From the Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06030
Received for publication, August 28, 2000, and in revised form, October 18, 2000
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ABSTRACT |
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The binding of The binding of The interaction between Bacterial Strains and Growth Conditions--
The
Escherichia coli strains used were JM83 (ara
Construction of Genes Encoding SspC N-terminal Variants--
The
genes encoding all SspC deletion variants were generated by the
polymerase chain reaction (PCR) using plasmid pPS708 (10) as the
template and the following oligonucleotide primers. The upstream
primers were: SSPC (5'-CCATGGCTCAACAA AGTAGATC),
SSPC Construction of sspB Promoter-sspC N-terminal Deletion Variant
Gene Fusions--
To achieve high level expression of SspC variants in
spores of B. subtilis, the genes encoding the variants were
fused to the strong, sporulation-specific promoter of the
sspB gene, which is expressed at the same time in
sporulation as sspC (16). An NcoI restriction
site was first introduced at the initiating methionine codon of the
sspB gene by the PCR megaprimer method (17). The first round
of PCR used the upstream primer SSPB1 (5'-ACGGCTAAGCTTTTTTTATTTCTC), complementary to nt 173-196 of the sspB sequence (15), and
the downstream primer SSPB-NCOI
(5'-GAGTTTTGGTTAGCCATGGGTAAAATCTCC), complementary to nt
357-386 of the sspB sequence (15) to generate a DNA
fragment containing the sspB promoter with a site-directed mutation (underlined residue) creating the NcoI site. This
PCR product was agarose gel-purified and used as the upstream primer in
a second round of PCR with the downstream primer SSPB2
(5'-GGATCCCTTTTTTTCTAGGATATGTGGAGCAGG), complementary to nt
639-665 of the sspB sequence (15) to generate a complete
sspB gene with an added 3' BamHI site (underlined
residues). This fragment was ligated into SmaI-digested
pUC19 to generate plasmid pPS2798. The identity of the insert in
plasmid pPS2798 was confirmed by DNA sequencing.
NcoI-BamHI fragments of all sspC variants were ligated to NcoI-BamHI-digested
plasmid pPS2798 generating the sspB promoter-sspC
gene fusions.
To introduce the sspB promoter-sspC gene fusions
into B. subtilis, these fragments were first subcloned into
a pBluescript-pUB110 fusion plasmid (pPS2952). Plasmid pPS2952 was
constructed by ligating a pBluescript (Stratagene, La Jolla, CA)
derivative (a SmaI-HincII fragment was deleted)
to a pUB110 derivative (plasmid pUB-B, containing the sspB
gene at the unique EcoRI site (10)) at their
BamHI sites. The sspB
promoter-sspC-containing pPS2798 plasmids were first
digested with BamHI, the ends filled with the large fragment of E. coli DNA polymerase I, and then digested with
HindIII to remove HindIII-end-filled
BamHI fragments containing the sspB promoter-sspC gene fusions. These fragments were ligated to
plasmid pPS2952 that had been digested with HindIII and
HpaI. Finally, the plasmid pPS2952 clones containing the
sspB promoter-sspC gene fusions were digested
with BamHI to remove pBluescript DNA, the pUB110 derivative
fragments agarose gel-purified, religated, and used to transform
B. subtilis strain PS356
( Purification of
Plasmid DNA (pUC19) was purified by two rounds of CsCl equilibrium
density centrifugation, and linearized by digestion with EcoRI. Poly(dG)·poly(dC) and poly(dA-dT)·poly(dA-dT)
were obtained commercially (Sigma). All polynucleotides were dialyzed
exhaustively in Spectra/Por 3 tubing (molecular mass cut-off 3500 Da)
against 10 mM sodium phosphate (pH 7.5).
Chemical Modification of
SspC (1 mg/ml) was digested with endoproteinase Asp-N (Sigma) in 10 mM sodium phosphate (pH 7.5) at an enzyme to substrate ratio of 1:200 (w/w) at 22 °C for 6 h. SspC (1 mg/ml) was
digested with the germination protease (recombinant GPR from
Bacillus megaterium) (22) at an enzyme to substrate ratio of
1:200 (w/w) in 10 mM Tris-HCl (pH 7.4), 2 mM
CaCl2 at 22 °C for 3 h. The C-terminal proteolytic
fragments from both digests were purified by reverse phase-HPLC as
described (11).
Analysis of SspC Variants in Vitro and in Vivo--
DNA
binding was assessed in vitro by DNase I protection assays
(5) and by circular dichroism (CD) spectroscopy (6). DNA binding of
purified
Thermal denaturation of SspC variant-DNA complexes was performed on
pre-equilibrated, stirring solutions containing 5 µM SspC variant complexed with 23 µM (in bp) poly(dG)·poly(dC)
in 10 mM sodium phosphate (pH 7.5) (6). This amount of DNA
is sufficient to bind all wild-type SspC (6). Denaturation was
monitored by measuring ellipticity at 222 nm at 0.5 °C intervals as
a function of temperature from 20 °C to 90 °C. The midpoint of
each transition (defined as TM) was determined
by taking the first derivative of ellipticity at 222 nm with respect to
the inverse of the absolute temperature as described (26). Experimental
error was estimated at ±1 °C, based on duplicate measurements of
poly(dG)·poly(dC) complexes with SspC,
SspC
Equilibrium binding titrations by CD spectroscopy were performed as
described using EcoRI-linearized pUC19 plasmid,
poly(dG)·poly(dC), and poly(dA-dT)·poly(dA-dT) (6). A DNA binding
site size of 4 bp was directly determined for SspC,
SspC
SspC variants were chemically cross-linked in the presence of
EcoRI-linearized pUC19 plasmid DNA with
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and cross-linked
products purified by SDS-PAGE as described (11). Purified cross-linked
and monomeric proteins were digested with trypsin as described (11) and
the digests (100 µl) dialyzed in 100-Da cut-off DispoBiodialyzersTM
(Spectrum) against two 1-liter changes of 50 mM
NH4HCO3 at 4 °C for 18 h each. Dialyzed
tryptic digests were directly analyzed by matrix-assisted laser
desorption ionization-time of flight (MALDI-TOF) mass spectrometry to
identify cross-linked peptide species (11).
