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(Received for publication, August 17,
1995; and in revised form, September 29, 1995) From the
The herpes simplex virus type 1 protease is expressed as an
80,000-dalton polypeptide, encoded within the 635-amino acid open
reading frame of the UL26 gene. The two known protein substrates for
this enzyme are the protease itself and the capsid assembly protein
ICP35 (Liu, F., and Roizman, B.(1991) J. Virol. 65,
5149-5156). In this report we describe the use of a rapid and
quantitative assay for characterizing the protease. The assay uses a
glutathione S-transferase fusion protein containing the
COOH-terminal cleavage site of ICP35 as the substrate (GST-56). The
protease consists of N
The existence of a herpesvirus-specific protease was first
reported in 1991, when the protein encoded by the UL26 gene of herpes
simplex virus I (HSV-1) ( In early studies involving
expression of recombinant protein in Escherichia coli, the
HSV-1 protease was shown to cleave two distinct sites, both contained
within the protease polypeptide itself (6, 7) . Both
occur between Ala and Ser residues, at positions 247 and 248 and
positions 610 and 611(7) . This specificity is shared by the
CMV enzyme(2, 3) . The carboxyl-terminal cleavage
occurs at a site common to both the protease and a capsid scaffolding
protein ICP35 (M site) ( A major barrier in the study of
the HSV-1 protease has been the extremely low activity level of the
enzyme when assayed in vitro. In one study, DiIanni et al.(16) surveyed several peptide substrates and reported k
Following the purification of an active form
of the protease(12) , a need arose for a rapid and quantitative
assay for activity. An acid solubilization assay was originally
developed in which metabolically labeled
[
Figure 1:
Schematic of the fusion protein
substrate GST-56, depicting GST (light box) and M site 56-mer
peptide (dark box) domains. Cleavage of the 56-mer by the
HSV-1 protease releases a TCA-soluble 25-mer peptide containing two of
the protein's twelve methionines.
Figure 2:
HSV-1 protease assay, showing the salt
dependence for cleavage of 4 µM
[
In parallel studies using the homologous
protease from HCMV, a similar stimulation was seen (Table 1).
HSV-1 N
The stimulation of
HSV-1 N
Figure 3:
Comparison of different salts on the
stimulation of the HSV-1 protease. Assays were performed as described
under ``Materials and Methods'' using 1 µM [
Given the lack of
activity by NaCl, additional antichaeotrophic or salting out agents (21, 22) were examined (Table 3). None of the
chlorides had any effect, even when added at 2.5 M. However,
all of the antichaeotrophs stimulated activity, in parallel with the
lyotrophic series of anions, the most potent being the phosphates,
followed by the sulfates and the more weakly antichaeotrophic acetates.
KPO
In addition to salts, several other solutes that
stabilize proteins were examined to look for HSV-1 protease
stimulation(23) . Sucrose at 34% and glycerol at up to 62%
produced marginal, if any stimulation, whereas results using larger
polymers were inconclusive. Ficoll 400 and dextran sulfate rendered the
solutions too viscous to permit pelleting of the substrate in the TCA
assay, whereas polyethylene glycols (400-8000 molecular weight
range) precipitated everything. However, when activity was measured
using the coupled translation/cleavage assay, polyethylene glycols of
average molecular weights 600-3350 produced a slight stimulation when
present at 10-20% (results not shown).
