumin and chymotrypsinogen were the standards.
Centrifugation was in a Beckman SW50.1 rotor at 48,000 rpm for 26 h at
4 °C.
Physical Parameters
Methods of calculation for the
Stokes radius, sedimentation coefficient, frictional ratio, and
Mof native SoxR were as in Weiss
(19) . A
value of 0.736 was assumed for the partial specific volume of SoxR
(20) .
Metal Analyses
Precautions were taken to reduce
contaminating metal ions
(21) . Metal analyses were performed by
inductively coupled plasma atomic emission spectrometry in the
Department of Geological Sciences at the University of Michigan. An
ultrafiltrate served as a blank sample; it contained <10% as much
iron as the SoxR solution.
Reductive Titrations
Titrations of SoxR with
sodium dithionite
(22, 23) were performed at 4 °C
under argon. Residual oxygen was removed with protocatechuic acid and
protocatechuate dioxygenase (a generous gift of D. Ballou)
(24) , which were added to 100 µM and 1
µM, respectively. The dithionite solution was standardized
with FAD.
EPR Spectra
Methods used for EPR spectroscopy
(25) and computer programs for data reduction
(26, 27) were as described previously.
Cloning of soxR in an Expression Vector
A
promoterless DNA fragment containing the ribosome binding site and
complete coding sequence of soxR was inserted into plasmid
pET-11, thereby placing the soxR gene downstream from a phage
T7 promoter under lac operator control (Fig. 1). The
plasmid was unstable in high density cultures in which ampicillin,
which is readily destroyed by -lactamase, was used as the
selective agent
(12) . Therefore, a kanamycin resistance gene
was inserted. The resulting strain was >99% stable as determined by
its ability to be killed by IPTG
(12) .
Figure 1:
Structure of
pET11K- soxR, a plasmid used for the overexpression of SoxR.
The open bars are DNA segments that were cloned into the
expression vector pET-11. One contains the soxR gene on a
620-bp BamHI fragment of plasmid pHE6:: soxR, which
was inserted into a BamHI site. The other contains the
kanamycin resistance gene of plasmid pUC4K (28) on a 1.3-kb
EcoRI fragment that was cloned into the PstI site
within the bla gene. Tis a phage transcriptional
terminator. The sequence shows the fusion of the T7 gene 10 promoter/ lac operator region (P
) of the
vector to the cloned segment containing the soxR ribosomal
binding site ( RBS) and structural gene. The DNA to the left of
the BamHI site is from pET-11. The sequence in brackets is a BamHI- SmaI fragment from pHE6 (13).
Downstream of the soxR gene are 61 additional base pairs of
contiguous chromosomal DNA connected by (dG)
to an 87-bp
EcoRI- PvuII segment of M13mp18 DNA
(8).
Activity in Vivo of the Cloned soxR Gene
To ensure
that our cloned soxR gene had not mutated, we tested for its
ability to restore to a soxR9::cat mutant
(6) the
capacity of its zwf (glucose-6-phosphate dehydrogenase) gene
to be induced by paraquat, a function that is mediated by the soxRS regulon
(1, 2) . The plasmid enabled a 10-fold
induction of the enzyme, comparable to that seen in wild type cells.
Overproduction of SoxR
The use of a vector with an
IPTG-inducible rather than a thermoinducible promoter, enabled the
overexpression of SoxR at low temperature, which favors the synthesis
of correctly folded, soluble, overexpressed proteins
(29) . At
30 or 37 °C, >50% of the SoxR produced from plasmid
pET11K- soxR was insoluble; however, at 15 °C, almost all
of the SoxR protein produced could be recovered in a soluble fraction
(Fig. 2).
Figure 2:
Overproduction ( A) and
purification ( B) of SoxR as monitored by SDS-PAGE. A,
a fermentor culture of strain BL21 (pET11K- soxR, DE3) was
sampled at 0, 18, and 23 h after treatment with IPTG at 15 °C.
Samples of equal cell mass were applied to the gel after lysis and
denaturation. B, lane I, sonicate supernatant;
II, polyethyleneimine supernatant; III, ammonium
sulfate precipitate; IV, phosphocellulose eluate. Lanes I to
III each contained proteins derived from 1 µl of sonicate. Lane IV
contained 10 µg of SoxR. Markers were chymotrypsinogen (25
kDa) and lysozyme (14 kDa).