Spores of B. subtilis that overexpress variants of SspC were
prepared by nutrient exhaustion in 2× SG medium (27). Spores were
purified by sonication and distilled water washing at 4 °C (28), and
all spores used were >98% pure. Purified spores were analyzed for
resistance to both wet heat and UV radiation as described (19, 29).
SspC N-terminal Variants Bind to DNA in Vitro--
Amino acid
sequence alignment of all identified
Variants were designed in which blocks of approximately five amino acid
residues were progressively removed from the N terminus of SspC (Fig.
1). In addition, SspC
All Thermal Stability and Equilibrium Binding Affinity of SspC
Variant-DNA Complexes--
The DNase I protection assays mentioned
above indicate that all SspC N-terminal variants (with the exception of
the C-terminal GPR fragment) are able to bind to plasmid DNA. However,
these experiments are performed at protein concentrations in excess of
the dissociation constants for the complexes, and thus it is difficult
to detect differences in
The thermal stability of SspC N-terminal variant complexes with
poly(dG)·poly(dC) was determined by monitoring the CD at 222 nm for
each protein-DNA complex as a function of temperature. Each
We next wanted to determine the apparent equilibrium binding constants
(K Protein-Protein Contacts between DNA-bound SspC N-terminal
Variants--
Previously, we used chemical cross-linking to identify
amino acid residues that form close contacts between adjacent DNA-bound
All N-terminal variants except the GPR C-fragment were cross-linked by
EDC in a DNA-dependent fashion, although a very small amount of cross-linking was also seen in the absence of DNA (Fig. 3). DNA-independent cross-linking is
probably nonspecific and occasionally occurs during lyophilization of
the samples if EDC is not completely removed by prior dialysis. The
smaller SspC N-terminal variants tended to cross-link with somewhat
lower efficiency than wild-type SspC, whereas the cross-linking of
SspC The
To further confirm that charged residues at the N terminus of
Analysis of the Effects of SspC N-terminal Variants on Spore
Properties--
The in vitro analysis of SspC N-terminal
variants described above demonstrates that up to 14 amino acid residues
may be deleted from the N terminus of SspC while still maintaining DNA
binding ability, albeit at lower affinities than full-length protein. Therefore, we wanted to determine whether SspC N-terminal variants are
able to confer heat and UV resistance on spores in vivo, as does the full-length protein. SspC N-terminal variant proteins were
overexpressed in The results reported here indicate that a substantial portion of
the N terminus of SspC (~20% of total amino acid residues) may be
deleted without destroying DNA binding function. These residues are
therefore probably not directly involved in contacting the DNA,
consistent with the low level of sequence conservation in this region
of Thermal stability and equilibrium binding analysis of the SspC
N-terminal variant-DNA complexes indicate that
SspC/
-type small, acid-soluble
spore proteins (SASP) to DNA of spores of Bacillus species
is the major determinant of DNA resistance to a variety of damaging
treatments. The primary sequence of
/
-type SASP is highly
conserved; however, the N-terminal third of these proteins is less well
conserved than the C-terminal two-thirds. To determine the functional
importance of residues in the N-terminal region of
/
-type SASP,
variants of SspC (a minor
/
-type SASP from Bacillus
subtilis) with modified N termini were generated and their
structural and DNA binding properties studied in vitro and
in vivo. SspC variants with deletions of up to 14 residues
(~20% of SspC residues) were able to bind DNA in vitro
and adopted similar conformations when bound to DNA, as determined by
circular dichroism spectroscopy and protein-protein cross-linking.
Progressive deletion of up to 11 N-terminal residues resulted in
proteins with progressively lower DNA binding affinity. However,
SspC
14 (in which 14 N-terminal residues have
been deleted) showed significantly higher affinity for DNA than the
larger proteins, SspC
10 and
SspC
11. The affinity of these proteins for
DNA was shown to be largely dependent upon the charge of the first few
N-terminal residues. These results are interpreted in the context of a
model for DNA-dependent
/
-type SASP protein-protein
interaction involving the N-terminal regions of these proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
-type small, acid-soluble spore proteins
(SASP)1 to DNA of spores of
Bacillus species is the primary mechanism by which spore DNA
is protected from the damaging effects of heat, peroxides, and UV
radiation (1, 2). The
/
-type SASP are nonspecific DNA-binding
proteins, whose synthesis is developmentally regulated such that these
proteins are only synthesized within the developing forespore during
sporulation (3, 4). The
/
-type SASP accumulate to very high
levels, which are sufficient to saturate the spore chromosome, and DNA
within this nucleoprotein complex is protected from a variety of
environmental insults (1, 2, 4). Accordingly, the
/
-type SASP are
important determinants of long term spore survival, and spore
resistance to heat, peroxides, and UV radiation. Spores of
Bacillus subtilis that lack the majority of their
/
-type SASP, termed
spores,
are much more sensitive to these treatments than are wild-type spores
(1, 2). The
/
-type SASP are encoded by a number (4-7) of
monocistronic genes in Bacillus species. Generally, in each
species there are two
/
-type SASP that are expressed at very high
levels (the major
/
-type SASP), and a variable number of other
/
-type SASP expressed at much lower levels (the minor
/
-type SASP) (4). The amino acid sequences of these proteins are
highly conserved, both within and between species; however, significant
differences in DNA binding affinity between
/
-type SASP have been
demonstrated (5-7). During spore germination, the
/
-type SASP
are rapidly degraded by a sequence-specific endoproteinase, termed the
germination protease (GPR), which recognizes and cleaves within a
pentapeptide sequence found within all
/
-type SASP (4).