Figure 4:
Fluorescence emission spectra of 0.5
µM HSV-1 N
If the 465 nm fluorescence were due to the specific binding
of ANS to the substrate binding pocket of the protease, it might be
possible to quench this fluorescence through competition for this site
by a specific protease ligand. For this purpose, the R cleavage site
peptide YLQASEKFKMWG (rP4-P8`), previously shown to inhibit
[ In the initial characterization of the HSV protease, an in vitro translation and cleavage assay (1) surprisingly revealed that activity could be greatly
stimulated by high concentrations of Na The
above findings led to the question of whether the solvent effect might
be on the substrate or the enzyme. The substrate was initially examined
using spectroscopic methods to probe secondary structure. The 56-mer
domain of GST-56, which parallels authentic ICP35 in its cleavage
behavior, was isolated and purified under native conditions and then
submitted to analysis by circular dichroism and NMR spectroscopy. In
physiological concentrations of salt, the peptide was primarily random
coil. We were unable to obtain spectra in the concentrations of
Na The effects of salt
on the protease were also examined. Peptide cleavage experiments (not
shown) suggested that the K To examine the
possibility that ANS was binding to the active site of N A consequence of our above results is the question of just why a
protease should be stimulated by unusually high concentrations of
antichaeotrophic salts. One explanation involves the local environment
of the protease during cleavage. Recent work on the assembly and
maturation of the HSV-1 capsid suggests that the uncleaved form of
ICP35 (i.e. containing the COOH-terminal 25 amino acids) is
required for the formation of ``sealed''
capsids(29, 30, 31) . Because the processed
form of this protein is the predominant form found when immature B
capsids are isolated from infected
cells(32, 33, 34, 35) , cleavage by
the protease most likely occurs within the capsid. Furthermore, based
on the ultrastructure and protein stoichiometry calculations of Brown
and colleagues(36, 37) , a major fraction of a B
capsid volume can be accounted for by ICP35 protein. This suggests that
the water activity (38) must be very low, a state that is
perhaps approximated in an in vitro assay by the addition of
molar concentrations of an antichaeotrophic salt.
Parts of this work were presented at the 18th International
Herpesvirus Workshop, Pittsburgh, PA, July 25-30, 1993.
Volume 270,
Number 50,
Issue of December 15, 1995 pp. 30168-30172
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, the NH
-terminal 247
amino acid catalytic domain of the UL26 gene product, also expressed as
a GST fusion protein. Upon cleavage with N, a single 25-mer
peptide is released from GST-56, which is soluble in trichloroacetic
acid. Using this assay, the protease displayed a pH optimum between 7
and 9 but most importantly had an absolute requirement for high
concentrations of an antichaeotrophic agent. Strong salting out salts
such as NaSO
and KPO (
1 M) stimulated activity, whereas NaCl and KCl had no effect.
The degree of stimulation by 1.25 M NaSO and KPO were 100-150- and 200-300-fold,
respectively. Using the fluorescent probe 1-anilino-8-naphthalene
sulfonate, the protease was shown to bind the dye in the presence of
1.25 M NaSO or KPO, but
not at low ionic strength or in the presence of 1.25 or 2.2 M NaCl. This binding was most likely at the protease active site
because a high affinity cleavage site peptide, but not a control
peptide, could displace the dye. In addition to cleaving GST-56, the
herpes simplex virus type I protease also cleaved the purified 56-mer
peptide. Circular dichroism and NMR spectroscopy showed the peptide to
be primarily random coil under physiological conditions, suggesting
that antichaeotrophic agents affect the conformation of the substrate
as well as the protease.
)was shown to process both itself
and the precursor form of a capsid scaffolding protein ICP35 ((1) ; also known as VP22a, p40). Shortly afterwards, other
reports demonstrated the existence of similar proteases in simian (2) and human (3) cytomegaloviruses (CMV). These
processing systems are reminiscent of those in bacteriophage, whereby a
specific phage protease processes a scaffolding protein during capsid
maturation(4, 5) .)and releases a 25-residue,
COOH-terminal peptide. ICP35 was recently shown to be a product of the
UL26.5 gene and to be in-frame and entirely contained within the
carboxyl-terminal 329 amino acids of the protease(9) .
Functionally, the processing of ICP35 by the protease appears to be an
essential viral event, because a ts mutant that fails to
process ICP35 at the nonpermissive temperature also fails to package
DNA(10) . Cleavage at the site proximal to the amino terminus
(R site) results in the release of N, an
NH-terminal, 247-residue polypeptide that contains the
proteolytic activity(11, 12) . This processed form of
the protease (also known as Prn or VP24) is known to be a constituent
of the viral capsid (13) and has been shown to be a serine
protease(14, 15) . and K
values of
0.2 min
and 190 µM, respectively, for
the cleavage of ALVNASSAAHVDV (M site peptide mP5-P8`). In a subsequent
study, Darke et al.(17) reported k
and K
values of 2.0
min
and 0.88 mM, respectively, for the
cleavage of the R site peptide HTYLQASEKFKMW-amide (rP6-P7`) and about
twice the activity as DiIanni et al.(16) for mP5`P8`.
In the present report we make use of a quick and sensitive assay for
the HSV-1 protease to characterize a unique interaction with
antichaeotrophic salts. Most significantly, we find that molar
concentrations of Na
SO result in changes to
both the protease and substrate and stimulate activity over 100-fold.