Purification
Only one chromatography step was
needed to purify SoxR (Fig. 2 B). Its high pI enabled it
bind to phosphocellulose at a high salt concentration required for its
stability. The yield was 1.5 mg of purified SoxR per g of wet cells.
Its specific activity was 10 units/ng of protein (transcriptional
activation assay).
General Properties
Purified SoxR containing 1
mM dithiothreitol was stable only for about 1 week at 4
°C. Preparations containing 10 mM dithiothreitol were
stable for about 1 month at 4 °C under argon or for at least 2
months at 20 °C in 50% glycerol. At salt concentrations
<0.2 M or at temperatures >10 °C, the reddish brown
solution formed an insoluble white precipitate within a few minutes.
Fast-frozen samples that were stored at
80 °C lost
15%
of their activity on thawing. Although SoxR was reported to have a
solubility
200 µg/ml
(11) , under our conditions it was
soluble at >10 mg/ml.
DNA Binding Activity
Purified SoxR bound
specifically to a fragment of soxS DNA extending 62 base pairs
upstream of the transcriptional start site. The electrophoretic
mobility of the DNA was retarded 11%, even in the absence of RNA
polymerase. Under the conditions given (see ``Experimental
Procedures''), the binding constant of SoxR dimer to soxS DNA (determined as the concentration of SoxR required for
retardation of 50% of the DNA) was between 0.4 and 2.0
10
M. The results (not shown) were similar
to those obtained by others
(11) and confirmed the activity of
our preparation.
Transcriptional Enhancement
Purified SoxR was
tested for its ability to activate the transcription of the soxS gene. The 173-bp linear soxS DNA template started 67
nucleotides upstream of the transcriptional start site. The one-cycle
transcription assay employed heparin to block reinitiation during the
elongation phase, thereby increasing the relative yield of full length
transcripts. The results (Fig. 3) indicated that transcription
from the SoxS promoter was dependent on the addition of SoxR. If SoxR
were added after heparin, there was no transcriptional enhancement
(results not shown). Therefore, SoxR functions in transcription
initiation.
Figure 3:
Activation of soxS transcription
by purified SoxR. A one-cycle transcription assay was performed with a
soxS DNA template and increasing amounts of SoxR protein (see
``Experimental Procedures'').
In vivo, SoxR requires activation by treatment
of cells with oxidants. However, purified SoxR was active in vitro without further treatment (Fig. 3)
(11) . We were
unable to increase the activity of our preparation by incubating it
with potassium superoxide or with superoxide generating systems
(phenazine methosulfate plus NADPH and O, or
6-hydroxydopamine plus O
), even in the presence of catalase
to remove any H
O
that might inactivate the
protein (results not shown).
SoxR Is a Homodimer
Transcriptional activity and
protein comigrated in a single peak during gel permeation
chromatography. Although the molecular mass of the polypeptide is 17
kDa
(8) , the native protein had an elution volume between that
of pancreatic DNase I (31 kDa) and ovalbumin (43 kDa), and its
sedimentation rate was between that of ovalbumin and chymotrypsinogen
(25 kDa). The physical parameters of the native protein are as follows:
Stokes radius, 2.81 nm; diffusion coefficient ( D), 7.65
10
cm
s
;
sedimentation coefficient ( s
), 2.71 S;
frictional ratio ( f/f
), 1.26; M
(calculated from D and s
),
32,700. Thus, SoxR is a globular homodimer in its active form.
Metal Content
Our SoxR preparation contained 2.0
atoms of iron per polypeptide chain. Values for other metals were as
follows: zinc, a ubiquitous contaminant
(21) , 0.39; copper,
0.34; and cobalt, molybdenum, and magnesium,
0.10.
Optical Spectrum
At peak wavelengths, the
extinction coefficients (mMcm
)
for the SoxR polypeptide were as follows:
=
53.5,
= 24.5,
=
12.7,
= 12.4, and
(shoulder) = 8.0. The spectrum was characteristic of a
[2Fe-2S] protein
(30) . The ratio of the absorbance of
other peaks to that at 276 nm is higher than that previously observed
(11) , reflecting its higher iron content.
Reductive Titration with Dithionite
Reduction by
dithionite (Fig. 4) produced a decrease in absorbance above 300
nm as observed with [2Fe-2S] ferredoxins
(23) . When
the reduced protein was aerated, the original spectrum was almost
completely restored within 2 min (Fig. 4, curve d),
indicating that SoxR is readily autooxidized and that the spectral
changes had been caused by the reversible reduction of the protein and
not by its denaturation.