/
-type SASP and DNA has been studied in
detail, and several features of
/
-type SASP-DNA binding have been
characterized (5, 6, 8-10). DNA structure changes from B-DNA to an
A-like conformation upon binding to
/
-type SASP (7, 8), whereas
/
-type SASP undergo a transition from random coil to a largely
-helical conformation upon binding to DNA (6). The binding
interaction is significantly cooperative, with
/
-type SASP having
a 50- to ~600-fold greater affinity for contiguous DNA binding sites
than for noncontiguous sites depending upon the bound polynucleotide
and the salt concentration (5, 6). The binding cooperativity is thought
to be due at least in part to protein-protein interactions between
adjacent, DNA-bound
/
-type SASP (8, 11). Contacts between the
-amino group and the carboxylate side chains of three acidic
residues found within the N-terminal 40-50% of a variety of
/
-type SASP have been identified previously using a zero-length
cross-linking reagent (11). The N-terminal third of
/
-type SASP
varies both in length and amino acid sequence, and its sequence is
generally less well conserved than the C-terminal two-thirds of these
proteins (Fig. 1) (4). Because individual
/
-type SASP have
significantly different affinities for DNA, it seems reasonable to
speculate that these differences in binding affinity could be due to
differences in the length and sequence of the N-terminal third of these
proteins. Consequently, we have investigated the effects of
modifications of the N-terminal sequence upon the DNA binding
properties of
/
-type SASP. This analysis has further led to a
model for
/
-type SASP protein-protein interaction, which involves
an electrostatic interaction between the positively charged N-terminal
region of one protein and an acidic region on an adjacent, DNA-bound protein.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(lac-proAB) rpsL
80 lacZ
M15)
(12) and BL21(DE3) (T7 RNA polymerase under control of the
lac promoter) (13). The B. subtilis strains used were all derivatives of strain 168. E. coli strains were
routinely grown in Terrific broth (24 g of yeast extract, 12 g of
tryptone, and 4 ml of glycerol per 900 ml, plus 100 ml of 170 mM KH2PO4, 720 mM
K2HPO4) at 37 °C with shaking. For the
overexpression of cloned genes encoding
/
-type SASP from pET11d
vectors (13), the medium was supplemented with 200 µg/ml ampicillin
and 0.5% glucose. B. subtilis was transformed to kanamycin
resistance with pUB110 derivatives as described previously (14).
5 (5'- CCATGGCTAGATCAAACAACAATAATG), SSPC
10 (5'-
CCATGGCTAATGATTTACTAATTCC), SSPC
11 (5'-
CCATGGCTGATTTACT AATTCCTCAAGC), SSPC
11-D13N
(5'-CCATGGCTAATTTACTAATTCCTCAA GC), SSPC
11-D13K (5'-
CCATGGCTAAATTACTAATTCCTCAAGCAGG), SSPC
14 (5'-CCATGGCTATTCCTCAAGCAGCTTCAGC); the downstream primer
was SSPC2 (5'-AGCTGGATCCACCATTAGTTCTGTATGG),
complementary to nt 530-547 in the sspC sequence
(15). All upstream primers contained added NcoI restriction
endonuclease sites (underlined residues) and the downstream primer
contained a BamHI restriction site and 5'-flanking sequences
(underlined residues), which were used for cloning purposes. All PCR
products were ligated to SmaI-digested pUC19, followed by
subcloning into NcoI-BamHI-digested plasmid
pET11d for overexpression of SspC variants in E. coli strain
BL21(DE3). The identities of all pET11d clones were confirmed by DNA sequencing.
) (18) to kanamycin resistance as
described (14).
/
-type SASP and Polynucleotides--
All
SspC variants were overexpressed in E. coli strain BL21(DE3)
from pET11d-derived plasmids as described previously (13). SspC and
SspC
11-D13K were extracted from dry ruptured
E. coli cells with 3% acetic acid, 30 mM HCl
and purified as described previously (19).
SspC
10 and SspC
11
were acid-extracted as described above, but were purified by ion
exchange chromatography on a QAE-cellulose column equilibrated in 10 mM Tris-HCl (pH 8.0) at 4 °C. Proteins were eluted by a linear salt gradient from 0 to 200 mM NaCl in 10 mM Tris-HCl (pH 8.0) at 4 °C.
SspC
5,
SspC
11-D13N, and
SspC
14 were acid-extracted as outlined
above, but were purified by ion exchange chromatography on a
CM-cellulose column equilibrated in 10 mM NaOAc (pH 5.5) at
4 °C. Proteins were eluted by a linear salt gradient from 0 to 200 mM NaCl in 10 mM NaOAc (pH 5.5) at 4 °C.
Column fractions containing purified
/
-type SASP were pooled,
concentrated by lyophilization, and dialyzed exhaustively against 10 mM sodium phosphate (pH 7.5). All proteins were >95% pure, as judged by SDS-PAGE and staining with Coomassie Blue. The
concentrations of protein stock solutions were determined by
quantitative amino acid analysis.
/
-type SASP--
/
-type
SASP were chemically deaminated as follows; 120 µl of purified
protein (~1 mg/ml in 10 mM sodium phosphate (pH 7.5)) was
slowly added to 350 µl of 2.5 M NaOAc (pH 5.2), 50 mM sodium glyoxylate, 5 mM CuSO4
(20, 21). The resulting solution was incubated at 22 °C for 20 min,
followed by dialysis in Spectra/Por 3 tubing (molecular mass cut-off
3500 Da) against three 1-liter changes of distilled water at 4 °C
for 8 h each. Dialyzed, deaminated protein was frozen,
lyophilized, dissolved in ~50-100 µl of 8 M urea, and
dialyzed exhaustively against 10 mM sodium phosphate (pH
7.5) at 4 °C. This protein was used for in vitro DNA
binding assays.
/
-type SASP was assessed by the ability of protein to
protect EcoRI-linearized pUC19 plasmid from DNase I
digestion as described previously (5, 19). All CD measurements and
spectra were obtained on a Jasco 715 spectropolarimeter with a Jasco
PS-150-5 power supply, and all recorded spectra were the average of
three scans (6). Far UV (protein conformation) spectra were obtained
from solutions containing 25 µM
/
-type SASP and 115 µM (in bp) poly(dG)·poly(dC) in 10 mM
sodium phosphate (pH 7.5) at 21 °C. Difference spectra corresponding
to the
/
-type SASP component of the complex were obtained by
subtracting the spectrum of free DNA from the spectra of
/
-type
SASP-poly(dG)·poly(dC) complexes as described (6, 23, 24). Secondary
structure deconvolution was carried out on data from difference spectra using a web-based neural network algorithm, K2D (25).
5, and
SspC
11.