Protease Purification
An active form of
the HSV-1 protease was expressed from pGST247 in E. coli BL21(DE3) as a fusion protein of glutathione S-transferase coupled to the NH-terminal
247-residue domain of the UL26 gene product. The fusion protease
(GST-N) and the thrombin-released form (N) have
been described(12, 16) . The homologous protease from
human CMV (HCMV N) has also been described(18) .ICP35 Purification
Recombinant ICP35 was
purified from E. coli BL21(DE3) transformed with the plasmid
pT7ICP35 (6) as described by Weinheimer et
al.(12) . In brief, cells were lysed and then clarified by
centrifugation. The supernatant was successively fractionated with 25%
of saturation of (NH)SO, 1.7 M KCl, and chromatographed over DEAE Sepharose (Pharmacia Biotech
Inc.). [S]ICP35 was prepared from bacteria
metabolically labeled with [
S]methionine (DuPont
NEN). Cells were grown in minimal salts, supplemented with 0.2% glucose
and 2.5% methionine assay medium (Difco Laboratories). Following
induction with 1 mM
isopropyl-1-thio-
-D-galactopyranoside, cells were washed
and incubated with 0.2 mCi/ml [S]methionine in
1/10 the original volume for 3 h. The labeled protein was purified
using the above protocol, except that the chromatography step was
omitted. Due to the selective incorporation of the
[
S]methionine into ICP35 under the induction
conditions used, the protein was
90% radiochemically pure and
50% pure by protein weight.
GST Fusion Proteins
The HSV-1 fusion
protein substrate GST-56 was constructed by using a double-stranded DNA
fragment amplified from pT7HSV (6) by the polymerase chain
reaction. A fragment spanning amino acids 580-635 (mP31-P25`) was
generated with flanking BamHI and EcoRI restriction
enzyme sites and cloned into pGEX-2T (Pharmacia). The homologous HCMV
fusion protein substrate (cGST-51) was similarly constructed using a
fragment spanning amino acids 618-667 of HCMV UL80 (mP26-P25`).
Following expression in E. coli, the fusion proteins were
purified by glutathione affinity chromatography. Cells were lysed and
clarified as described (12) , with the exception that the
initial lysis buffer contained 1 mM phenylmethylsulfonyl
fluoride. The supernatant was adsorbed to a column of glutathione
agarose (Sigma), washed with 50 mM Tris-HCl, pH 8.0, 1
mM DTT, and eluted in the same buffer containing 5 mM reduced glutathione. Peak fractions were pooled, dialyzed
extensively to remove glutathione, and stored at -80 °C in
the above buffer containing 40% glycerol. Radiolabeling in E. coli with [S]methionine was carried out as
described for [
S]ICP35, and the proteins were
purified over glutathione agarose.
Preparation of the HSV-1 ICP35 Cleavage Site 56-mer
Peptide
Purified mP31-P25` 56-mer was obtained from GST-56
following treatment with thrombin. The reaction contained 2 mg/ml
GST-56, 150 mM Tris-HCl, pH 8.0, and 8 units/ml bovine
thrombin (Sigma). After 2 h at 30 °C, the material was adsorbed to
a MonoQ column (Pharmacia) equilibrated in 50 mM Tris-HCl, pH
8.0, and then eluted with a linear gradient of 0-400 mM NaCl. The 56-mer eluted at 75 mM NaCl, whereas the
GST eluted at 225 mM. Purified 56-mer appeared as a single
band when analyzed by HPLC. When analyzed on a 14% SDS gel, it migrated
as a diffuse 7000-dalton protein with specific immunoreactivity against
peptide antibodies raised against UL26 sequences (599-620 and
611-626) flanking the M cleavage site.
[
S]56-mer was purified by the same procedure,
using [
S]GST-56 as starting material.
Protease Assays
Protease activity was
measured in a 30-µl reaction containing 50 mM Tricine, pH
8.0, 10 mM DTT, 100 µg/ml bovine serum albumin, 1.25 M NaSO, and the indicated radiolabeled
substrate. Reactions were initiated by the addition of protease,
incubated at 30 °C for 1 h, and then quenched with 100 µl of
10% trichloroacetic acid. Following centrifugation, the supernatant was
removed, and the radioactivity was quantitated by liquid scintillation
counting. In some assays, the polypeptides were examined directly.