Figure 4:
Reductive titration of SoxR by sodium
dithionite. A solution of 0.28 mM sodium dithionite in 20
mM MOPS buffer (pH 7.6) was added incrementally to SoxR (0.37
mg/ml) under anaerobic conditions in a 1-cm path length cuvette.
Spectra were recorded: a, before treatment; b, after
partial titration; c, after full titration; and d,
after re-exposure to air for 2 min. Inset, Titration of a SoxR
solution containing 34 µM bound
Fe.
Oxidized [2Fe-2S] and
[4Fe-4S] centers each accept one electron from dithionite
(23) . When SoxR protein was titrated anaerobically with
dithionite, 0.5 reducing equivalent was accepted per gram atom of iron
(Fig. 4, inset). These results strongly suggest that
each Fe-S center contains 2Fe. Because there are only 2Fe and 4
cysteines per chain of SoxR, there should be one [2Fe-2S]
center per chain.
EPR Spectrum
Untreated SoxR produced no EPR
signal, thereby ruling out the presence of a [3Fe-3S] or
[3Fe-4S] cluster
(31) . A sample reduced by dithionite
gave EPR signals with g values of 2.01, 1.92, and 1.90
(Fig. 5), which are consistent with those of an S = 1/2
[2Fe-2S] center and close to those produced by the
[2Fe-2S] cluster in E. coli fumarate reductase
(2.03, 1.93, 1.92)
(32) . The almost axial spectrum resembles
that first described for the hydroxylase type of [2Fe-2S]
protein, which is exemplified by putidaredoxin and adrenodoxin ( g = 2.02, 1.94, 1.94)
(33) .
Figure 5:
EPR
spectrum (25 K) of SoxR after 50% reduction by dithionite. The data
( solid line) are superimposed by a simulation ( dashed
line) with g values: 1.903, 1.922, 2.007, and line widths
given in g units: 0.0086, 0.0065, and 0.0041, respectively.
The incident microwave power was 0.4 milliwatt; the field modulation
amplitude was 0.5 mT.
EPR spectroscopy was
performed on samples of SoxR that had been titrated
spectrophometrically with dithionite. For a fully titrated solution of
SoxR containing 45 µm of bound iron, the density of unpaired
electron spins ( i.e. the concentration of Fe-S centers) was 22
µM; for a half-titrated one, it was 11 µM.
These results confirmed that there are 2Fe per Fe-S cluster.
Sequence Similarities
Using Genetics Computer
Group software
(34) , we scanned the SWISS-PROT data base
(35) for Fe-S proteins with sequences similar to a 20-amino acid
segment of SoxR encompassing the cysteine cluster. The greatest
similarity was found with the [4Fe-4S] centers in bacterial
ferredoxins, almost all of which share the following consensus sequence
with SoxR around the first two cysteines:
D XC(I,L,V) XCG. However, the spacing of the cysteines
in SoxR (C XC XC X
C) is
unique; the bacterial ferredoxins, e.g. have two amino acids
separating the second and third cysteines. MerR, a Hg-binding protein
to which SoxR seems most closely related
(9) , has little
homology to SoxR in this region, has only three cysteines
(C XC X
C), and does not contain an Fe-S
cluster.
O
(37) , which primarily undergoes two-electron redox
reactions in vivo. It is therefore of great significance that
we found SoxR to contain [2Fe-2S] clusters, prosthetic groups
that are capable of reversible univalent redox reactions. The most
likely hypothesis is that SoxR is normally in a reduced
([2Fe-2S]
) state in vivo and is
activated by oxidation to the [2Fe-2S]
state. This can occur upon exposure of the cell to superoxide
generators and other univalent oxidants, or perhaps by any stress that
decreases the availability of the NADPH and reduced flavodoxin or
ferredoxins that may keep SoxR reduced
(4) . In vitro,
activation may occur rapidly by autooxidation. However, a crucial item
of supporting evidence is lacking, namely, that the protein is inactive
in its reduced form. So far, this demonstration has eluded us, as well
as others
(11) , because of the technical difficulty of
performing multistep microscale assays under stringent anaerobic
conditions.
-D-thiogalactopyranoside; MOPS,
3-( N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel
electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.