5,
SspC
11-D13N, and
SspC
11-D13K from stoichiometric forward
titrations of poly(dG)·poly(dC) in 10 mM sodium phosphate
(pH 7.5) at 21 °C, as was found previously for a number of
/
-type SASP with several DNAs (5, 6). Stoichiometric DNA binding
conditions could not be obtained for SspC
10,
SspC
11, Asp-N fragment, and
SspC
14, although data from forward
titrations of poly(dG)·poly(dC) with these proteins under no salt
conditions were entirely consistent with the same 4-bp site size.
Mean residue ellipticity, [
]222, values for DNA-bound
protein were determined from complexes containing 25 µM
protein and 115 µM (in bp) poly(dG)·poly(dC) under no
salt conditions after correction for DNA contributions to ellipticity at 222 nm. Further addition of poly(dG)·poly(dC) above 115 µM resulted in no additional change in
/
-type SASP
ellipticity at 222 nm. The binding site size (n) and the
mean residue ellipticities at 222 nm for free
([
u]222), and DNA-bound ([
b]222),
/
-type SASP were
used to calculate the percentage of bound and free protein at each
point in forward titrations according to a two-state model as described
(6). The McGhee-von Hippel model was fit to these data by an iterative
least squares method (using KaleidaGraph 3.0.2) to obtain apparent
binding constants (K
) for the interaction with pUC19, and
intrinsic binding constants (K) and cooperativity factors
(
) for the interaction with poly(dA-dT)·poly(dA-dT) (6). Errors in
the fits of K and
were determined by the model fitting program. The experimental error in K
determinations was
estimated to be ±15% as determined by duplicate titrations of pUC19
DNA with SspC and poly(dA-dT)·poly(dA-dT) with
SspC
11-D13K.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
-type SASP from
Bacillus, Sporosarcina, and
"Thermoactinomyces" species show that these proteins are
most highly conserved in the C-terminal two-thirds of the protein (Fig.
1) (4), and part of this region is
thought to be involved in directly contacting the DNA (30). In
contrast, the sequence of the N-terminal one third of
/
-type SASP
is less well conserved with variations in both length and amino acid
sequence (4). The identification of protein-protein contacts between
amino acid residues found within the N-terminal 40-50% of
/
-type SASP in the DNA-bound state (11) suggests that the N
termini of
/
-type SASP mediate protein-protein interactions which
may be important for binding cooperativity, and therefore the apparent
binding affinity of the DNA binding interaction. To explore this
question in more detail, a number of N-terminal variants of SspC, a
minor
/
-type SASP from B. subtilis, were produced and
their DNA binding properties analyzed. SspC was chosen for this study
because it is easily overexpressed and purified from both B. subtilis spores and E. coli, and its interaction with
and effects upon DNA have been studied extensively both in vitro and in vivo (5, 9, 10). Furthermore, SspC binds to DNA with a higher affinity than many other
/
-type SASP, and also has one of the longest N termini of all identified
/
-type SASP (4, 15).
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Fig. 1.
Amino acid sequences of SspC N-terminal
deletion variants. The amino acid sequences of SspC N-terminal
deletion variants are given in one-letter code.
Sequences are as follows: SspC (SspC),
SspC 5 (
5),
SspC
10 (
10),
SspC
11 (
11),
SspC
14 (
14), Asp-N
digest C-terminal peptide (Asp-N frag), and GPR digest
C-terminal peptide (GPR C-frag). Amino acid
residues marked by an asterisk (*) are completely conserved
in all
/
-type SASP identified from Bacillus,
Sporosarcina, and "Thermoactinomyces" species
(4, 31); underlined acidic residues have been identified as
sites of intermolecular EDC protein cross-linking (11). The
downward-pointing arrow indicates the peptide
bond cleaved by GPR.
11 was constructed to
assess the effect of an N-terminal deletion that removes an asparagine
residue conserved in all
/
-type SASP from Bacillus
species and their close relatives (Fig. 1). Each deletion variant was
designed such that the protein would contain an N-terminal alanine
residue after post-translational removal of the initiating methionine
residue (Fig. 1), since alanine is the N-terminal residue of most
/
-type SASP (4, 31). It was possible to overexpress in and purify
SspC
5, SspC
10,
SspC
11, and
SspC
14 from either E. coli, or
from spores of B. subtilis. However, smaller SspC variants
(SspC
21 and
SspC
25) could not be overexpressed in
E. coli nor in spores of B. subtilis (data not
shown); the reason for this is not clear, but these proteins may not
bind to DNA well and therefore they may be very unstable in
vivo, since DNA binding greatly stabilizes
/
-type SASP
against protease digestion (32). In addition to these genetically engineered N-terminal SspC variants, the C-terminal fragments of SspC
from endoproteinase Asp-N and GPR digests were also purified and
analyzed (Fig. 1). The ability of SspC N-terminal variants and
C-terminal proteolytic fragments to bind to DNA was first assessed by
DNase I protection assays. All SspC N-terminal variants as well as the
C-terminal Asp-N fragment of SspC gave approximately the same degree
and pattern of DNase protection to linear pUC19 plasmid DNA as did
wild-type SspC, whereas the C-terminal GPR fragment of SspC gave no
DNase protection to pUC19 (data not shown). These data indicate that up
to 14 residues of SspC (~20% of total residues) may be removed
without abolishing DNA binding.
/
-type SASP are essentially random coils in the absence of
double stranded-DNA, but become
-helical upon binding to DNA (6).
These changes in protein conformation are conveniently monitored by CD
spectroscopy, which was used to confirm the DNA binding properties of
the SspC N-terminal variants (Fig. 2).
The far UV CD spectra of all N-terminal SspC variants were indicative of unstructured polypeptides (Fig. 2 and data not shown), but upon
addition of poly(dG)·poly(dC) each variant except the GPR C-terminal
fragment of SspC became significantly
-helical (Fig. 2 and data not
shown). The
/
-type SASP bind to poly(dG)·poly(dC) very tightly
(5, 6), and the vast majority of the
/
-type SASP (~98%)
is bound to DNA under these conditions (25 µM
/
-type SASP and 115 µM (in bp)
poly(dG)·poly(dC)). Therefore, the secondary structure content of the
DNA-bound protein could be estimated using [
] values from
difference spectra in which the spectrum of free poly(dG)·poly(dC)
had been subtracted from the spectrum of the complex (6, 25). The
estimates of secondary structure content suggest that the great
majority of the removed residues are not part of regular secondary
structure in the full-length protein as the calculated number of
-helical residues is similar (44-50 residues) in each deletion
variant (data not shown).