Following removal of the supernatant, the pellet was solubilized in SDS
sample buffer and analyzed on a 12% SDS gel followed by autoradiography
or quantitation on a Betascope 603 Blot Analyzer (Betagen Corp).In Vitro Transcription and
Translation
UL26 RNA was transcribed in vitro from
pRB4090 (plasmid U in (1) ) using SP6 RNA polymerase (Life
Technologies, Inc.). This RNA was then purified and translated for 20
min in a reticulocyte lysate system (Promega) in the presence of
[S]methionine. The reaction was quenched by the
addition of cycloheximide (100 µg/ml) and then diluted 20-fold into
a test buffer (see ``Results''). Protease reactions were
typically incubated for 6 h at 30 °C and then quenched by the
addition of 10% TCA. Pellets were washed once with 100% ethanol,
solubilized in SDS sample buffer, and analyzed on 12% SDS gels.
Other Methods
Protein concentration was
measured using the method of Bradford(19) . SDS gel
electrophoresis was performed using 12 or 14% Laemmli gels (20) and then either stained with Coomassie Blue R250 or
transferred to nitrocellulose for immunoblot analysis with alkaline
phosphatase detection. Reverse phase HPLC analysis of peptides was on a
Vydac C4 column, using an acetonitrile gradient in 0.1% trifluoroacetic
acid. Analysis was with a Waters 991 Diode Array system and Radiomatic
A-250 radioactivity detector (Camberra). Fluorescence emission spectra
were recorded on a Spex Fluorolog-2 recording spectrofluorometer.
Spectra were recorded at 90 ° in a 200-µl fused silica micro
cell using an excitation wavelength of 280 nm and slit settings of 2
nm.
Development of an in Vitro Protease
Assay
Initial studies on the HSV-1 protease were performed
using the coupled in vitro translation/cleavage assay
described by Liu and Roizman(1) . UL26 RNA was translated as
described under ``Materials and Methods'' and then diluted
into buffers of varying composition for 6 h to allow cleavage to occur.
The protease exhibited maximal activity between pH 7 and 9; it was
virtually inactive below pH 6 or above 10. Surprisingly, it was
potently stimulated by buffers containing high concentrations of
NaSO or KPO but not KCl. (
)This last finding was essential for the development of the
assay described below.S]ICP35 could be cleaved by GST-N
to release a COOH-terminal, 25-amino acid peptide. This peptide
was soluble in 10% trichloroacetic acid. Because it contained two of
the protein's seven methionines, it could be easily quantitated
by liquid scintillation counting. An adaptation of this assay was to
use a fusion protein substrate in which the COOH-terminal 56-residue
peptide of ICP35 was fused to the COOH-terminal end of glutathione S-transferase (GST-56, Fig. 1). This protein was
expressed at high levels in E. coli and could be purified in
one step by glutathione agarose affinity chromatography in yields of
20-30 mg/liter of culture. The purified protein was free of
nonspecific proteolytic activity and was stable to prolonged
incubations at 30 °C. When purified from metabolically labeled
cells, [S]IGST-56 was obtained at >95% purity
with yields of 0.15 mCi of labeled protein/mCi of labeled culture
(15-fold greater yield than [
S]ICP35).
Effect of Na
Using the assay
described above, the salt stimulation of HSV-1 protease seen in the in vitro translation/cleavage assay was re-examined.
NaSOon HSV-1 Protease ActivitySO was initially selected for detailed study
because it potently stimulated HSV-1 protease activity in the
translation assay and avoided the buffering and chelating properties of
phosphate. Fig. 2A shows a titration of
NaSO into an assay for
[S]GST-56 cleavage by GST-N
. The
activity was virtually undetectable in the absence of
NaSO and increased only slightly upon the
addition of up to 750 mM. However, at 1.0 M and 1.25 M NaSO, the activity was about 75- and
150-fold greater than the level seen in the absence of this salt. When
the polypeptide products of the above cleavage reactions were analyzed
by SDS gel electrophoresis and autoradiography, the disappearance of
the [S]GST-56 polypeptide corresponded to the
appearance of a single cleavage product (Fig. 2B).