View larger version (22K):
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Fig. 2.
CD difference spectra of SspC N-terminal
variants bound to poly(dG)·poly(dC). Far UV difference spectra
of SspC N-terminal deletion variants bound to poly(dG)·poly(dC). The
CD spectrum of poly(dG)·poly(dC) was subtracted from the CD spectra
of complexes of SspC N-terminal deletion variant (25 µM)
and poly(dG)·poly(dC) (115 µM in bp), as described
under "Experimental Procedures" to obtain the presented difference
spectra.
/
-type SASP-DNA binding affinities using
this assay. Therefore, we conducted more sensitive and quantitative studies of SspC N-terminal variant-DNA binding, which were monitored by
CD spectroscopy. It has been demonstrated previously that corrected [
]222 values from
/
-type SASP-DNA mixtures can
be used to determine the relative concentrations of DNA-bound and
unbound
/
-type SASP in solution (6). Thus, [
]222
values are an index of
/
-type SASP-DNA binding, which can been
used to determine the thermal stabilities (TM)
and equilibrium binding constants (K
) for
/
-type SASP-DNA interactions (6).
/
-type SASP-poly(dG)·poly(dC) complex underwent a sharp thermal
dissociation transition over a characteristic temperature range, and
the midpoint of each transition (defined as TM)
was determined (Table I) (6). The thermal
stability hierarchy was as follows: SspC > SspC
5 > SspC
14 > SspC
11
Asp-N C-fragment > SspC
10 (Table I). This result was unexpected
in that the deletion of additional residues from
SspC
11 to SspC
14
actually increased the stability of the
/
-type
SASP-poly(dG)·poly(dC) complex.
Thermal stability of SspC N-terminal variant-poly(dG) · poly(dC) complexes
) for the interaction between SspC N-terminal deletion variants and DNA to confirm the relative binding affinities suggested by the thermal stability studies. Intrinsic binding constants (K) and cooperativity factors (
) may be extracted from
equilibrium binding titrations of homogeneous synthetic polynucleotides
by fitting the McGhee-von Hippel model of cooperative, nonspecific protein-nucleic acid binding to the experimental data (6, 33). The
cooperativity factor (
) is a dimensionless thermodynamic parameter,
which describes the relative affinity of a protein ligand for a
ligand-contiguous binding site versus an isolated binding
site (33). Therefore, differences between SspC variants in both their
intrinsic binding constants and binding cooperativity may be detected
and quantitated; this is potentially important as the N termini of
/
-type SASP are thought to be involved in DNA-dependent protein-protein interactions (11). Initial
equilibrium binding studies were conducted with the N-terminal variants
and poly(dG)·poly(dC); however, because the binding cooperativity is
low with this polynucleotide and the interactions are very tight (6),
it was impossible to accurately determine K and
values.
The interaction between
/
-type SASP and poly(dA-dT)·poly(dA-dT) is much more cooperative than with poly(dG)·poly(dC) (6).
Unfortunately, most of the SspC N-terminal variants did not bind to
poly(dA-dT)·poly(dA-dT) at concentrations that could be accurately
measured by CD spectroscopy. However, we were able to conduct
titrations of poly(dA-dT)·poly(dA-dT) with SspC and
SspC
5, and determined a significant decrease
in both K and
for the interaction with
SspC
5 compared with that with SspC (Table
II). To obtain binding constants for all
the N-terminal variants, additional equilibrium titrations were
conducted using linear pUC19 plasmid DNA, which tends to bind to
/
-type SASP more tightly than poly(dA-dT)·poly(dA-dT). Apparent
equilibrium binding constants (K
) from these titrations confirmed the hierarchy of binding affinities as determined by thermal
stability assays, although individual fits of K and
were
not possible because the McGhee-von Hippel model was not designed to
describe binding to polynucleotides of heterogeneous sequence (Table
II).
Equilibrium binding constants for SspC N-terminal variant-DNA
interactions
/
-type SASP (11). These residues are located within the
N-terminal 40-50% of the proteins, and all identified cross-links
occurred between the
-amino group of the protein and either of the
two glutamate residues of the GPR recognition sequence, or a relatively nonconserved acidic residue (aspartate 13 in SspC) found closer to the
N terminus of
/
-type SASP (11) (Fig. 1). Therefore, the N
terminus of a DNA-bound protein appears to be interacting with
negatively charged residues on an adjacent DNA-bound protein. This
proposed protein-protein interaction could partially explain the
unexpected increase in binding affinity seen in SspC N-terminal variants as additional amino acid residues are removed from
SspC
11 to SspC
14
(Table II). SspC
10,
SspC
11, and the Asp-N C-fragment all contain
a negatively charged aspartate residue near the N terminus (Fig. 1),
which could interfere with the postulated DNA-dependent
protein-protein interactions through electrostatic repulsion. This
N-terminal aspartate residue is absent in
SspC
14 and may therefore account for the
increase in DNA binding affinity over
SspC
10, SspC
11,
and the Asp-N C-fragment. To determine if the SspC N-terminal variants
made the same type of protein-protein contacts when bound to DNA, these
proteins were cross-linked in the presence of DNA and the cross-linking
sites identified.