Furthermore, when these bands were quantitated on a two-dimensional
radioactivity scanner, the decrease in [
S]GST-56
radioactivity was closely proportional to the increase in that of its
cleavage product (GST-31, Fig. 2C). A similar salt
dependence was seen when the assay was performed using
[
S]ICP35 as substrate or when N
was
used in place of GST-N (data not shown). Due to the limited
solubility of the proteins at high concentrations in 1.25 M
NaSO, reliable kinetic analysis was not
possible. Initial studies indicated a K
> 10
µM and k =
1
min
.
S]GST-56 by 0.3 µM GST-N
. A, release of the COOH-terminal,
25-mer peptide, as measured by liquid scintillation counting. B, analysis of the polypeptide cleavage by SDS gel
electrophoresis and autoradiography. Lane B, no enzyme was
present. C, quantitation of the polypeptides shown in B by two-dimensional radioactivity
detection.
and HCMV N were stimulated 60- and
130-fold, respectively, by 1.25 M NaSO when using saturating levels of radiolabeled fusion protein
substrates. Under these conditions, the HCMV enzyme was about an order
of magnitude more active that the HSV-1 enzyme.
activity by NaSO was
further examined using a peptide substrate.
[S]GST-56 from metabolically labeled cells was
treated with thrombin to produce GST and a 60-residue peptide. The
peptide, containing the 56-mer fused to a thrombin site linker
(Gly-Ser-Pro-Met) at its NH
terminus, was purified under
nondenaturing conditions. When assayed with HSV-1 N in the
presence of 0, 0.5, and 1.25 M NaSO and analyzed by HPLC, cleavage was seen only in the presence of
NaSO (Table 2). The appearance of a
single radiolabeled proteolysis product (13.3 min) was consistent with
the occurrence of a single cleavage event in a peptide where the only
two methionines were at the extreme COOH terminus. However, when the
56-mer was examined for secondary structure, by either circular
dichroism (in 100 mM KPO, pH 6.7) or NMR
spectroscopy (25 mM sodium acetate, pH 5.5), it exhibited
spectra characteristic of random coil (results not shown). Spectra were
not obtainable in 1.25 M NaSO.
HSV-1 Protease Is Stimulated by Antichaeotrophic
Agents
The initial finding that NaSO and KPO but not KCl stimulated protease activity in
the translation assay was reproduced using purified N in
the in vitro TCA precipitation assay (Fig. 3, note
replacement of KCl by NaCl). Due to the limited solubility of
NaSO, an attempt was made to see whether the
more highly soluble antichaeotrophic salts,
(NH)SO or KPO, would
further stimulate the protease at higher concentrations (Fig. 3). Again, both salts greatly stimulated protease
activity, with maxima in the 1.25-2.0 M range. The
potency was greatest for KPO, which produced twice the peak
activity level of either NaSO or
(NH)SO.
S]GST-56 and 0.1 µM N
. Indicated salts were present in the assays at the
concentrations shown: NaSO (
), KPO (
), (NH)SO (
),
and NaCl ().
and (NH)PO were about 75% more
potent than NaSO. (NH)SO and MgSO were about 3-fold less potent, whereas
guanidine sulfate was nearly inactive. Among the acetates, only
Mg(OAc) showed activity at 1.25 M. However,
because this salt contains two acetate anions/mol, the other acetates
were assayed at 2.5 M. All showed weak to moderate
stimulation.
Fluorescence Emission Analysis of HSV-1
N
To learn whether or not the protease
stimulation by antichaeotrophic agents was due to a conformational
change in the vicinity of the substrate binding pocket, the chromophore
reagent 1-anilino-8-naphthalene sulfonate (ANS) was used to probe the
protein's topology. ANS is known to be nearly nonfluorescent in
aqueous buffers but to partition into hydrophobic sites within
proteins, resulting in the appearance of a strong emission band around
450 nm(24) . Fig. 4A, shows the fluorescence
emission spectra of N in 50 mM Tricine, pH 8.0,
either in the presence or absence of ANS. In both instances, the major
feature was a broad emission peak at 340 nm, attributable to the
protease's two tryptophan residues. When a similar experiment was
performed in the same buffer containing 1.25 M
Na
SO, the addition of ANS produced a second
fluorescent peak, centered at 465 nm (Fig. 4B). This
second peak was not due to N itself but rather to ANS. It
was not present when ANS alone was examined in
NaSO, and was not a general phenomenon of
protein and salt because (i) it was not seen when carbonic anhydrase
was used in place of N, and (ii) it was not present when
N and chromophore were examined in 1.25 M or 2.2 M NaCl (Fig. 4C). However, when N was examined in the presence of ANS in the antichaeotrophic salt
KPO, the 465 nm fluorescence peak was again present (not
shown).