14 was significantly reduced compared
with the larger variants (Fig. 3). To determine whether cross-linking
was occurring between the same amino acid residues in the SspC
N-terminal variants as in wild-type SspC, EDC cross-linked and
monomeric proteins were purified by SDS-PAGE, digested with trypsin,
and the total digests analyzed by MALDI-TOF mass spectrometry. MALDI
maps of tryptic digests of wild-type SspC EDC dimer showed three
additional peptides not seen in tryptic digest mass maps of EDC-treated
SspC monomer (Fig. 4, A and
B). The mass of one of these peptides (Fig. 4B,
labeled 2745.12 Da) corresponded to that of a previously characterized EDC cross-linked peptide in SspC (Ala1-Arg5 × Ser8-Lys27), which contains an isopeptide
cross-link between the
-amino group of alanine 1 and the
-carboxyl of aspartate 13 (Table III) (11). The mass of a second unique peptide (Fig. 4, labeled 2490.75 Da)
corresponded to that predicted for a peptide containing a cross-link
between the
-amino group of alanine 1 and the
-carboxyl of either
glutamate 29 or 33 (Ala1-Arg5 × Leu28-Arg45) (Table III); these
glutamate residues are located within the GPR recognition and cleavage
site (Fig. 1). This type of cross-link has previously been identified
in other
/
-type SASP (SASP-A and SASP-C from B. megaterium, and Bce1E10K from Bacillus
cereus) treated with EDC (11), and therefore it was presumed that
this peptide has the same structure. The mass of the third unique
peptide was consistent with a peptide containing a cross-link between
the
-amino group of alanine 1 and the C-terminal carboxyl group
(Fig. 4, labeled 1802.55 Da). This third cross-linked species is
probably a minor cross-linking product, as it has never been detected
in reverse phase-HPLC tryptic maps of cross-linked SspC (11);
therefore, we have not been able to purify this peptide nor confirm its
structure. MALDI tryptic digest maps of EDC-cross-linked
SspC
5 and SspC
10
indicated that the same amino acid residues were cross-linked as in
wild-type SspC (Table III and data not shown). The MALDI tryptic map of
SspC
11 dimer only showed two cross-linked
peptides corresponding to cross-links between the
-amino group and
aspartate 13 and the C terminus (Table III). It is possible that the
other expected cross-linked peptide from
SspC
11 failed to ionize efficiently and
therefore was not detected by mass spectrometry. Only two cross-linked
peptide species were expected in SspC
14
dimer trypsin digests because the aspartate residue involved in
cross-linking in the other proteins (aspartate 13 in wild-type SspC)
has been deleted in this variant (Fig. 1). We were only able to detect
one cross-linked peptide, corresponding to a cross-link between the
-amino group and the C terminus, in the trypsin digest of
SspC
14 dimer. However, we hypothesize that
additional cross-linking occurs between the
-amino group and the
glutamates of the GPR sequence, and that this cross-linked peptide
probably does not ionize efficiently during mass spectrometry. These
data, in conjunction with the estimates of DNA-bound N-terminal variant
secondary structure content, suggest that the nature of the
protein-protein interactions formed by the N-terminal variants while
bound to DNA is very similar to those formed by wild-type SspC.
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Fig. 3.
EDC cross-linking of SspC N-terminal deletion
variants. SspC N-terminal deletion variants (0.5 mg/ml) with or
without linear pUC19 plasmid DNA (0.1 mg/ml) were treated with EDC (25 mM) as described under "Experimental Procedures."
Aliquots of reactions were run on Tris-Tricine SDS-PAGE and stained
with Coomassie Brilliant Blue R-250. Numbers in the
left margin indicate the positions of molecular
mass markers in kDa.
View larger version (21K):
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Fig. 4.
Tryptic peptide mapping of cross-linked
peptides by MALDI-TOF mass spectrometry. EDC-treated monomeric
(panel A) and dimeric (panel
B) SspC were purified by SDS-PAGE as shown in Fig. 3,
digested with trypsin, and subjected to MALDI-TOF mass spectrometry as
described under "Experimental Procedures." Cross-linked peptides
are labeled in panel B, and peaks labeled
T are derived from trypsin.
Identification of cross-linked tryptic peptides by MALDI-TOF mass
spectroscopy
-Amino Group and N-terminal Charge
/
-type SASP Are
Important for DNA Binding--
Amino acid residues within the
N-terminal half of
/
-type SASP have been shown to be in close
contact with one another while in the DNA-bound state by cross-linking
and all identified cross-links involved the
-amino group of the N
terminus (11). The data presented thus far in this report also suggest
that residues in the N-terminal region of
/
-type SASP are
important in determining the strength of protein-protein interactions
and therefore DNA binding affinity. Therefore, we sought to determine
whether the
-amino group of
/
-type SASP is important for
binding of these proteins to DNA. The N-terminal
-amino group of
proteins can be specifically converted to an
-keto group by a
nonenzymatic transamination reaction with glyoxylate and copper(II)
ions, resulting in an oxidative deamination of the
-amino group (20,
21). Several
/
-type SASP were deaminated quantitatively as
determined by polyacrylamide gel electrophoresis at acid pH (34), which demonstrated a net loss of positive charge (data not shown). The transamination reaction was specific as no other modifications were
detected by reverse phase-HPLC peptide mapping of trypsin and
endoproteinase Glu-C digests of unmodified and deaminated
/
-type
SASP (data not shown). DNase I protection assays showed that deaminated
SspC conferred somewhat less protection to plasmid DNA than did
untreated SspC (Fig. 5, lanes
3 and 6). CD-based equilibrium binding studies
also demonstrated that N-terminal deamination significantly reduced the
affinity of SspC for pUC19 with a >6-fold decrease in the apparent
binding constant (Table III). The effect of N-terminal deamination was
even more dramatic for another
/
-type SASP, SASP-A, a major
/
-type SASP from B. megaterium, which binds less
tightly to DNA than does SspC; deaminated SASP-A provided much less
DNase I protection to plasmid DNA than untreated SASP-A (Fig. 5).
N-terminal deamination of SspC
10 and
SspC
11 had the same effect upon the ability
to provide DNase I protection to plasmid DNA as was seen with SASP-A
(Fig. 5 and data not shown), whereas the effect was less dramatic
comparing SspC
5 and deaminated
SspC
5 (Fig. 5).
View larger version (93K):
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Fig. 5.
The -amino groups of
/
-type SASP are important for DNA binding.
/
-type SASP
were chemically deaminated to the
-keto form and used in DNase I
protection assays as described under "Experimental Procedures." The
arrows labeled a and b indicate the
positions of 2.3- and 0.56-kilobase pair DNA size markers,
respectively. Each reaction contained 0.12 mg/ml pUC19 plasmid DNA with
unmodified or
-keto protein at 0.15 mg/ml (lane
1), 0.3 mg/ml (lane 2), or 0.6 mg/ml
(lane 3). Some lanes contain bands at ~2.7
kilobase pairs, which probably arise from microdrops of plasmid DNA,
which escaped exposure to DNase I.