± 10 µM ANS in
buffers of varying composition. A, 50 mM Tricine, pH
8.0. B, 50 mM Tricine, pH 8.0, 1.25 M
NaSO. C, 50 mM Tricine, pH
8.0, 2.2 M NaCl. D, same conditions as B. In
the experiment denoted by the dotted curve 50 µM
rP4-P8` was added to the reaction.
S]GST-56 cleavage (IC
=
5-10 µM)
, was used as a competitor. In Fig. 4D, the addition of rP4-P8` to a solution of
N and ANS in 1.25 M NaSO buffer resulted in a large increase in emission from tryptophan
(peptide Trp at P7`) but a decrease of the 465 nm peak. Such an effect
was not seen using a control peptide, ASNAEAGALVNAS (mP12-P1`),
previously implied to be noncleavable (16) and shown to be
noninhibitory. Thus, the presence of an antichaeotrophic
salt results in the formation of a hydrophobic area on the protease,
possibly the substrate binding site, which is not present at low ionic
strength or at high concentrations of a neutral salt.
SO and
KPO but not KCl. This finding was confirmed in a more
defined system, using purified proteins for both the protease and
substrate. Most striking, however, was the requirement for molar
concentrations of an antichaeotrophic salt; little effect was seen at
physiological ionic strength. In addition, NaCl did not affect activity
between 0 and 2.5 M (Fig. 3). These studies were
extended by surveying a variety of salts (Table 3), spanning the
lyotropic series of anions and cations. Most conclusive was the finding
that the potency of stimulation paralleled the order of stabilizing
anions, which preferentially hydrate
proteins(21, 22) : chloride acetate < sulfate
< phosphate. The effect of cations was less pronounced. Sodium ion
appeared to be slightly more potent than ammonium, but guanidine, which
binds tightly to protein(25) , was inhibitory. Several other
reagents known to stabilize proteins were also examined. Glycerol (26) and sucrose(27) , both known to preferentially
hydrate proteins, were ineffective. This was somewhat surprising,
because glycerol was earlier shown to stimulate the activity of the
related HCMV protease(28) , which behaved like the HSV-1 enzyme
toward Na
SO. Polyethylene glycols, which have a
more pronounced effect on water activity, were potent precipitants in
the TCA precipitation assay. They did, however, slightly stimulate
activity in the in vitro translation/cleavage assay.SO or KPO required for optimal
cleavage by the protease. Nevertheless, these studies suggested that
one role of an antichaeotrophic agent might be to induce some unique
feature of secondary structure into the cleavage site region. Earlier
studies on peptides suggested a requirement for secondary structure on
the P` side of the scissile bond(16) . for substrates
decreased when assays were performed in high concentrations of
NaSO. This led to the notion that the effect
might involve changes in the structure at the active site. For this
purpose, the fluorescent dye ANS was used to probe protease topology.
Stryer (24) showed that it bound to apomyoglobin with a
dissociation constant of 10
M but not
to myoglobin. Furthermore, it could be displaced from the apoprotein by
the addition of hemin. When added to solutions of HSV-1 N
,
ANS bound protein only in the presence of 1.25 M
NaSO or KPO but not NaCl (Fig. 4). These conditions paralleled those required for optimal
activity, suggesting that a conformational change had occurred upon
introduction of these antichaeotrophic salts. In contrast, carbonic
anhydrase did not bind ANS under any conditions., a
high affinity substrate peptide, rP4-P8`, was used as competing ligand.
Addition of this peptide to the ANS bound protein solution resulted in
a decrease in ANS fluorescence, consistent with a model that the
salt-induced hydrophobic site was indeed the substrate binding site.
)
))-SSAA will be denoted
mP4-P4`, etc.
)
We thank Dr. William J. Metzler and Valentina Goldfarb
for the NMR studies and Dr. Harry Brittain for the CD studies. We also
thank Dr. Fenyong Liu for help and advice during the initial
translation/cleavage studies and Mr. Patrick McCann III for critical
reading of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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