/
-type SASP are important for high affinity DNA binding, the aspartate 13 residue of SspC
11 was changed
to either an asparagine or a lysine residue. These changes were chosen
because asparagine (or glutamine) and lysine residues are found at this
position in other
/
-type SASP (4, 31). Far UV CD spectroscopy of
the resulting proteins, SspC
11-D13N and
SspC
11-D13K, bound to an excess of
poly(dG)·poly(dC) indicated that these proteins contain essentially
the same amount of secondary structure as
SspC
11 in the DNA-bound state (data not
shown). As predicted, SspC
11-D13N- and
SspC
11-D13K-poly(dG)·poly(dC) complexes
were significantly more thermostable than the
SspC
11-poly(dG)·poly(dC) complex (Table
I). Accordingly, SspC
11-D13N and
SspC
11-D13K also bound to DNA with
significantly higher affinity than SspC
11
(Table II). SspC
11-D13N had ~10-fold
greater affinity for pUC19 plasmid DNA than did SspC
11, whereas
SspC
11-D13K bound to pUC19 and
poly(dA-dT)·poly(dA-dT) more tightly than even wild-type SspC (Table
III). EDC cross-linking of SspC
11-D13K and
subsequent MALDI mass spectrometry analysis confirmed that this protein
(and presumably also SspC
11-D13N) makes the
same DNA dependent protein-protein contacts as the other SspC
N-terminal variants (Table III).
spores of B. subtilis (which lack 75-80% of their
/
-type SASP and are
therefore very sensitive to heat and UV radiation) (18), and the
resistance properties of these spores studied (Fig.
6, A and B). SspC,
SspC
5, SspC
10,
and SspC
11 were expressed at similar high
levels in these spores. Spores expressing SspC,
SspC
5, and SspC
10
were essentially equally resistant to UV radiation, whereas spores expressing SspC
11 were slightly, yet
significantly, more sensitive to UV radiation (Fig. 6A).
Similar results were obtained when heat resistance at 85 °C was
determined, except that spores expressing
SspC
5 and SspC
10
appeared to be less resistant to heat killing than spores expressing wild-type SspC (Fig. 6B). The resistance properties of
spores expressing SspC
14 could not be
directly compared with the other spore strains because SspC
14 was only expressed at ~50% the
levels of wild-type SspC (data not shown). These data indicate that,
although SspC
5 and
SspC
10 have a lower affinity for DNA
in vitro, they still function nearly as well as wild-type
SspC in vivo.
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Fig. 6.
Heat and UV radiation resistance of spores
expressing SspC N-terminal deletion variants. Spores of various
strains were purified, and their resistances to UV radiation at 254 nm
(panel A) and heat at 85 °C (panel
B) resistance were determined as described under
"Experimental Procedures." The following spores were tested:
SspC (
),
SspC
5
(
),
SspC
10 (
),
SspC
11
(
),
pUB110 (no
/
-type SASP,
).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
-type SASP. However, SspC N-terminal deletion variants do
show reduced binding affinity for DNA, indicating that the deleted
residues contribute significantly to the SspC-DNA binding interaction.
Our inability to overexpress shorter SspC N-terminal deletion variants,
and the failure of the GPR C-terminal fragment of SspC to bind DNA,
suggest that proteins smaller than SspC
14 do
not interact with DNA very strongly, and are presumably rapidly degraded in vivo. Additionally, it appears that the N
terminus of wild-type SspC is considerably flexible because protein
cross-linking demonstrated that the N termini of the SspC N-terminal
variants all make similar close contacts with acidic residues on
adjacent DNA-bound proteins. Indeed, heterotypic cross-links have also been identified between SASP-A and SASP-C from B. megaterium, whose N termini vary greatly in length (4, 11). These
conclusions are consistent with the variability in N-terminal length
and sequence seen in the amino acid alignment of all identified
/
-type SASP; in fact, the first significantly conserved amino
acid residue in wild-type SspC is asparagine 12 (Fig. 1). However,
although not conserved or required for DNA binding, residues glutamine 2 through asparagine 11 in wild-type SspC are important in that they
significantly increase the affinity of this
/
-type SASP for DNA.
14 binds to DNA with higher affinity
than SspC
10,
SspC
11, and the large Asp-N fragment of
SspC. The DNA binding affinity of these proteins seems to be related to
the net charge in the N-terminal regions. These findings, in
conjunction with the EDC cross-linking data showing that the N terminus
of each protein is in close contact with negatively charged residues on
adjacent DNA-bound protein (11), may be explained by a model of
DNA-dependent protein-protein interaction which involves a
significant electrostatic interaction. According to this model, a
flexible and positively charged N terminus from each DNA-bound protein
interacts with a proposed acidic patch (formed by aspartate 13 and
glutamates 29 and 33) found on a neighboring DNA-bound protein (Fig.
7). If the N terminus is arbitrarily
defined as the first five amino acid residues, the net charge of the N
terminus of wild-type SspC at pH 7.0 is +2, and is conferred by
arginine 5 and the
-amino group (Fig. 1). By the same criteria, most
of the N-terminal deletion variants of SspC have a less positively
charged N terminus: SspC
5 is +2,
SspC
10 is 0, SspC
11 is 0, Asp-N C-fragment is 0, and
SspC
14 is +1 (Fig. 1). These changes in
N-terminal charge are due to the removal of arginines 5 and 7, and the
increasing proximity of aspartate 13 to the N terminus as amino acid
residues are deleted from wild-type SspC (Fig. 1). In particular, the
presence of the negatively charged aspartate 13 residue at the N
terminus (in SspC
10,
SspC
11 and the Asp-N fragment of SspC)
appears to interfere with protein-protein interaction and therefore
with DNA binding affinity, because when this residue is removed (along
with leucines 14 and 15) in SspC
14, the DNA
binding affinity is significantly increased. Therefore, we hypothesize
that electrostatic repulsion between the now N-terminal aspartate 13 and the acidic patch on an adjacent DNA-bound
SspC
11 (or
SspC
10) contributes to the lower DNA binding
affinity. Consistent with this model, derivatives of
SspC
11 that contain either an asparagine or
a lysine residue in place of aspartate 13 have significantly higher
affinity for DNA than SspC
11.
View larger version (22K):
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Fig. 7.
Model of SspC protein-protein contacts in the
DNA-bound state. Unbound SspC are depicted as random coil peptide
chains that interact with one another and the DNA binding site for
productive binding to occur (6). Significant structural rearrangements
occur during binding (DNA-bound SspC are represented as ovals), and
protein-protein interactions between the positively charged N-terminal
region (labeled + and N+) and a proposed negatively charged
patch are shown.
The model presented above (Fig. 7) is probably applicable to all
/
-type SASP; the evidence for this is as follows. First, DNA-dependent EDC cross-links identical to those found in
SspC have been identified in three other
/
-type SASP from
different species (11). Second, higher
/
-type SASP-DNA binding
affinity roughly corresponds to a net positively charged N terminus (5, 6, 11). Third, deamination of SASP-A and SspC and its variants reduces
the N-terminal net positive charge and results in significantly lower
DNA binding affinity. Of course, we cannot exclude the possibility that
the N-terminal
-keto group in deaminated
/
-type SASP actually disrupts binding interactions. However, the electrostatic component of
/
-type SASP protein-protein interactions has been shown to be
thermodynamically significant because the cooperativity parameter (
)
of the
/
-type SASP-DNA interaction decreases with increasing salt
concentration (6), suggesting that DNA dependent protein-protein interactions are destabilized by salt.
Although the deletion of 10 amino acid residues from the N terminus of
SspC resulted in a protein (SspC10) that
showed significantly lower affinity for DNA in vitro, it was
still able to confer almost full resistance to UV radiation and
significant protection from heat to
spores. This is not particularly surprising because SASP-
, the
/
-type SASP in B. subtilis most responsible for spore
UV resistance, has a much lower affinity for DNA than does wild-type
SspC (5, 7). In fact, it is thought that high affinity for DNA is not necessary for
/
-type SASP function because the major
/
-type SASP are present at very high concentrations (~1-2 mM)
within the spore core (4). At these concentrations, ~80-90% of
/
-type SASP would be DNA-bound at binding constants as low as
1.0-2.0 × 104 M1. In
addition, the spore core is very dehydrated compared with the
corresponding cell cytoplasm, and most of the divalent cations are
probably chelated by the enormous level of dipicolinate in the spore
core (1); both of these spore core environmental factors should tend to
favor
/
-type SASP-DNA interaction. However, although
SspC
11 has a similar or even higher affinity
for DNA in vitro compared with
SspC
10, it was less able to confer UV and
heat resistance to
spores. The
reason for this is not clear, but may be due to the removal of the very
highly conserved asparagine 12 residue, which is found in all
/
-type SASP identified from Bacillus species (4, 31).
Perhaps this conserved asparagine residue plays an significant
structural role in vivo.
It appears that the function of amino acid residues glutamine 2 through
asparagine 11 in wild-type SspC is to increase the DNA binding affinity
of the protein. Although this increased affinity is not necessary for
/
-type SASP function in vivo, it may reflect the
actual role SspC plays in wild-type (
+
+)
spores. SspC is a minor
/
-type SASP and is only present as ~10% of total
/
-type SASP within wild-type B. subtilis spores (4), with SASP-
and SASP-
comprising most
(~80%) of the remaining
/
-type SASP (18). SspC has a higher
affinity for DNA than both SASP-
and SASP-
, and therefore SspC
may have been selected to bind regions of the spore chromosome that are
not bound efficiently by SASP-
or SASP-
. Although there is no
significant decrease in spore heat or UV radiation resistance in the
laboratory when the sspC gene is inactivated, the role for
SspC proposed above could confer a selective advantage to spores. This
proposed function for SspC may be generalizable because all
Bacillus examined to date contain a number of minor
/
-type SASP (4, 31).
The results in this report also have practical value in aiding in the
designing of a minimal high affinity /
-type SASP for biophysical
studies. Because all
/
-type SASP are largely unstructured in the
absence of double-stranded DNA (6), a well defined
/
-type SASP-oligonucleotide complex will be required to obtain a high resolution structure. This complex should be small to avoid potential problems with degeneracy arising from the nonspecific nature of
/
-type SASP-DNA binding. In addition, only small complexes (<25 kDa) are routinely tractable by multidimensional NMR methods. For these
reasons, the identification of minimal
/
-type SASP that maintain
high affinity for DNA is desirable. This study indicates that ~1.2
kDa of SspC may be removed while retaining DNA binding, although there
is an attendant loss of binding affinity. However, we have been able to
produce a truncated
/
-type SASP,
SspC
11-D13K, which binds to DNA with higher
affinity than even wild-type SspC. Small, double-stranded
oligonucleotides that bind to
/
-type SASP have previously been
identified by CD spectroscopic studies (6), and these oligonucleotides
are currently being tested with SspC
11-D13K
in an attempt to determine the solution structure of an
/
-type SASP-DNA complex by multidimensional NMR studies.
![]() |
ACKNOWLEDGEMENT |
---|
All mass spectrometry was performed by Dr. John Leszyk at the Laboratory for Protein Microsequencing and Mass Spectrometry at the University of Massachusetts School of Medicine.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM19698.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Dept. of Biology, Massachusetts Institute of
Technology, Cambridge, MA 02139.
§ Current address: Dept. of Biochemistry, Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional, Mexico City, Mexico 11340.
¶ To whom correspondence should be addressed. Tel.: 860-679-2607; Fax: 860-679-3408; E-mail: setlow@sun.uchc.edu.
Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M007858200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
/
-type SASP,
/
-type small acid-soluble spore protein(s);
bp, base pair(s);
CD, circular dichroism;
DNase I, deoxyribonuclease I;
EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide;
GPR, germination
protease;
K, intrinsic binding constant;
MALDI, matrix-assisted laser desorption/ionization;
TOF, time of flight;
nt, nucleotide(s);
[
], mean residue ellipticity;
, cooperative
binding factor;
HPLC, high performance liquid chromatography;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain reaction;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
---|
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